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

* In Have We “Met the Enemy”?, Science 3 February 2012: Vol. 335 no. 6068 pp. 540-541, Dr. David Relman, who had been vice-chairman of the NAS Committee, explains:

Posted by DXer on March 5, 2012


David Relman


“By 2010, when the Amerithrax investigation was closed by the U.S. Department
of Justice, there were two components of the case: the possible linkage
between the material in the letters and a flask of B. anthracis spores at the
U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) in
Fort Detrick, Maryland, and the assessment about who might have used the
contents of the flask to prepare and send the letters. Had the case gone to
trial, the strengths of the two components would have been weighed and
integrated by the prosecution and then challenged by the defense. But
because the lead suspect, Bruce Ivins (a microbiologist at USAMRIID),
committed suicide, we do not have the benefit of court proceedings. Without
such, we must refrain from trying this man in the courtroom of public


In early 2011, the U.S. National Academy of Sciences released the results of
a 20-month review of the science used to build the first component of the
case along with nearly 10,000 pages of supporting documents previously held
by the Department of Justice (1). The National Research Council (NRC)
committee found that it is impossible to arrive at a definitive conclusion
about the origin of the letter spores based on the available scientific
evidence alone. The scientific data generated by and on behalf of the
Federal Bureau of Investigation (FBI) provided leads as to a possible source
(flask RMR-1029) but did not rule out other sources. In contrast, the
Department of Justice’s Amerithrax Investigative Summary states conclusively
that the flask was the source of the spores in the letters.  …


There were problems with the representativeness
of the FBI repository of B. anthracis samples against which the letter
material was compared. The FBI failed to aggressively pursue an alternative
explanation: that some of the mutations in the letters might have arisen by
parallel evolution rather than by derivation from the flask. Similar kinds
of mutations are known to occur with large-scale production of anthrax
spores using bulk serial passaging (1). Guillemin also ignores other NRC
criticisms, including questions about the reliability of the genetic assays
used to look in the repository for the mutations found in the letters. In
addition, she describes some of the details of the science-based
investigation incorrectly, such as the number of positive samples from a
clandestine effort to assess a possible overseas source of the spores and
the number of collection missions that yielded positive samples. Although
seemingly minor, these incorrect descriptions of the scientific findings may
lend credence to the Department of Justice’s overstated conclusions …”


63 Responses to “* In Have We “Met the Enemy”?, Science 3 February 2012: Vol. 335 no. 6068 pp. 540-541, Dr. David Relman, who had been vice-chairman of the NAS Committee, explains:”

  1. DXer said

    There are many distinguished people who should guide consideration of the Amerithrax whodunnit. As Senator Leahy has said, “the case is not closed.”

    For starters, let’s see what we learn from KSM and Hambali’s trial next year at Guantanamo. They were both involved in Dr. Ayman Zawahiri’s anthrax program.

  2. DXer said

    “For example, we ultimately found the crude laboratory in Kandahar, Afghanistan, where Pakistani biologist Rauf Ahmed stashed equipment he had ordered to Ayman Zawahiri’s specifications. We went to the hospital laboratory in Kandahar where Malaysian “Anthrax CEO” Yazid Sufaat claimed to have isolated a virulent form of bacillus anthracis. There, a joint FBI-CIA-military team collected forensic samples and evidence of biological weapons-related activity, precisely as Sufaat had claimed under interrogation by Malaysian authorities.”

    How to Get Terrorists to Talk
    A former CIA interrogator on the “do’s” and “don’ts” of interrogation.
    Rolf Mowatt-Larssen
    February 18, 2015

  3. DXer said

    Anthrax detection: Analyses and select studies of sampling methods (with accompanying DVD) (2013)

    Arnaud, C.M.

    View references (47)


    A workgroup, led by the U.S. Department of Homeland Security (DHS) and including the Centers for Disease Control and Prevention (CDC), the Environmental Protection Agency (EPA) and the Federal Bureau of Investigation (FBI), have taken actions to validate environmental sampling methods for detecting Bacillus anthracis-the bacteria that cause anthrax. This book provides an overview of select studies in sampling methods and analyses with regard to anthrax detection. © 2013 by Nova Science Publishers, Inc. All rights reserved.

  4. DXer said

    Application of High-Throughput Sequencing: Discovery of Informative SNPs to Subtype Bacillus anthracis Guillaume Girault, Simon Thierry, Emeline Cherchame, Sylviane Derzelle Affiliation(s)

    University Paris-Est, ANSES, Animal Health Laboratory, Bacterial Zoonoses Unit, Maisons-Alfort, France.


    Single Nucleotide Polymorphisms (SNPs) are the most common and abundant genetic variation found in the genome of any living species, from bacteria to humans. In bacterial genotyping, these evolutionarily stable point mutations represent important DNA markers that can be used to elucidate deep phylogenetic relationships among worldwide strains, but also to discriminate closely related strains. With the advent of next generation sequencing (NGS) technologies, affordable solutions are now available to get access to the complete genome sequence of an organism. Sequencing efforts of an increasing number of strains provide an unprecedented opportunity to create comprehensive species phylogenies. In this study, a comparative analysis of 161 genomes of Bacillus anthracis has being conducted to discover new informative SNPs that further resolves B. anthracis SNP-based phylogenetic tree. Nine previously unpublished SNPs that define major groups or sub-groups within the B. anthracis species were selected and developed into SNP discriminative assays. We report here a cost-effective high-resolution melting-based genotyping method for the screening of 27 canonical SNPs that includes these new diagnostic markers. The present assays are useful to rapidly assign an isolate to one sub-lineages or sub-groups and determine its phylogenetic placement on the B. anthracis substructure population.

    SNPs, Bacillus anthracis, Genotyping, HRM, Phylogeny Cite this paper

    Girault, G. , Thierry, S. , Cherchame, E. and Derzelle, S. (2014) Application of High-Throughput Sequencing: Discovery of Informative SNPs to Subtype Bacillus anthracis. Advances in Bioscience and Biotechnology, 5, 669-677. doi: 10.4236/abb.2014.57079.

  5. DXer said

    The DNA detected was Ames, not Vollum or some other strain.

  6. DXer said

    Pregnancy Hormone Sniffs Out Anthrax
    Fri, 08/22/2014
    To mitigate these risks, Sandia National Laboratories developed a credit-card sized device based on the lateral flow assay for detection of B. anthracis in ultra-low resource environments: BaDx (Bacillus anthracis diagnostics). BaDx is a low-cost, disposable device that requires no power, instrumentation or equipment to operate, and no refrigeration to maintain efficacy. It requires little training to operate, detecting B. anthracis in the field with an accuracy that rivals laboratory analysis. Further, the device “self-destructs” by sterilizing all contents upon assay completion. This greatly improves safety for the operator, while preventing the dangerous bacteria from being recovered.

  7. DXer said

    5 Questions: David Relman on risks of creating new pathogens

    Biosecurity expert David Relman, MD, asserts that a better approach is needed for assessing the risks and benefits of research involving the creation of new and dangerous infectious agents.–david-relman-on-risks-of-creating-new-pathogens.html

  8. DXer said

    If the Department of Army cannot find what it did with the attachments to its response to the February 2002 subpoena in Amerithrax, then it doesn’t inspire confidence in its ability to find a starter culture at Yazid Sufaat’s Kandahar lab. The lab is known by the Army to have been subject to attempts to destroy equipment and evidence. (I am happy for Agent Borelli, though, that he enjoyed the tea and cookies served by Rauf Ahmad at the ISI safehouse).

    If the USG cannot find the smallpox it keeps in an unlocked FDA storage room, maybe they should have checked the bombed broom closet in Kandahar a little more closely.

    Differences as to what Yazid Sufaat had available to him aside, the USG i(ncluding USAMRIID and the Department of Army) is obligated under the rule of law to comply with FOIA.

    The Administration’s “White House equities” memorandum seems quite Nixonian. As ISIS announces it plans to attack the US, the amount of golf that President Obama has played this term reminds me of the nearly permanent vacation President Bush took before 9/11.

    Click to access ECF-No.-1-Complaint.pdf

  9. DXer said

    “Depending on the scenario, efforts to investigation a case of alleged use would face substantial practical challenges. There could be issues of access to sites, availability and conditions of samples, analytical capability on the ground, concealment of evidence or deliberate efforts to mislead investigators, and concerns for their safety (Casagrande presentation, 2013) Depending on the mandate for an investigation, there would almost certainly be questions related to the sensitivity of information and the willingness and capacity of various actors to share, and at present no international agreement or standard governs what will or will not be shared ina given set of circumstances. ” (p. 18)

    Comment: Dr. Ayman’s plan was to paint the lab walls so they could be wiped down with insecticide.

    • DXer said

      Drs. Randall Murch, Bruce Budowie, and Paul Keim three of the [FBI’s] pioneers of the emerging field of microbial forensics, collaborated on a list of unmet needs — and questions — in microbial forensics, addressing methodologies, technologies, applications, and practices. This list was presented in the opening plenary session of the Zagreb workshop to encourage the other participants to define their own ideas about needs and questions throughout the workshop. As Murch, Keim, and Budowie see it, the challenges include: ***

      “Sampling and forensic characterization of any relevant microbial background to provide key context for microbial forensic analyses, interpretation, communication, and resulting decision making. There is insufficent understanding of microbial diversity and endemism to inform assessment of where an attack effort may have been developed or perpetrated, or how perpetrators may have exploited the microbial background. And as technologies become more sensitive, it is more likely that organisms that may not have been known to be endemic may be discovered to reside in a particular egion. False positives may increase and methods for assessing the signficance of false positives and false negatives are necessary. With higher throughput systems, it is conceivable that background sampling could be performed when an event occurs to attempt to define what may be endemic. Of course such an approach requires access to the geographic location of an event, which may not always be feasible. Determining the probative value of a a “small signal” (microbe of interest) in a “big noise” (highly cluttered with other material or microorganisms, or “dirty”) sample, with defined confidence. Environmental samples are very dirty samples. What if we find one cell of interest, but not understand the background — what does that cell really mean? What can the clutter tell us? How should scientific and legal significance be determined and supported when the agent of interest is a minority constituent in a “probative sample”? How much of the threat agent of interest must be contained in a sample to be considered significant?” (p. 21)

      • DXer said


        “Exploiting the “clutter” (microbiota other than the threat agent of interest) in metagenomic samples for forensic sources. In metagenomic samples (including highly complex samples), can the “clutter” provide more power to microbial forensic analyses? Can it meet “forensic standards”? What is required to demonstate viability of this approach, including the limits on analysis conclusions?”

      • DXer said


        “- Avoiding the filtering of data on the basis of individual preferences and biases.

        – Instituting processes to inform decision makers in a way that ensures that the science is properly understood. Many nonscientists who make decisions based on forensic science make those decisions based in large part on media, such as television.” (p. 22)

      • DXer said

        “The major difference between the two approaches is that the public health investigator’s goal is to manage the public health response and protect the public’s health and safety, whereas law enforcement’s is to provide safety and security by apprehending and convicting those who committed the attack.” (p.24)

  10. DXer said

    Former senior FBI scientist Dr. Randall S. Murch, in a June 2014 article on microbial forensics, explains:

    “Space does not permit an extensive review of the literature of this still new and emerging field. However, the textbooks cited earlier (Breeze et al. 2005; Budowle et al. 2011; Cliff et al. 2011), reviews of the science developed and used in major cases (National Research Council 2011), strategic assessments of the status and needs of the discipline (Budowle et al. 2005a, b, c; Fletcher et al. 2006) and a host of legacy and newer method—and application-specific papers provide for good grounding in the past, present and near-term future of the field.

    The scientific disciplines that microbial forensics draws from in addition to forensic science are different than what one would find in traditional forensic disciplines and includes: epidemiology; genomics and metagenomics; bioinformatics; microbial, host, reservoir and vector ecology; biostatistics and population genetics; bacteriology, virology and mycology; plant pathology; biomedical and public health sciences; veterinary medicine and pathology; food science and microbiology; analytical chemistry and biochemistry; microscopy; materials science; process engineering; physical sciences and aerobiology. Many of the analytic methods and technologies used in microbial forensics are common to these other fields but are used to address different questions and purposes.

    Microbial forensics is still a young discipline; many challenges exist in spite of the advancements that have been made since its origination. Unlike traditional forensic science, wherein the “known universe” is well understood, the field of microbial forensics is confounded by considerable fundamental and applied uncertainties, from genomics to the ecology of microbes of interest and many aspects between these. Usually, identifying and performing at least some characterization of a pathogen or toxin is not difficult, using methods that are derived from clinical microbiology and related fields. The more difficult and challenging forensic issues relate to the attribution or “where did it come from and how confident are we” questions. Only a few of the most likely infectious disease agents and biological toxins to be used as weapons are sufficiently well understood and with validated methods available to allow for forensic analysis with defined confidence according forensic and legal requirements, within the limits of what the science will allow and acceptable to the community of stakeholders.

    Even with intense discussion about or science performed to support forensic investigation involving those agents on “threat agent” lists (Centers for Disease Control and Prevention 2014), much remains to be done to bring microbial forensics to maturity such that all phases of an investigation and its resolution can leverage knowledge and methods that are available on demand, not just with agents on threat lists, but any one of thousands of pathogens or biological toxins that could be used as weapons, depending on the intentions, goals, objectives, resources and capabilities of an adversary.”

  11. DXer said

    Byrne further pointed out that even if you use a process to kill the spores, you are likely to have enough intact DNA to ID the anthrax with PCR.

  12. DXer said

    The GAO should obtain and publish details of the FBI and Army testing of Afghanistan labs in 2002 and 2003 — and not merely in 2004 and 2005.

    First Case of Bioterrorism-Related Inhalational Anthrax in the United States, Palm Beach County, Florida, 2001

    Marc S. Traeger*† , Steven T. Wiersma†, Nancy E. Rosenstein*, Jean M. Malecki‡, Colin W. Shepard*, Pratima L. Raghunathan*, Segaran P. Pillai§, Tanja Popovic*, Conrad P. Quinn*, Richard F. Meyer*, Sharif R. Zaki*, Savita Kumar‡, Sherrie M. Bruce*, James J. Sejvar*, Peter M. Dull*, Bruce C. Tierney*, Joshua D. Jones*, Bradley A. Perkins*, and Florida Investigation Team1
    Author affiliations: *Centers for Disease Control and Prevention, Atlanta, Georgia, USA; †Florida Department of Health, Tallahassee, Florida, USA; ‡Palm Beach County Department of Public Health, West Palm Beach, Florida, USA; §Florida Department of Health, Miami, Florida, USA;

    B. anthracis was identified in 2 of 12 specimens obtained on October 5: from the index patient’s computer keyboard and his mailbox in the company mailroom.
    Workplace interviews regarding mail exposure showed that the index patient rarely handled or opened workplace mail, but co-workers recalled that he had examined a piece of stationery containing a fine, white, talc-like powder on September 19. The patient was observed holding the stationery close to his face as he looked at it over his computer keyboard.

    The index patient’s infection most likely occurred from inhalation of B. anthracis spores following a primary aerosolization, i.e., spores released into the air after opening a spore-containing letter. This scenario is consistent with co-workers’ recollections that the index patient held a letter containing powder over his computer keyboard, as well as environmental samples showing contamination at his keyboard, an incoming-mail desk near his workspace, and his mailroom mailbox.

  13. DXer said

    Detection of Bacillus anthracis DNA in Complex Soil and Air Samples Using Next-Generation Sequencing
    • Nicholas A. Be,

    • James B. Thissen,

    • Shea N. Gardner,

    • Kevin S. McLoughlin,

    • Viacheslav Y. Fofanov,

    • Heather Koshinsky,

    • Sally R. Ellingson,
    • Thomas S. Brettin,

    • Paul J. Jackson,

    • Crystal J. Jaing mail

    • Published: September 09, 2013


    Bacillus anthracis is the potentially lethal etiologic agent of anthrax disease, and is a significant concern in the realm of biodefense. One of the cornerstones of an effective biodefense strategy is the ability to detect infectious agents with a high degree of sensitivity and specificity in the context of a complex sample background. The nature of the B. anthracisgenome, however, renders specific detection difficult, due to close homology with B. cereusand B. thuringiensis. We therefore elected to determine the efficacy of next-generation sequencing analysis and microarrays for detection of B. anthracis in an environmental background. We applied next-generation sequencing to titrated genome copy numbers of B. anthracis in the presence of background nucleic acid extracted from aerosol and soil samples. We found next-generation sequencing to be capable of detecting as few as 10 genomic equivalents of B. anthracis DNA per nanogram of background nucleic acid. Detection was accomplished by mapping reads to either a defined subset of reference genomes or to the full GenBank database. Moreover, sequence data obtained from B. anthracis could be reliably distinguished from sequence data mapping to either B. cereus or B. thuringiensis. We also demonstrated the efficacy of a microbial census microarray in detecting B. anthracis in the same samples, representing a cost-effective and high-throughput approach, complementary to next-generation sequencing. Our results, in combination with the capacity of sequencing for providing insights into the genomic characteristics of complex and novel organisms, suggest that these platforms should be considered important components of a biosurveillance strategy.

    • DXer said

      Although this article is lucidly written, I would need the authors or an expert or such as Dr. Relman or GAO to explain the implications of the article for analysis of the testing done in Amerithrax.

      Detection of Bacillus anthracis DNA in Complex Soil and Air Samples Using Next-Generation Sequencing
      • Nicholas A. Be,

      • James B. Thissen,

      • Shea N. Gardner,

      • Kevin S. McLoughlin,

      • Viacheslav Y. Fofanov,

      • Heather Koshinsky,

      • Sally R. Ellingson,
      • Thomas S. Brettin,

      • Paul J. Jackson,

      • Crystal J. Jaing mail

      • Published: September 09, 2013



      The life cycle and virulence of B. anthracis distinguish it as an organism with a high potential for use as a bioterrorism agent, as well as being problematic as a natural human and animal pathogen in many parts of the world. Efforts toward detection of B. anthracis are fraught with multiple difficulties, including the presence of extraneous contaminants, low bacterial numbers, and non-cultivable bacilli within suspect samples [21]. Studies using a variety of methods for detection of B. anthracis have been performed in the past, and have been effectively summarized in several reviews [8,11,21]. Methods applied include RT-PCR, microarray, ELISA-based immunoassays, spectroscopy, mass spectrometry, biosensor assays, and Sanger sequencing. This study demonstrates that analysis of next-generation sequencing data and detection microarrays represent sensitive and specific complementary techniques for B. anthracis identification.

      The increasing availability and falling cost of next-generation sequencing technology provide an opportunity to perform whole genome analyses of pathogenic microbes, providing a breadth of information not available from more limited and focused genetic protocols such as PCR or Sanger sequencing. Next-generation sequencing has been used to characterize isolates of B. anthracis and successfully identified SNPs corresponding to distinct strains [22]. The Amerithrax investigation of the 2001 anthrax letters used whole genome sequencing and comparative analyses to identify unique genomic characteristics of the B. anthracis strain sent in the letters, validating the forensic potential of this technology [8,23]. These and other similar efforts have used purified DNA from isolated Bacillus strains [24,25], identifying unique and minute genetic characteristics [25]. Analysis of next-generation sequencing data for detection of biothreat organisms has not, however, been validated in the context of an environmental background [8]. Since these are the sample types most likely to be encountered in the event of bacillary release, screening for B. anthracis genomic DNA in aerosol and soil backgrounds was performed after addition of titrated genomic equivalents to determine detection efficacy. A whole genome approach was employed to increase species resolution and provide comprehensive breadth of organism detection.


      This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under contract DE-AC52-07NA27344.

      The acute and potentially lethal nature of aerosol infection by B. anthracis, in combination with the resilience of its spores upon exposure to heat and radiation, contributes significantly toward the possibility of this pathogen being used as an aerosolized bioweapon. During the months of October to November of 2001, B. anthracis spores were sent via public mail to government and news organizations, resulting in 22 cases of anthrax and 5 deaths [4]. In the decade following these attacks there has been an increased interest and investment in surveillance to detect bioterrorism agents, particularly B. anthracis, in complex samples.

      One of the most important aspects of an effective response to a biologically-based attack is the ability to detect potentially infectious agents with a high degree of sensitivity and specificity within a complex mixture.


      A number of methods, ranging from basic to complex in their application, have been employed for detecting B. anthracis. Most simply, the bacilli may be cultured and identified on blood agar. Microbiological techniques are, however, slow and require personnel trained in recognizing bacterial morphology. Moreover, while culture methods can provide extensive information about a microbe, these procedures are often lengthy and do not provide a genetic signature for the isolate being characterized. Identification based on biochemical characteristics has been examined, including assays for lipids characteristic of certain bacilli [6] and intact cell mass spectrometry for the construction of a spectrum typifying B. anthracis[7]. Such methods, however, often require pure cultures, specific growth conditions, and depend on the quality of available lipid and proteomic databases [8]. Numerous immunoassays have been developed to detect B. anthracis antigens and toxins [9–11], but their utility is limited by low sensitivity and reduced specificity due to significant antigenic homology between B. anthracis and some B. cereus isolates [11]. The current gold standard for detection and quantification of bacterial species in environmental samples is real-time quantitative PCR. Numerous such assays have been evaluated and tested for detection of B. anthracis [12–14]. Advantages of this approach include relatively high sensitivity and a protocol performed in most standard laboratories. These assays do not, however, provide in depth genomic detail on the strain detected, and may suffer from non-specific detection of near-neighbor species. All of the above techniques currently in place have limited potential for characterization of novel or previously uncharacterized isolates.


      In targeted approaches, the 16S small ribosomal subunit is often used due to its broad applicability to bacterial species and relative evolutionary conservation. 16S ribosomal DNA (rDNA) sequencing yields high-level taxonomic data regarding the composition of a microbial community. It is not, however, likely to yield strain or species-specific resolution [15]. Additionally, 16S rDNA-based methods will not identify bacterial plasmids, or distinguish between samples which do or do not contain plasmid DNA, which is particularly relevant in the case of the pXO1 and pXO2 plasmids of B. anthracis. Finally, instances may arise when it would be useful to attain comprehensive detection of both bacterial and non-bacterial organisms in conjunction, including fungi, parasites, DNA viruses, and some RNA viruses. Performing shotgun metagenomic sequencing, paired with specialized bioinformatics analyses, has the potential for making distinctions at a higher resolution, as well as for identifying non-prokaryotic species. Due to our interest in identifying highly similar Bacillus strains with enhanced specificity in environmental samples, we employed the shotgun metagenomic approach.

      We added defined copy numbers of the B. anthracis genome to whole nucleic acid extracted from aerosol and soil particulates, simulating a complex environmental background, to assess the efficacy of whole genome next-generation sequencing for detection of B. anthracis in the environment. We sequenced this material using the Illumina and 454 platforms and processed the resultant data using multiple bioinformatics approaches. Finally, we processed the same B. anthracis-spiked samples using a previously developed microbial census microarray to compare the sensitivity of this more cost-effective and higher-throughput approach to next-generation sequencing.


      Addition of Bacillus anthracis Ames DNA to environmental samples

      B. anthracis Ames DNA was acquired from the LLNL select agent laboratory and confirmed to be free of viable cells or spores by plating of 1/10 the total DNA volume on blood agar plates followed by incubation at 37°C for 72 hours. DNA concentration was determined by measurement using a Qubit fluorometer and the number of femtograms/genome equivalent was determined based on GenBank chromosomal and plasmid genome sizes. B. anthracisgenomic DNA was added to nucleic acid extracted from the two environmental sources in six amounts (1, 10, 100, 1000, 10000, and 100000 genomic equivalents of B. anthracis DNA). B. anthracis DNA was mixed with 100 pg of DNA extracted from aerosol filters or 1 ng DNA extracted from the combined Oakland and San Francisco soil extracts.


      Specificity of B. anthracis Ames-detection via next-generation sequencing

      Following the detection analysis above, studies to determine the specificity of sequencing detection were conducted. The percentage of sequence reads mapping to the B. anthracisAmes genome and the pXO1 and pXO2 plasmids was determined and compared to the percentage of reads mapping to two close relatives: B. thuringiensis Al Hakam, including the pALH1 plasmid, and B. cereus biovar anthracis strain CI, including the pCI-X01, pCI-X02, and pBAslCI14 plasmids (Table 2).

      The proportion of reads mapping to B. anthracis Ames was only slightly higher than the proportion of reads mapping to B. thuringiensis or B. cereus in the soil and aerosol background samples spiked with B. anthracis DNA (Figure 3). At the highest spiked-in genome copy number of 100,000, a 1.3-fold increase in the proportion of reads mapping to B. anthracisrelative to B. thuringiensis, and a 1.2-fold increase relative to B. cereus was observed. Nearly identical ratios were observed for soil samples spiked with B. anthracis DNA. Such narrow distinctions are problematic when aiming for specific detection of B. anthracis. The ability to discriminate between strains was thus limited using the approach of comparing all mapped reads due to a high degree of sequence similarity (97.2%) between B. anthracis and these two close relatives.


      These results confirm our previous observation that the unique mapping analysis approach is a robust method for distinguishing between very closely related Bacillus species in complex environmental samples. A potential limitation of this approach is that B. anthracis detection is likely to be challenging in samples rich in closely related organisms, such as agricultural samples containing large volumes of B. thuringiensis.


      Microarray-based detection of B. anthracis in aerosol and soil samples

      The detection sensitivity of a microbial census microarray, developed at Lawrence Livermore National Laboratory (LLNL), was tested to determine its applicability for detection of biothreat agents from environmental samples. This array contained two distinct probe categories: census and detection. Detection probes were designed to be conserved across multiple sequences from within a family, but not across families or kingdoms. Such probes aim to detect known organisms or discover novel organisms exhibiting some homology to species whose genomes have previously been sequenced, particularly in those regions known to be conserved. The Lawrence Livermore Microbial Detection Array (LLMDA) was previously designed using this approach [20]. In contrast, census probes represent the least conserved regions, and are the most strain or isolate-specific probes (Table S7). Such census probes should provide higher level resolution among known species and strains to facilitate forensic discrimination.

      The census array was tested using the same serially-diluted B. anthracis Ames genomic DNA spiked into either aerosol or soil samples, followed by whole genome amplification. DNA from each sample was fluorescently labeled and hybridized to the census array in duplicate experiments. A statistical method was developed to analyze the array data, by estimating the log likelihood of the observed probe intensities as a function of the combination of targets present in the sample, and performing greedy maximization to find a locally optimal set of targets.

      In the presence of 100 pg aerosol background DNA, 100 genome equivalents of B. anthracisDNA were required for successful microarray detection of these sequences in both replicates (Table 3), while 10 genome copies were detected in one replicate. In the presence of 1 ng soil background DNA, 1000 genome equivalents were required for detection in both replicates, while 100 genome copies were detected in one replicate.


      The life cycle and virulence of B. anthracis distinguish it as an organism with a high potential for use as a bioterrorism agent, as well as being problematic as a natural human and animal pathogen in many parts of the world. Efforts toward detection of B. anthracis are fraught with multiple difficulties, including the presence of extraneous contaminants, low bacterial numbers, and non-cultivable bacilli within suspect samples [21]. Studies using a variety of methods for detection of B. anthracis have been performed in the past, and have been effectively summarized in several reviews [8,11,21]. Methods applied include RT-PCR, microarray, ELISA-based immunoassays, spectroscopy, mass spectrometry, biosensor assays, and Sanger sequencing. This study demonstrates that analysis of next-generation sequencing data and detection microarrays represent sensitive and specific complementary techniques for B. anthracis identification.

      The increasing availability and falling cost of next-generation sequencing technology provide an opportunity to perform whole genome analyses of pathogenic microbes, providing a breadth of information not available from more limited and focused genetic protocols such as PCR or Sanger sequencing. Next-generation sequencing has been used to characterize isolates of B. anthracis and successfully identified SNPs corresponding to distinct strains [22]. The Amerithrax investigation of the 2001 anthrax letters used whole genome sequencing and comparative analyses to identify unique genomic characteristics of the B. anthracis strain sent in the letters, validating the forensic potential of this technology [8,23]. These and other similar efforts have used purified DNA from isolated Bacillus strains [24,25], identifying unique and minute genetic characteristics [25]. Analysis of next-generation sequencing data for detection of biothreat organisms has not, however, been validated in the context of an environmental background [8]. Since these are the sample types most likely to be encountered in the event of bacillary release, screening for B. anthracis genomic DNA in aerosol and soil backgrounds was performed after addition of titrated genomic equivalents to determine detection efficacy. A whole genome approach was employed to increase species resolution and provide comprehensive breadth of organism detection.

  14. DXer said

    What does Al-Barq, one of Yazid Sufaat’s assistants, say about what strain they were using? (He was vaccinated as protection against use of the virulent strain according to that Yazid Sufaat told KSM).

    Family Al Qaeda Terrorist Held by Israel Denies Charges
    Terrorist’s mother claims son ‘just wanted to study microbiology’; officials say he planned to release anthrax into urban center.

    By Tova Dvorin
    First Publish: 11/19/2013, 11:31 AM

    Illustration: Islamist fighters in N. Africa

    The mother of Samer Halmi Abdel Latif Al-Barq, the Senior al-Qaeda terrorist revealed to have been held in Israel for the past three years, has insisted in an interview with Israeli media that her son was “never” involved in building biochemical weapons.

    Al-Barq, 39, was detained by security forces as he attempted to enter Israel from Jordan via the Allenby Bridge. He faces allegations of planning a large-scale biological weapons attack against Jews via Jordan, and officials say he had elaborate plans to recruit a suicide bomber to release anthrax in a major urban center.

    Al-Barq’s family – Kuwaiti nationals – lives in the Palestinian Authority settlement of Jayyous, near Qalqiliya. While reports have been surfacing across the nation of her son’s capture, Al-Barq’s mother vigilantly denies his involvement, Channel 10 / Nana revealed Monday.

    “I know my son,” Al-Barq’s mother claims. “If he only got the chance to say his side of the story, there would be no reason to continue the administrative detention he is in now.”

    Al-Barq’s family claims that the terrorist’s odyssey from Arab country to Arab country was a quest to learn the best biology at local universities – nothing more. In his youth, he left Kuwait for Pakistan, ostensibly to study, then continued to Afghanistan.

    There, officials say, he began to prepare biochemical weapons. Al-Barq has reportedly told Israeli investigators that he performed initial tests in Afghanistan, using nerve gas on a dog. “Within seconds, the dog died,” he told them coolly. “I began talking to friends about possible plans to go back to the West, and use weapons like this against Israel.”

    Al-Barq also described how he was recruited into the terror organization, Nana reports, by the organization’s leader, Ayman Al-Zawahiri. “I met him in Afghanistan. He said I should be in touch with him to learn about producing anthrax,” al-Barq stated. “We talked about the possibility of a suicide attack by releasing anthrax into a major urban center.”

    While he has yet to be charged with a specific crime in Israel, al-Barq can be legally detained indefinitely if shown that he poses a threat. On Monday, the State told the High Court that the terrorist must remain in jail for the time being.

  15. DXer said

    J Forensic Sci. 2013 Oct 22. doi: 10.1111/1556-4029.12283. [Epub ahead of print]
    Detection of Anthrax and Other Pathogens Using a Unique Liquid Array Technology.
    Schweighardt AJ, Battaglia A, Wallace MM.

    Graduate School and University Center, The City University of New York, 365 Fifth Avenue, New York, NY, 10016.

    A bead-based liquid hybridization assay, Luminex® 100™, was used to identify four pathogenic bacteria, Bacillus anthracis, Clostridium botulinum, Francisella tularensis subsp. tularensis, and Yersinia pestis, and several close relatives. Hybridization between PCR-amplified target sequences and probe sequences (located within the 23S ribosomal RNA gene rrl and the genes related to the toxicity of each bacterium) was detected in single-probe or multiple-probe assays, depending on the organism. The lower limits of detection (LLDs) for the probes ranged from 0.1 to 10 ng. Sensitivity was improved using lambda exonuclease to digest the noncomplementary target strand. All contributors in 33 binary, ternary, and quaternary mixtures in which all components were present in a 1:1 ratio were identified with an 80% success rate. Twenty-eight binary mixtures in which the two components were combined in various ratios were further studied. All target sequences were detected, even when the minor component was overshadowed by a tenfold excess of the major component.
    © 2013 American Academy of Forensic Sciences.

  16. DXer said

    Dr. Majidi is not persuaded by Dr. Relman’s points. Dr. Relman was the Vice-Chairman of the NAS Committee reviewing the matter.

  17. DXer said

    The FBI culled the production of Al Qaeda-anthrax documents provided to the NAS and public (Dr. Majidi explains) — providing only what they wanted.

    For example, Yazid Sufaat explained to KSM that he and his assistants were vaccinated to work with the virulent anthrax they were using. Yet Dr. Majidi withheld the documents relating to that information from NAS.

    What was Dr. Majidi’s justification for not informing the NAS that Yazid Sufaat was vaccinated to protection against his work with virulent anthrax?

  18. DXer said

    Vahid Majid in his new manuscript attempting to defend the FBI’s investigation of the Fall 2001 anthrax mailings — and falling far short — he says that the FBI selectively culled documents that it did not think needed to be provided to the NAS.

    In addition to the documents that he considered “owned” by other agencies, for example, they withheld anything that they found to be “hyperbole.”

    In producing documents, it is not for the party producing the documents to do such culling. Such selective production in civil discovery would be sanctionable and subject to an award of monetary damages.

    An example of documents withheld by the FBI include all the documents relating to Sufaat’s decontamination.

    If you don’t understand the decontamination that was done, then you aren’t likely to be able to reliably assess your findings.

    Can J Vet Res. 2013 Apr;77(2):100-104.
    Influence of temperature and organic load on chemical disinfection of Geobacillus steareothermophilus spores, a surrogate for Bacillus anthracis.
    Guan J, Chan M, Brooks BW, Rohonczy L.
    Ottawa Laboratory (Fallowfield), Canadian Food Inspection Agency, Ottawa, Ontario K2H 8P9.
    Abstractin English, French

    This study evaluated the influence of temperature and organic load on the effectiveness of domestic bleach (DB), Surface Decontamination Foam (SDF), and Virkon in inactivating Geobacillus stearothermophilus spores, which are a surrogate for Bacillus anthracis spores. The spores were suspended in light or heavy organic preparations and the suspension was applied to stainless steel carrier disks. The dried spore inoculum was covered with the disinfectants and the disks were then incubated at various temperatures. At -20°C, the 3 disinfectants caused less than a 2.0 log10reduction of spores in both organic preparations during a 24-h test period. At 4°C, the DB caused a 4.4 log10 reduction of spores in light organic preparations within 2 h, which was about 3 log10 higher than what was achieved with SDF or Virkon. In heavy organic preparations, after 24 h at 4°C the SDF had reduced the spore count by 4.5 log10, which was about 2 log10 higher than for DB or Virkon. In general, the disinfectants were most effective at 23°C but a 24-h contact time was required for SDF and Virkon to reduce spore counts in both organic preparations by at least 5.5 log10. Comparable disinfecting activity with DB only occurred with the light organic load. In summary, at temperatures as low as 4°C, DB was the most effective disinfectant, inactivating spores within 2 h on surfaces with a light organic load, whereas SDF produced the greatest reduction of spores within 24 h on surfaces with a heavy organic load.

  19. DXer said

    Dr. Vahid Majidi dismisses the points raised by Dr. Relman because … well, let’s see… it’s not because he knew Dr. Ivins or the lab and equipment available…

    and it’s not because he thinks the moon landing was faked….

    Wait… Dr. Relman was the guy who was privy to the Rauf Ahmad and Zawahiri correspondence as early as 2002 — and Dr. Majidi didn’t even mention Rauf Ahmad (as far as I’ve seen).

    Could it be that Dr. Majidi, as he pointed out, is not the smartest dude in the room?

    Maybe he should rely on Peter Weber on the association of silica and iron.

    Maybe he should rely on top military aerosol scientists like John Kiel on why iron makes the anthrax more lethal in the lungs. And the nature of microencapsulation — not relating to floatability but making the anthrax more difficult to detect.

    Maybe he should actually read the documents detailing what Dr. Ivins was doing in the lab those nights instead of engaging in totally bullshit conjecture and surmise.

  20. DXer said

    Among the most downloaded Forensic Science International articles in the last 90 days is an article on the validation of new methods. The GAO has considerable expertise on the validation of methods used in sampling anthrax.

    6. Validation of new methods
    17 January 2007
    Frank T. Peters | Olaf H. Drummer | Frank Musshoff

    Abstract: Reliable analytical data are a prerequisite for correct interpretation of toxicological findings in the evaluation of scientific studies, as well as in daily routine work. Unreliable analytical data might not only be contested in court, but could also lead to unjustified legal consequences for the defendant or to wrong treatment of the patient. Therefore, new analytical methods to be used in forensic and/or clinical toxicology require careful method development and thorough validation of the final method. This is especially true in the context of quality management and accreditation, which have become matters of increasing relevance in analytical toxicology in recent years. In this paper, important considerations in analytical method validation will be discussed which may be used as guidance by scientists wishing to develop and validate analytical methods.

  21. DXer said

    Strengthening Forensic Science in the United States: A Path Forward – mars-1:hrs01JUD2141_090513b – Rayburn 2141 – Committee on the Judiciary – 2009-05-13 –

    Subcommittee on Crime, Terrorism, and Homeland Security.

    Witness List:

    Kenneth Melson, Acting Director Bureau of Alcohol, Tobacco, Firearms and Explosives, Former Director, Executive Office for the United States Attorneys, U.S. Department of Justice, Washington, DC;

    Peter M. Marone, Director, Virginia Department of Forensic Science, Richmond, VA;

    John W. Hicks, Director, Northeast Regional Forensic Institute, The University at Albany, State University of New York, Albany, NY;

    Peter Neufeld, Co-Director, The Innocence Project, New York, NY.

  22. DXer said

    Syria retaliation: Could Assad strike back in response to U.S. military action?

    With President Obama asking for congressional approval for military action in Syria, top U.S. Officials and analysts are exploring Syria’s capability for retaliation.

    In a report, CNN detailed some of Bashar al-Assad’s government’s military capabilities that could be brought to bear against the United States in response to a strike:

    • P-800 Oniks/Yakhont anti-ship missiles: Syria has at least 20 with a range of 62 to 186 miles. They fly low and strike at supersonic speed, leaving small vessels vulnerable.
    • Naval capabilities: Syria has naval fast-attack craft and anti-submarine warfare helicopters at its disposal.
    • Scuds: Syria is thought to possess more than 500 surface-to-surface weapons, with a range of 186-435 miles. They could pose a threat to nearby countries like Turkey, Jordan or Israel.
    • Aircraft: Syria has many attack helicopters, as well as MiG 21/25 air-to-air combat aircraft, MiG 23/29 attack aircraft and SU-22/24 attack aircraft in its hangars.
    • Chemical weapons: Analysts believe Syria has a large stockpile of these, as well as the capability to deliver chemical weapons agents by a variety of methods.

    But how much of a threat do these pose?


    As we ponder why people cannot resolve their differences peacefully, I think the United States and UN should release as much scientific evidence as possible and the UN should encourage Russia into publicly responding to the evidence on the scientific merits.

  23. DXer said

    25 AUGUST 2013 – 20H40
    Syria chemical attack evidence may have been destroyed: Hague

    AFP – British Foreign Secretary William Hague on Sunday warned that any evidence of a chemical attack by the Syrian regime may have already been destroyed.

    “The fact is that much of the evidence could have been destroyed by that artillery bombardment,” he cautioned during a press conference after Damascus gave its green light to a mission by UN inspectors.

    Comment: GAO, what scientific analysis did the FBI do relating to the destruction of evidence at the Al Qaeda anthrax labs:

    (1) due to the passage of years;
    (2) the decontamination of the lab and equipment;
    (3) and the bombing of the labs.

    What testing was done in 2001?
    What testing was done in 2002?
    What testing was done years later that could have been done in 2001 or 2002?

    • DXer said

      Here is an article this week out of the FBI’s laboratory on the collection and analysis of chemical warfare agent surrogate and degradation components.

      Application of a High Surface Area Solid-Phase Microextraction Air Sampling Device: Collection and Analysis of Chemical Warfare Agent Surrogate and Degradation Compounds.
      Stevens ME Jr, Tipple CA, Smith PA, Cho DS, Mustacich RV, Eckenrode BA.
      Anal Chem. 2013 Aug 29. [Epub ahead of print]
      PMID: 23902152 [PubMed – as supplied by publisher]

      Visiting Scientist Program, Oak Ridge Institute for Science and Education, Counterterrorism and Forensic Science Research Unit,Federal Bureau of Investigation Laboratory , Quantico, Virginia 22135, United States.


      This work examines a recently improved, dynamic air sampling technique, high surface area solid-phase microextraction (HSA-SPME), developed for time-critical, high-volume sampling and analysis scenarios. The previously reported HSA-SPME sampling device, which provides 10-fold greater surface area compared to commercially available SPME fibers, allowed for an increased analyte uptake per unit time relative to exhaustive sampling through a standard sorbent tube. This sampling device has been improved with the addition of a type-K thermocouple and a custom heater control circuit for direct heating, providing precise (relative standard deviation ∼1%) temperature control of the desorption process for trapped analytes. Power requirements for the HSA-SPME desorption process were 30-fold lower than those for conventional sorbent-bed-based desorption devices, an important quality for a device that could be used for field analysis. Comparisons of the HSA-SPME device when using fixed sampling times for the chemical warfare agent (CWA) surrogate compound, diisopropyl methylphosphonate (DIMP), demonstrated that the HSA-SPME device yielded a greater chromatographic response (up to 50%) relative to a sorbent-bed method. Another HSA-SPME air sampling approach, in which two devices are joined in tandem, was also evaluated for very rapid, low-level, and representative analysis when using discrete sampling times for the compounds of interest. The results indicated that subparts per billion by volume concentration levels of DIMP were detectable with short sampling times (∼15 s). Finally, the tandem HSA-SPME device was employed for the headspace sampling of a CWA degradation compound, 2-(diisopropylaminoethyl) ethyl sulfide, present on cloth material, which demonstrated the capability to detect trace amounts of a CWA degradation product that is estimated to be less volatile than sarin. The rapid and highly sensitive detection features of this device may be beneficial in decision making for law enforcement, military, and civilian emergency organizations and responders, providing critical information in a contaminated environment scenario when time is of the essence.

