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An Obscure Weapon of the Cold War Edges Into the Limelight

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Science  10 Oct 2003:
Vol. 302, Issue 5643, pp. 222-223
DOI: 10.1126/science.302.5643.222

The tularemia bacterium sickened thousands during World War II and was stockpiled by the superpowers. Now researchers are racing to comprehend this potential bioterror threat

BATH, U.K.—In the winter of 1999 in war-torn Kosovo, scores of people started coming down with headaches and sore throats that hung on much longer than a normal flu. In many cases, the patients' lymph nodes swelled to gigantic proportions and sometimes broke through the skin to form hideous open sores. It took half a year for scientists from the World Health Organization to make a surprising diagnosis: tularemia, a rare bacterial infection most often seen in North American rabbit hunters, villagers in central Sweden, and farmers in southern Russia. Tularemia succumbs readily to antibiotics, and by May the outbreak had subsided after 327 cases, none of which were fatal.

But although the bug was vanquished, a disconcerting question remained. Tularemia had never been spotted before in Kosovo. Had someone deliberately spread the bacterium?

This was not an idle fear. Japan tested the bacterium Francisella tularensis as a potential weapon during World War II, and the United States and the Soviet Union stockpiled it during the Cold War. Although those munitions are supposedly long gone, experts argue that tularemia remains a bioterror threat. It is one of the most infectious organisms known: Inhaling as few as 10 of the microbes can cause debilitating illness. A few grams of a virulent strain of F. tularensis dispersed in a city could quickly sicken thousands, says Anders Sjöstedt of the University of Umeå in Sweden.

For rogue governments and terrorists, tularemia's allure is that of a weapon of mass disruption, not mass murder. This possibility, combined with the bug's checkered past, has made it one of the latest targets of the U.S. government's massive R&D effort to defend against bioweapon attacks. Civilian tularemia research, once a backwater, is now flush with cash, and new researchers are rushing in. The fourth International Conference on Tularemia, held here from 15 to 18 September, drew nearly 200 participants, more than twice as many as previous gatherings.

There are plenty of puzzles for the recent converts to tackle. It's still unknown where the bacterium lives in the wild, how exactly it is transmitted, or even how it makes people sick. At the meeting, however, scientists reported tantalizing clues to the bug's virulence from its nearly finished genome sequence as well as new leads to track it in the environment and after it infects people.

Rabbits and reservoirs

Tularemia was first identified in California in 1911 and was soon recognized as the cause of epidemics in Russia and Scandinavia. In North America, the disease was associated primarily with hunters who skinned and ate infected animals; as hunting has declined, so has the disease. There are still several dozen cases of tularemia in the United States each year, however. One of the highest risk factors—blamed for recurrent outbreaks on Martha's Vineyard island in Massachusetts and for a cluster of cases this summer in Nebraska—is lawn mowing. Unsuspecting yard workers who run over a sick or dead rabbit can unleash tularemia-laden droplets into the air. Two of Germany's three reported cases in 2001 were a father and daughter who had eaten a rabbit the father had hit with his car.

No ordinary gardeners.

Scientists with the U.S. Centers for Disease Control and Prevention attempt to track the source of a tularemia outbreak on Martha's Vineyard, Massachusetts.

CREDIT: RUSSELL ENSCORE/CDC

Fortunately, the bacterium does not spread from person to person, but it has a wide repertoire of ways into the body. In some cases, doctors have attributed infections to tick or mosquito bites, although it is unclear whether the bacterium thrives in such insects. Tainted milk was blamed for an outbreak in Moscow in 1995; other outbreaks have been traced to contaminated wells. Epidemiologists have yet to pinpoint the source of the Martha's Vineyard cases. As for Kosovo, epidemiologists say a terror attack is an unlikely explanation. They now believe that a runaway population of rats and mice gorging on unharvested crops and contaminating human food and water supplies triggered the outbreak.

One of the largest known epidemics sickened tens of thousands of soldiers and civilians during the battle of Stalingrad in the winter of 1942 to 1943. In his 1999 book Biohazard, Ken Alibek, a former top official in the Soviet bioweapons program, alleges that the Soviet army unleashed airborne tularemia bacteria on German troops during the battle, which was a turning point of World War II. But many medical historians doubt that claim, arguing that miserable conditions for the besieged German soldiers, rampant rodents, and contaminated water and food combined for an unprecedented but natural outbreak. “To my knowledge, there is no hard proof for the deliberate spread of these organisms,” says Sjöstedt. The Stalingrad outbreak followed the epidemiological pattern of previous outbreaks in the region, he says: “People were relying on water sources from rivers or lakes, and there were huge numbers of dead rodents.”

