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5
Microbiological and Genetic Analyses of
Material in the Letters
5.1 INTRODUCTION
As discussed in Chapter 2, the bacterium Bacillus anthracis (B. anthracis)
is the causative agent of the disease anthrax. Anthrax generally affects graz -
ing mammals (e.g., cattle, sheep, horses). Anthrax in humans, particularly
inhalational anthrax, is rare and occurs mainly in individuals with occupations
involving the handling of hides, hair, or bone from infected animals. Inhala -
tional anthrax can also be a sign of a biological attack with B. anthracis spores,
especially in individuals without likely exposure to infected animals or their
products. 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). Identification of B. anthracis
as the cause of the 22 cases of illness and five deaths in 2001 was determined by
clinical laboratory means (CDC, 2001a, b, c). Because these illnesses and deaths
appeared to have resulted from a bioterrorist attack, immediate efforts were
undertaken to identify a common source for the outbreak, including molecular
genetic analysis of the causative agent.
5.2 IDENTIFICATION OF THE B. ANTHRACIS STRAIN
The first step in the search for a source of the anthrax-causing powders
in the 2001 mailings was to identify which “strain” (or strains) of B. anthracis
was used in the attack. Disparate cases of human anthrax in Connecticut, New
York, Florida, and Washington, D.C., and contaminated environmental loca -
tions in the last three of these sites were all linked when a single B. anthracis
97
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98 SCIENTIFIC APPROACHES USED TO INVESTIGATE THE ANTHRAX LETTERS
strain, the Ames strain, was identified in all of these cases and associated
locations (Keim et al., 2008). As noted in Chapter 2, B. anthracis is one of the
most genetically homogeneous microorganisms known (Keim et al., 2000).
Nonetheless, even in the most homogeneous species there are usually some dif -
ferences in genome sequences among populations. These sequence differences,
although few in number, are sufficient to characterize subgroups, or “strains.”
Strains are members of the same species, but their differences reflect the diver-
gence of sublineages as they evolved over time (Keim et al., 2000). Among B.
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.
anthracis. In the mid-1990s, work by Hendersen and colleagues (1995) and
Anderson and colleagues (1996) led to the identification of a 12-nucleotide
variable number tandem repeat (VNTR) sequence (called vrrA for “variable
repeat region A”) that provided the first molecular marker that distinguished
among B. anthracis strains. The basis of this marker was shown to be differences
in the number of repeated sections of this genetic sequence, and five different
variations were detected. Subsequently, VNTR analysis at multiple genetic loci
(Multiple Locus VNTR Analysis or MLVA) enabled the characterization of 426
B. anthracis isolates with 89 distinct genotypes (Keim et al., 2000).
Another approach, amplified fragment length polymorphism (AFLP)
analysis, has been particularly useful for examining differences between B.
anthracis and close relatives, such as B. cereus and B. thuringiensis (Keim et al.,
1999, 2008; Hoffmaster et al., 2002; Keim et al., 2008). The AFLP technique
had been used to identify about 30 variable regions and provided an ability
to profile portions of the genome of a large number of diverse B. anthracis
strains. In addition, the pagA gene, which encodes the protective antigen (PA)
protein (one of the three anthrax toxin proteins discussed in Chapter 2) also
had been sequenced (Price et al., 1999). Because of the importance of pagA in
the development of immunity to anthrax, this gene was of interest in determin -
ing whether a particular strain might have been genetically altered, or “engi -
neered,” for increased effectiveness as a weapon (Hoffmaster et al., 2002).
These new molecular approaches, combined with the creation of a collec -
tion of strains from many of the world’s geographic regions, greatly enhanced
scientific capabilities for identifying genetic variations among anthrax strains at
that time. Using these methods, Keim and colleagues (1999, 2000) had estab -
lished a picture of the evolutionary lineages of B. anthracis. These methods for
rapid, reliable molecular subtyping were also critical in determining the identity
of the clinical and environmental isolates in the 2001 anthrax attacks. Although
a complete genome sequence provides the most effective genetic signature
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MICROBIOLOGICAL AND GENETIC ANALYSES OF MATERIALS IN THE LETTERS
for identifying an organism, at the time of the attack mailings no B. anthracis
genomes had yet been completely sequenced and published (Keim et al., 2008).
Using MLVA at eight loci identified by Keim and his colleagues, scientists
from the Centers for Disease Control and Prevention’s (CDC’s) Laboratory
Response Network subtyped 135 B. anthracis isolates (samples) collected from
the attack victims, letter powders, and environmental samples, and determined
that all these isolates were likely to have been derived from a common source
(Hoffmaster et al., 2002). In addition, CDC scientists sequenced the pagA genes
from a subset of these isolates and concluded that none of the isolates appeared
to have been engineered (Hoffmaster et al., 2002). (The use of additional
tests performed under the aegis of the FBI to assess the possibility of genetic
engineering is discussed below.) All attack-associated isolates were identified
as MLVA genotype 62 and PA genotype I. Genotype 62 is the genotype of the
Ames strain commonly used worldwide for laboratory research for vaccine
development. The PA I genotype was also identical to the Ames strain PA geno -
type. These results led CDC to conclude that the B. anthracis strain used in the
attacks was indistinguishable from the Ames strain (Hoffmaster et al., 2002).
As early as October 2001, samples from the spore-laden envelopes and clin -
ical isolates (including one from Robert Stevens, the deceased Florida patient
and index case) were also sent to Paul Keim’s laboratory at Northern Arizona
University. The Keim laboratory had already established the B. anthracis MLVA
sequence database that contained information on more than 1,000 samples
from around the world. This database proved useful for identifying the B.
anthracis strain in the forensic samples.