  24. DXer said

    “Al-Zawahiri worked with the Red Crescent across the border in Pakistan, treating wounded mujahedin fighters with rudimentary medicine like the use of honey to sterilize wounds.”

    Dr. Evil: Terror Alert Puts Bin Laden’s Successor Back In The Spotlight
    Read more:

    I am looking forward to an exhibit “Wild Medicine” at the New York Botanical Garden on medicinal herbs where there is recreation of an Italian renaissance garden.

    Medicine’s Wild Roots On Display At NY Botanical Garden
    NY1 ‎- 21 hours ago
    A new exhibit at the New York Botanical Garden titled “Wild Medicine, healing plants from around the world” explores more than 500 medicinal plants that can …

    Did they use medicinal herbs in Afghanistan? In the US Civil War circa 1860?

    Al Qaeda was using virulent Ames anthrax in 2001.

    Testing was have been problematic because, as the documents show was planned, the labs were decontaminated with insecticide. (That was why the walls were painted — so they could be wiped down).

    Sweet Soraya and her mom should have long ago urged to Yazid that Yazid Sufaat gains nothing by not addressing all the historical details of his anthrax program. He has served his time for his work in Afghanistan. The legal system is what it is.

    Yazid told me he could work magic and I believe him. The apple doesn’t fall far from the tree and I know the work ethic he instilled in his daughter.

  25. DXer said

    See pubmed for new GABRI method of detecting low levels of contamination — which avoids effect of environmental contamination. Sorry I haven’t been providing links of late — am making do with device. Question for coauthor MHJ: might it avoid effect of Yazid’s decontamination using insecticide? See classified reports.

    • DXer said

      MHJ revised the experimental design of this study of the GABRI method. The authors note that it makes things a lot easier if a farmer points you to where he buried his animals. Similarly, it makes it a lot easier if you ask Yazid Sufaat, the Al Qaeda anthrax lab director, where he buried the bodies of the animals used in experiments. Yazid was captured in December 2001. He was first interviewed by the FBI in November 2002. GAO, what explains the 11 month delay? By the time of the Director’s visit to Malaysia in March, Malaysian officials had been told that Yazid had been part of an earlier secret Malyasian biological weapons program. Did the FBI Director Mueller know that at the time of his visit? In my interviews of Yazid, he does not deny Al Qaeda’s responsibility for the anthrax mailings. Instead, he purports to invoke the Fifth Amendment.

      Click to access 1471-2180-13-167.pdf

  26. DXer said

    J Vet Sci. 2013 Jun 30. [Epub ahead of print]
    The determination of the genetic diversity of Korean Bacillus anthracis isolates from soil through the use of single nucleotide repeat analysis.
    Kim SH, Jung KH, Kim SK, Kim SJ, Kim JC, Cho SY, Chai JC, Lee YS, Chai YG.


    Bacillus anthracis, the etiological agent of anthrax, is one of the most genetically monomorphic bacteria species in the world. Due to the very limited genetic diversity of this species, the classification of isolates of this bacterium requires methods with high discriminating power. Single nucleotide repeat (SNR) analysis is a type of variable-number tandem repeat (VNTR) analysis that assesses regions with very high mutation rates. To subtype a collection of 21 isolates that were obtained during a B. anthracis outbreak in South Korea, we analyzed four SNR marker loci using nucleotide sequencing analysis. These isolates were obtained from soil samples and from the Korean Center for Disease Control and Prevention (KCDC). The SNR analysis was able to detect the existence of 13 sub-genotypes, which allowed for the detailed investigation of the examined Korean isolates. Our study demonstrated that the SNR analysis was able to discriminate among the strains with the same MLVA genotypes. Through this study, we obtained SNR results for four SNR marker loci from newly examined strains in Korea; this discovery will prove to be helpful for the creation of marker systems because it identifies markers that could be used for future forensic studies.

  27. DXer said


    As far as the people who support him or don’t think that he did it say one of the most glaring things is that no spores were ever found. The spores weren’t found in the house. The spores were not found in the cars or in any piece of equipment.

    [lead investigator Montooth]

    … [It] didn’t necessarily mean anything if we found them or we didn’t find them, because he worked with them.

    If we found spores there, his comment would be: “Well, I work with them. Somehow when I cleaned myself as I was exiting the hot suite, I must not have done it properly, and I must have carried it home.” …

    But if the spores had had the same signature in some ways as the attack spores, it would have been a smoking gun.

    Not necessarily. He works with it.

    But the attack anthrax he didn’t work with.

    He worked with RMR-1029, which was the parent [strain]. …

    But the attack stuff had silicon, for instance. … The spores were different because they seemed to have been grown [over] a couple of generations. So there would have been something.

    Could have.

    It would have been a possibly suspicious thing.

    Yes. But once again, you wouldn’t have been able to really go one way or another positively, because he worked on it. He handled the letter, right? They’ve handed him samples when it was in the hot suite. He’s still going to be able to say maybe it came home with him then.


    Agent Montooth explained that although they found no spores in Dr. Ivins home or car, it would not have been a smoking gun in any event given he worked with the stuff.

    On the other hand, if it were found in Sufaat’s lab, it would be a smoking gun because Dr. Zawahiri had announced an intention to use anthrax to retaliate for the rendering of senior EIJ leaders, to include Blind Sheik Abdel-Rahman. The announcement had been made by the blind sheik’s lawyer Montasser Al-Zayat.

  28. DXer said

    Anyone well read on the subject understands that the detainee assessments confirm Yazid Sufaat was working with virulent anthrax. Yazid explained to KSM that it was safe because he and his assistants had been vaccinated.

    Wikileaks / Guantanamo : Doesn’t the United States know the strain of the virulent anthrax used in one or more of the Al Qaeda labs, for which Sufaat and his assistants were vaccinated, based on interrogation of the individuals? Why didn’t the FBI simply provide the NAS with the answers to its question about the strain of the anthrax based on interrogation of Yazid Sufaat or his anthrax lab assistants? Will it answer the same questions for GAO?

    Posted by Lew Weinstein on April 27, 2011

  29. DXer said

    An Interview By Malaysiakini With Yazid Sufaat (Former ISA Detainee), The Experience Of Meeting With Sheikh Usamah Bin Ladin

    Jamadi-ul-Awwal 03, 1433 A.H, Tuesday, March 27, 2012

    JOHOR, Malaysia – Yazid Sufaat is a biochemist and former Army captain, accused of aiding the 11th September 2001 attacks in the US, and was imprisoned in the Internal Security Act political prison of Malaysia since 2002, and then was freed in 2008. He told that he had met with Sheikh Usamah bin Ladin rahimahullah and it was an honour for him. Below is the article and interview by Malaysiakini with Yazid Sufaat in the series, ISA by Fathi Fathi Aris Omar, Aidila Razak and Salhan K Ahmad, published by Malaysiakini on (20/3/2012).


    A simple Google search on Yazid Sufaat will return the following results – militant, bombmaker, biological weapons expert, and one of the longest serving detainees under the Internal Security Act (ISA).

    “They call me the CEO of Anthrax.”

    Shrugging as he says this, the father of four who was released from ISA detention in November 2008 after seven years – five of which were in solitary confinement – wears the title almost like a badge of honour.

    “They (the accusers) call me that because I only have a ‘cabok’ (simple) Bachelor of Science but my students were PhDs, Masters’ degree holders,” he told Malaysiakini in an extensive interview at his home last week.

    His “students” are now inmates in Cuba’s Guantanamo Bay, which he discovered when the Federal Bureau of Investigation showed him photographs of them during questioning at the Kamunting detenion centre where he was held.

    “I am Yazid Sufaat. I am not going to hide myself, my face, my name. Why should I? I am handsome, no?” asked the 48-year-old Johor native, laughing.

    His devil-may-care attitude may lead one to believe that Yazid’s “I love Osama Laden” proclamation on his Facebook page as a sort of joke.

    But the unrepentant Yazid, who is one of seven Malaysians on the United Nations ‘travel, asset and arms deal’ sanction list for alleged involvement with the infamous al-Qaeda, really loves Osama.

    Recalling his time in Afghanistan in 2000-2001, Yazid admitted having met the now deceased al-Qaeda leader and considered it “an honour”.

    “Of course, I met him, it’s an honour to meet him. How many people have?” he asked.

    Yazid, who crossed the the Pakistan-Afghan border on learning of the Sept 11, 2001 attacks on the US, said he trained as a militant for six months under the one-time most wanted man in the world.

    “That was the opportunity to face them (the enemy). Of course, they came… I think the closest bomb that fell was 15m from me. That was the experience; (I) wanted it, Allah give (me) a chance,” he said.

    Under Osama, he sharpened his shooting skills, learned to walk in the dark and navigate using the stars and how to withstand the brutal desert combat conditions -losing about 18kg in the first two months.

    “I am military trained but trained in a different terrain. (In Afghanistan) there is no jungle, all desert.

    “Of course we could not run away from the Qur’an. I learned Arabic, listened to (Osama’s) lectures, his usrah (discussion to instill loyalty and brotherhood).”

    When Kabul fell to the Northern Alliance forces in November 2001, it was Osama who advised him to leave the war-torn country and return when “we have won back Afghanistan”.

    “At that time I thought, if they catch me out here, I would be heading to Guantanamo. If I go home, it’s only ISA. So I called a friend at Bukit Aman and he said: ‘Come back. At least here it’s only ISA’.

    “My wife didn’t believe I would be arrested, so I said let’s cross the border and see. We crossed Bukit Kayu Hitam (Thai-Malaysian border) and I was arrested ,” he said of the December 2001 incident.

    The journey to Afghanistan

    A Royal Military College alumnus and a retired army captain, Yazid’s journey to Afghanistan began when he started seeking answers about his religion.

    He said that he became an adult in the “sin cities” of the US, having graduated from California State University as a biochemist at the age of 23.

    “When I returned, I was still wild. My mother-in-law said I should go study religion, but I didn’t want to learn about the solat or how to perform the haj.”

    I wanted something different. So (people said) go meet this ustaz and (when I did) I thought, ‘this is good, this is something different’.”

    “Addicted” to the lessons, Yazid began delving more and more into the teachings of Islam only to be left unsatiated.

    “I thought, ‘this can’t be it’. (I) wanted to graduate, so I started reading more and when you have knowledge you want to ‘do’ (something). I am a scientist… I want to prove the theory.”

    In 1995, Yazid performed the umrah and vowed to only return to perform the haj when he could understand the Qur’an. He returned in 1998.

    The same year, he went to Ambon, Indonesia, at the height of the Christian-Muslim conflict – to experience the “real jihad”.

    “I had a bit of money. If I left my family, they can sustain themselves. I wanted to see the real thing. What is so special about it. I wanted to face it… jihad in terms of qital , which means war,” he said.

    Going into Ambon “blind” on his own steam on the first fact-finding mission, Yazid and a friend met with Muslims there to understand their urgent needs and returned home to build a network of assistance.

    At the time, Yazid was operating a pathology lab, running medical tests for up to 600 clinics and it was his clients that he approached to get the rudimentary medical supplies to send to Ambon.

    “It was humanitarian. There you could find all sorts of non-Muslim humanitarian groups like the Red Cross. The Muslims were people like me, who wanted to help. They were called Mujahideen (freedom fighters), now they are called terrorists.

    “In a conflict area, you have to defend yourself. You don’t just go like that Mavi Marmara ship. It’s stupid!” he said.

    Yazid would later be charged for funding sectarian violence in Ambon – one of the five charges which kept him under the ISA.

    “Mercy (Malaysian NGO) was there. Umno (Malaysia’s ruling party – ed.) guys were there. Both were funded. Yazid Sufaat was there, self-funded, but this is a threat to national security,” he said.

    The ‘project’ in a Kandahar lab

    His desire to “help (his) fellow brothers” also saw Yazid finding himself criss-crossing the Afghan-Pakistan border – this time to build a hospital.

    He used his experience in running a pathology laboratory to train staff at the hospital and to set up a laboratory next to the hospital in the Taliban stronghold of Kandahar.

    It was at this laboratory that he was accused of having developed biological weapons – something which he terms “the so-called project”.

    Initially hesitant to reveal what went on in the lab, which he claims he started before Sept 11, 2011 and was bombed out following the World Trade Centre attacks, Yazid let out that it was a defence strategy.

    “Of course (there was research), we are scientists (laughs) . We have to do it, but the lab wasn’t that sophisticated. It was bare bones, that was what we could manage. We do what we can and leave the rest to Allah.

    “If (the other side) use ‘bugs’, you must understand that bug in detail so we can counter any biological weapon that they use. You must know your enemy,” he said.

    But was he really the ‘CEO of Anthrax’ and a senior al-Qaeda leader as alleged?

    “I never expected to be accused of doing this thing… if you read the stuff they wrote about me, it’s impressive. But (what I did) was really nothing.

    “If people want to write bad things, they will write bad things. (If) they want to write good things they would. That is just perception. I don’t care. What do I need to hide?

    “My name is Yazid Sufaat. I did not do anything wrong. I don’t feel guilty at all,” he declared.


    Translated and Submitted by a Mujahid

  30. DXer said

    “Former ISA detainee Yazid Sufaat, 49, has been charged with inciting terrorist acts that “threatened the public in Syria”.

    Although he was among the first three people to be arrested under the newly enacted Security Offences (Special Measures) Act 2012, he was charged under Section 130G(a) of the Penal Code.

    His wife’s religious teacher, Halimah Hussein, 52, was charged with abetting Yazid in inciting the acts at a house in Ampang between August and October 2012.

    The third person, Mohd Hilmi Hasim has been remanded and did not appear in the Ampang magistrate’s court yesterday.

    The charges against Yazid and Halimah come just three months after two other Malaysians, Razif Mohd Ariff, 30 and Muhamad Razin Sharhan Mustafa Kamal, 21, were arrested in Lebanon on suspicion of terrorism.”

    Do the Affidavits provided defense counsel in advance of a scheduled May 6 hearing on the pending charge recount any detail provided by Razif Mohd Ariff and Muhamad Razin Sharhan Mustafa Kamal?

    Did Yazid talked about his work with anthrax years earlier in Afghanistan on wiretaps?

  31. DXer said

    None of these Facebook posts by Yazid could be part of a case of recruitment to Jihad.

    For one thing, besides being protected free speech, they were posted after the young men were arrested.

