I
n the fall of 2001, letterscontaining anthraxspores were mailed tonews media and congres-sional ofces, killing ve people and sickening 17others. In response to theattack, the Federal Bureauof Investigations (FBI)launched an extensiveinvestigation that spannedsix continents, involvedover 10,000 witness inter-views, and resulted in the issuance of some5,750 grand jury subpoenas. During the investi-gation, the FBI worked with other federalagencies to coordinate and conduct scienticanalyses of the anthrax spore powders, envi-ronmental samples, clinical samples, andsamples collected from laboratories that mayhave been the source of the letter-associatedspores. In addition, several government,university, and commercial laboratoriesassisted in conducting the scientic analysesthat were a central aspect of the investigation.Although it is not unusual to use science toinvestigate evidence in a criminal case, tracingthe source of the anthrax spores used in themailings would require the development of newscientic techniques. In addition to informingFBI investigators about possible leads, much of the science used in the investigation formed the basis of a rapidly developing but still nascentscientic eld called micro- bial forensics, which servesto determine the geneticidentity of a microbial agentused for nefarious purposes.In 2008, seven years intothe investigation, the FBIasked the National ResearchCouncil to conduct anindependent review of thescientic approaches usedduring the investigation of the anthrax mailings. Acommittee of experts evaluated the scienticfoundation for the experiments conducted by, andon behalf of the FBI, to determine whether theymet appropriate standards for scientic reli-ability and for use in forensic validation, and todetermine whether the FBI reached appropriatescientic conclusions from its use of thesetechniques. The committee did not undertake anassessment of the probative value of the scien-tic evidence and did not offer any view on theguilt or innocence of any individual(s) inconnection with the 2001 mailings.
Looking for Leads
Investigators analyzed the physical andchemical characteristics of the powders recov-ered, performing experiments to study the sizeand granularity of the particles, to assess thechemical content and age of the spores, and toidentify any other chemical signatures that
It is not possible to reach a denitive conclusion about the origins of the
Bacillus anthracis
usedin 2001 anthrax mailings based on the available scientic evidence alone, this National ResearchCouncil committee nds. Scientic analysis played a central role in the FBI’s investigation of theanthrax mail attacks. To help investigators narrow their search for the source of the attack material, researchers characterized its physical, chemical, and biological properties. In thisreport the committee reviews the scientic approaches used during the investigation, and evalu-ates whether the FBI reached appropriate scientic conclusions from the use of these techniques.
Review of the Scientic ApproachesUsed During the FBI’s Investigationof the 2001 Anthrax Letters
Figure 1.
Letter containing anthrax mailedto Senator Patrick Leahy on October 9, 2001.
Source: FBI
might provide clues related to the source or produc-tion processes used. The committee found that theseanalyses were appropriate and were carried out properly, and agreed with the FBI and Departmentof Justice that these analyses proved to have limitedforensic value in identifying the source of the anthrax.Researchers also carried out tests to determineif the spores had been weaponized by coating themwith a chemical such as silicon to help the sporesdisperse into the air once the envelopes were opened.Although silicon was present in the letter powders,the committee found no evidence of the intentionaladdition of silicon-based dispersants.
Tracing the Anthrax
There are many different strains of
B. anthracis
,each with distinct genetic features, which haveevolved over time. Before the anthrax mailings took place, scientists had developed molecular methodsto search the genes of anthrax spores for markers todifferentiate the various strains. Using these tests,researchers identied the dominant organism inthe letters as the Ames strain of anthrax. Becausethe Ames strain had been widely distributed amonglaboratories around the world for research andcountermeasure development (see box 2), thisnding suggested that the attack material had come,either directly or indirectly, from one of the labora-tories that possessed the Ames strain. Further analysis of the genome sequence of the letter-associated anthrax spores determined that it wasunlikely that this strain had been geneticallyengineered—a concern for investigators who fearedthe strain may have been altered to enhance anti- biotic resistance or to increase virulence.
Searching for the Source
Having determined that the anthrax used inthe letters was the Ames strain, the FBI worked toestablish a repository of Ames strain samples heldin laboratories around the world. In all, 20 laborato-ries provided a total of 1070 samples of Ames strainanthrax stocks to the FBI.To identify the source of the letter materials,researchers searched for a link between the materialrecovered from the letters and samples in the reposi-tory. Microbiologists at USAMRIID had observedthat some spores from the attack material grew to produce bacterial colonies with distinctive appear-ances, different from typical Ames strain anthraxcolonies. These variants were suspected to be theresult of genetic mutations that had spontaneouslyarisen in the attack spore population.Researchers determined the nature of themutations associated with some of the unusualcolonies in the attack material, and then designedtests to search the FBI’s repository of anthraxstocks for samples that contained these samemutations. The tests showed that 8 samples from
Box 2. What is the Ames strain?