Beginning in January 2002, Keim also began conducting genetic testing
at the request of the FBI on isolates of B. anthracis provided by the United
States Army Medical Research Institute for Infectious Diseases (USAMRIID),
which had received evidentiary samples from the FBI. The first 18 eviden -
tiary samples (designated Batch E0001) received by the Keim laboratory were
handled according to FBI chain of custody requirements and were initially not
identified. An initial MLVA-8 analysis found that all but two of the samples
(“Connecticut samples”) provided by USAMRIID were identical to and con -
sistent with the Ames strain genotype (Keim, 2002a). An expanded analysis
of 15 MLVA loci (Keim, 2002b) yielded similar results, with all but 3 forensic
samples matching the Ames strain. One sample differed from the Ames strain
in that it had lost the pXO2 plasmid (see Chapter 2), but was otherwise identi -
cal to the others. Keim noted that plasmids are commonly lost during culture
and that this loss may have occurred prior to shipment of the sample to his
laboratory, but was not to be interpreted as necessarily indicating that the
original forensic sample was pXO2 negative. The two Connecticut samples
proved to be distinct from the other evidence, but were identical to each other.
Their genotypes matched 10 isolates from China in the Keim laboratory data -
base that did not have a genotype or strain designation at that time, but most
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100 SCIENTIFIC APPROACHES USED TO INVESTIGATE THE ANTHRAX LETTERS
closely resembled reference strain Genotype 61. At the time, Keim noted that
these samples clearly did not match the Ames strain. These samples proved to
be from a separate case (Malakoff, 2002) and not from the elderly Connecticut
patient who died from inhalational anthrax. The FBI was initially interested in
these samples until it was determined that they were not related to the anthrax
letter investigation. The Keim laboratory continued to receive and strain type
the B. anthracis samples submitted to the FBI Repository (see Chapter 6) over
the next six years.
5.3 WAS THE B. ANTHRACIS IN THE LETTERS
GENETICALLY ENGINEERED?
An important investigative issue was whether the B. anthracis strain used
in the mailings had been genetically engineered. For example, the FBI was
interested in whether antibiotic resistance genes or virulence factors had been
introduced into the attack strain’s genome from other strains or species, and
whether there were any other mutations in the sample, engineered or otherwise,
that might help investigators determine its source.
To address some of these questions, Paul Jackson and colleagues at the
Los Alamos National Laboratory (LANL) analyzed samples from the attack
envelopes from October 2001 through mid-2002. 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. The
samples were tested for possible indications of genetic engineering using DNA
sequencing (see Box 5-1) and polymerase chain reaction (PCR) amplification
(see Box 5-2) to look for (1) the presence of genes encoding resistance against
the antibiotics ciprofloxacin, tetracycline, erythromycin, bleomycin, kanamycin,
and chloramphenicol; (2) modifications of the protective antigen, edema fac -
tor, and lethal factor protein genes; and (3) inserted sequences derived either
from cloning vectors (plasmids) known from the literature to have been used
to engineer B. anthracis or from the insertion of the cereolysin genes of B.
cereus, reported (Pomerantsev et al., 1997) to have conferred upon the strain
an ability to evade protective immunity induced by some anthrax vaccines (FBI
Documents, B1M4D2). The LANL scientists reported that “none of the isolates
assayed showed any indication of genetic manipulation based on the presence
of markers normally associated with genetic manipulation of B. anthracis.”
It should be noted that the LANL investigators did not look for other less
obvious alterations that also might have indicated that the organisms in the evi -
dentiary samples had been genetically engineered. Indeed, they acknowledged
that a well-financed laboratory could exploit or develop other cloning vectors
and other methods for genetic manipulation without leaving clear molecular
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MICROBIOLOGICAL AND GENETIC ANALYSES OF MATERIALS IN THE LETTERS
BOX 5-1
Genome Sequencing
Until the 1970s, DNA was the most difficult molecule in biology to analyze because
of its enormous length and its “monotonous” chemical structure. The DNA molecule is
a string of chemical building blocks—the nucleotides or “bases” adenine (A), thymine
(T), cytosine (C), and guanine (G). DNA sequencing is the process of determining the
exact order of these building blocks in a piece of DNA. For example, “ATCGGCTAA”
is part of a DNA “sequence.” Today, DNA sequencing has become indispensable for
basic research and for numerous applications, such as disease diagnostics, biotech-
nology, systematics, and forensic biology. The earliest DNA sequencing methods were
developed in the 1970s and were laborious and very slow. But the Human Genome
Project, which began in 1990 and was largely completed in 2003, greatly stimulated
the development of new sequencing technologies. These are largely automated and
are orders of magnitude faster than earlier efforts. For example, in 2001 it could take
about a year to sequence the genome of a bacterium like B. anthracis, whereas today
such a process requires only a few hours.
Genome sequencing generally requires that the genome being studied first be
broken into smaller pieces. This process is usually carried out by enzymes that “cut”
the DNA into short fragments. In an alternate approach called “shotgun” sequencing,
the DNA of interest is mechanically broken into random overlapping fragments. Each
fragment is sequenced numerous times, and the genome is reassembled using com-
putational methods to order the fragments based on the regions of overlap. If there is
sufficient overlap, the genome sequence can be considered complete or “closed.” In
bacteria, which usually have circular chromosomes, this means that the entire circle
of the sequence is known. This technique works particularly well for small genomes,
such as those of bacterial species that do not have extensive regions of repetitive
nucleotide sequences. For the much larger genomes of animals and plants, the DNA
of interest may also be broken into pieces and then cloned by inserting the fragments
into bacteria, which make copies of the DNA when they divide and reproduce. The
cloned fragments are then mapped against the genome being sequenced. The map-
ping reduces the likelihood that regions containing repetitive sequences (much more
common in eukaryotic genomes) will be assembled incorrectly.