    Chomel has said that the young men contacted Yazid after his interview about his interview with BIn Laden. In that filmed interview, Yazid affably said the murder of the 3000 innocents was useful marketing for Islam.

    He told me that knew that might very likely get him in trouble. But he said he was going to tell all and people could judge.

    Telling (with corroboration) the genetic strain of anthrax he and his two assistants were using, on the other hand, likely would only get the US FBI and CIA in trouble.

    Then the FBI and CIA investigators and scientists could explain why the strain known to have been used (from theinterrogation of Yazid’s two PhD assistants) was not disclosed to the public.

    They haven’t even disclosed the lab that Rauf Ahmad visited on his quest for Ayman Zawahiri.

    Ironically, if Yazid wants to retaliate against the US CIA and FBI, the way to do it is to tell everything he knows about Al Qaeda’s anthrax program so that the US public can compare it to what we’ve been told. In the US, lying is a firing offense.

    If Yazid turns out to be the truth-teller and the USG officials prove to have been the liars, well they will be the one to bear the consequences. At the same time, Yazid will assume his place in history as the CEO of Al Qaeda’s anthrax program rather than assistant at a drinks stall facing 30 years for some friendly chat over meals.

    So far the government has succeeded in making out Yazid to be the liar — on issues such as the purpose of the 4 tons of ammonium nitrate, whether he knew hijacker Nawaf, etc.

    • DXer said

    January 29, 2013 at 9:41 am
    Yazid Marwan Hadeed today writes from near Kuala Lumpur, Wilayah

    “Strive hard for your place in Jannah….cant get there cara lenggang kangkong, tepuk tangan dan carpet merah, brother….”

    I think he may be saying you can’t obtain your place in Jannah by focusing on applause and the red carpet.

    • DXer said

    January 13, 2013 at 11:20 pm
    Yazid Marwan Hadeed (Yazid Sufaat) writes by Blackberry 19 hours ago:

    “Silence and smiles are two powerful tools. A smile is the way to solve many problems & silence is the way to avoid many problems.”

    • DXer said

    January 3, 2013 at 7:38 am
    Yazid Marwan Hadeed (Yazid Sufaat) today writes:

    Hari ini dlm sejarah: Di hujung hayat kuasanya, Shah Iran tabur wang dari helikopter — apalah sgt kasi rebate HP. Rakyat tak peduli & Shah tetap tumbang. Should learn from history, berjimat itu rahmat.

    DXer said

    December 23, 2012 at 8:46 am

    Most recently Yazid writes:

    “Aside from their 32 brains, leeches also have 3 mouths and each of which are filled with hundreds of teeth.”

  32. DXer said

    Of course, the doubts are expressed not merely by the major media, but the scientists who led the NAS panel.

  33. DXer said

    Here is the audio of the welcoming remarks of David Relman who was Chair of the event on microbial threats in June 2012 at the National Academy of Sciences.
    [audio src="" /]

  34. DXer said

    David Relman gave welcoming remarks at the June 2012 conference at which Dr. Fraser-Liggett and Dr. Bruce Budowie spoke.

    He was Vice-Chairman of the panel that reviewed the science used in the investigation.

    National Academy of Sciences casts doubt on FBI’s Anthrax Investigation

    OCT. 28, 2011
    Scientific Case Still Open On 2001 Anthrax Attacks

  35. DXer said

    Anthrax: DHS Faces Challenges in Validating Methods for Sample Collection and Analysis

    GAO-12-488, Jul 31, 2012

    Timothy M. Persons
    (202) 512-3000

    A workgroup—led by the U.S. Department of Homeland Security (DHS) and made up of DHS and the Centers for Disease Control and Prevention (CDC), the Environmental Protection Agency (EPA), the Federal Bureau of Investigation (FBI), and the National Institute of Standards and Technology (NIST)—has attempted to address GAO’s recommendations to (1) validate environmental sampling methods for detecting Bacillus anthracis and (2) conduct studies to develop probability-based sampling approaches for indoor environments. This workgroup has taken some actions to validate environmental sampling methods (collection, transportation, preparation, analysis) and develop statistically based sampling approaches that will provide confidence statements when test results are negative. These activities were projected to be completed by fiscal year 2013, but delays are now expected.

    While progress has been made in validating sampling methods for detecting Bacillus anthracis spores in indoor environments, their validation is not yet complete.

    • DXer said

      The documents indicate that Ayman planned to have the lab in Afghanistan wiped down with insecticide. Both Rauf Ahmad and Yazid Sufaat could confirm this. Validation of methods relating to a clandestine lab needs to factor in attempted decontamination. By analogy, finding no detection of anthrax of the lyophilizer (that turns out to have been unavailable to Dr. Ivins on those nights) was negative — but they assumed he had decontaminated it.

      In contrast, when faced with a lab where they know anthrax was being developed for use against the United States, some didn’t take into account what the documentary evidence shows was the plan for decontamination of the location.

  36. DXer said

    David Relman thinks the FBI has not proved its case — he was the Vice-Chair of the NAS panel.

  37. DXer said

    J Appl Microbiol. 2012 Jul 2. doi: 10.1111/j.1365-2672.2012.05381.x. [Epub ahead of print]
    Laboratory studies on surface sampling of Bacillus anthracis contamination: summary, gaps, and recommendations.

    Piepel GF, Amidan BG, Hu R.

    Pacific Northwest National Laboratory, Richland, Washington, USA.


    This article summarizes previous laboratory studies to characterize the performance of methods for collecting, storing/transporting, processing, and analyzing samples from surfaces contaminated by Bacillus anthracis or related surrogates. The focus is on plate culture and count estimates of surface contamination for swab, wipe, and vacuum samples of porous and nonporous surfaces. Summaries of the previous studies and their results were assessed to identify gaps in information needed as inputs to calculate key parameters critical to risk management in biothreat incidents. One key parameter is the number of samples needed to make characterization or clearance decisions with specified statistical confidence. Other key parameters include the ability to calculate, following contamination incidents, the 1) estimates of Bacillus anthracis contamination, as well as the bias and uncertainties in the estimates, and 2) confidence in characterization and clearance decisions for contaminated or decontaminated buildings. Gaps in knowledge and understanding identified during the summary of the studies are discussed. Additional work is needed to quantify 1) the false negative rates of surface sampling methods with lower concentrations on various surfaces, and 2) the effects on performance characteristics of: aerosol versus liquid deposition of spores, using surrogates instead of B. anthracis, real-world versus laboratory conditions, and storage and transportation conditions. Recommendations are given for future evaluations of data from existing studies and possible new studies. © 2012 © No claim to US Government works Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.


    This review of the literature relates to areas contaminated by an attack — not the laboratory where the anthrax was processed and a decontaminating agent had been used. Where is the FBI’s science on detecting whether an insecticide was used at the Al Qaeda labs to wipe down the lab? As I vaguely recall, Dr. Ayman contemplated painting the labs and using paraformaldehyde — and moving them every 3 months so as to avoid detection.

  38. DXer said

    Science 11 May 2012:
    Vol. 336 no. 6082 pp. 669-670
    DOI: 10.1126/science.336.6082.669-b
    Presumed Guilt in the Anthrax Case—Response
    Related Resources
    In Science Magazine
    Presumed Guilt in the Anthrax Case
    Jeanne Guillemin
    Science 11 May 2012: 669.

    In my review of American Anthrax, I sought to highlight the valuable contributions of this book to the public understanding of this complex and controversial case, as well as the book’s shortcomings. It is true that Guillemin makes no explicit statement about her position regarding the guilt or innocence of Ivins. However, statements such as, “the FBI had solid scientific proof that the spores in the anthrax letters matched those in a flask, labeled RMR (Reference Material Receipt)-1029, that was in Ivins’ keeping at the Army’s medical institute at Detrick” (p. xxii), and “a criminal—either Ivins or someone else—had used the institute’s Ames anthrax spores to commit murder” (p. 251), misrepresent the strength of the scientific evidence that points to this flask as the source of the spores in the letters (and by inference, Ivins and/or the Army’s institute at Fort Detrick, Maryland). A variety of weaknesses in the FBI’s scientific investigation are discussed in detail in the NRC Report (1) (issued by a committee on which I served as vice chair), but unfortunately, these weaknesses are not given much coverage in her book. Guillemin is correct: I served as vice chair of the NRC committee that issued this report.

    [Corrected 14 May 2012 to replace earlier version posted in error.]

    David A. Relman
    + Author Affiliations

    VA Palo Alto Health Care System, Stanford University, Palo Alto, CA 94304, USA.
    1.↵ National Research Council, “Review of the scientific approaches used during the FBI’s investigation of the 2001 anthrax letters” (National Academies Press, Washington, DC, 2011);

