DEPT. OF EPIDEMIOLOGY

The Ames Strain

How a sick cow in Iowa may have helped to create a lethal bioweapon.

by Peter J. Boyer

On the evening of October 12th, a group of scientists and academics at Iowa State University's veterinary college, in Ames, Iowa, gathered in one of the school's laboratories for a procedure involving the university's collection of Bacillus anthracis, the bacteria that causes the disease anthrax. The school's anthrax collection was noteworthy both for what was known about it and for what was merely speculated. What was known was that over the years Iowa State's veterinary microbiologists had accumulated more than a hundred vials containing various strains of anthrax, some dating back to 1928. In 1978, a fondly remembered professor named R. Allen Packer had uncorked one of the fifty-year-old vials and, after a couple of tries, was able to coax the bacillus back to life. The experiment, a testament to the remarkable durability of anthrax spores, had lent a certain distinction to the collection.

What was speculated about the Iowa State anthrax was even more compelling. One week earlier, on October 5th, a Florida photo editor named Bob Stevens (case 5), at American Media Inc., had died of anthrax, the first bioterror fatality in what has come to be known as "the homeland." Early news reports suggested that the F.B.I. had traced the anthrax to a laboratory in Ames, from which the bacteria had perhaps been stolen or otherwise obtained by terrorists.

The reports of an Ames connection to the anthrax terrors caused much excitement in Iowa, and the College of Veterinary Medicine was suddenly fielding scores of calls from reporters wanting to know about the deadly "Ames strain" of anthrax. The trouble was, nobody at the school knew anything about an "Ames strain" -- whether it was the strain of anthrax infecting the mail, whether the Iowa State lab had ever possessed it, or even whether there was such a thing as an "Ames strain." None of the vials were identified as "Ames," but then the labels were cryptic, some bearing only numbers or dates.

The scientists and teachers at Iowa State's veterinary school had not been incautious with their anthrax specimens, but neither had they been obsessed with security. The school's anthrax collection had been stored in cabinets in the teaching laboratory, the doors of which were routinely locked at night. In the context of the academy, this relative casualness was not unusual, especially in the heart of the farm belt, where science was employed as a plowshare rather than as a sword. When an associated laboratory nearby, run by the United States Department of Agriculture, had outgrown its building space a few years earlier, it had moved some of its work on anthrax and mad-cow disease to a rented space in an Ames strip mall. But all of that was before the Florida incident.

On October 10th, Governor Tom Vilsack ordered law-enforcement officers to stand guard over the Iowa State laboratory and at the state's other labs with anthrax (including the Agriculture Department's lab in Ames and labs at the University of Iowa). The Iowa State anthrax collection was beginning to seem like more trouble than it was worth, and the college's dean, Norman F. Cheville, after consultation with the lab's director and a school health-and-safety group, decided to do something about it.

Around 5:30 P.M. on October 12th, college staff members wearing biosafety gloves removed the anthrax specimens from the laboratory cabinet and placed them in an autoclave, a steam sterilizer about the size of a filing cabinet. The scientists knew that an hour or so in the autoclave would do the trick, but they let the machine run all night. At eight-thirty the following morning, the bacilli, although certainly dead, were placed in an incinerator for good measure. Some of the biologists and academics who attended the destruction felt a trace of regret. "We said to each other, 'This is kind of sad we have to destroy this,' " Dr. Jim Roth, an assistant dean for international programs at the school, recalls. "Especially the cultures we'd had since 1928."

Less than two weeks later, Tom Ridge, the director of Homeland Security, announced at a Washington press conference that investigators had identified the anthrax that had been sent through the mail as belonging to the Ames strain. It now seemed likely that there was an Iowa State connection to the Ames strain, and that the original culture of the Ames isolate was sterilized and incinerated with the rest of the veterinary school's collection. Jim Roth had wondered about that possibility, and the school had contacted the F.B.I. and the Centers for Disease Control before killing the specimens. Both agencies approved the destruction. "They may be having some second thoughts about that, but it's too late now," Roth says.

In its way, the uncertainty about the Iowa State anthrax reflects the larger puzzlement facing federal officials as they have tried to work out the provenance of the anthrax that killed Bob Stevens and at least three others (case 15, case 16, case 22 and case 23). As investigators try to determine who is behind the bioterror, alternating between theories that its source is foreign or domestic, state-sponsored or freelance, Dr. Roth and his colleagues in Iowa have tried to unravel the problem of the origins of the Ames strain.