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. These millions of short sequences are
then ordered by computer programs, enabling the identification of single nucleotide
polymorphisms (SNPs) and other genetic variants.
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102 SCIENTIFIC APPROACHES USED TO INVESTIGATE THE ANTHRAX LETTERS
BOX 5-2
The Polymerase Chain Reaction Technique
The PCR technique provides a rapid means to amplify (increase the number
of copies of) DNA segments of interest. Knowledge of the DNA sequence to be
amplified is used to design two specific, but fairly short, synthetic DNA strands, or
oligonucleotides, that are complementary to the sequence of DNA to be amplified.
These oligonucleotides serve as “primers” for DNA synthesis and determine the seg-
ment of DNA amplified. In PCR, the original double-stranded DNA is first heated to
separate the strands. The separated strands are then cooled in the presence of an
excess of the two primers, which hybridize with the complementary sequences in the
strands of DNA being studied. The mixture is then incubated with the nucleotides that
are the raw materials for DNA synthesis and an enzyme called “DNA polymerase.”
This enzyme synthesizes new DNA starting from the primers and copying the DNA
strand to which the primer is bound. The result of the first cycle of PCR produces two
new double-stranded DNA molecules that each contains one strand of the original
DNA and one strand from the primer. This cycle of denaturation, hybridization, and
synthesis is then repeated many times, increasing the number of copies of the DNA
sequence of interest. Each cycle generates fresh templates for further amplification,
and only the sequences bracketed by the primers are amplified, while regions that
lack priming sites are not. After a number of these cycles, a substantial proportion of
the reaction mixture corresponds to the amplified DNA.
SOURCE: Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., and P. Walter.
(2002). Molecular Biology of the Cell, 4th ed. New York: Garland Science.
signatures, or at least the signatures that the investigators sought. 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). As
noted by Read and colleagues (2002), identification of genes that have been
altered or inserted deliberatively in potential bioweapons agents is facilitated by
complete genome sequencing. No further testing related to the issue of genetic
engineering of the attack powders was performed after mid-2002 aside from
the genome sequencing.
Prior to the 2001 letter attacks, the Institute for Genomic Research (TIGR)
had begun sequencing the genome of the “Porton Down” isolate of B. anthracis
(Read et al., 2002). The genomes of many bacteria have multiple parts, typically
including a single large circular chromosome and one or more smaller plasmids.
In particular, most B. anthracis strains carry two plasmids, called pXO1 and
pXO2, that encode proteins required for virulence but that are not essential
for the bacteria to grow under laboratory conditions. However, the Porton
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MICROBIOLOGICAL AND GENETIC ANALYSES OF MATERIALS IN THE LETTERS
isolate had been rendered avirulent by “curing” (eliminating) both plasmids
from the Ames strain (Read et al., 2003), making it a less than optimal choice
for a reference genome. Shortly after the letter attacks, the National Science
Foundation provided critical funding that allowed TIGR to sequence to draft
quality the isolate from the spinal fluid of the deceased Florida patient, Robert
Stevens (Ames “Florida”). Here, “draft quality” refers to a genome sequence
for which a small fraction of the nucleotide bases remains uncertain, there are
remaining small gaps in the coverage of the complete genome, or both. This
work was described in a paper by Read and colleagues in 2002, and included a
comparison of the Florida and Porton isolates and an examination of a group
of isolates of the Ames strain obtained from various laboratories prior to 2001
as well as an isolate from a Texas goat obtained in 1997. The latter was the only
other isolate of the Ames strain known to have been collected in the field aside
from the 1981 dead cow “Ancestor” isolate. In this initial set of comparisons, 11
sequence differences were found between the chromosomes of the Porton and
Florida isolates (Read et al., 2002); however, thesel differences were also found
in all the other Ames isolates tested. From these data, and the understanding
that the Porton Down strain was derived from earlier isolates at USAMRIID
(Read et al., 2002), it was inferred that the mutations in the Porton strain
occurred after the 1982 transfer of the strain to Porton Down. No sequence dif-
ferences appeared to distinguish the isolate of the Florida victim from many of
the isolates of the Ames strain present in various laboratories before the attack.
In spring 2003 TIGR also initiated the sequencing of the Ames Ancestor
and completed this work in October 2003 (Ravel, 2010). Unlike the Florida
isolate, which was sequenced only to draft quality, the Ames Ancestor sequence
was “closed,” that is, assembled into one contiguous sequence. Annotation
and analysis of the Ames Ancestor sequence continued until mid-2004 and
it was released to GenBank on June 1, 2004. (The paper announcing the
Ames Ancestor sequence was not, however, published until 2009; see Ravel et
al., 2009). 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).
The apparent absence of chromosomal differences between the attack
strain and Ames strains had important implications, both positive and nega -
tive, for the investigation. On the positive side, the findings strongly supported
the inference that the attack strain had come—directly or indirectly—from a
laboratory that possessed the Ames strain. Also on the positive side, the find -
ings supported the conclusion that the attack strain had not been engineered
to make it resistant to treatment or more virulent. On the negative side, the
absence of distinctive sequences in the attack strain seemed to mean that it
would not be possible to use genetic markers to trace the attack material to
one particular source among the various institutions (and laboratories within
the institutions) that possessed the Ames strain.