  39. DXer said

    Professor Guillemin spoke on April 2, 2012 at one of my favorite buildings — the Madison Building on Capitol Hill which has the current periodicals loom. In her book, the sociologist specialized in biodefense matters provides another much welcome overview of a range of important issues in advance of the GAO report The book excels in its readability. The strong point in Laurie Garrett’s book, in contrast, was a more detailed and incisive discussion of the NAS findings. Professor Guillemin’s overall conclusion stated in her book is that we may never know the perpetrator of the anthrax mailings. Let’s hope she’s wrong and that, in the meantime, the GAO is more able to be more substantively probing due to its greater access to materials. Laurie Garrett appeared to recognize that an Ivins Theory merely Hatfill Theory Redux. The “Hatfill Theory” was part of the same unstoppable train wreck as the “Ivins Theory.” There was a change of cars (investigators), but it was the same flawed train of reasoning and the investigators never overcame the earlier truncated emphasis of the investigation. Professor Guillemin’s value judgments are less sharply felt — or at least better disguised in an approach that comes across as academic. Anthrax Redux: Did the Feds Nab the Wrong Guy? March 24, 2011 Laurie Garrett recognized that too much is at stake to be content with the latest investigators’ position that they do not know the what, how or why of the anthrax mailings. In contrast, Professor Guillemin emphasizes the lack of acknowledgment of the lapse in biosecurity. (She draws from her own experience with the investigation of Sverdlosk and the lack of forthright acknowledgment of responsibility). Actually, more to the point (that she totally overlooks), is that Bruce Ivins gave virulent Ames to a former Zawahiri associate and Ali Al-TImimi, who was coordinating with Anwar Awlaki, shared a suite with Ames anthrax researcher Ken Alibek who famously did a duet with Professor Gullemin’s husband in June 2002 on the issue of silica. The New York Times review of her book on Sverdlosk had complained that it was neither a detective story or a scientific paper. The response at the time in one letter to the editor was that should not matter: The author is still highlighting an important issue and providing a useful historical narrative. The same criticism and the same response apply to AMERICAN ANTHRAX. On the other hand, given the importance of solving the mystery, this blog favors reaching the critical true crime facts as revealed by documents. Anything else is mistakenly thinking it is time to write the history when it instead is the time to press for documents. For example, the investigators were privately convinced of Dr. Ivins’ guilt partly because of what they learned in mid-July from the notes from Dr. Ivins’ first counselor. Those claims were heavily relied upon by a panel of psychiatrists led by the psychiatrist who had guided the FBI’s approach to Ivins. The likely author, Ronald Schouten, spoke along with Professor Guillemen at a November 2011 conference of life sciences. Dr. Schouten’s tone certainly warranted the characterization “presumption of guilt.” He certainly is trying Dr. Ivins in the court of public opinion. Moreover, I think David Relman’s point about Professor G’s presumption of guilt is illustrated by a question and answer with a European blogger last month when I had to urge to Professor Guillemin that her assumption as to what Dr. Ivins’ motive would be was contradicted by the evidence. Her response was that perhaps some other document not yet made public supported her point. But that’s the problem. People are making presumptions of guilt — assumptions about documents that in fact do not support the presumptions. People are making factual assertions contradicted by numerous documents withheld by the DOJ — with now some of them produced by USAMRIID after a two-year struggle. Professor G graciously deleted — or the journalist — deleted the mistaken factual point. But it was wrong to presume that a document she had not seen supported her presumption as to what Dr. Ivins’ motive would have been. (It related to a mistaken claim about lack of future funding). The investigators and psychatrists in 2008 could not have known that in 2009 Dr. Ivins’ first therapist, Judith M. McLean, would write of how she acquired her psychic abilities in her book available for sale — from a being from another planet … In addition to helping the FBI with Amerithrax, the psychic relied upon the government prosecutors and investigators helped with 911 by her astral travelling and retrieval of etheric body parts at Ground Zero … She reports she was granted her psychic abilities by a being claiming to be an extraterrestrial … I am still waiting for the author or journalist that interviews the members of the EBAP panel that relied on the first counselor — to see if they say “oopsie.” I wrote Dr. Schouten (and David WIllman and Professor Guillemin today) and got no response. They have never corrected themselves on the point and then continue to rely on the stories told by the first counselor. She does not note that Dr. Greg Saathoff, who gave the key psychiatric report about Dr. Ivins and after his death justified their approach arguing that Dr. Ivins likely was guilty, is a longtime partner of FBI Quantico and instead spins his report as independent. In fact, the Professor is perhaps too refined to get into matters relating to semen stained panties that were the subject of the FBI’s DNA swabbing in July 2008 and threat to call Dr. Ivins’ family in front of the grand jury. I’ve always liked her precisely because she is so refined. An experienced sociologist married to an eminent biodefense scientist who consulted for the FBI in this matter, Professor Guillemin knowledgeably describes the cast of characters. She does not get into the details to the extent of press this Spring did on the genetics, where the FBI’s own experts explained the holes in the FBI’s case. Instead, now she dismisses the NAS panel as the “B Team” while blowing right pass key points made by none other than Claire Fraser-Liggett. Claire Fraser-Liggett: the genetic analysis of the spores in Ivins’ flask do not indicate Ivins is guilty Disturbing questions haunt the anthrax killings inquiry Like Laurie Garrett, I don’t see that Professor Guillemin addresses the documentary evidence of Agent Lambert’s concern … that the compartmentalization of the investigative squads ordered by Director Mueller would prevent investigators from connecting the dots. Posted by Lew Weinstein on March 23, 2011 Professor Guillemin nowhere addresses the documentary evidence produced in May 2011 that now shows what Dr. Ivins was doing in the B3 and instead bought into the FBI’s mistaken narrative that Ivins had no reason to be in the B3 on those nights. I am less interested in an academic saying the perpetrator may never be identified than some journalist writing up the notes that were withheld for three years (along with other even more revealing lab notes). Given Laurie Garrett’s expertise lies in science writing, and she likely is not daunted by lab notebook pages, she could usefully turn to them now to our great benefit. Similarly, given Professor Guillemin’s contacts among some FBI and non-FBI biodefense experts, she too could provide credible discussions of what the notes (and notes still to be released) reveal. How could the longtime FBI Quantico psychiatrists or the author not have requested from the FBI the record showing what Dr. Ivins was actually doing on the nights that the investigators, without basis, speculated he was making a powdered anthrax? Professor Guillemin has an interesting detail in which she notes that the DRES biodefense researchers — who had shown that mailed anthrax immediately dispersed from the envelope and travelled across the room — briefed a military audience at the time the anthrax was being discovered at the Senate building. Others in the US had been briefed months earlier and the FBI should have obtained the list of those briefed. While I don’t think it material to the true crime analysis, it is interesting to see the fresh (to me) detail. As another example, she notes Ben Garrett’s expertise at the FBI prior to 9/11, pointing out another possible interview that the GAO should have conducted. Professor Guillemin, to my eye, nowhere notes or even mentions that anthrax in the New York Post letter was 10% silica or silicates but importantly does emphasize that the government had long been told that if they find the person growing anthrax in silicates, they may have found their perpetrator. On the silica issue, she returns us to the days of what I’ll call the Meselson v. Matsumoto debate… when 7 years later discussion should have moved on to what I’ll call the Velsko/Weber position. Both government consultants, they say that given its probative importance, the issue and reason for the Silicon Signature needs to be further explained. Writers and journalists need to leave behind the Meselson/Matsumoto/Spertzel framing of the issue — it obscures rather than illuminates. My friend Barry Kissin is still mistakenly equating silica with weaponization when it also instead could be equally probative but point to other things. As “weaponization,” many commentators overlook that it increases resistance to sunlight. And using it in the growth media — in the event that was what was done — is not at all the end of the question. Indeed, Matt Meselon’s friend Ken Alibek specifically had invented the patent in Spring 2001 to concentrate anthrax by using silica in the growth media! If a journalist is not interviewing and citing the Lawrence Livermore experts Weber and Velsko, then they are living in the past. Indeed, to be expert on the issue, one needs to have actually done controlled experiments with and without silicon such as John Kiel, head of the Air Force lab. So at the end of the day I favor the view of Professor Guillemin’s co-author on Sverdlosk — Martin Hugh-Jones. It is not to say that Professor Guillemin is mistaken in what she says, it is that she is missing the point. The Silicon Signature needs to be understood because it is potentially highly probative. The “Red Team” conclusion should not have been so readily accepted. (Moreover, those experts should have been identified consistent with FOIA). . The Technical Review Panel Summary notes that the NY Post sample had apparently been treated with hydrophilic silica. The term “weaponization” is used by Professor Guillemin as a straw man to avoid the potential key probativeness of the silicon signature. . She nowhere suggests that the USG has explained how Dr. Ivins’ processing could have resulted in the Silicon Signature. . . To credit that the silicon signature did not relate to “weaponization” – as Professor Guillemin and many of us do — does not avoid the fact that it is potentially highly probative, and without more tends to be exculpatory of Dr. Ivins. For example, if it relates to “microencapsulation” using hydrophilic silica. That might be a huge lead. It is important to recognize that none other than government-funded experts Weber and Velsko, key experts on the nonmicrobiological signature signature, think that further study is warranted to determine the source of the Silicon Signature. She nowhere mentions the 302 interview statement that checking the health of the animals typically would take 2 hours and was a one person job. This is important background in understanding the lab notes produced on May 11, 2011. Given the lead times involved in publishing, she does not address the sworn deposition testimony in the Stevens v. United States case of Patricia Worsham or Stephen Little casting doubt on the FBI’s Ivins Theory. She mentions the lyophilizer but does not address the Speed Vac. The DOJ’s reliance on the lyophilized was utterly destroyed. Professor Guillemin does not address the fact that US Attorney Taylor in explaining Ivins’ overtime in Fall 2001, including November and December, did not realize that new rules in 2002 precluded such overtime, working alone in B3. In his FOIA to the Army, David Willman did not seek access records from the earlier or later period and I don’t see that Ms. Garrett or Professor Guillemin submitted any FOIAs to Army (and I don’t know offhand about DOJ). Source: “An eye on safety” by Alison Walker … “Better enforcement … In 2002, USAMRIID officials mandated a two-person rule, which creates peer pressure to follow safety protocol by requiring material be handled by two people of equal experience, training and qualification. USAMRIID is phasing out the rule due to space and staff limitations, replacing the physical presence of another person with video surveillance.” She does not mention, but it is important to note, that Dr. Ivins had no access to the filters and thus there would have been traces in the filters if the anthrax had been made in that B3. Professor Guillemin does not address the weaponized anthrax that Dr. Ivins says he had heard had been shipped to Ft. Detrick and then went missing though she makes passing mention of CIA experiments involving Battelle and Dugway early on. She mentions that James Burans learned how to culture anthrax from Dr. Ivins. He was the lead Navy fellow and Al-Timimi had a security clearance to work on a Navy project while at SRA in 1999 — all this talk about missing weaponized anthrax prompts me to wonder what experiments the Navy was doing in Spring 2001. I know (but perhaps shouldn’t) that there were aerosol experiments involving ships. (I presume simulants but have no information; Greg Knudson, who had obtained the Ames originally, then went to work for NMRC and the CIA, I believe. At page 214 she explains that USAMRIID’s John Ezzell, the FBI’s anthrax expert, prior to 9/11, made a dried aerosol using Ames supplied by Bruce Ivins and sent to Johns-Hopkins Applied Physics.“murder-weapon”-to-borrow-us/ Where does she address Ivins’ email of 6-28-05 that discusses powder deemed closest to attack anthrax … in which Ivins says, “but I told ??? we didn’t make spore powder” Who was the FBI’s anthrax expert who told Dr. Ivins not to get his “panties in a bind over this”? Was that Dr. Jahrling rather than Dr. Ezzell as I had once inferred? She nowhere addresses the fact that the FBI removed the original of Lab Notebook 4010 (and other notebooks that were subpoenaed) without leaving a copy. Why won’t the FBI produce the relevant pages from the lab notebooks it took? . She nowhere explains that Daniel Seikaly pled the Fifth Amendment in connection with the leaks relating to Hatfill or notes that his daughter represented “anthrax weapons suspect” Ali Timimi pro bono. . She nowhere addresses why the US Attorney and AP created the impression that the Federal Eagle stamp was uniquely sold in Ivins’ post office (near USAMRIID) when it in fact was sold throughout Maryland and Virginia. This misstatement by the US Attorney (picked up by AP) was as great as any misstep in connection with a “Hatfill Theory”. . She nowhere addresses why the FBI failed to disclose that the photocopier mentioned in the Amerithrax Summary could be excluded as the source of the Amerithrax letters. That is the sort of evidence that makes for a strong scientific case — or demolishes one. This is different from the less the much less significant issue of “tracks” made by the photocopier gripper. . The best I recall, she nowhere addresses why the FBI let USAMRIID General John Parker’s false claim that USAMRIID did not make dried powder stand — when the FBI and the scientists overseeing the investigation knew its own expert had made dried powdered aerosol using Ames. . She nowhere addresses the identity of the colleague with whom Dr. Heine says he did research regarding antifoam in creating aerosols or Dr. Heine’s report that the FBI falsely told Dr. Ivins that Dr. Heine had accused him of the anthrax mailings. This is a huge issue because the investigators then used Dr. Ivins’ rage as proof of his guilt — rather than evidence of his innocence. . She nowhere addresses why the FBI never disclosed the email withheld for 2 years that shows Dr. Ivins knew that 5 ml of virulent Ames had been taken from Building 1412. She nowhere addresses the email asking about weaponized anthrax that came to Detrick and then was shipped out and some was missing. She does not emphasize that the FBI estimates that up to 377 had access required elimination (allowing for some duplication who had access in both 1425 and 1412). US Taylor falsely claimed that only 100 needed to be eliminated — only those with access at Building 1425. Although she may not get into the particulars, she does a good job on the issue of genetics and speaks with authority on such issues — but she does not discuss the reason the location of the flasks (initially there were two flasks) was carefully whited out so as to change its location from Building 1412 to Building 1425. That change violated USAMRIID protocol about record-keeping. She nowhere addresses to whom Dr. Ivins was writing about the Ames missing from building 1412 and the autoclaving of samples there. She nowhere addresses what happened to the other slant sent from Texas, or interviews the original researcher who obtained the slants from Texas who then went to work for the CIA. On a minor note, she is mistaken that the inventory destroyed in Iowa was only a single sample (I interviewed the professors involved in the destruction). She nowhere addresses Dr. Ivins’ concern expressed to a superior that he was missing samples — only to be told to shut up. He never identifies the superior telling Ivins that everything was under control. She nowhere addresses when Southern Research Institute first obtained virulent Ames and from whom. She nowhere addresses where the research on the corona plasma discharge and sonicator on Ames spores supplied by Bruce Ivins was conducted for DARPA. She nowhere addresses where else the DARPA aerosol studies using dried powder were done. Given the performance of the dried aerosol, the technical question of whether the floatability is due to use of a CPD or sonicator should be addressed by the scientific experts. She nowhere addresses the fact that the only expert interviewed by the FBI about the code in the letters for which documents were produced disagreed with the FBI’s theory of code in the letters and that all the letters needed for the FBI’s interpretation of the code were NOT in fact double-lined. Once this understood, one realizes what a crock of a case the FBI concocted. She nowhere addresses why the FBI was asking everyone whether they had seen olive oil in one of the aerosol rooms. She does not addresses whether olive oil was what the bloodhounds smelled at Denny’s when the FBI assumed they were tracking Steve Hatfill who had visited the day before. She nowhere uses the name Ayman Zawahiri — the man whose close associates had announced he was going to use anthrax against US targets to retaliate for the rendering and detention. This sort of omission makes it a fine entry for a public library but not for anyone interested in determining whether Dr. Ivins in fact committed the crime. … which should be the point of the entire exercise. The book’s treatment is academic in the best and worst sense. She nowhere addresses the 16 pages which were not obtained by the FBI until February 2005 — those pages involved distribution of Ames to a former Zawahiri associate. I don’t mind that reporters and academic don’t write of such things — indeed, given the history that Amerithrax took with the hyping of the Hatfill story by the chief prosecutor whose daughter came to represent Ali Al-Timimi, I totally understand it. But the fact that they don’t make relevant phone calls supports the view that investigative reporting is dead in this country. Nowadays, writing up a filed court document or reading documents the FBI submits to NAS is deemed a substitute. For broadcast, getting interviews of people journalists have quoted suffices. And of course, when you are channeling investigators, interviewing the dead guy’s prom date is what passes for investigation. Heaven forbid that someone would pick up the phone and seek an interview with Rauf Ahmad who was working with Dr. Zawahiri.’-rmr-1029-anthrax-more-questions-for-um-and-lsu-researchers/ She nowhere addresses the work that Yazid Sufaat and his lab assistant did at Omar Hospital in May 2001 while the equipment was en route to the lab being established at Kandahar. (Yazid Sufaat does not deny to me that he was working with virulent Ames on his Facebook page posts or in private chats.) The Detainee Assessments on this subject are part of the public record and so why did they not inform the discussion? Is the GAO report going to be similarly bereft of highly relevant information? She nowhere addresses that FBI experts found that Dr. Ivins handwriting does not match the handwriting in the anthrax letters but is understandably highly skeptical of the FBI’s sorority theory. Don’t get me wrong. On her overall approach to an Ivins Theory, I totally agree with her just as I did Laurie Garrett (although there are key differences in the approach of the two authors). I am using this approach to assessing the book to highlight uploaded documentary evidence that can further advance things. Because determining the confidence level we should have in the FBI’s speculative Ivins Theory is very important. Professor Guillemin nowhere addresses a “Waly Samar” who was a microbiologist connected to the WTC 1993 participants based on phone records and reportedly lived in the Trenton area in 2001 — an estimated 20 miles from the mailbox. I called and left a message last month but hadn’t hear back when I last checked for messages. And, again, it is totally understandable for a journalist or sociologist and so this is not a fault. It all comes down to one’s personal assessment about what is at stake. And I think on that question — lapses in biosecurity — no one should disagree with the Professor. But that in fact has been acknowledged at the expense of actually solving the whodunnit. Professor G. nowhere addresses the claim that a pharmacist Najmut Tariq in New York City was connected to Al Qaeda anthrax program and apparently no effort was made to contact him in Pakistan. But someone like the Wash Po correspondent in Pakistan should attempt an interview. She does not address how the FBI was able to exclude Abderraouf Yousef Jdey as the mailer if the FBI doesn’t know where he was and, according to former top CIA analyst Rolf Mowatt-Larssen, Jdey was released before the mailings. Intelligence analysis, for example, would point out that Dr. Assaad was Coptic Christian and the Blind Sheik’s group primary mission is to persecute Coptic Christians. For people including JG to miss this is to miss the possible significance of that anonymous letter that some view as an intentional “red herring” pointing to Dr. Assaad. (Dr. Assaad, for his part, thinks that the claims against Dr. Ivins are part of a vicious plot).…-who-was-the-real-anthrax-mailer-the-key-people-in-the-anthrax-mailings-were-not-bruce-ivins-or-steven-hatfill-his-predecessor-as-the-fbis-target-instead-they-app/ She nowhere addresses experiments led by Egyptian Abu Khabab killing rabbits with poisons under during the month before 9/11 at a camp outside Kabul. She nowhere addresses the training in late 2001 at the training camp outside Kandahar to introduce poison into water systems. Given that the recent claims about ricin and the lack of any direct evidence that the planning in fact related to bombs, this sort of consideration is very important in any book on the subject of biodefense and the threat faced by the country. Professor G. nowhere addresses the capture of Mustafa Hawsawi and his laptop containing anthrax spray drying documents. In her notes she relegates to passing mention in a footnotes the issue of subtilis (see footnote to conclusion) that full deserved the much greater importance placed on the issues by the McClatchy journalist. You’ll recall that it was McClatchy that emphasized the potentially critical importance of b. subtilis contaminant found in the Brokaw and New York Post anthrax letters … not connected to Dr. Ivins … and substantially ignored by the FBI. The public is expecting great things from McClatchy/Frontline/ProPublica — let’s just hope on the silica issues they move things along to what should be the proper frame of the issue — the probative importance of the Silicon Signature. The framing of the debate in 2003 missed the point entirely. The key consideration is what processing might Dr. Ivins have used that would have resulted in the signature. Professor G. nowhere addresses the fact that Anwar Aulaqi was coordinating with Ali Al-Timimi who shared a suite with the two leading Ames researchers. Heck, if she doesn’t so much as name the head of Al Qaeda’s specific anthrax planning and sending of scientists to attend USAMRIID conferences, this is not surprising. But Rauf Ahmad’s attendance of Porton Down conferences is far more relevant to the issue of biosecurity — on which she organizes her thesis — than it is that Dr. Ivins attended a presentation she gave at Ft. Detrick on Sverdlosk in 2002. People need to address the issue and stop being ostriches with their head in the sand. That is precisely how lapses in biosecurity occur. Ironically, she and the psychiatrists are part of the problem — not part of the solution. She nowhere addresses the documents from peer reviewed literature in Ayman Zawahiri’s possession. She nowhere addresses the spraydrying documents on Al-Hawsawi’s laptop. She nowhere addresses Rauf Ahmad’s notes and handwritten letter (he was one of the scientists working for Ayman Zawahiri). She nowhere addresses the typed correspondence from a later visit by Rauf Ahmad indicating that he had successfully achieved the targets. (And, no, Milton L. did not systematically refute the matter in discussing the correspondence in his book – he avoided quoting this critical language altogether!) She nowhere addresses Ali Mohammed, the head of intelligence for Egyptian Islamic Jihad who had a document on his computer seized by the FBI that outlined principles of cell security that would be followed, trained Dahab, a Cairo medical drop-out, to make deadly letters. She nowhere addresses the Egyptian visitor in the B3 who was the lifelong friend of a former Egyptian Islamic Jihad member, a schoolmate, recruited by Ayman Zawahiri. She nowhere addresses the fact that Dr. Bruce Ivins hosted one Egyptian visitor in the B3 who was the lifelong friend of a former Egyptian Islamic Jihad member, a schoolmate, recruited by Ayman Zawahiri and that the FBI not obtain the relevant documents until February 2005. She nowhere addresses the fact that this document seized in Afghanistan pointed to infiltration of US biodefense. To what was the author referring? She nowhere addresses this Zawahiri correspondence with infiltrating scientist that was part of parallel compartmentalized cell operation. Who else did Ayman attempt to recruit (besides the schoolmate and close friend of Bruce Ivins’ co-worker)? She nowhere addresses the documents dating from April 1999 showing that Ayman Zawahiri’s plan was to recruit a specialist. Who else did Ayman Zawahiri succeed in recruiting? She nowhere addresses the fact that the lifelong friends of Dr. Tarek Hamouda, supplied virulent Ames by Bruce Ivins, actively denounce their former medical school associate Ayman Zawahiri as a fanatic – one serving as President of CAIR-St. Louis and the other as author of INSIDE JIHAD. After the FBI first obtained in 2005 the documents relating to Dr. Hamouda’s work with Dr. Ivins, did they contact Dr. Hamid who reports he was recruited into the Egyptian Islamic Group by Ayman Zawahiri while in medical school? Did they contact his brother who publicly announced that he could not identify a sleeper cell if he did not know about it? She nowhere addresses why the FBI failed to disclose that Jdey was detained and released as the same time as Moussaoui. She nowhere addresses the fact that Ayman Zawahiri had an extensive recruiting network for his anthrax planning and the announcement of his plans in March 1999, including the blind sheik’s son who spoke alongside Ali Al-Timimi and was on Al Qaeda’s 3-member WMD society. Did the blind sheik’s son recruit Ali Al-Timimi? She nowhere addresses the fact that Ayman Zawahiri used “school” to refer to the Egyptian Islamic Jihad but does poke fun at the FBI’s theory as to why “school” was used. She nowhere addresses the documentary evidence showing that Ayman Zawahiri used “school” as code and not Bruce Ivins. She nowhere addresses the fact that while the US government focuses on Anwar Al-Aulaqi, the media continues to overlook Aulaqi’s connection to fellow Falls Church imam, a scientist sharing the suite with the leading bioweapons Ames anthrax researchers with whom defense counsel says Aulaqi was coordinating. She nowhere addresses the fact that Ali Al-Timimi had unfettered access to the largest microbiological repository in the world where the bacteriology collection scientist was the future head of the Amerithrax science investigation who would guide the NAS review and the production of documents from the FBI to NAS. … Essentially, Laurie Garret’s book asked: given all the uncertainties, isn’t there a very real chance that an Ivins Theory is just a Hatfill Theory redux — and had the same effect of narrowing the investigative focus? (Laurie suggests FBI higher-ups by Spring of 2002 never meaningfully pursued an Al Qaeda theory.) Professor Guillemin, in contrast, just sticks to very safe territory and the rubber never hits the road. That serves very well for an academic overview that is well-suited to grace library shelves. But what we need is investigative reporting and a GAO report that is written by investigators who understand the importance of obtaining the most probative documentary evidence — for example, emails. If you merely pay “lip service” to “ambiguities” in the DOJ’s proof — when the unanswered questions are so important — then you may not be in a comfortable position if the GAO ever lets loose with a fire hose of documentary evidence.
    • DXer said

      The New York Post also highlighted the role of the “Red Team” in a different context. The names of the people who provided such important input to the investigation should be disclosed under FOIA.

      FBI’s anthrax case blows apart–Editorial –…/anthrax_and_the_fbi_36aNmZJGENZCAPCmv...
      Last Updated: 4:24 AM, October 23, 2011

      Read more:

      What’s more, the baseline evidence linking Ivins to the anthrax spores was inconclusive, according to a “Red Team” of outside scientists the FBI called in to review its work — but then utterly ignored.

      The feds “deviated from traditional lab practice in this particular case,” said Jenifer Smith, former section chief at the FBI’s Weapons of Mass Destruction Directorate. “There were some political things going on behind the scenes, and it was embarrassing not to have this solved.”


      Truth is, the case has been a mess since Day One. G-men first named and shamed biowarfare specialist Steven Hatfill as a person of interest but later exonerated him — and coughed up a $5.8 million settlement for ruining his life.

      And while the FBI says the new paper is wrong, it clearly can’t be trusted to judge cases that reflect badly on its own conduct. Indeed, its ability to pursue sensitive investigations at all is in doubt.

      To cite just one example, in the months before Nidal Malik Hasan massacred 13 people at Fort Hood in 2009, the FBI intercepted e-mails Hasan had sent to al Qaeda imam Anwar al-Awlaki. But it sat on clear evidence the unhinged Hasan was quickly boiling over — and let the killer-in-waiting go on his fatal shooting spree.

      Given the FBI’s troubled anthrax history, it’s good to see that Congress’ oversight body, the Government Accountability Office, is conducting its own review of the FBI’s work and looking into the possibility that Ivins had help in growing the anthrax or acquired it from another lab.

      We hope the FBI is right about Ivins, and that Americans can sleep soundly. But hope doesn’t cut it in bioterrorism.

  40. DXer said

    Profssor Guillemin responds:

    Science 11 May 2012:
    Vol. 336 no. 6082 p. 669

    “Presumed Guilt in the Anthrax Case”

    In his review of my book American Anthrax on the 2001 anthrax letter attacks (“Have we ‘met the enemy?,’” 3 February, p. 540), D. A. Relman accuses me of imposing a “presumption of guilt” on the FBI’s prime suspect, U.S. Army microbiologist Bruce Ivins. In fact, I described many ambiguities in the case against Ivins and took no personal position on his guilt or innocence. I relied on the report of the National Research Council committee that evaluated the FBI science, and, regarding a possible foreign source for the letter spores, I accepted its conclusion that “We consider these data to be inconclusive regarding the possible presence of B. anthracis Ames at this undisclosed overseas site” (1). Relman served as vice chair of the committee that reached this conclusion.

    [Corrected 14 May 2012 to replace earlier version posted in error.]

    Jeanne Guillemin

    Security Studies Program, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

    • DXer said

      For some of the backstory, you need to understand that last fall, Professor Guillemin spoke at the American Association of the Advancement of Science and called for a commission to review the scientific consisting of the “A Team” — implying that the A Team wasn’t on the NAS committee. She says the Commission should be granted access to classified information. Respectfully, a more basic start would be to disclose the members of the “Red Team” who opined that the silicon signature was not worth pursuing. Her husband is Matt Meselson who addressed the issue with Ken Alibek in the June 2002 New York Times — at the time, Ken had not even known of the AFIP findings until I told him. The “A Team” would be people who have actually made anthrax and anthrax simulants such as John Kiel who did controlled studies with and without silanizing solution and sent me the images. I urged that he be called to testify. As for access to information, nothing — nothing at all — warranted the FBI withholding of any information relating to the overseas testing until 9 months after the last presentation. It is a canard to equate the silicon signature with weaponization — when instead the key is its probativeness as a signature (such as characteristics of the mailed powder).

      Ronald Schouten also spoke and discussed the uncertainties in the case. What a bunch of crock. What he did not disclose was what he now knows about the reliability of the central witness, Judith McLean, in the EBAP report. (He may actually have been its author). He did not advise the federal court to whom his report was submitted of changes that needed to made. So paying lip service to uncertainties in criminal prosecutions is merely part of his public and formal advocacy based on Dr. Ivins’ presumed guilt in the absence of any direct evidence implicating him. If a prosecutor did what the psychiatrists did here, they likely would be savaged by a District Court who had been presented the witness statements.

      Dr. Guillemin and Dr. Schouten pay disengenuous lip service to uncertainties in the DOJ’s Ivins Theory in the same breath where they do not address the real issues. They appear not to understand them. For example, they evidence no knowledge of the rabbit documents showing that Dr. Ivins’ time in the lab was not in fact unexplained. Professor Guillemin is focused on the irrelevant qualifications about the genetic connection to the 4 morphs — while totally overlooking the more fundamental issue of sample collection being voluntary and the fact that the 4 morphs did not even narrow the field at USAMRIID, given as a practical matter everyone had access to both the samples with the morphs and those without. Nationally, it only limited things from 700 to 200-300.

      So, we don’t need to view the NAS committee as the B team — on the theory that the A team was too busy with real work of making money. What we need is the GAO to arrange for the disclosure of all information that it is not exempt from production. And the GAO should study how the FBI is overclassifying information that is over 10 years old and not appropriately withheld from public view. GAO in consulting scientists should not continue the FBI’s rich tradition of tolerating and not disclosing conflicts of interest.

      • DXer said

        We don’t need the views of sociologists or psychiatrists in addressing a true crime matter.

        We need evidence — and the FBI has not provided any establishing Dr. Ivins guilt under any standard.

        The judge would not even hear the evidence Dr. Schouten evidence — he would be too enraged about what Dr. Schouten and Dr. Saathoff had not disclosed that central witness explained that she was psychotic at the time (2000) and having auditory hallucinations about murderous fiends she imagined trying to kill her. She received her instructions each night from an alien who had implanted a chip in her butt. The pair have continued to promote their report without any changes.

        The most reliable evidence — absent any pertinent scientific evidence — is documentary evidence.

        The GAO can make that happen so long as they have adequate resources for the necessary staff to marshall and upload the documents.

        • DXer said

          The best insight into Professor Guillemin’s thinking is her talk at the Madison Building on April 2, 2012. An uploaded transcript would avoid playing the game of piercing carefully constructed sentences to a renown journal like SCIENCE.

  41. DXer said

    Here is a May 2012 article by S. Leppla on the role of mutations in the Amerithrax investigation.

    Microbes and Infection
    Volume 14, Issue 5, May 2012, Pages 387–391

    Occurrence, recognition, and reversion of spontaneous, sporulation-deficient Bacillus anthracis mutants that arise during laboratory culture

    Inka Sastalla, Stephen H. Leppla,
    Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 33, Bethesda, MD 20892-3202, USA
    Received 20 October 2011. Accepted 17 November 2011. Available online 28 November 2011

    Bacillus anthracis is a spore-forming, soil-dwelling bacterium. This review describes the occurrence of spontaneous mutations leading to loss of sporulation and the selective pressures that can lead to their enrichment. We also discuss recognition of the associated phenotypes on solid medium, thereby allowing researchers to employ measures that either prevent or favor selection of sporulation-deficient mutants.

    Bacillus anthracis; Sporulation; spo0A; Mutation; Amerithrax


    1. Introduction
    The ability to form endospores sets the Bacillus genera apart from many others. This trait allows the bacteria to survive for long periods in harsh and unfavorable conditions, such as heat, desiccation, and low nutrient availability. In nature, Bacillus anthracis spores can reside in the soil for decades before they are inhaled by grazing animals, which initiates germination and vegetative growth, followed by production of capsule and anthrax toxins. The anthrax letter attacks in 2001, which became the subject of the FBI’s “Amerithrax” investigation, involved mailing of B. anthracis spore preparations to several news organizations and to two U.S. Senate offices, leading to the death of five people and infection of many others. In nature, the formation of spores, usually induced by starvation signals, is an essential part of the developmental life cycle of B. anthracis. However, in the rich media used in laboratory cultures, where nutrient availability is not obviously limited and the pressure to sporulate is low, spontaneous, and initially silent, mutations in genes involved in sporulation pathways can accumulate, leading to loss of the ability of Bacillus to initiate or complete the sporulation cycle.

    2. Sporulation-deficient mutants spontaneously formed by Bacillus have a distinct phenotype on solid medium

    The frequency of spontaneous mutations occurring in B. anthracis was estimated to be between 10−7 to 10−9 per base pair per generation [1]. Mutations can have many origins, including replication errors, DNA damage caused by mutagens, and ineffective repair mechanisms [2]. Because of the low frequency with which these errors occur, they are generally lost during propagation of a population. However, it was shown that in certain liquid growth media, asporogenic bacteria can quickly accumulate and become a large fraction of a population, as was observed for several different Bacillus species [3], [4] and [5]. It appears that in these cases the growth or passage conditions imposed an unrecognized selective pressure that favored the growth or survival of sporulation-defective mutants. As long as growth was continued in a liquid culture, the identification and elimination of the spontaneous, asporogenic members of the population was not possible.