What they do know is that it all began with a sick cow, probably somewhere in a pasture in the western part of Iowa, probably in 1979 (error: origin in Texas). In all likelihood, a farmer encountered his stricken beast after it was already dead, and had not been witness to the sudden fever, the clumsy staggering, the trembling, and, finally, the convulsions that preceded the animal's death. Anthrax seizes and consumes its victims quickly. The farmer might have suspected anthrax, a diagnosis probably confirmed by his veterinarian immediately upon encountering the carcass. In most ways, the beast would have looked good, even healthy, except for the blood streaming from its nostrils, ears, and rectum.

The recommended procedure in Iowa, as elsewhere, is not to disturb the carcass of an animal killed by anthrax. Veterinarians almost never perform an autopsy, because opening the beast's body would expose the bacteria to air, triggering the organism's self-preservation mechanism. Bacillus anthracis is a spore-forming bacterium, which is to say that, when faced with an environmental challenge, it forms a kind of shell, allowing it, acorn-like, to lie dormant for years, even decades. When Professor Packer opened that 1928 vial of anthrax at Iowa State, the jellylike medium inside the tube had turned hard and crusty, but the anthrax spores inside were still alive. (Packer had put the anthrax in a fresh medium, sealed it back up, and left instructions for some future Iowa State microbiologist to try to revive it again in 2028.)

The stricken Iowa cow had probably contracted anthrax by consuming spores that had settled into the Iowa soil, perhaps after an anthrax outbreak in 1950-52, caused by feeding contaminated bonemeal to livestock. The spores from those afflicted animals had gone to ground, until they were ingested, probably with a clump of grass, by the cow in 1979. Once inside the warm, moist environment of the cow's digestive system, the spores came back to life, releasing their bacteria, which grow at phenomenal rates -- each organism replicating itself every fifteen to twenty minutes. As the bacteria grew, they excreted a toxin that, in essence, caused the animal's immune system to go into hyperdrive, leading to shock and near-instant death.

The veterinarian would have disposed of the carcass immediately, either by burning it or, if it could be moved without rupture, by burying it after covering it with quicklime. In either case, before disposal, the vet salvaged a specimen from the diseased animal, either cutting off an ear and sealing it in a bag or drawing blood from the cow's jugular. The vet would have sent that specimen to the nearest state veterinary diagnostic center, which in this case was almost certainly the lab at Iowa State's College of Veterinary Medicine.

Iowa State microbiologists would have seen under the microscope big, rod-shaped bacteria that turned blue when introduced to an identifying substance called a Gram stain. Further biochemical tests would have proved positive for anthrax. At that point, a subculture would have been grown and sent down the street to the Department of Agriculture's National Veterinary Services Laboratory for confirmation. The original culture was probably put in a vial, which somehow found its way to the cabinet that held the Iowa State collection.

Here the story of the Ames strain leaves the realm of speculation, because what happened next is precisely known.

The National Veterinary Services Laboratory in Ames serves as the diagnostic center for the entire nation; it is a repository for all manner of germs and diseases that afflict American livestock. That is why the U.S. Army wrote to the N.V.S.L. in late 1980 requesting a sample of an anthrax culture. The Ames lab made a subculture of the anthrax and sent it to the Army's Medical Research Institute of Infectious Diseases --USAMRIID  -- at Fort Detrick, near Frederick, Maryland, along with the information that the isolate had come from a dead cow. The Army named it the Ames strain.

USAMRIID has long been familiar with anthrax, as far back as the days when it was the Army Medical Unit and was associated with the Biological Warfare Laboratories at Fort Detrick. The mighty lethality of anthrax has been appreciated by mankind since classical times, and its potential as a weapon has been intuited by warriors since 1876, when the bacteriologist Robert Koch discovered that the disease had a bacterial cause. During the First World War, German agents were injecting anthrax into American livestock. In the nineteen-thirties, Japan tested anthrax as a weapon in Manchuria. In the forties, the United States, Britain, and the Soviet Union also took up the challenge of weaponizing anthrax.

But if anthrax is the perfect killer, silent and invisible, it is not, as it exists in nature, a perfect weapon. It is a livestock disease, and when humans contract it under normal conditions it is through contact with diseased animals or their hides. The commonest form of human infection (ninety-five per cent) is through skin contact -- cutaneous anthrax. Lesions form, followed by a black scab, but, while potentially deadly, cutaneous anthrax is highly treatable by antibiotics. For people, by far the deadliest form of anthrax is that which is contracted by breathing spores into the lungs --inhalation anthrax. But, as the spores are not airborne under natural conditions, inhalation anthrax has been a rarity. According to the Center for Nonproliferation Studies, between 1900 and 1978 only eighteen cases of inhalation anthrax were reported in the United States, and two of those were contracted in a laboratory.