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104 SCIENTIFIC APPROACHES USED TO INVESTIGATE THE ANTHRAX LETTERS
Nonetheless, an important clue as to the source of the strain in the letters
came from microbiologists at USAMRIID who discovered that the attack mate-
rial contained at low to moderate frequency several subtypes of B. anthracis that
produced morphologically distinct colonies. These colony morphological vari -
ants (or “morphotypes”) retained their distinctive appearance when single cells
from the colonies were regrown into new colonies. This persistence meant that
the morphotypes were genetically distinct from the standard (wild-type) Ames
strain in the samples; that is, they apparently contained a mutation or muta -
tions that caused them to produce their distinctive-looking colonies. That sev -
eral morphotypes could be distinguished indicated that different morphotypes
contained different mutations. The morphotypes appeared to be spontaneous
mutants that arose during the preparation of the batches of spores that were
eventually used in the attacks.
As described and discussed later in this chapter and in Chapter 6, TIGR
also sequenced the genomes of several of these morphotypes to identify the
mutations that distinguished them from the wild-type Ames strain and from
each another (FBI Documents, B1M5D1-2). These mutations played a central
role in the investigation (USAMRIID, 2005; FBI Documents, B1M2D12;
Worsham, 2009), helping the FBI to trace attack materials to a possible
source.
The investigation summaries provided to the committee in December 2010
refer to the presence of a pE03 vector referred to as an ‘Israeli cloning vector’
among certain repository isolates (B3D1). On the January 11, 2011 meeting
the FBI was asked to clarify what was known about the vector. The committee
was told that this vector was a derivative of the commonly used cloning vector
pBR322 that was found in some isolates, and that it had no forensic value to
the investigation (FBI/USDOJ, 2011).
5.4 B. SUBTILIS CONTAMINATION OF THE NEW YORK SAMPLES
A finding that was initially of high forensic interest was the discovery, based
on cultivation techniques, that the powder from the letters sent on September 18
to two New York City addresses (the New York Post and Tom Brokaw at NBC)
contained a mixture of B. anthracis and, at a frequency of about 1 to 5 percent,
a non-B. anthracis bacterium. The contaminating bacterial species was identified
by the CDC as B. subtilis on the basis of 16S rRNA gene sequencing and later
whole genome sequencing (CDC, 2001a; FBI Documents, B2M1D2). B. subtilis
is a ubiquitous bacterial species that is readily isolated from environmental
samples from around the world. The identification of the contaminant as B.
subtilis was at first considered of potential importance because certain strains
of B. subtilis are widely used in academic and industrial laboratories. Hence, if
the contaminant had proved to be a particular laboratory strain, it might have
provided a clue to the origin of the New York City powders.
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MICROBIOLOGICAL AND GENETIC ANALYSES OF MATERIALS IN THE LETTERS
Whole genome sequencing analysis carried out and reported in 2004 by
TIGR of an isolate referred to as GB22 from the New York Post letter showed
high (98 percent) similarity, but not identity, to the published sequence of the
standard laboratory strain B. subtilis 168 (Kunst et al., 1997). In 2006, TIGR
developed 95 PCR assays for 23 B. subtilis loci in the evidentiary sample that
differed from the reference (B. subtilis 168) genome. The amplified DNA
regions were compared using gel electrophoresis. (DNA sequencing of these
amplified regions would have been a more definitive approach.) The B. subtilis
isolates from the New York Post and Brokaw letters were identical to each other
at all 23 loci, indicating that they were the same strain. (The whole genome
sequence of the B. subtilis from the Brokaw letter was not determined, however,
so their identity was not definitively demonstrated.)
Because the 95 PCR assays would have been cumbersome to perform on
larger collections of samples, the FBI Laboratory next identified four genetic
markers in the GB22 letter strain, three of which (designated ID 65, ID 91, and
ID 107 by TIGR) were rare in a survey of 72 B. subtilis strains isolated from
around the world. These strains were obtained from the NRRL (formerly the
Northern Regional Research Laboratory) collection of the U.S. Department of
Agriculture and the American Type Culture Collection, and they were meant
to represent a geographically and genetically diverse collection (FBI Docu -
ments, B2M4D2). The three rare markers distinguished the GB22 strain from
the other strains. The fourth marker (sboA) was common to all the B. subtilis
strains examined. This combination of markers was designed first to determine
whether any B. subtilis was present in additional samples based on the presence
of the sboA marker and, second, to determine whether such samples contained
a strain that might be similar to or the same as the GB22 strain from the New
York letters.
The FBI Laboratory developed TaqMan (see Box 5-3) real-time PCR
(RT-PCR) assays for the four markers, and these assays were provided to the
Naval Medical Research Center (NMRC) and the National Bioforensic Analysis
Center (NBFAC) at the Department of Homeland Security’s National Bio-
defense Analysis and Countermeasures Center, where the assays were validated
by blind testing. NMRC and NBFAC used the assays to evaluate over 300 evi -
dentiary samples. Only two B. subtilis strains from these samples were found
that matched GB22 at all four loci. But when TIGR followed up by further
characterizing these two samples using the complete set of 95 PCR assays, they
proved to be genetically different from GB22 (FBI, 2009). NBFAC later (2007)
also tested all Ames strain samples in the FBI Repository (see Chapter 6) for
the presence of B. subtilis contamination (see NBFAC analytical result reports,
November 2006-December 2007, FBI Documents, B2M4D3-15). Although
322 out of 1057 repository samples tested positive for the sboA nucleic acid
sequence, further testing showed that none of these 322 samples was posi -
tive for the rare ID 65 marker in GB22 (NBFAC, 2007; FBI Documents,
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106 SCIENTIFIC APPROACHES USED TO INVESTIGATE THE ANTHRAX LETTERS
BOX 5-3
The TaqMan Technique
The TaqMan® technique is highly sensitive (Easterday et al., 2005a) and reliable
as a diagnostic tool (Easterday et al., 2005b), and allows the detection of genetic dif-
ferences between samples even at the level of SNPs (Van Ert et al., 2007b). It uses
an oligonucleotide probe that anneals to both the wild-type and mutant target DNA.
