Friday, April 2, 2010

Anthrax Investigation: Silicon Mystery Endures in Solved Anthrax Case/ Science

Science 19 March 2010:
Vol. 327. no. 5972, p. 1435
News of the Week
Yudhijit Bhattacharjee

What about the silicon?

That question has confounded investigators throughout the probe into the 2001 anthrax letter attacks, which the U.S. government formally concluded in February. Scientists inside and outside the government say there is clear evidence that the high levels of silicon found in the anthrax came not from anything added to "weaponize" the anthrax spores—as researchers had suggested early in the probe—but from the culture in which the spores were grown. That evidence may have settled the issue of whether the anthrax was weaponized, at least for scientists familiar with the case. But it raises a different question: Why did the mailed anthrax have such a high proportion of spores with a silicon signature in comparison to most other anthrax samples?

The answer, according to academic scientists who helped with the case, probably would not change the FBI's conclusion that the attacks were the sole handiwork of now-deceased U.S. Army researcher Bruce Ivins. But it could help illuminate exactly how the attack material was prepared. Resolving the mystery might also pave the way for new techniques using trace elements in a bioterrorism agent to link it to its source.

"There's tremendous interest in using metal signatures as a forensic tool," says Adam Driks, an anthrax researcher at Loyola University Chicago in Illinois. But the science to do that is lacking: "We know very, very little about the diversity of elemental composition within spores when they are produced in different ways."

Figure 1Closer look. Michael (above) saw silicon well inside individual spores, visible as the green lining in the top right image.


The FBI's scientific case against Ivins rests on DNA tests showing that the mailed anthrax came from a flask under Ivins's control at the U.S. Army Medical Research Institute of Infectious Diseases in Frederick, Maryland. Investigators also had the attack material chemically analyzed, first at the Armed Forces Institute of Pathology (AFIP) in Washington, D.C., within weeks of the attack. Examining the spores under a scanning electron microscope, AFIP scientists detected silicon and oxygen and concluded that the spores had been coated with silica to make them float easily, enhancing their power to kill.

A more detailed analysis by Joseph Michael and Paul Kotula of Sandia National Laboratories in Albuquerque, New Mexico, contradicted that conclusion. Studying individual spores with a transmission electron microscope, they found that the silicon was located within the spore coat, well inside the cell's exosporium (outermost covering). By contrast, when they looked at surrogate spores weaponized with silica, the silicon was clearly outside the exosporium.

But the Sandia study, presented last September to a National Academies panel reviewing the science behind the investigation, still leaves questions. Out of 124 spores from a letter mailed to Senator Patrick Leahy of Vermont, Michael found the silicon-and-oxygen signature in 97—78% of the sample. The signature was present in 66% of a sample from a letter to former Senator Tom Daschle and in 65% of spores from a letter sent to the New York Post.

Out of nearly 200 other anthrax samples from different labs, none came close to displaying such a prominent silicon signature. The highest, in a sample from Dugway Proving Ground in Utah, was 29%. The researchers couldn't find silicon in the coat of a single spore out of some 300 taken from RMR-1029, the flask in Ivins's lab identified as the source of the bacteria used in the attacks; they concluded that all the silicon had come from the culture.

The unusually high percentage of silicon-bearing spores in the attack material "is a bit of a strange thing," says Michael. "We have no way of knowing how they were really grown." An anthrax researcher who did not wish to be named calls it "awfully weird" and "a particularly inconvenient exception" because it leaves a gap in the case. However, neither scientist thinks the anomaly casts doubt on the broader investigation.

The key to the mystery likely lies in the culture medium the perpetrator used to grow the anthrax spores, says Michael. In a recent study, Japanese researchers grew colonies of Bacillus cereus—a close relative of the anthrax bacterium, B. anthracis—in culture media with and without added silicate. Spores grown in the silicate-containing culture showed silicon within the spore coat. In the absence of silicate, there was no silicon, the group reported in January in the Journal of Bacteriology.

One of the study's authors, microbiologist Akio Kuroda of Hiroshima University in Japan, says the precise amount of silicon in individual spores from the anthrax letters could offer clues about the medium. "If the anthrax spores contained a high amount, the suspect must have used a medium that was supplemented with silicon or that intrinsically contained a lot of silicon," Kuroda says. "If a thorough testing of various media sold in the U.S. identifies a few that contain higher amounts of silicon, those could become an investigative clue."


DXer said...

Kathryn Crockett, Ken Alibek’s assistant — just a couple doors down from Ali Al-Timimi — addressed this issue of microencapsulation in her 2006 thesis, “A historical analysis of Bacillus anthracis as a biological weapon and its application to the development of nonproliferation and defense strategies.” She expressed her special thanks to bioweaponeering experts Dr. Ken Alibek and Dr. Bill Patrick. (Dr. Patrick also reviewed the 1993 OTS report). Dr. Patrick consulted with the FBI in Amerithrax. Dr. Crockett successfully defended the thesis before a panel that included USAMRIID head and Ames strain researcher Charles Bailey, Ali Al-Timimi’s other Department colleague. In 2001 he said he did not want to discuss silica because he did not want to give terrorists any ideas. Oops! Too late. The scientist coordinating with the 911 imam and Bin Laden’s Sheik was in the same suite, just a few feet away.