What happens to a human being who does develop inhalation anthrax, however, is what inspired bioweaponeers. Once someone has breathed anthrax spores deep into the lungs, symptoms soon appear that seem very much like those of the common flu. There is a fever, cough, and aches, at which point aggressive antibiotic treatment can still offer patients a fighting chance. Otherwise, the fever suddenly elevates, breathing becomes labored, and shock seizes the body. After the onset of this severe stage, it is almost always too late for a cure.

Broadly posed, the trick of weaponizing anthrax is to make it breathable. A clump of infected soil might contain billions of anthrax spores, but a clump of soil is unlikely to be inhaled. So the first task in weaponizing anthrax is to purify it, producing a concentration of spores. This is done by creating a suspension, in which the anthrax spores are separated from the material surrounding them in the sample -- water, material from the growth medium, and so on. No particle of anything much bigger than five microns is likely to get past the mucous membranes and reach deep into the lungs, and each anthrax spore is itself less than two microns in size. Purifying and concentrating the spores requires real laboratory skill.

Purification and concentration, however, is not enough. In even the purest concentrate, anthrax spores, like most small particles, will clump together, owing to natural electrostatic force. "If you just grow up spores in a test tube and then you remove the liquid, you'll have a kind of a clump," says Philip S. Brachman, a legendary epidemiologist and an old anthrax hand. "Now, that clump won't go anywhere -- it'll fall to the ground." The next grand step in weaponizing anthrax is to cause those purified spores to separate, like individual sprinkles of a fine powder, so they can linger in the air and be inhaled.

Such anthrax becomes a weapon of unfathomable potency, but for years bioweapons scientists searched vainly for an efficient means by which to turn clumpy anthrax spores into airy, inhalable anthrax. Finally, in the early nineteen-sixties, a man named William C. (Bill) Patrick III, chief of product development for the American biowarfare program, found the answer. Patrick discovered that a certain combination of ingredients formed a handy anti-caking material, which, when combined with anthrax spores, allowed the spores to separate into a fine dry mist of unagglomerated poisons. "You want a free-flowing powder containing the agent that is electrostatic-free, so that it flows very nicely," Patrick explains calmly, as if he had developed a product to keep laundry static-free in a clothes dryer. "And when energy is applied to the powder, it breaks up into small particles."

A single gram of powdered anthrax can contain as many as a hundred billion anthrax spores. Conventional medical wisdom holds that inhalation of just eight to ten thousand spores is needed to trigger infection. The letter sent to Senate Majority Leader Tom Daschle's office last month contained two grams of purified, powdery anthrax spores -- potentially enough to kill twenty-five million people if it were efficiently distributed.

A letter sent through the mail is not a maximally efficient means of distribution, although, as government officials were surprised to discover, the automatic sorting machines at postal centers can, in jostling a tainted letter, cause a lot of human damage. Two postal workers at a mail-distribution center near Washington have died of inhalation anthrax. "When a person opens a letter, that represents the munition," Patrick says. "When letters go through that high-speed sorting-out process in the post office, you are talking about a huge amount of energy. And you really have a munition."

Patrick's process for making static-free anthrax spores was secretly patented by the government, but he switched over to defensive work when Richard Nixon announced, in 1969, that the nation would unilaterally end its biological-warfare programs. Two and a half years later, the United States -- and ultimately some hundred and fifty other nations -- became a signatory to the international Biological and Toxin Weapons Convention. By most accounts, the United States actually did stop making these weapons, although the Soviet Union continued with a huge program of germ- and chemical-weapon development until at least 1992. Iraq, another signatory to the convention, admitted in 1995 to having produced two thousand gallons of liquid anthrax, and is believed to have an ongoing biowarfare program. Israel never signed the accord. Patrick says, "I think that the Israelis, if truth be known, have an extremely advanced program in biological warfare, because it's too good a weapons system to give up."

After the American program ended, the research on biological and chemical weaponry was taken up by the Army's Medical Research Institute of Infectious Diseases. The unit's chief purpose turned from making biological weapons to devising defenses against them, through such means as developing and testing vaccines.

It was an Army scientist, George Wright, who developed the human anthrax vaccine, which was field-tested in the nineteen-fifties by Philip Brachman. To test the vaccine, Brachman went to the one place in the country where human anthrax infection, including the inhalation form, was most likely to occur -- the animal-hide-processing industry in New England. (Anthrax was once called woolsorter's disease.) Brachman recruited volunteers from four mills whose workers regularly contracted anthrax at a rate much higher than the average population -- an annual rate of 1.2 infections for every hundred workers. He divided the volunteers into two groups, vaccinated one group, and administered a placebo to the other. The results proved the efficacy of the vaccine. That same formula is, in its essence, the vaccine now administered to the American armed forces and other people at risk, and the one that the government intends to make available to the broader public. (Before that can happen, the private firm in Lansing, Michigan, that holds the license on the formula must meet requirements imposed by the F.D.A.)

