* 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
opinion.
******
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 …”
******
CASE CLOSED ... what really happened in the 2001 anthrax attacks? 
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.
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
http://nationalinterest.org/feature/how-get-terrorists-talk-12270
DXer said
Anthrax detection: Analyses and select studies of sampling methods (with accompanying DVD) (2013)
(Book)
Arnaud, C.M.
——————————————————————————-
View references (47)
Abstract
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.
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.
ABSTRACT
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.
KEYWORDS
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.
DXer said
The DNA detected was Ames, not Vollum or some other strain.
DXer said
http://www.rdmag.com/award-winners/2014/08/pregnancy-hormone-sniffs-out-anthrax
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.
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.
http://med.stanford.edu/news/all-news/2014/09/5-questions–david-relman-on-risks-of-creating-new-pathogens.html
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
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)
http://www.nap.edu/openbook.php?record_id=18737&page=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)
http://www.nap.edu/openbook.php?record_id=18737&page=21
DXer said
[relatedly]
“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
[relatedly]
“- 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)
http://www.nap.edu/openbook.php?record_id=18737&page=24
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.”
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.
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.
http://wwwnc.cdc.gov/eid/article/8/10/02-0354_article.htm
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.
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
Abstract
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.
http://www.amerithrax.wordpress.com
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
Highlight:
Discussion
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.
***
Discussion
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.
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
Reuters
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.
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.
Source
Graduate School and University Center, The City University of New York, 365 Fifth Avenue, New York, NY, 10016.
Abstract
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.
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.
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?
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.
Source
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.
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.
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.
http://www.journals.elsevier.com/forensic-science-international/most-downloaded-articles/
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.
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.
DXer said
Syria retaliation: Could Assad strike back in response to U.S. military action?
http://www.syracuse.com/news/index.ssf/2013/09/syria_retaliation_could_assad_strike_back_against_us_military_action.html
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?
Comment:
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.
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.
Abstract
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.
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: http://swampland.time.com/2013/08/07/dr-evil-terror-alert-puts-bin-ladens-successor-back-in-the-spotlight/#ixzz2bZVB2HOq
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.
http://www.nybg.org/
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.
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
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.
Abstract
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.
DXer said
http://www.pbs.org/wgbh/pages/frontline/criminal-justice/anthrax-files/edward-montooth-the-mandate-was-to-look-at-the-case-with-fresh-eyes/
[interviewer]
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.
Comment:
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.
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
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.
(arrahmah.com)
Translated and Submitted by a Mujahid
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?
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.”
DXer said
Of course, the doubts are expressed not merely by the major media, but the scientists who led the NAS panel.
http://www.amerithrax.wordpress.com
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="http://www.iom.edu/~/media/Files/Activity%20Files/PublicHealth/MicrobialThreats/2012-JUN-12/June-2012-Audio/101-Welcoming-Remarks.mp3" /]
DXer said
David Relman gave welcoming remarks at the June 2012 conference at which Dr. Fraser-Liggett and Dr. Bruce Budowie spoke.
http://www8.nationalacademies.org/publicmeeting/meetingview.aspx?meetingid=338
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
http://www.sciencefriday.com/segment/10/28/2011/scientific-case-still-open-on-2001-anthrax-attacks.html
DXer said
http://www.gao.gov/products/GAO-12-488
Anthrax: DHS Faces Challenges in Validating Methods for Sample Collection and Analysis
GAO-12-488, Jul 31, 2012
Timothy M. Persons
(202) 512-3000
personst@gao.gov
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.
DXer said
David Relman thinks the FBI has not proved its case — he was the Vice-Chair of the NAS panel.
DXer said
http://www.ncbi.nlm.nih.gov/pubmed/22747878
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.
Source
Pacific Northwest National Laboratory, Richland, Washington, USA.
Abstract
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.
Comment:
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.
DXer said
Science 11 May 2012:
Vol. 336 no. 6082 pp. 669-670
DOI: 10.1126/science.336.6082.669-b
•Letters
Presumed Guilt in the Anthrax Case—Response
Related Resources
In Science Magazine
Letters
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.
E-mail: relman@stanford.edu
References
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); http://www.nap.edu/catalog.php?record_id=13098.
DXer said
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 – NYPOST.com
http://www.nypost.com/…/anthrax_and_the_fbi_36aNmZJGENZCAPCmv...
Last Updated: 4:24 AM, October 23, 2011
Read more: http://www.nypost.com/p/news/opinion/editorials/anthrax_and_the_fbi_36aNmZJGENZCAPCmvdbIrK#ixzz1vkbkE5Sy
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.
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.
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
Review
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
Abstract
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.
Keywords
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].
DXer said
http://www.adn.com/2012/03/15/2372022/stevens-prosecutors-intentionally.html
Report on Stevens prosecution tells of ‘systematic concealment’
By SEAN COCKERHAM and RICHARD MAUER
Anchorage Daily News / adn.com
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: http://www.adn.com/2012/03/15/2372022/stevens-prosecutors-intentionally.html#storylink=cpy
Comment: Delays or failure to produce happen for widely varying reasons. But producing the documents, even late, is the wise course.
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
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.
DXer said
One of the virulence plasmids in the mailed anthrax was inverted. See Keim et al, Science.
DXer said
Years ago, I asked ACLU Attorney Annette L when Aafia went to Houston in 2001 and she didn’t know.
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
Abstract
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
English
Author keywords
Amerithrax; Bacillus anthracis; Mutation; spo0A; Sporulation
References
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.”