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 genotype. 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 clinical 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 evidentiary 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 consistent 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 identical 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 database that did not have a genotype or strain designation at that time, but most