Review of the Scientific Approaches Used During the FBI's Investigation of the 2001 Anthrax Letters (2011) / Chapter Skim
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5 Microbiological and Genetic Analyses of Material in the Letters
5 Microbiological and Genetic Analyses of Material in the Letters
Pages 97-124
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From page 97... ...
This was the case in 2001, when a number of cases of both cutaneous and inhalational anthrax were diagnosed among media and postal employees and others after the delivery of letters containing suspicious powders in several places. In October 2001, clinical reporting of human anthrax cases spurred a broad epidemiological investigation to identify the source of the illnesses as well as any other infected parties (see Chapter 3 for a timeline and a more detailed discussion of this epidemiological investigation)
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From page 98... ...
anthracis populations, a variety of strains had already been recognized even before sequencing technology enabled detailed characterizations of genome differences among strains. Work performed by Paul Keim and others well before the 2001 anthrax attacks had resulted in the development of several molecular methods to detect genetic differences among Bacillus species as well as among isolates of B
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From page 99... ...
anthracis provided by the United States Army Medical Research Institute for Infectious Diseases (USAMRIID) , which had received evidentiary samples from the FBI.
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From page 100... ...
The materials tested included samples of the progenitor Ames strain isolated from the dead cow in 1981 (Ames "Ancestor") ; an isolate from the deceased Florida patient, Robert Stevens; isolates from the Brokaw, New York Post, and Daschle letters; and several stocks from USAMRIID, including a sample from flask RMR-1029.
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From page 101... ...
The enormous increases in speed and efficiency of DNA sequencing have led to a revolution in scientists' ability to identify mutations quickly and precisely. Recently, the term "deep sequencing" was coined to describe this approach of simultane ous sequencing of massive numbers of short fragments derived from a mixture of genomes, such as might exist in an evolving population derived from a single cell that has, over time, accumulated genetic variants.
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From page 102... ...
However, the subsequent completion of the genomic sequences of the Ames Ancestor and later of the letter isolates (see Section 5.5.5 below) strongly supported the findings of the LANL group (see also Box 5-1 on genome sequencing)
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From page 103... ...
. The Ames Ancestor sequence served as the high-quality reference genome needed for the comparative genomics work that TIGR later performed on colony morphotypes identified from the attack letter materials (see below)
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From page 104... ...
subtilis on the basis of 16S rRNA gene sequencing and later whole genome sequencing (CDC, 2001a; FBI Documents, B2M1D2)
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From page 105... ...
subtilis contamination (see NBFAC analytical result reports, November 2006-December 2007, FBI Documents, B2M4D3-15)
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From page 106... ...
5.5 IDENTIFICATION AND CHARACTERIZATION OF COLONY MORPHOLOGICAL VARIANTS IN THE EVIDENTIARY MATERIAL 5.5.1 Why Was the FBI Interested in Colony Morphotypes? Any microbial geneticist can attest to the fact that close scrutiny reveals unusual individual variants in a population of microbes.
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From page 107... ...
This process represents, in effect, rapid evolution leading to a population becoming better adapted to its particular laboratory conditions. Analyses of the spore samples from the attack letters from as early as November 2001 (FBI Documents, B1M2D7)
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From page 108... ...
Poorly sporulating colonies do not accumulate the dye, resulting in a white appearance that can be easily identified by eye. Further study of the white colony morphotypes revealed that they were sporulation deficient.
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From page 109... ...
anthracis phenotypic variants, Worsham and her colleagues were readily able to apply methods for the study and characterization of phenotypic variants in the letter samples. In early analysis of the Leahy letter sample, spores were plated on sheep blood agar and incubated for three days.
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From page 110... ...
These conditions were employed to allow the detection of variants that might be apparent only under particular growth conditions. While the characteristics of colony morphotypes are most obvious following incubation for 48 hours or more after nutrient exhaustion and sporulation, these starvation conditions were avoided in order to diminish the likelihood of the selection of new variants that were not present in the initial evidentiary samples.
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From page 111... ...
This is a standard microbiological practice for the maintenance of strains under conditions that prevent the further accumulation of genetic variation. The remainder of the recovered material was again streaked onto a fresh sheep blood agar plate and incubated at 37°C for 18 to 22 hours in order to produce numerous colonies for each candidate morphotype.
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From page 112... ...
anthracis cells on blood agar. The colony on the top displays the mor bitmap phology designated "Type E." The colony on the bottom displays the typical wild-type morphology.
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From page 113... ...
