Distinct signature found in '01 anthrax

In a possible break for the FBI's investigation of the anthrax letters of 2001, scientists have discovered that the mailed anthrax was a mix of two slightly different samples, giving the bacteria a distinct signature that might make it easier to match with a source, according to two non-government experts who have been told of the finding.

The discovery that bacteria taken from the letters all grew in the double pattern was made at least a year ago, and it is not known whether the FBI's hunt for a matching sample has succeeded. The bureau and its scientific consultants are screening dozens of anthrax samples collected all over the United States and in some foreign countries, seeking the closest match to the spores used in the attack, according to a scientist who advises the FBI.


The revelation of the double pattern of the mailed anthrax comes as the FBI is due Tuesday to give a Washington judge a secret progress report on the investigation the bureau calls Amerithrax, which is well into its third year without visible results.

The FBI progress report, requested in March by U.S. District Judge Reggie B. Walton, is likely to be less about old-fashioned police work than newfangled science, according to statements made in court by FBI and Justice Department officials. The case of the anthrax-laced letters that killed five people and sickened at least 17 others in 2001 has become the first major test of an emerging scientific discipline called bioforensics, the use of genetic analysis and other modern laboratory tools to track germs used in an attack back to their origin.


FBI spokeswoman Debra Weierman declined last week to discuss the investigation, except to say that tests on the anthrax powder have not been completed. But experts on the fast-developing science of biological sleuthing say it should by now have helped the bureau to substantially narrow the search.

"I think we have the science now to trace the anthrax to a particular lab," said Babetta L. Marrone, a cell biologist at the Los Alamos National Laboratories and a member of an FBI advisory group on bioforensics.

Still, she says, finding the source lab will not by itself identify a perpetrator, only reduce the number of potential suspects. "If I had to guess, I'd say what has the FBI stumped is the non-scientific stuff," Marrone said.

Walton is presiding over a lawsuit filed by former Army biowarfare expert Dr. Steven J. Hatfill, who says the government wrecked his career and upended his life with a deliberate campaign of leaks falsely suggesting that he was the anthrax mailer. Hatfill's lawyers want to obtain FBI documents and question government officials to support his claims.

In successfully seeking to put on hold Hatfill's lawsuit and another filed by the widow of a Florida anthrax victim, the FBI claimed in court early this year that the investigation was at a "critical stage" and that the litigation could endanger investigators' work.

Impressive effort

An affidavit filed by FBI Inspector Richard L. Lambert in January described a major scientific effort to define the "specific forensic signature" of the attack anthrax.

"To forensically characterize the anthrax evidence ... the FBI has contracted with 19 government, commercial and university laboratories which are performing research, analyses and evaluations to assist the FBI Laboratory," Lambert wrote. "Most of these scientific initiatives are scheduled to be completed within the next six months. If successful, these initiatives will ... facilitate the attribution of the anthrax used in the attacks to one or more U.S. and foreign laboratories."


Of 30 FBI special agents and 13 U.S. postal inspectors working full-time on the case, officials say, eight of the FBI agents have a doctorate "in a scientific discipline related to the investigation."

"I've been impressed with the patience and perseverance of our partners in the FBI," said Claire M. Fraser, director of the Institute for Genomic Research in Rockville, a private organization that is carrying out much of the genetic analysis for the bureau.

Fraser said she could not discuss specific findings related to the investigation. But she said that after initial skepticism, she has been pleasantly surprised by the progress made in the past year in finding forensically useful ways to distinguish samples of anthrax from one another.

Still, she said genetic analysis of samples of pathogens is not nearly as advanced as the human DNA testing that has become routine in investigations of murder, rape and other crimes. "We're not even close to being at that level," she said.

Ronald Kessler, a Washington author who has written several books on the FBI, said the Amerithrax investigation represents the largest mobilization of inside and outside scientists in the bureau's history.

The emphasis on first-rate science is in part a reaction to a scandal in the 1990s that discredited some work by the FBI laboratory, Kessler said. "That definitely led to a realization that they had to get outside accreditation and hire outside scientists, not just rely on agents who had worked their way up," he said.


But the nature of the anthrax crimes - the first major bioterrorist attack on U.S. soil - is driving the reliance on research. "You're talking about inventing a new science here," Kessler said.

The finding that the attack anthrax came from a combination of two distinct samples is one small step in that new science. Like other bacteria, anthrax grown in the laboratory forms tiny colonies of bacteria that can have particular physical characteristics. Colonies from different strains or samples can be larger or smaller, have more or less uneven edges or form distinctive shapes.

While all the anthrax used in the attacks is a variant of the Ames strain, scientists found that the spores recovered from the envelopes grew into two slightly different kinds of colonies.

That might mean part of the original sample was removed from a freezer and grown for a period of days, allowing very slight genetic mutations, and then recombined with the original sample, according to the two outside experts familiar with the tests. The perpetrator might then have taken a sample from the mixed batch and used it to grow the bacteria used in the attack. The double pattern gives the mailed anthrax one more distinctive characteristic to be compared with possible sources.

"Potentially, that could be very useful," one scientist said.

Rapid development


The developing science of bioforensics, also known as microbial forensics, encompasses far more than the genetic fingerprinting or patterns of growing colonies.

Advanced testing on samples of anthrax or other organisms can also reveal clues to the location of the water used to grow them, because isotopes of oxygen and other elements in water vary from place to place. Germs also usually carry traces of the growth medium, or nutrient mixture, used to produce them, yielding another potential clue.

Some bioweapons, including anthrax spores, can contain chemical additives to permit the germs to float freely in the air. Experts who have seen the mailed anthrax have been divided on what additive it contained or whether it contained one at all.

All such markers will be studied at a bioforensics unit being created by the Department of Homeland Security as part of its planned $200 million biodefense center at Fort Detrick in Frederick. Currently occupying temporary space at the Army's biodefense laboratory, the National Bioforensic Analysis Center will maintain a reference library of biological samples and data to be consulted in the event of a biological attack, according to written plans for the center.

Abigail A. Salyers, a University of Illinois professor who organized one of the first meetings on bioforensics while president of the American Society for Microbiology in 2002, said she has been impressed with the field's rapid development.

She's pessimistic, however, that the anthrax case will be solved. "The case would be compelling if they found in someone's apartment or home some of this same anthrax mixture," Salyers said. Nearly three years after the attacks, that's unlikely, she said.


But she said the quest is building scientific knowledge and laboratory techniques that will be critical in understanding and tracing the source of any future biological attack.

"Even if they don't solve the anthrax case, that doesn't mean it's a dead loss," Salyers said. "We've developed an awful lot of very useful information in trying to solve it."


Also known as microbial forensics, bioforensics is a new scientific discipline that has emerged since the anthrax mail attacks of 2001. It seeks to identify the likely source of pathogens in a terrorist attack by using several laboratory tools.

Genetic analysis: Different samples of the same strain of anthrax and other organisms have slightly different genetic fingerprints, possibly permitting scientists to track germs used in an attack to a matching sample in a particular laboratory.

Water: Even dried anthrax spores, like all living things, contain water. CIA-funded research has developed a way to trace water to particular geographic areas by analyzing isotopes of oxygen and other elements.


Growth medium: Bacteria and viruses are usually grown in the laboratory using a nutrient mixture, traces of which can be found with the germs.

Additives: To make anthrax spores float more freely in the air, so that victims can inhale them, silica or other substances might be added.