Over the years, the Army and civilian scientists at USAMRIID have tested new variations of the vaccine, and it was for just such a test that the Army requested a strain of anthrax from the Department of Agriculture lab in Ames in 1980. What they received was a subculture of the anthrax that had killed the cow in western Iowa the year before.

In working with their new isolate, the scientists discovered something remarkable about it: the Ames strain excreted an especially potent toxin. Ames became known as a "hot," or highly virulent, strain, and by the late nineteen-eighties it had become the gold standard for anthrax strains. "It's hot, so people like to challenge their animals with the Ames to determine how well their vaccine or their treatment modality is working," Patrick says.

The Ames strain's reputation among laboratory scientists created a demand for it, and the demand was handily met. Philip Brachman says that if he had wanted to get hold of an anthrax strain, he could have simply written to a laboratory that had it and they would have sent it to him. Germ banks around the world maintain and sell from collections of bacteria, and hundreds of university and research laboratories freely exchange strains of various organisms. There are some limitations, particularly in the United States. The 1996 Anti-Terrorism Act requires that anyone dealing in dangerous pathogens must show a legitimate scientific purpose and must register with the Centers for Disease Control. But the law does not prohibit possession of those dangerous pathogens by non-scientists. Nor are background checks, such as those required before the purchase of a handgun, conducted upon the hiring of technicians who have access to pathogens in laboratories.

In any event, restrictions can be avoided through private exchanges between scientists. With anthrax, there is plenty to share. "You streak it out on a petri dish until there's one individual live bacterium at a particular spot on the plate," Jim Roth explains. "You grow it overnight, one bacterium turns into millions."

"It is an isolated case," Tommy Thompson, the Secretary of Health and Human Services, said after Bob Stevens died of anthrax on October 5th. "There is no terrorism." Such dissonance, which has characterized official pronouncements about the unfolding bioterrorism, is both alarming and, in a way, understandable. In recent weeks, even medical professionals have been made to realize how little they know about anthrax. The military establishment's research centers have been out of the bioweapons business for so long that they have had to rely partly on the advice of the last generation with real hands-on experience.

When anthrax outbreaks in New York, New Jersey, and Washington made it clear that the Florida occurrence was not an isolated case, it became imperative to determine the provenance of the bacteria that was being sent through the mail. A particular strain cannot be identified merely by peering at it through a microscope -- all bacilli anthracis look more or less the same on a slide. To establish the genealogy of the poison-letter anthrax, the government turned to a young civilian scientist named Paul Keim, who is associated with Northern Arizona University, in Flagstaff, and whose wizardry in the field of DNA sequencing is fabled. It was Keim who determined that the unsuccessful anthrax attacks mounted by the Aum Shinrikyo cult in Tokyo in 1993 failed partly because the bacteria used by the terrorists was of the Sterne strain -- an avirulent (nontoxic) bacteria that is used in an anthrax vaccine.

Keim has assisted American intelligence for some time, though he refuses to talk about it. He and an associate, Martin Hugh-Jones, a microbiologist at Louisiana State University, have compiled a formidable collection of pathogens. "It was set up way back when, 'To be ready for ...' " Hugh-Jones says. "It went from 'Let's look at thirty samples' to 'Gosh, do you think we could get two hundred?' to now we have something like between twelve hundred and thirteen hundred."

Samples from the anthrax letters were sent to Keim at his laboratory in Flagstaff, where he put the bacteria through genetic-sequencing tests and compared them to known strains. Soon, he had a match: it was the Ames strain.

When Tom Ridge announced, on October 25th, that the strain had been identified, it seemed like a breakthrough. Officials now knew that they were dealing with a highly toxic strain. This, in turn, suggested something more ominous. "The fact that they have selected the Ames strain, a hot strain of anthrax, indicates to me that they know what the hell they are doing," Bill Patrick says.

But, in a way, identifying the anthrax only clouded the picture. Because of its popularity in laboratories, Ames had become a sort of stock strain, untraceable through its genetics alone to any particular source. "Being Ames doesn't tell me anything, except that somebody got ahold of a stock strain without any difficulty," Philip Brachman says.