    In contrast to the situation in liquid medium, B. anthracis mutants grown on solid media that fail to initiate sporulation are easily recognizable by their unique colony morphologies. In comparison to their sporogenic counterparts, colonies consisting of asporogenic bacteria are more translucent and larger [6], or can have a yellow or yellow-gray color [7]. The addition of certain indicator dyes such as Congo Red to solid media can enhance the differences in appearance between sporulating and non-sporulating colonies [8] (Fig. 1). We have also noticed this atypical colony phenotype of B. anthracis in less than 1% of colonies after 2 days of growth on some solid media such as Luria-Bertani, while it was less obvious on other media [9]. Additionally, factors such as the age of colonies, the number of passages performed for a single colony, and the type of solid medium on which bacteria are maintained can greatly influence the frequency of mutants altered in sporulation pathways. In particular, we found frequent mutations of various kinds in the global regulator Spo0A, which is involved in the initiation of sporulation [6]. Loss of Spo0A function leads to a complete block of sporulation.
    3. Key role of sporulation-impaired B. anthracis mutants in the Amerithrax investigation
    The accumulation of mutant bacteria within a laboratory culture as described above played a key role in solving the Amerithrax investigation [7]. The microbial forensics studies performed in support of the investigation produced a unique fingerprint of the culture used in the anthrax letter attacks and allowed it to be traced back to the presumed perpetrator’s laboratory. Rare colonies within the population were noted to have aberrant morphologies (“morphotypes”). The genomes of these variants were sequenced and found to be highly similar to the Ames Ancestor strain, which is studied in laboratories worldwide [10]. Furthermore, these analyses showed that four chromosomal loci harbored mutations unique to the variants present in the spore preparations of the anthrax letters. Of these four loci, three could be linked to Spo0F, a bacterial response regulator that, like Spo0A, is activated at the onset of sporulation [11], [12] and [13]. Unlike the totally sporulation-deficient Spo0A mutants that we obtained [9], the mutants identified in the anthrax letter cultures retained a limited ability to form spores, as expected considering their isolation from spore preparations. Interestingly, some of the mutations occurred through mechanisms like those that caused the Spo0A mutations we found. For example, both groups of mutants included mutations caused by illegitimate recombination events [14] and [15] that occurred between short direct repeats, resulting in deletion of a larger region of the genome. Thus, it appears that the culture that was the origin of the spores used in the letter attacks had been repeatedly passaged and/or expanded in a way that enriched for variants that sporulated poorly. We discuss below mechanisms that may have led to this enrichment.

    4. Possible reasons for the selection of asporogenic mutants during laboratory culture
    Laboratory manipulations as simple as growth in a specific medium unintentionally impose selective pressures that can lead to the enrichment of bacteria harboring particular mutations. Thus, cultures passed repeatedly can quickly acquire a genetic fingerprint unique to a bacterial culture that is kept in a particular laboratory. However, the mechanisms that drive induction, selective growth, and enrichment of asporogenic mutants are not well understood. While a shortened lag phase could be an explanation for the larger colony size observed in asporogenic colonies of B. anthracis[9], in Bacillus subtilis it was found that asporogenic mutants with mutations in particular sporulation-dependent genes have an improved overall growth rate and higher biomass yield [16]. The higher biomass yield in those B. subtilis mutants could result from a loss of the cannibalistic behavior described for B. subtilis, whereby sporulating bacteria eliminate their non-sporulating siblings to acquire nutrients [17] and [18]. Asporogenic bacteria with a mutated Spo0A would not be able to kill their neighbors, resulting in a higher overall biomass.

    An alternative phenomenon that might select for sporulation-defective mutants also depends on competition for nutrients. When bacteria grow on agar plates, it is likely that the very dense population of bacteria in a colony (>107 organisms in a 2–3 mm colony) will lead to severe competition for the nutrients that slowly diffuse into the colony from the surrounding agar. The resulting nutrient deficiency will be recognized as a signal to sporulate, especially by bacteria at the center of the colony, so that the wildtype members of the population will stop dividing and sporulate. Any rare spontaneous mutant bacteria that fail to recognize or successfully act on the signal to sporulate will continue to grow, perhaps making use of nutrients released by lysis of mother cells of nearby spore-forming bacteria. In this way, the sporulation mutants can become enriched as the colony ages.

    5. Advantages and disadvantages of sporulation-deficient mutants for research
    Bacillus mutants defective at various stages of sporulation have proven invaluable for the analysis of biochemical markers characteristic of sporulation, the identification of genes involved in sporulation, and for the elucidation of complex sporulation pathways. For example, early studies comparing sporogenic and asporogenic colonies of B. subtilis showed that certain proteases and antibiotics are only secreted by sporulating bacteria [19] and [20]. Similarly, the many genomic loci required for sporulation were identified by transduction analyses (for reviews see [6] and [21]) and later by cloning of genes involved in these pathways [22], [23], [24], [25] and [26]. The rate with which Bacillus loses the ability to sporulate in liquid culture has further been useful for the analyses of mutation frequencies as a model for calculating the rate with which trait losses occur during evolution [27] and [5].

    Bacillus species are valuable as expression hosts for production of industrial enzymes (e.g., proteases), as recently reviewed [28], and are offered commercially as expression hosts to the research market (e.g., Bacillus megaterium by MoBiTec, Inc.). The currently licensed (non-recombinant) anthrax vaccines are partially purified supernatants of attenuated B. anthracis strains [29], and some candidate second generation, recombinant anthrax vaccines are produced in improved B. anthracis host strains. An improvement relevant to this discussion is to render the expression host unable to sporulate. Strains that are sporulation-deficient can be isolated as spontaneous mutants using the simple morphological screens mentioned above and then validated by sequencing [9] and [8], or can be engineered by intentional deletion of specific sequences [30]. Thus, strains derived by these methods and specifically deleted in the spo0A gene are being used to produce candidate anthrax vaccine proteins [31], [8], [32] and [33], although whether mutations in spo0A or other sporulation-related genes enhance toxin production has not yet been determined. Nevertheless, in this and several other laboratories, Spo0A-deleted strains are routinely used to produce recombinant anthrax toxin proteins [34] and [35].

  42. DXer said

    Report on Stevens prosecution tells of ‘systematic concealment’
    Anchorage Daily News /
    Published: March 15th, 2012

    WASHINGTON — A court-appointed special prosecutor’s report made public on Thursday details findings that Justice Department attorneys intentionally withheld information from the defense in the bungled prosecution of former Alaska Sen. Ted Stevens.

    “The investigation and prosecution of U.S. Senator Ted Stevens were permeated by the systematic concealment of significant exculpatory evidence which would have independently corroborated Senator Stevens’ defense and his testimony, and seriously damaged the testimony and credibility of the government’s key witness,” the report said.

    The 514-page report by Washington lawyer Henry Schuelke was released Thursday by order of U.S. District Judge Emmet Sullivan, who presided over Stevens’ 2008 trial and hired Schuelke to evaluate whether anyone on the Stevens prosecution team should be charged with criminal contempt of court.

    Read more here:

    Comment: Delays or failure to produce happen for widely varying reasons. But producing the documents, even late, is the wise course.

  43. DXer said

    Experts in the field could weigh in on the potential probativeness of the inquiry into the ink used on the envelopes to include but not limited to Derek L. Hammond, U.S. Army, Criminal Investigations Lab, Ryan C. Tomcik or Gerald M. LaPorte, United States Secret Service, Forensic Services Division, Albert H. Lyter, Federal Forensic Associates, James F. McClelland, Jeffrey S. Sweterlitsch and Roger W. Jones, Iowa State University, or Walter F. Rowe, George Washington University, Department of Forensic Services.

    The AUSAs failed to disclose the most important factual issue of all to their superiors — Dr. Ivins work with the 52 rabbits that week in the space as explaining why he was in the lab.

    Let’s at least obtain and disclose the relevant scientific evidence such as examination of the ink used on the envelopes. In the hundreds of handwriting exemplars seized by the FBI from his residence and office, Dr. Ivins never used the pen used to address the envelopes. It is part of the science relied upon by the FBI in its Amerithrax investigation and should be examined by the GAO.

    • DXer said

      As an example, it could be compared to the black felt tip pen used in Afghanistan in the anthrax planning session.

      On the floor, there was what appeared to be a disassembled rocket alongside a helium canister, as well as two bags of powder. A detailed diagram scrawled in black felt tip pen on a white board shows what appears to be a balloon rising at various trajectories, alongside a fighter jet that is apparently shooting at the balloon.

      Beside the jet are the words, “You are dead, bang.”

    • DXer said

      It is even the stuff of science fair projects.

      Using Paper Chromatography

      Students will use paper chromatography to separate ink molecules and identify the pen used on an …. Relate the ink back to ink on the anthrax letters.

      Click to access paper_chrom.pdf

  44. DXer said

    Dr. Leppla and his co-author explain:

    “We found that accidentally asporegenic mutants can be “repaired” and that complex genetic strain construction projects can be rescued by applying phage transduction in B anthracis, where it has enabled transfer of the virulence plasmid x02 and marked mutation fom one strain to another.”

    Back in 2001, the University of Houston had a $100,000 grant from the CIA to study the persistence of the growth of anthrax in soil. As a layperson, I understand soil to be silica. As I explained to Dr. Leppla, most science is over my head and so the scientists can correct me if I am mistaken.

    I wrote Dr. Koehler in 2002 to ask her whether that lab had virulent Ames. The lab upgraded to BL-3 in March 2001. As she explained to NPR in Fall 2001, the security at the lab was not what it might have been. There weren’t even locks on the door as I recall her description to NPR in Fall 2001.

    Then a tropical storm wiped out the lab in June 2001, flooding the lab with millions of gallons of water and leading to the propping open of doors and the tragic death of many lab animals.

    When I asked Dr. Koehler why the lab upgraded to BL-3 in March 2001 — and everyone was vaccinated — she explained that the scientists at the lab were inserting virulence plasmids into avirulent strains, rendering them virulent. Do it yourself, if you will.

    Now in March 2001, Dr. Ivins fedexed virulent Ames to Rick Lyon’s lab at UNM. Why was it sent in March if the UNM lab was not completed until December 2001?

    Back at Houston, then graduate student Melissa Drysdale was a key scientist doing the insertion of virlence plasmids, rendering avirlent strains virulent. What does her PhD thesis say about her work?

    After finishing her thesis, when did Aafia travel to see her brother’s family in Houston? She is associated with the address in Houston as is her sister and mother. Aafia’s sister-in-law, Dr. Khawaja, had an office down the hall in that building from Dr. Koehler. Aafia used to go to classes in that building. Did she visit Houston in June and go to that building to visit her old haunts? Years ago I emailed Aafia’s sister-in-law, Dr. Khawaja, to ask but got no reply.

    The FBI cannot fault Mr. Cohen of NYPD intelligence for wanting to check the FBI’s work on Amerithrax given that the DOJ and FBI provably failed to acknowledge that 52 rabbits were delivered to Dr. Ivins B3 in late September 2001. PC PR may loom large to the FBI but everyone is going to have to take a deep breath while the Volvo-driving soccer moms are separated fom the Al Qaeda operatives working for KSM and Atef and Dr. Ayman.

    In advancing worthy public interest causes like civil liberties, ACLU and other organizations always have a choice of particular defendants in advancing the issue. Sometimes they choose wrong.

  45. DXer said

    GAO: Please take note of this article that is on the subject of mutations and has as one of its key words “Amerithrax.”

    Occurrence, recognition, and reversion of spontaneous, sporulation-deficient Bacillus anthracis mutants that arise during laboratory culture

    Sastalla, I., Leppla, S.H.

    Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 33, Bethesda, MD 20892-3202, USA


    Bacillus anthracis is a spore-forming, soil-dwelling bacterium. This review describes the occurrence of spontaneous mutations leading to loss of sporulation and the selective pressures that can lead to their enrichment. We also discuss recognition of the associated phenotypes on solid medium, thereby allowing researchers to employ measures that either prevent or favor selection of sporulation-deficient mutants.

    Language of original document


    Author keywords

    Amerithrax; Bacillus anthracis; Mutation; spo0A; Sporulation


    • DXer said

      All this science stuff is over my head. But early learning regarding the effect of nutritional stress and mutants (in work done using bacillus subtilis) was done by the fellow who was in phone contact with the phone associated with the WTC 1993 bomber / Ramzi Youssef (KSM’s nephew) right up to the moment of the Blind Sheik’s arrest. The one who in 2001 lived in the New Brunswick area.

      Mutation under stress in Bacillus subtilis: Is it transcription – Induced or is it due to Gene Amplification R. Rudner D. White, A. Murray and W. Samarrai, Abstract :Functional Genomic of Gram-Positive Microorganisms 12th International Conference of Bacilli Baveno, Italy 2003

      3. Differential Response of Bacillus subtilis Ribosomal RNA Promoter to Nutrition Stress., W. Samarrai, E. Shorn, R. Rudner Abstract: ASM 103 General Meeting 2003.

      Michal Grop, E. Eizenman, G. Glaser, W. Samarrai and R. Rudner, A relA (s) suppressor mutant allele of Bacillus subtilis which maps to relA and responds only to carbon limitation. Gene, 140 (1994).

      For some reason, the AUSA thought it more probative that Dr. Ivins handed a female co-worker a dildo on her last day of work.

      Everyone is predisposed to certain thinking based on their life’s experiences.

      My life experience was shaped by going to Tysons Corners mall.

      Amerithrax represents the greatest failure in intelligence analysis in the history of the United States.

    • DXer said

      Laurie Garrett makes mention of Dr. Leppla’s expertise:

      “When the Daschle letter reaches Ft. Detrick, a team of 90 USAMRIID scientists, assembled virtually, awaits it. Their names and expertise are listed, and their willingness to assist in the FBI investigations noted. Collectively they represent the world’s top anthrax team, including Dr. Arthur Friedlander, whose animal studies of inhaled anthrax are the primary source of all medical treatment decisions for people suspected of having been exposed to the microbe. Friedlander has studied anthrax for 25 years. Also on the team are scientists John Ezzell, Stephen Leppla, Perry Mikesell, Bruce Ivins and Susan Welkos, who have co-authored many significant studies of anthrax toxins and vaccines, dating back to 1983. Ivins has been been working on anthrax since 1980.”

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