As reports of anthrax attacks across the United States multiplied earlier this week, researchers scrambled to identify the strains of bacteria involved. Such information will be vital in shaping the investigation of the attacks, and the political response to them.
Investigators hope that new analytical approaches will shed light on the origins of anthrax spores sent through the mail. As Nature went to press, envelopes containing spores had already been confirmed in Florida, New York, Nevada and at the offices of majority senate leader Tom Daschle (Democrat, South Dakota) in Washington. If they all involve the same strain, for example, it would indicate that they are part of a coordinated attack.
As the number of reported attacks rose over the weekend, health secretary Tommy Thompson said that the White House would ask Congress for an additional $1.5 billion to counter bioterrorism and purchase antibiotics.
According to sources close to the investigation, the laboratory of Paul Keim, a geneticist at the Northern Arizona University (NAU) in Flagstaff, has taken an early lead in identifying the strains of the causative bacterium, Bacillus anthracis, involved in the attacks.
The authorities turned to Keim's lab immediately after the Florida attacks because of its published record of describing anthrax strains and its role in developing analytical techniques to identify the strains.
In 1998, Keim's team played a key role in proving that a deadly outbreak of anthrax at Sverdlovsk in Russia involved multiple strains of the bacterium, and so was related to an escape of material from germ-warfare facilities and not a natural outbreak, as Russia had claimed (P. J. Jackson et al. Proc. Natl Acad. Sci. USA 95, 1224–1229; 1998).
Although Keim declined to discuss specifics of the latest investigation or the results of his tests, authorities have said the anthrax spores in the Florida attack are believed to be a derivative of the virulent 'Ames strain'. This was commonly used in laboratories for research, developing vaccines and tests, after its original isolate was removed from a dead animal in the 1950s near Ames, Iowa.
More than 1,200 strains of B. anthracis have been identified around the world over the years, and the NAU laboratory has used AFLP (amplified fragment length polymorphism) DNA analysis to examine all of them.
This technique uses enzymes to cut the bacterium's genome into random fragments, and then uses the polymerase chain reaction (PCR) to amplify some of the fragments. Sequence variation between strains, and variation in the length of regions in which small stretches of sequence are repeated, results in a different pattern of amplified fragments for each strain. Data derived from the AFLP technique are held on databases at the NAU and at the Los Alamos National Laboratory in New Mexico.
But Keim's lab has also adapted a more precise test called multi-locus VNTR (variable-number tandem repeat) analysis, or MLVA, for use with microorganisms. With MLVA, researchers use PCR to amplify regions of the genome containing repeated sequences and so develop a genetic fingerprint for a strain or species. MLVA has so far been performed on about 400 of the 1,200 known strains of B. anthracis.
The genome of B. anthracis contains short sequences of DNA that, depending on the strain, are repeated a different number of times at certain loci. Keim's group has published findings on eight such markers used to probe these loci, using a technique that is similar to that used in genetic fingerprinting for paternity suits or criminal investigations. Keim's team has a total of about 50 B. anthracis markers at its disposal, and is working on more markers for the roughly 1,000 VNTR loci in the bacterium's genome. “This is a highly precise method that also can be used for tuberculosis, Escherichia coli and other pathogens,” says Keim.
It takes about 12 hours for Keim's lab to analyse an anthrax sample. Once a strain is identified, investigators can try to match it to its original source — but this is not always easy. The Ames strain, for example, has been passed around the world by researchers — the sample held by the NAU came from Britain's chemical and biological defence facility at Porton Down, which received it from the US Army Medical Research Institute of Infectious Diseases at Fort Detrick in Maryland.
In the future, analysis of anthrax will get a boost from the sequencing of a derivative of the Ames strain by The Institute for Genomic Research (TIGR) in Maryland. Timothy Read, a bacterial genomicist at TIGR, says he expects to close the final gaps in the strain's sequence in the next few months. Other strains will also be sequenced in the near future.
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Dalton, R. Genetic sleuths rush to identify anthrax strains in mail attacks. Nature 413, 657–658 (2001). https://doi.org/10.1038/35099687