There are other ways to trace the bacteria's source. The anthrax sent to Senator Daschle's office was weaponized -- that is, it had been pulverized by the method that Bill Patrick pioneered almost forty years ago. (Twenty-eight of the forty people in the area where the letter was opened tested positive for anthrax exposure.) The fact that it was weaponized means that the powder contained not only anthrax spores but the anti-caking material that allows the spores to float free. Identifying that material --which has been described as a fine, brownish particulate -- could help to pinpoint the source.

Early analyses suggested that Iraq could be the source of the anthrax. Bill Patrick says that when he was in Iraq as a member of the United Nations Special Commission charged with dismantling Iraq's weapons-of-mass-destruction programs, he saw batches of a substance called bentonite -- a readily available material, brownish in color, that can be used to separate the anthrax spores into powdery particles. "It's not the material that we added to our weaponized agent, because we added a much better material," Patrick says. "But it will prevent, to a certain degree, sticking of the spores."

By last week, though, the head of the U.S. Army lab had ruled out bentonite as an ingredient in the anthrax letters. This seemed to rule out Iraq, even as the Czech government confirmed that the presumed lead hijacker in the September 11th attacks, Mohamed Atta, had met with an Iraqi spy in Prague last April.

In announcing the discovery that an anti-cling agent had been added to the anthrax sent through the mail, intelligence officials declared that only three nations in the world had the capacity to weaponize anthrax in that manner: the United States, the former Soviet Union, and Iraq. According to the Washington Post, an unnamed government official also said that "the totality of the evidence in hand suggests that it is unlikely that the spores were originally produced in the former Soviet Union or Iraq."

To Bill Patrick, the assertion that only three nations are capable of producing weaponized anthrax is laughable. "How in the hell he arrived at that conclusion I don't know," Patrick says. "I think the Iranians have a very advanced program in biological warfare. The Israelis ... And we feel that China has an advanced program."

However, close parsing of the official's statement, an exercise that may be warranted just now, reveals that his pronouncement is literally true: the "spores" were not "originally produced" in the Soviet Union or Iraq; they were produced in the belly of a cow in western Iowa.

But the larger implication was somewhat puzzling. If the anthrax sent to Daschle came from one of three state-sponsored bioweapons programs, and if the former Soviet Union and Iraq are discounted as suspects, that leaves the biowarfare program of the United States, which officially ended its biological weapons program in 1969. At that time, government scientists destroyed their stores of weaponized anthrax, kept in Arkansas and Utah, by putting them through autoclaves, just as Iowa State killed its anthrax collection. Also, the Ames strain wasn't isolated until nearly a decade after the American program was supposed to have ended.

In a sense, Army scientists at USAMRIID have, in recent years, "weaponized" the Ames strain whenever they have tested anthrax vaccines on monkeys. They make an aerosol of the Ames strain, spray it into the monkeys' containment area, and await the results. But after each experiment, according to Caree Vander Linden, a USAMRIID spokeswoman, the aerosolized anthrax not inhaled by the monkeys is destroyed. "The aerosolized sample is contained within an airtight cabinet," she explained. "The air and the cabinet are decontaminated after the exposure to destroy all spores that were not inhaled. Any spores that are not inhaled are trapped by an all-glass impinger of water" -- and destroyed.

The notion that only a state-sponsored biological-weapons program could produce the dangerous, powdery anthrax is one of the assumptions now being seriously challenged. The White House spokesman Ari Fleischer said last week that the anthrax may have been produced by "a Ph.D. microbiologist" and that "it could be derived at a small, well-equipped microbiology lab" -- the nutty-professor scenario. Fleischer may be right. The scientific know-how involved in weaponizing anthrax is formidable, but it is out there, and the technology is available. One of the steps in making a powdered, airy form of anthrax is freeze-drying the spores, along with the anti-caking material, in a lyophilizer, or freeze-drying machine. A new-model tabletop lyophilizer can be bought for less than eight thousand dollars.

"It's very easy to do on a lab scale," Patrick says. "Small production. We're talking about milligram quantities, as opposed to when you expand your process to get industrial-sized production."

It may be that Paul Keim can answer the state-sponsorship-or-lone-terrorist question through DNA sequencing, which might be capable of determining whether the anthrax was part of a small batch (the handgun scenario) or a vast store (the biological equivalent of a nuke).

One of the paradoxes of scientific inquiry in such circumstances, especially in the early stages, is that each answer only poses new questions. Paul Keim's DNA sequencing established that the terror anthrax is natively American -- the Ames strain -- but this knowledge seemed to widen the range of possible suspects rather than narrowing it, because Ames is now so common. Similarly, the discovery that an additive had been applied to anthrax spores would prove little, even if it had turned out to be bentonite, the material identified with the Iraqi program. Bentonite is a common substance with a wide range of uses, both in the laboratory and in household products, including cat litter.