The probe is labeled with both a fluorescent tag and a fluorescence quencher and
binds tightly to the exact complementary sequence in the target DNA. PCR is initi-
ated using primers that anneal nearby. One of these primers is designed such that it
anneals only to a template containing the specific allele to be detected. Two primer
sets are generally used, one that is specific for the wild-type allele and a second that
is specific for the mutant allele. If the primer anneals and Taq polymerase synthesizes
a new strand along the template, then the bound fluorescent TaqMan probe will be
digested by the exonuclease activity of the advancing polymerase, thereby releasing
the fluorophore and producing a signal that indicates the presence of the particular
allele. If the primer does not anneal and Taq polymerase does not synthesize DNA,
then the oligonucleotide probe will remain bound and intact, and the fluorescent tag
will not emit detectable fluorescence due to the close proximity of the quencher.
B2M4D13). (If any samples had been positive for the presence of the ID 65
marker, analyses for the ID 91 and ID 107 markers would have also been per-
formed.) In short, many repository samples were contaminated with B. subtilis,
although apparently not by the same strain as in the New York Post letter. Ulti-
mately, the FBI concluded that the testing for B. subtilis did not provide useful
information leading to the source of the New York letter materials—GB22 is
apparently an environmental strain of unknown origin that could not be traced
to any particular source.
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. Such variants often
carry genetic alterations that produce noticeable phenotypic changes in physi -
ology, behavior, or morphology. When the variants are observed at the level of
the physical appearance of colonies as they grow on agar plates, those variants
are often referred to as “morphotypes.” As the selective pressures for rapid
growth under laboratory conditions may differ from those experienced by
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MICROBIOLOGICAL AND GENETIC ANALYSES OF MATERIALS IN THE LETTERS
organisms in their natural environment, genetic variants (mutants) may arise
that replicate faster and, over time, replace the “wild-type” strain during
repeated cycles of laboratory culture. 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) revealed the presence of colony
morphotypes whose stability suggested that they resulted from the presence of
genetically distinct subpopulations. The specific set of genetic alterations in a
population might provide a useful profile for that population and if it can be
demonstrated to be present in two different sample populations, might suggest
that the two were derived from a common source. With sufficient knowledge
about the identities of such genetic variations and the frequencies with which
they arise in the population under specific culture conditions, the statistical
significance of the similarities between the two populations might, in principle,
be calculated. Under some circumstances, the chance of two independent popu-
lations containing the same genetic variant subpopulations might be so small
that it could be concluded with high confidence that they were derived from a
single source. With this goal, the FBI pursued detailed characterization of the
phenotypic and genetic variation among the evidence samples.
5.5.2 Background Information on Morphotypes
Given the rapid generation times (many generations per day) and large
populations (often billions of cells) typically observed in laboratory bacterial
cultures, it is highly likely that genetic variants will arise in cultured popula -
tions. If a genetic variant in a population is able to initiate growth more quickly,
grow more rapidly, or sustain growth longer as conditions become less favor-
able, it tends to increase its frequency in the population. This process can
lead to cultures with multiple genetically distinct subpopulations. While this
basic process of selection drives evolution in nature, it can present problems
for genetic studies in the laboratory, as the characteristics of an organism may
change over the time frame of a scientific study (Elena and Lenski, 2003).
Microbiologists usually seek to avoid this phenomenon using two primary
methods. First, stock cultures are stored under nongrowth conditions in either
a dried or frozen state, since most mutations do not arise in the absence of
growth. Fresh samples of that nonvarying stock are then used to initiate each
new set of analyses. Second, cultures retrieved from the stock are streaked on
growth medium in such a way that individual colonies are obtained and each
colony contains a population derived from a single cell (i.e., a clonal popula -
tion). Some genetic variants that make up a minor proportion of the stock
population may be recognizable based on their atypical colony morphology,
and those variants can be avoided in the generation of cultures for further uses.
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114 SCIENTIFIC APPROACHES USED TO INVESTIGATE THE ANTHRAX LETTERS
FBIR samples was deemed an impractical method for several reasons. First,
phenotypic screens of the types described above are relatively slow, labor inten-
sive, and highly dependent on the trained eyes of the investigator to identify
variant colonies. 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. Third,
phenotypic screens are insensitive and do not reliably detect rare variants. Iden-
tification of the specific mutations associated with each phenotypic variation
was required for the development of definitive assays to detect the presence of
shared mutations in multiple strains within the repository. Such DNA-based
assays are rapid, sensitive, compatible with high-throughput methods, and
definitive to the level of nucleotide sequence.
Scientists selected representative morphotype isolates as well as control
wild-type isolates from each of three letter samples for detailed genetic analysis.
Several criteria were used for this selection. First, the scientists needed to be
able to distinguish the variants from the wild-type colonies on plates. Second,
these particular morphotypes must have been present at a high enough fre -
quency for the scientists to identify them repeatedly. The third essential crite -
rion was the apparent presence of the morphotype in each of the three letter
samples (Leahy, Daschle, New York Post) that were subjected to this analysis.
The final selection of morphotypes focused on four variants: A, B, C/D, and
E. There were other morphotypes found in the letter materials, but they were
not used for further forensic testing. Worsham and colleagues at USAMRIID
quantified the percentages of variants by randomly picking about 370 isolated
colonies from plates made using dilutions from the Leahy letter. These colonies
were 79 percent wild-type morphology, 6.7 percent C/D morphotype, 1.1 per-
cent B morphotype, 1.3 percent A morphotype, and 4.9 percent E morphotype
(other morphotypes accounted for the remaining fraction) (FBI Documents,
B1M2D12). It is important to note that two identical-looking morphotypes
need not, and often did not, have the same genotype. Indeed, as discussed in the
next section, two independent isolates exhibiting similar colony morphotypes
might have mutations in different genes or even different mutations in the same
gene. Also, some colonies identified as morphotypic variants may not have had
any mutation, as the distinction between genetic and nongenetic variation is not
always clear. Thus, it was crucial to identify differences in nucleotide sequence
as an unambiguous signature of different mutant subpopulations.