Dr. Crockett in her PhD thesis says that scientists who analyzed the powder through viewing micrographs or actual contact are divided over the quality of the powder. She cites Gary Matsumoto’s “Science” article in summarizing the debate. She says the FBI has vacillated on silica. The AFIP data, if released, would point to the high level of silica in the first batch of letters. The FBI’s science investigation is being led by Jason Bannan, the former collections scientist at the Bacteriology Division at the American Type Culture Collection which, located at GMU, co-sponsored Al-Timimi’s program and later successfully bid on the Critical Reagent Program handling virulent Ames for government researchers.

On the issue of encapsulation, Crockett reports that “many experts who examined the powder stated the spores were encapsulated. Encapsulation involves coating bacteria with a polymer which is usually done to protect fragile bacteria from harsh conditions such as extreme heat and pressure that occurs at the time of detonation (if in a bomb), as well as from moisture and ultraviolet light. The process was not originally developed for biological weapons purposes but rather to improve the delivery of various drugs to target organs or systems before they were destroyed by enzymes in the circulatory system” (citing Alibek and Crockett, 2005). “The US and Soviet Union, however, ” she explains, “used this technique in their biological weapons programs for pathogens that were not stable in aerosol form… Since spores have hardy shells that provide the same protection as encapsulation would, there is no need to cover them with a polymer.“ She explains that one “possible explanation is that the spore was in fact encapsulated but not for protective purpose. Encapsulation also reduces the need for milling when producing a dry formulation.”

The former State Department analyst, Ken Dillon, PhD, notes:

“Interested readers should study the contributions of Barbara Hatch Rosenberg (Sept. 9) and Serguei Popov (Sept. 24) at I find quite intriguing Rosenberg’s reference in footnotes 21 and 22 of her analysis to U.S. Patent Application number 09/805,464 by Charles Bailey and Ken Alibek, March 14, 2001. The patent (#6,649,408) was issued on Nov. 18, 2003. Silica is mentioned repeatedly in the patent, and the instructions look very much like they could have been used by the preparer of the anthrax in the letters. Rosenberg suggests that the patent application was available to 100+ potential anthrax attack suspects well before the anthrax attacks, but it is not clear that the application was made public before the attacks started.” Dr. Popov does not dismiss the possibility but instead refers to it merely as “hypothetical.”

One military scientist who has made anthrax simulants described the GMU patents to me as relating to a silicon encapsulation technique which serves to increase the viability of a wide range of pathogens.

DXer said...

I find Peter Setlow’s commentary on the recent Japanese article about silicon encapsulation to be thoughtful and would have preferred that he address the issue before the NAS.

Once the AFIP data is released, experts like Peter Setlow can consider the source of the reason for the silica such as whether it was putting virulent Ames soil (silica) suspension such as the FBI scientist John Ezzell did in 1996 for DARPA when he made dry powdered anthrax at Ft. Detrick. Or we can turn to the “Microdroplet Cell Culture” patent filed by Ali Al-Timimi’s Discovery Hall colleagues at the DARPA-funded Center for Biodefense and see if there is a connection. The international application involves a pharma aerosol application and the co-applicant is the founder of the Aerosol Techniques in 1955, which had a central role in the US offensive program. Under the GMU patent, the silica would be in the culture medium and then would be removed by repeated centrifugation.

Or we can explore the other hypotheses relating to the reason for the Silicon Signature, such as it being due to rice hull contamination (silicon) in a spraydryer. Or whether it is due to use of a silicone sealant sprayed on the inside of the envelope such as the Al Qaeda chapter on “poisonous letters” instructs be used (to avoid killing the mailman).

I’m not a scientist which is why it seems that the data and pictures need to be released so that we can have experts like the Center for Biodefense’s Sergeui Popov and the government’s John Kiel review it. If we learned anything from 9/11, it is that there are times that information needs to be shared so that people can connect the dots. This is such a time. Any one with a conflict of interest should recuse himself from the particular aspect of Amerithrax. Anyone without the relevant qualification to address an issue should sit down and have a qualified expert, who has done controlled experiments, address the question. John Kiel is such a scientist. Head of the Air Force lab, his lab did controlled experiments on the issue independent of the FBI and could report to Congress on the issue.

Anonymous said...

I found Judith Miller's article of Sept 4, 2001 compelling for a few reasons. It was published only a few days before the attacks and it talks about America's new secret enhanced anthrax and other research which was created with either implanted genes from Bacillus cereus or a Bacillus cereus growth medium as described by Russian and then UK scientists. The Japanese research confirmed how this works.