Screening of samples from the Daschle and New York Post letters revealed morphotype classes that were phenotypically similar to those from the Leahy letter. (Due to a scarcity of material from the Brokaw letter, no screening of samples for colony morphotypes was performed on that material [Hassell, 2010]
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From page 114... ...
Second, similar phenotypic variations can be associated with different genetic alterations located in either the same or widely separated genetic loci. The presence of similar colony morphotypes in two samples would not provide direct genetic evidence to link the two sample populations.
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From page 115... ...
TIGR ID FBI ID Origin Morphotype Sequencing status GBA Ames Porton Porton Down Wild type 12X – closed GB6 Ames Ancestor Texas/USAMRIID Wild type 12X – closed GB8 LL10/E3 Leahy letter A 8X – closed GB9 LL9/E2 Leahy letter B 8X GB10 LL1/E1 Leahy letter Wild type 8X GB11 PL10/E6 New York Post letter A 8X GB12 PL9/E7 New York Post letter B 8X – closed GB13 PL1/E9 New York Post letter Wild type 8X – closed GB15 DL10/E4 Daschle letter A NS GB16 DL9/E5 Daschle letter B NS GB17 DL1/E8 Daschle letter Wild type NS GB18 LL6/E10 Leahy letter C 12X GB19 LL7/E11 Leahy letter D NS GB23 LL18 Leahy letter E 12X GB24 LL19 Leahy letter E NS NS = not sequenced SOURCE: FBI Documents, B1M5D1-2. extracted from the colony morphotypes identified by USAMRIID was provided by Paul Keim to TIGR, where it was prepared for genome sequence analysis.
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From page 116... ...
The first was a SNP TABLE 5-3 Further Genetic Characterization of the Morphotype Isolates Genotype Assay Morphotype Affected Type of examined in method class locus mutation greater detail Letter source developed Leahya A One copy Insertions A1, 2024 bp qPCR Daschleb of 16S in different New York Post b rRNA gene sites overlapping New York Posta A2, 2608 bp Not used a 16S rRNA Leahy b A3, 823 bp qPCR gene Daschlea New York Post b Leahya B SNP in B Not used spo0F New York Posta promoter intergenic region Leahya C Sensor his SNP C Not used kinase producing stop codon Leahya D Sensor his 258 bp D Taqman + kinase deletion PCR Leahya E Putative 9 or 21 bp E, 9 bp Taqman + Daschlec response deletion, or deletion PCR New York Postc regulator SNP (plasmid) NOTE: bp = base pair; qPCR = quantitative polymerase chain reaction; SNP = single nucleotide polymorphism.
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From page 117... ...
, while the morphotype isolate from the Daschle letter was studied using PCR. The sequencing revealed that the Leahy and New York Post letters contained an identical SNP in the noncoding region between the spo0F gene and an adjacent gene, and this SNP was not present in the Ames Ancestor or Ames Porton reference samples.
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From page 118... ...
TIGR tested these samples using the assays developed for the various genotypes. These additional analyses showed that only the colony morphotype samples themselves contained the specific polymorphisms identified by the TIGR team, which was interpreted to mean that these genotypes represented TABLE 5-4 Distribution Among the Anthrax Letters of the Genotypes Selected for FBIR Screening Letter Genotype A1 Genotype A3 Genotype D Genotype E Leahy + + + + Daschle + + NT + + + NT + New York Post Brokaw NT NT NT NT NT = not tested SOURCE: Hassell (2010)
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From page 119... ...
The A1 genotype had a 2024 bp insertion and was originally identified in an isolate from the Leahy letter. During assay development by CBI this allele was also detected in DNA derived from bulk Daschle and New York Post letter spores (Hassell, 2010)
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From page 120... ...
The IITRI and MRI assays for the D genotype were both accepted by the FBI and used to screen the FBIR samples. The D genotype had a 258 bp deletion and was originally identified in an isolate from the Leahy letter.
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From page 121... ...
anthracis Ames strain in the letters had undergone deliberate genetic engineering or modification was timely and appropriate, though necessarily incomplete. The genome sequences of the letter isolates that became available later in the investigation strongly supported the FBI's conclusion that the attack materials had not been geneti cally engineered.
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From page 122... ...
Finding 5.4: Multiple colony morphotypes of B anthracis Ames were present in the material in each of the three letters that were examined (New York Post, Leahy, and Daschle)
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From page 123... ...
Future cases may not allow for a time frame as lengthy as that of the anthrax letters investigation. Assay development and validation took almost two years in some cases, for reasons that are not clear to the committee.
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