5.5.5 Whole Genome Sequencing of Morphotype Isolates
To determine whether the genetic alterations associated with each colony
morphotype might be suitable for use as forensic markers, the genome sequences
of multiple morphotype isolates were determined (Table 5-2). Genomic DNA
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MICROBIOLOGICAL AND GENETIC ANALYSES OF MATERIALS IN THE LETTERS
TABLE 5-2 B. anthracis Isolates Analyzed by the Institute for Genomic
Research (TIGR)
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 pro-
vided by Paul Keim to TIGR, where it was prepared for genome sequence
analysis. Plasmid libraries were produced from the genomic DNAs and shotgun
sequencing was carried out to produce approximately 8X (in some cases 12X)
average coverage of the genome. However, most of these genome sequences
were not closed (i.e., assembled into one contiguous sequence). In some cases
(e.g., the samples from the Daschle letter), no sequencing was performed. In
these cases the TIGR scientists used PCR to test whether the same genetic
differences in the sequenced samples were also present in the unsequenced
ones (FBI Documents, B1M5D1). Again, as previously noted, efforts were not
undertaken to identify these or other morphotypes in the Brokaw letter.
Control wild-type isolates from each letter were found to possess no genetic
differences from the Ames Ancestor strain (FBI Documents, B1M5D1-2). The
genome sequences of each of the chosen morphotype isolates did, however,
exhibit differences from the wild-type isolates in particular genetic regions
of the B. anthracis chromosome or, in the case of the E morphotype, in the
pXO1 plasmid. Some samples of morphotype A contained a single nucleotide
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116 SCIENTIFIC APPROACHES USED TO INVESTIGATE THE ANTHRAX LETTERS
polymorphism (SNP) while others carried a duplication. SNPs were also found
for morphotypes B and C, while D contained a chromosomal deletion and
E had a deletion in the pXO1 plasmid. Following the identification of these
sequence differences, unsequenced morphotype isolates were further tested
for the presence of the same sequence differences using PCR amplification and
sequencing of the amplified DNA. The results are summarized in Table 5-3.
These data revealed that multiple isolates of some of the morphotypes (e.g., B)
were associated with a single genetic change while others (e.g., isolates of the
A morphotype) exhibited several different sequence variations in the same
chromosomal region.
The genotypes of the A morphotype isolates from three letters (Leahy,
New York Post, and Daschle) were of two general kinds. 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.
a Source of original morphotype isolate with this genotype.
b Sample subsequently found to contain DNA carrying this genotype.
c Source of subsequent morphotype isolate demonstrated to have this genotype.
SOURCE: FBI Documents, B1M5D1-2.
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MICROBIOLOGICAL AND GENETIC ANALYSES OF MATERIALS IN THE LETTERS
in a gene encoding a K+ uptake protein. This SNP was found only in the New
York Post letter material, but not in the Porton Down, Ames Ancestor, or Leahy
genomes, and no further forensic use was made of this mutation. The second
kind of A morphotype genotype involved large insertions in one of the eleven
copies of the B. anthracis 16S rRNA gene. A 2024 bp (base pair) insertion (later
termed the A1 genotype) and an 823 bp insertion (A3 genotype) were both
found in the materials from the Leahy, Daschle, and New York Post letters, and
both were subsequently chosen for the development of two separate assays to
be used to screen the FBI Repository of Ames strain samples (see Chapter 6). A
2608 bp insertion (A2 genotype) was found only in the New York Post letter and
an assay was developed for the detection of this genotype by Commonwealth
Biotechnologies, Inc., (CBI) in Richmond, Virginia. In validation testing, the
performance of the A2 assay was surpassed by that of the A1 and A3 assays.
Consequently, the FBI decided not to use the A2 assay for evidence analyses.
Eventually, many more variants ranging from 822 to 2608 bp were found among
other samples provided to TIGR (FBI Documents, B1M5D4).
B morphotype isolates from the Leahy and New York Post were fully
sequenced (Table 5-2), 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. PCR amplification and resequenc-
ing of the amplicon confirmed the presence of the same SNP in the same
region of the Daschle morphotype B isolate. The SNP was the replacement of
a thymine (T) with a cytosine (C). The two open-reading frames are divergently
transcribed, so this intergenic region likely contains the promoters for these
genes, one of which (spo0F) plays a key role in governing entry into sporulation.
This mutation may explain why the morphotype is sporulation deficient. The
gene expression patterns of these mutants were not examined because these
kinds of experiments were considered outside the scope of the investigation.
The consistent presence of the B SNP provided a second potential genotypic
signature for comparing FBIR samples to the letter materials, but it was not
used by the FBI to screen repository samples because multiple efforts by con -
tractors to develop assays for this SNP failed (see below), nor were any other
SNPs representing single base pair mutations used to that end (see Chapter 6).
The morphotype C and D isolates shared a very similar phenotype. For
the C morphotype, only material from the GB18 sample from the Leahy letter
was sequenced (Table 5-2). Again, the TIGR team’s reasoning was that other
samples did not need to be sequenced because any polymorphisms found in
GB18 could be tested by PCR later. The GB18 C morphotype analysis found
one SNP corresponding to a nucleotide change from G to A, creating a stop
codon in a histidine sensor kinase (“his kinase”) gene, a member of a family
of proteins that regulate gene expression. When the D morphotype sample
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118 SCIENTIFIC APPROACHES USED TO INVESTIGATE THE ANTHRAX LETTERS
GB19 from the Leahy letter was subsequently examined for the presence of
the C SNP in the same gene sequence, a 258 bp deletion was found instead.