The Ames strain of
B. anthracis
was rst isolated froma dead cow in Texas in 1981. Texas A & M Universityshipped this new isolate to the United States ArmyMedical Research Institute of Infectious Diseases(USAMRIID) in Fort Detrick, Maryland. Because the box used for the shipment bore a previously-usedAmes, Iowa, address label, the strain became knownas the “Ames” strain. Subsequently, the Ames strainwas shared with laboratories and research institutionsthroughout the United States and the world.
Figure 2.
Anthrax powder recovered from the
New York Post
letter.
Source: FBI
Box 1. What is anthrax?
Anthrax is a disease caused by the bacterium
Bacillusanthracis.
It is generally a disease of herbivores such ascows and sheep, which acquire the infection by grazingon contaminated soils. Highly resistant toenvironmental degradation,
B. anthracis
spores cansurvive for long periods of time in soil, their naturalreservoir. The disease occurs worldwide and there areoccasional outbreaks in livestock in the United States.Humans can acquire
B. anthracis
infection via theskin, gastrointestinal tract, or by inhalation. Mostcommonly, this occurs by eating the meat or handlingthe hides of infected animals.
B. anthracis
infections caused by the inhalation of
anthracis
spores are the most lethal. The high mortalityrate, and the fact that with appropriate preparation, prodigious quantities of the spores can be produced andconverted to an easily inhaled powder, mean that
B. anthracis
spores are effective agents of biologicalwarfare and bioterrorism.
two laboratories contained the same set of four mutations that had been selected from the attack material as the basis for subsequent tests. The FBI,through its other investigatory efforts, stated thatall of these samples derived from a ask labeledRMR-1029, found in a laboratory at USAMRIID.
Evaluating the Science
The committee agreed that genetic analysis of the FBI’s repository was consistent with the ndingthat the spores in the attack letters came from ask RMR-1029; but the analyses did not denitivelydemonstrate such a relationship. Overall thecommittee found that the scientic link betweenthe letter material and ask RMR-1029 is not asconclusive as stated in the Department of Justice’sInvestigative Summary, for the following reasons:
•
The FBI’s repository was not optimal. For example, instructions provided to laboratoriesfor preparing samples for the FBI’s repositorywere not precise enough to ensure that all thelaboratories would follow the same procedure.The lack of consistently prepared samples limitsthe strength of comparisons of the repositorysamples and the letter material. Furthermore,the instructions were not sufcient to ensurethat samples of atypical Ames colonies, such asthose found in the anthrax letters, would have been submitted to the repository by recipientsof the FBI’s instructions.
•
It is possible that some of the mutations identiedin the attack materials and in ask RMR-1029could have arisen independently, by parallelevolution. This particular type of mutation isknown to arise frequently and be enriched duringlarge-scale
B. anthracis
growth procedures andspore preparation. This possible explanation for the genetic similarity between the spores in theletters and in RMR-1029 was not rigorouslyexplored during the course of the investigation.
•
Flask RMR-1029, identied by the U.S.Department of Justice as the “parent material”for the spores in the attack letters, was not theimmediate source of the spores used in theletters. As noted by the FBI, one or morederivative growth steps would have beenrequired to produce the anthrax in the attack letters. Furthermore, the contents of the NewYork and Washington letters had different physical properties.The committee found the FBI’s scientic data provided leads as to the origin of the anthrax sporesin the letters, but these data alone did not rule outother possible sources. The committee was notcharged with reviewing, nor was it given access to,the ndings from the criminal investigation compo-nent of this case; therefore, the committee could notfully assess the potential value of additional scien-tic investigation with respect to establishing thesource of the anthrax spores.Late in its study, in discussions with FBI andDepartment of Justice leadership, the committeewas made aware of additional information thatwould require review of classied material. Dueto the timing of this revelation and the desire thatall materials considered in this study be publiclyavailable, the committee did not review thesematerials. A separate review of the classiedmaterials offered by the FBI and Department of Justice should be conducted.
Looking to the Future
While much of the committee’s effort wasfocused on a review of the science performed insupport of the investigation of the 2001 anthrax
A bacterial colony is a visible cluster of bacteria growing on a solid medium such asan agar plate. All the cells in one colony aredescendents of a single cell, and are thereforegenetically identical.
Figure 3.
Bacterial colonies formed by growth of
Bacillusanthracis
cells on blood agar. The colony on the topdisplays a “bull’s eye,” typical of
anthracis
colonies. The bottom colony displays an appearance more typical of theAmes strain.
Source: U.S. Army Medical Research Institution of Infectious Diseases
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Review of the Scientific Approaches Used During the FBI's Investigation of the Anthrax Letters
It is not possible to reach a definitive conclusion about the origins of the Bacillus anthracis in the mailings based on the available scientific evidence alone, this National Research Council report finds. Scientific analysis played a…
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