Replies

The mixed genotype and inverted plasmid points not to Ivins’ flask but a stream of 8 isolates to which a reported 100 known people had access.

Dr. Read, a scientist helping with the Amerithrax investigation in the DNA sequencing, long ago published the news that the anthrax was a 50/50% mixture of genotype 62 (Ames) and genotype 62 with an inversion on the plasmid. This would mean two distinct nucleic acids were detected in the sample. This means that some of the Ames had a segment of DNA that is inverted, or flipped, relative to the remainder of the plasmid. One expert advises me that no properly trained microbiologist would propagate or archive a mixture (I am unaware of why Dr. Ivins did but he probably had a reason). Standard microbiological procedure calls for isolation of single colonies - i.e., single, unmixed cells and their clonal, unmixed progeny — at each step. Inversions are not an uncommon class of mutational events, however. It would only be especially probative if it were a rare inversion and if samples were to be present among samples collected from laboratory archives. It turns out, thankfully, that there was a match — with 8 isolates that were collected to which 100 people were known to have access (and an unknown number of additional people).

Barbara Hatch Rosenberg, who runs the Federation of American Scientists’ chemical and biological arms control program, announced in December 2001. “I’m certain it’s someone connected with a government program, or who works in a laboratory connected with a government program,” she said. “The grapevine has it that the results of an experiment on genetic variation at certain locations suggest that this material was made in a very small batch, and that suggests that the material was not made in some old weapons program on a large scale,” she said, citing sources inside and outside the government. “All the available information is consistent with a U.S. government lab as the source, either of the anthrax itself or of the recipe for the U.S. weaponization process,” wrote Rosenberg on a webpage.

In August 2007, scientists working on the FBI Amerithrax investigation wrote “Role of Law Enforcement Response and Microbial Forensics in Investigation of Bioterrorism” in the Croat Medical Journal. The FBI scientist’s article explained that there

“are a variety of genetic markers and methods that allow highly specific and accurate characterization of microbial diversity. For forensic purposes, assaying rapidly evolving markers enables better affiliation to recent common sources, while more stable markers provide better lineage-based evolutionary interpretations, such as strain and sub-strain definition. Since bacteria, viruses, and some fungi reproduce asexually, their genomes are considered to be clonal and portions of their genomes may be very stable and uninformative for distinguishing samples. Therefore, it may not be possible to identify the source of a sample by genetic analysis alone (as often is accomplished in human DNA identity testing). Since many microbial genomes have relatively short generation times, in an overnight culture, a single microbe could have reproduced its genome over a million times, increasing the chance of mutation that may be seen within the culture. Thus, some variation, and hence a forensic signature, may occur during asexual reproduction.”

The authors explained: “The forensic comparison of a genetic profile from a reference sample with that of an evidentiary sample can have three possible general outcomes: match or inclusion, exclusion, or inconclusive. With microbial genetic information, it is less likely to have a prescribed interpretation policy for what constitutes a match and what does not. Some questions may be difficult to answer unequivocally based on extant data. Uncertainty is greater than what is experienced for human DNA identity testing because of unknown diversity, limited databases, unknown manipulations, and limited genetic testing. However, the power of microbial forensic tools is increasing rapidly with ever advancing technology.”

Selected sources:

Budowle, B., M. D. Johnson, C. M. Fraser, T. J. Leighton, R. S. Murch, and R. Chakraborty. 2005. “Genetic analysis and attribution of microbial forensics evidence,” Crit. Rev. Microbiol. 31:233-254.
http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=retrieve&db=pubmed&list_uids=16417203&dopt=AbstractPlus

Budowle B, Murch R, Chakraborty R., “Microbial forensics: the next forensic challenge,” Int J Legal Med. 119(6):317-30 (Nov 2005).
http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15821943&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

Budowle, B., S. E. Schutzer, M. S. Ascher, R. M. Atlas, J. P. Burans, R. Chakraborty, J. J. Dunn, C. M. Fraser, D. R. Franz, T. J. Leighton, S. A. Morse, R. S. Murch, J. Ravel, D. L. Rock, T. R.

Slezak, S. P. Velsko, A. C. Walsh, and R. A. Walters. “Toward a system of microbial forensics: from sample collection to interpretation of evidence,” Appl. Environ. Microbiol. 71:2209-2213 (2005).
http://aem.asm.org/cgi/content/full/71/5/2209?ijkey=11f63da16d84d14221469a04d0917d00b4ae7e74&keytype2=tf_ipsecsha

Budowle, B., S. E. Schutzer, A. Einseln, L. C. Kelley, A. C. Walsh, J. A. L. Smith, B. L. Marrone, J. Robertson, and J. Campos. Building microbial forensics as a response to bioterrorism. Science 301:1852-1853 (2003).
http://www.sciencemag.org/cgi/content/summary/301/5641/1852?ijkey=6c5eda5d0b0d4dec11807281f555d5087c756235&keytype2=tf_ipsecsha

Keim et al., “Microbial forensics: DNA fingerprinting of Bacillus anthracis (anthrax),” Analytical Chemistry, 2008 Jul; 80 (13): 4791-9
http://pubs.acs.org/subscribe/journals/ancham/80/i13/html/0708feature_keim.html

Legal admissibility:

Budowle B, Harmon R., “HIV legal precedent useful for microbial forensics,” Croat Med J. 46(4):514-21 (Aug 2005).
http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16100753&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum