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 differences 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 divergence 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 determining whether a particular strain might have been genetically altered, or “engineered,” for increased effectiveness as a weapon (Hoffmaster et al., 2002).

These new molecular approaches, combined with the creation of a collection 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 established 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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