This genetic deletion resulted, in turn, in a deletion of 86 amino acids in the
same his kinase protein. The SNP found in GB18 is located in the chromosomal
region that is deleted in GB19. Thus the similarity in the C and D phenotypes
could be explained since both the C SNP and the D deletion likely produced
a nonfunctioning protein from the same his kinase gene, which plays a role in
sporulation. The New York Post and Daschle letters were not tested for the C
and D morphotypes.
The morphotype E isolate from the Leahy letter (GB23) was sequenced
and had no chromosomal mutations. Instead, this strain had a 21 bp deletion
in the pXO1 plasmid. The deletion was located in a gene encoding a putative
gene expression regulator. A PCR/resequencing assay was used to test for the
presence of the same deletion in the GB24 Leahy letter sample. This sample
contained a 9 bp deletion in the same genetic locus. The PCR/resequencing
analysis was run on a series of other blind samples relevant to the investiga -
tion according to TIGR’s 2005 report (FBI Documents, B1M5D4). Some of
these additional strains carried the 9 or 21 bp deletions, but others contained
a SNP representing a single point mutation (CGT → TGT) in the same locus
that appeared to create defects in the corresponding proteins severe enough
to interfere with normal function, although it was beyond the scope of TIGR’s
work to test this.
Table 5-4 provides a summary of the distribution among the case letters
of the morph genotypes that were ultimately used for screening of the FBIR.
TIGR completed this stage of the study by analyzing a set of samples (listed
in Table 7, FBI Documents, B1M5D4) that included additional evidentiary
samples as well as noncase strains from the Keim laboratory’s scientific collec -
tions. 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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MICROBIOLOGICAL AND GENETIC ANALYSES OF MATERIALS IN THE LETTERS
unique markers suitable for further forensic use. The A1, A3, D, and E geno -
types were employed for the development of validated assays that were used
to screen samples of the Ames strain collected by the FBI from all domestic
and foreign sources that it was able to identify. The details of this screening are
provided in Chapter 6.
5.5.6 Development and Application of Assays for the Genotypes
Genotypes A1 and A3
CBI was the contractor selected by the FBI to develop the genetic assays for
the A1 and A3 morphotypes and test the FBI Repository samples (Chapter 6).
CBI began work for the FBI in mid-2002 (FBI Documents, B2M5D2). The A
morphotypes that were analyzed most thoroughly were found to contain large
insertions overlapping a 16S rRNA gene (FBI Documents, B1M5D1). Although
the insertion was of a different size in each of the three A morphotypes, all
three had insertions in the same locus. The A1 genotype had a 2024 bp inser-
tion 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). The A3 genotype
contained an 823 bp insertion that was originally identified in an isolate from
the Daschle letter, but during assay development by CBI this allele was also
detected in DNA derived from bulk Leahy and New York Post letter spores
(Hassell, 2010). The A2 genotype had a 2608 bp insertion and was originally
identified in an isolate from the New York Post letter. No acceptable assay for
A2 was developed, and it is not known whether the A2 allele was present in
spore material from the other letters. The assays developed by CBI used the
TaqMan analytical technique (Didenko, 2001), which is an adaptation of PCR
(see Boxes 5-2 and 5-3).
CBI completed its validation studies in February 2004. Limits of detection
were estimated at 0.005 percent for the A1 genotype assay and 0.001 percent
for the A3 assay in a background of 20 nanograms (ng) of Ames Ancestor DNA.
Appropriate reaction controls were also developed. Sequencing of amplicons
served as a final confirmatory step. The A1 and A3 genotype assays were chosen
by the FBI for use in analyzing the FBIR samples (see Chapter 6) as problems
(e.g., high number of false positives) with the validation of the assay for geno -
type A2 ultimately caused this assay to be abandoned. It should be noted that
assay development and validation took almost two years.
Genotypes B and D
Three companies—CBI, IIT Research Institute (IITRI), and Midwest
Research Institute (MRI)—were hired to develop assays for the B and D geno -
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120 SCIENTIFIC APPROACHES USED TO INVESTIGATE THE ANTHRAX LETTERS
types. None of the contractors was successful in developing a reliable B assay.
In addition, the FBI expressed a preference not to use assays directed at SNPs
(FBI, 2009). Consequently, the FBIR samples were not screened for the B
genotype. 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. It was
never determined whether this allele was present in the spore populations in
the other evidentiary letter samples. Assay development and validation in each
case took almost one year.
IITRI assay (FBI Documents, B2M7): A technical proposal for assay develop-
ment for the D genotype was submitted by IITRI in July 2004 and validation
of this assay was completed in April 2005. Using TaqMan/PCR (Boxes 5-2 and
5-3) this assay detected the genotype D when it was present at levels as low as
0.01 percent relative to the Ames Ancestor background. The repository screen -
ings using the IITRI assay for the D genotype began in May 2005 and were
completed in early 2007.
MRI assay (FBI Documents, B2M8): MRI submitted its technical proposal for
development of the D deletion assay to the FBI in July 2004 and assay develop -
ment was completed in June 2005. It used RT-PCR and had a detection limit of
0.01 percent in the Ames Ancestor background. Three approaches were used to
increase sensitivity: closely spaced primers, short annealing time (15 seconds),
and confirmation of reaction amplicon with melt curve and fragment size
analysis. This second D assay also went forward for screening the repository
and other samples. Screening began in December 2005 and was completed in
October 2007.
