and 5,750 grand jury subpoenas were served. Twenty-nine government, university, and commercial labs assisted with the scientific analyses that were a central aspect of the investigation (U.S. Department of Justice, 2010). Note that, at the time of the anthrax letters mailings, the tools and technologies that were readily available were not adequate and the science of microbial forensics was in its infancy and limited to a few pioneering laboratories (Clements remarks, 2013). The Amerithrax investigation accelerated the development of microbial forensics, resulting in remarkable development and applications of new techniques and approaches for using laboratory tools to pinpoint the genetic identity of a microbial agent (Tucker and Koblentz, 2009). Microbial forensics became an essential part of the scientific investigation, which was combined with physicochemical analyses and other evidence to narrow the search for the source of the B. anthracis used in the attacks.
An important point to be made here is that B. anthracis is extremely stable genetically. One reason for this stability is that the organism’s life cycle includes long periods of dormancy in the form of spores, rendering the genome highly homogeneous (Pilo and Frey, 2011). Paradoxically, reconstruction of the evolutionary history of B. anthracis has been challenging because of the same stability that allowed investigators to track its use as a biological weapon (Van Ert et al., 2007). Very early in the investigation the anthrax spores in the letters and the environmental and clinical isolates were identified as the “Ames strain” by Dr. Paul Keim and members of his team at Northern Arizona University (NAU). At the Zagreb workshop, Keim summarized what enabled them to accomplish this identification in the era before whole-genome sequencing (WGS) became commonplace. Research in the mid-1990s to differentiate B. anthracis strains had been limited to working with small sections of genomic DNA. The genomes of very few bacteria had, at that point, been fully sequenced. Keim and his colleagues had focused on hypervariable regions in the genetic material of B. anthracis called variable number tandem repeat (VNTR) regions. His team was able to identify eight loci in B. anthracis that had multiple alleles (i.e., different versions of the same genetic marker). They developed a typing system called multiple-locus VNTR Analysis, or MLVA. In fact, B. anthracis was one of the first bacteria upon which this subtyping was performed. In 2001, NAU had a database of about 400 different B. anthracis strains and could differentiate these isolates into approximately 90 different genotypes. Both Keim’s lab at NAU and the CDC lab independently produced the same result: the MLVA8 genotype was found to be consistent only with the Ames strain genotype that Keim and colleagues had identified earlier (Keim et al., 2000).
The Ames strain was a known laboratory strain used by the U.S. military and other vaccine development teams. The strain was origi-