It seems very likely to me that had Bruce Ivins not died, the analysis carried out by a panel from the National Academy of Sciences in assessing the scientific evidence tying Ivins to the 2001 anthrax attacks would have led to reasonable doubt on whether Ivins carried out the attacks. For this post, let us concentrate only on the NAS response to FBI claims on the spores used in the attack, especially with regard to how the spores were prepared.
What we do know from the report is that the spores used in the attacks did not come directly from Bruce Ivins’ RMR-1029 flask, but had to undergo a culturing step if RMR-1029 was even the source. Also, the spore material in different individual mailings differed in purity and particle size. Silicon was present in the spores, but was not added as a step to “weaponize” the spores. Instead, the silicon was incorporated into the coating of the spores themselves. Finally, the NAS panel did not feel there was sufficient evidence to support the FBI claim that a highly skilled person had prepared the material.
Overall, the importance of the primary conclusion of the NAS report cannot be overstated (p. 4 of the report as marked, all references will use internal page numbers, not pdf numbers from my pre-publication copy):
It is not possible to reach a definitive conclusion about the origins of the B. anthracis in the mailings based on the available scientific evidence alone.
A good defense attorney probably would need no more than that conclusion to establish reasonable doubt in a trial. But the details on how the panel reached that conclusion are important.
In this post, I discussed whether Ivins could have produced all of the material used in the attacks with the equipment he had available and without drawing attention to himself. That post opened with a discussion of how many spores were known to have been present in the attacks. I looked at what was in the FBI’s report, filled in some gaps with my experience in microbiology and came to the conclusion that somewhere between four and seventy liters of liquid culture could have produced the attack material. The NAS report comes to a similar conclusion on page 62:
Thus, cultivation in the range of 2.8 to 53 liters of liquid medium would have been required to produce the spores required for the letters (see Table 4.2).
Interestingly, the panel also calculated that it would have required between 463 and 1250 agar plates if the attack material had been produced on solid medium. It seems highly unlikely Ivins could have cultured this many extra Petri dishes without someone in the lab taking note and reporting it to the FBI once the investigation began.
The photo above shows how dramatically different the highly purified material in the letter sent to Senator Patrick Leahy’s office was when compared to the material mailed to the New York Post.
There had been much speculation early on in the press that the material sent to Senator Leahy’s office had been “weaponized” by the addition of materials including silicates to make tiny particles remain suspended in the air so that they could be inhaled. The report documents that although silicon is found in the spores used in the attacks, the silicon is localized in the spore coats. This point is driven home very clearly in this photo, where the silicon can be seen “lighting up” within the outer lining of the spores:
Many experiments were carried out in an attempt to match the silicon content, the particle size distribution and the degree to which the final material would remain suspended in air. I will rely here on the entire summary the panel provided on the issue of silicon in the spores (page 71):
The substantial effort devoted to the characterization of silicon in Bacillus spore coats resulted in new fundamental insight into microbial processes and the development of new or enhanced analytical measurement technology. (Table 4.4 presents a summary of the analytical results.) Elemental analysis of the letter samples showed that 1) the silicon content was high, 2) most of the silicon was incorporated in the spore coat, 3) the majority of spores in the samples contained silicon in the coat, and 4) no silicon was detected in the form of a dispersant in the exosporium.
The bulk silicon content in the Leahy letter could be completely explained by the amount of silicon incorporated in the spores during growth. (Not enough material was available to make this comparison for the Daschle letter.) In contrast, the New York Post letter had significant bulk silicon content, far exceeding that contained in the spores.
No studies have considered the effect of the chemical form of silicon (e.g., silicate impurity versus polydimethylsiloxane antifoam agent) on uptake. The inability of laboratory experiments to reproduce the silicon characteristics of the letter samples is not surprising given the complexity of the uptake mechanism.
A few spores analyzed from RMR-1030 contained silicon in the coat, but none of the spores analyzed from RMR-1029 contained silicon in the coat. Therefore, the letter samples could not have been taken directly from the flasks—a separate growth preparation would have been required.
The material in the Daschle and Leahy letters was reported to have “a high level of purity” and to have electrostatic properties that caused it to disperse readily upon opening of the letters. These properties should be regarded as qualitative observations since they were not based on quantitative physical measurements. The committee received testimony (Martin, 2010) stating that some Dugway preparations, particularly those utilizing lyophilization but no dispersant, gave products with similar appearance and electrostatic dispersibility as the letter samples, suggesting that these properties were not necessarily connected to an intentional effort to increase dispersibility through addition of a dispersant. Exogenous silicon and bentonite, which enhance the dispersibility of spore preparations, were not found in the Leahy and Daschle letters.
Note that this analysis provides the strongest evidence to date that the spores used in the attacks did not come directly from the RMR-1029 flask, because the spores in the flask do not have the silicon content as those in the attack material. Note also that growth in the presence of polydimethylsiloxane-containing antifoam agents is seen as one route that needs to be investigated for how silicon can be incorporated at elevated concentration in the spores, just as I suggested in this post.
It is also worth noting that highly purified spores produced at Dugway did have the aerosolizing quality of the attack material. No additional treatments besides purification were needed for these spores to disperse in air, so I suspect that is one of the primary reasons that the NAS panel could not support the FBI claim that someone with a very high level of expertise must have prepared the material used in the attacks.