Sjöstedt and his colleague Mats Forsman of the Swedish Defense Research Agency in Umeå are hoping to track down the source of the perennial outbreaks in central Sweden; nearly 500 people have fallen ill this year alone. Few Swedish patients report recent contact with rabbits or other small mammals, leading doctors to speculate that mosquitoes may be transmitting the bacterium. In Bath, Forsman noted progress in flushing out Francisella's Swedish retreat. It has been cultured from streams and lakes, but researchers suspect that its reservoir is a water-dwelling host, perhaps a protozoan. Forsman says that F. tularensis indeed grows in lab cultures of amoebae as well as several freshwater species of flagellates and ciliates common in central Sweden. But the team has yet to detect the bacterium in wild microorganisms.

Genetic insights

Researchers have made a bit more progress unraveling Francisella's genetic secrets. A consortium from Sweden, the United States, and the United Kingdom is nearly finished sequencing the genome of two strains: a weakened strain used in an experimental vaccine from the 1960s and a virulent one. Complementing that effort are new techniques for making mutant versions of the finicky organism to assess the importance of various genes. “Having tools to generate mutants makes a huge difference,” says Karen Elkins of the U.S. Food and Drug Administration (FDA) in Rockville, Maryland. “You can knock something out and test whether it still makes an animal sick.”

Researchers are especially intrigued by a possible “pathogenicity island,” a DNA region that looks like it might have found its way into the genome relatively recently. The island includes several genes that appear to have something to do with the bacterium's ability to enter and grow inside other cells, Francis Nano of the University of Victoria in British Columbia, Canada, told those attending the meeting. And Igor Golovliov of Umeå University reported that he and his colleagues at the State Research Center for Applied Microbiology in Obolensk, a former bioweapons lab near Moscow, have modified a version of the live vaccine strain, stripping it of a protein in the region called iglC. The knockout bacteria are not as good at multiplying in cell lines, and they make mice only mildly ill. (For some reason, mice readily succumb to the vaccine strain, which doesn't sicken humans.)

Sordid history.

Francisella tularensis can cause open sores. It was weaponized by Japan, the United States, and the Soviet Union.

CREDITS: (TOP TO BOTTOM) KARI LOUNATMAA/PHOTO RESEARCHERS INC.; PUBLIC HEALTH IMAGE LIBRARY/CDC

One aim of such work is a smarter tularemia vaccine. Several strains that prompt immunity but don't cause disease were isolated in Russia in the 1930s. A mixture of those strains was brought out of the Soviet Union by a researcher with the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) in the 1950s. Scientists at USAMRIID cultured the mixture in the lab and in mice, eventually recovering a single effective vaccine strain that was used to inoculate lab workers and military personnel for decades. But in 2001 the FDA halted use of the vaccine because of concerns about the stability and potency of the vaccine strain; there is currently no vaccine available outside Russia. Knowing how the bacterium causes severe disease would allow researchers to design a more reliable vaccine, says Elkins. “You need to know if the crippled bacteria are going to revert to wild type.” If manufacturers could test a strain for the absence of specific virulence genes, they could reasonably assure a vaccine's safety.

Mining the bacterium's genome should also help speed the development of diagnostic tests. Because tularemia is so rare, it often goes undiagnosed for weeks. “No one thinks Francisella unless you're in central Sweden,” says Elkins. Standard tests involve culturing the bacterium or checking whether a patient has antibodies. But antibody tests work reliably only several weeks into an infection, and because the bacterium is so infectious, few labs are willing or able to handle it. Such tests “are 20 years out of date and are only applied very late in the disease—after doctors have tested for everything else they can think of,” Elkins says. “We now have genomics information that can be used within a day or two” in polymerase chain reaction tests for the bacterium.

Some tularemia experts confess that a disease causing fewer than 50 deaths a year worldwide may not merit all the money and attention it's getting. Nonetheless, the effort to understand tularemia won't go unrewarded, argues Elkins: “There's new biology lurking everywhere”—not to mention a once and possibly future bioweapon threat.

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