Genotype E
The E morphotype was identified from the Leahy, Daschle, and New York
Post letters (FBI Documents, B1M5D4). Although there were apparently sev -
eral different mutations that produced the “opaque” phenotype, all appeared to
involve the same gene on the pXO1 plasmid. One isolate from the Leahy letter
material carried a 21 bp deletion in this gene, and another isolate also from the
Leahy letter had a 9 bp deletion in the same region of that gene. Both of these
deletions were also found in E isolates from the Daschle and New York Post
letters using PCR and sequencing of this locus (Hassell, 2010). Other E isolates
contained a single bp substitution in the same gene.
The 9 bp deletion was chosen for the development of an assay for use in
screening the FBIR samples. The assay was developed in 2005 using TaqMan
technology, validated, and applied to the repository by TIGR under contract
from the FBI in 2007 (FBI Documents, B2M9). Preparations of purified mutant
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MICROBIOLOGICAL AND GENETIC ANALYSES OF MATERIALS IN THE LETTERS
and wild-type DNA were mixed in amounts covering a 1,000,000-fold range
of ratios of mutant-to-wild-type DNA, and the assay was shown to reliably
detect the mutant genome when present at 0.01 percent of the total DNA in
the sample. In addition, the assay did not produce false positives for the pres-
ence of the mutant allele using varying amounts of wild-type DNA. This assay
was approved by the FBI for testing repository samples, which was performed
from June to August of 2007.
5.6 COMMITTEE FINDINGS
Finding 5.1: The dominant organism found in the letters was correctly and
efficiently identified as the Ames strain of B. anthracis. The science performed
on behalf of the FBI for the purpose of Bacillus species and B. anthracis strain
identification was appropriate, properly executed, and reflected the contem -
porary state of the art.
Finding 5.2: The initial assessment of whether the B. 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.
In the first few months following the attacks, isolates from the letters
and other sources were examined only for the presence of some obvious and
expected signs of genetic engineering. This examination was not exhaustive
and would have missed less obvious or less well recognized signatures of
deliberate genetic alteration. Had the case not involved the Ames strain of B.
anthracis, with its relatively brief history and high degree of characterization,
this limitation could have been a serious one.
Finding 5.3: A distinct Bacillus species, B. subtilis, was a minor constituent
of the New York Post and Brokaw (New York) letters, and the strain found
in these two letters was probably the same. B. subtilis was not present in the
Daschle and Leahy letters. The FBI investigated this constituent of the New
York letters and concluded, and the committee concurs, that the B. subtilis
contaminant did not provide useful forensic information. While this contami -
nant did not provide useful forensic information in this case, the committee
recognizes that such biological contaminants could prove to be of forensic
value in future cases and should be investigated to their fullest.
Although the B. subtilis isolates in the two New York letters appeared
to be closely related, the B. subtilis isolate in the Brokaw letter was not fully
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122 SCIENTIFIC APPROACHES USED TO INVESTIGATE THE ANTHRAX LETTERS
sequenced, and therefore the presumed identity of the two isolates was not
definitively demonstrated. Although B. subtilis was found in several hundred
repository samples, the strains in these samples did not match the isolates found
in the New York letters. Biological contaminants could prove to be of great
forensic value and should be investigated to their fullest in future cases.
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), and each of the phenotypic morphotypes was found to
represent one or more distinct genotypes.
This important discovery greatly facilitated the subsequent laboratory
investigation and is a testament to the critical importance of attentive, thought -
ful scientists who were prepared to explore unexpected results in the setting of
a forensic investigation.
Finding 5.5: Specific molecular assays were developed for some of the B.
anthracis Ames genotypes (those designated A1, A3, D, and E) found in
the letters. These assays provided a useful approach for assessing possible
relationships among the populations of B. anthracis spores in the letters and
in samples that were subsequently collected for the FBI Repository (see also
Chapter 6). However, more could have been done to determine the perfor-
mance characteristics of these assays. In addition, the assays did not measure
the relative abundance of the variant morphotype mutations, which might have
been valuable and could be important in future investigations.
In the course of developing the assays that were used to screen the FBIR
samples for the four genotypes, procedures were employed to examine both the
specificity and sensitivity of the assays, including analyses of defined mixtures
of genotypes at known proportions. However, the repository included both
homogeneous and heterogeneous samples, in unknown proportions, and the
extent of genetic diversity in the heterogeneous samples was also unknown.
More could have been done to determine the performance characteristics,
including reproducibility of results, under the actual conditions associated with
the repository samples.
In addition, these assays were not used to quantify the relative abundance
of the genotypes in the FBIR samples and the evidentiary materials. Measure -
ment of relative abundance of genotypes might have helped clarify the relation-
ship between the evidentiary spore samples and whether they were derived
from the same or different cultivation events.
Finding 5.6: The development and validation of the variant morphotype muta -
tion assays took a long time and slowed the investigation. The committee
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MICROBIOLOGICAL AND GENETIC ANALYSES OF MATERIALS IN THE LETTERS
recognizes that the genomic science used to analyze the forensic markers
identified in the colony morphotypes was a large-scale endeavor and required
the application of emerging science and technology. Although the committee
lauds and supports the effort dedicated to the development of well-validated
assays and procedures, looking toward the future, these processes need to be
more efficient.
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. The
committee recognizes that the experience gained in the case, as well as faster
and greatly improved technologies, could help speed future investigations.
These factors alone, however, may not be sufficient for all contingencies. In
particular, future cases could involve less well documented or less easily grown
species and strains, and precious investigation time could be lost because of
the need to establish basic information about the relevant organism’s biology
and population genetics. In addition, original attack material (in this case, the
powder in the anthrax letters) may not be available in all bioterrorism scenarios.
Also, in some future cases of bioterrorism the attacks may continue until the
perpetrators are identified and apprehended.
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