From Squirrels to Biological Weapons: The Early History of Tularemia

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FROM SQUIRRELS TO BIOLOGICAL WEAPONS: THE EARLY HISTORY OF TULAREMIA J.V. Hirschmann MD PII: DOI: Reference:

S0002-9629(18)30218-0 10.1016/j.amjms.2018.06.006 AMJMS 685

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The American Journal of the Medical Sciences

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Please cite this article as: J.V. Hirschmann MD , FROM SQUIRRELS TO BIOLOGICAL WEAPONS: THE EARLY HISTORY OF TULAREMIA, The American Journal of the Medical Sciences (2018), doi: 10.1016/j.amjms.2018.06.006

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FROM SQUIRRELS TO BIOLOGICAL WEAPONS: THE EARLY HISTORY OF TULAREMIA

J.V. Hirschmann, MD Emeritus Professor, Department of Medicine, University of Washington, Seattle WA

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Mailing address: 2623 Mt. St. Helens Place,Seattle WA 98144, Telephone: 206 310 3326 Email: [email protected]

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I have no conflict of interest or source of funding for this paper.

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Abstract After George McCoy accidentally discovered a new infection in 1911 while investigating bubonic plague in squirrels, he transmitted the disease to experimental animals and isolated

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the causative organism. He called it Bacterium tularense, after Tulare County, California. In 1919, Edward Francis determined that an infection called “deer-fly fever” was the same disease, naming it “tularemia.” He demonstrated that it occurred in wild rabbits and

inadvertently showed that it was highly infectious, for he and all his laboratory assistants

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contracted the illness. This characteristic led to studies of its potential as a biological weapon, including involuntary human experimentation by Japan among civilian, political and military prisoners, and its probable use in warfare during World War II. Later, in the United States,

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voluntary human experimentation occurred in the 1950s-60s with penitentiary inmates and

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non-combatant soldiers. Soviet Union scientists allegedly developed a vaccine-resistant strain,

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which they tested as a biological weapon in 1982-3.

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Keywords Tularemia; Francisella tularensis; Edward Francis; George McCoy; Human experimentation; Biologic warfare

INTRODUCTION The initial discovery of the infection, tularemia, and the isolation of its causative

organism occurred in the United States early in the 20th Century, and, within a few years, American scientists, sponsored by the government under the auspices of the United States 2

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Public Health Service (USPHS), had delineated the clinical features of the infection, determined its epidemiology, described the mechanisms of its acquisition, and identified the vectors of disease transmission. Later, investigators infected volunteers or unwilling subjects with the tularemia bacterium to evaluate its potential as a biological weapon or to assess vaccines

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devised to protect combatants against its use in warfare. This early history of tularemia helps illuminate the process of scientific discovery, the role of chance in investigations, the benefits of federal sponsorship of biomedical research, the ethics of human experimentation, and the

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use of infectious organisms as weapons. DISCOVERY OF THE DISEASE

The history of tularemia begins with the third pandemic of bubonic plague. The first

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pandemic, the Plague of Justinian, lasted from 541 to approximately 750. The second started in 1346, included the “Black Death” that ravaged Europe from 1346-1353, and ended about

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1750.1 In 1894 the third pandemic began as plague flourished in southern China and spread to Hong Kong.2 In the next few years, the disease, carried aboard ships by rats and their fleas,

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appeared in other Asian countries, including India, Japan, Taiwan, and the Philippines.2(pp17-82) It

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had never occurred before in the Western Hemisphere, but in 1899, cases were reported from ports in Argentina, Brazil, and Paraguay.2(pp133-82) Originating from an Asian country and

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possibly traveling via Honolulu, where it had arrived in 1899,2(183-211) bubonic plague appeared in San Francisco in 1900.2(pp213-41) The first sign was increased numbers of dead rats in Chinatown, followed by human cases in March. A potential consequence of the incursion of this infection into an environment where it was previously unknown was that the plague bacillus would become a permanent member of 3

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the North American ecology if fleas from affected rats transmitted the disease to wild animals. Suggestive evidence for that eventuality came in 1903 when a plague-like disease almost annihilated the population of ground squirrels in Contra Costa County, just east of San Francisco.3 Although the cause of this epizootic was unconfirmed, two men in the area died of

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plague that same year after shooting or eating squirrels. In 1908, another fatal human case occurred there, and cultures from a dead squirrel found nearby grew the plague bacillus.

Studies by William B. Wherry (1874-1936), a physician temporarily assigned to the USPHS,

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demonstrated that four of 432 ground squirrels collected in the county were infected. 4 In 1909, George W. McCoy (1876-1952) began investigating the geographic extent of this disease in squirrels. McCoy, who had joined the USPHS in 1900 immediately after graduating from the University of Pennsylvania Medical School, had met Wherry in the Philippines, where they both

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had become interested in bubonic plague.5 McCoy headed the U.S. Plague Laboratory in San

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Francisco from 1908 to 1911, and by September 1910, his team had shot and autopsied over 150,000 squirrels, discovering the disease in 402 animals from 10 of the 58 California counties. 6

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In this study, McCoy unexpectedly found another disorder, but with no detectable

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pathogen, in 42 squirrels from 9 counties. Like plague, this novel infection caused enlarged lymph nodes, called “buboes” (Greek for “groin,” where they often appear in humans), and

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involved some internal organs, particularly the spleen. Using blood or tissue emulsions from affected squirrels, he transmitted the disease to guinea pigs, rabbits, mice, rats, gophers, and monkeys by rubbing the infected material onto the skin, injecting it subcutaneously, dripping it into their nasal passages, inoculating it into the abdominal cavity, or feeding it to these animals. For them, the illness was usually lethal, but dogs, cats, and pigeons seemed less susceptible. 4

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Bacteremia in sick guinea pigs was intense—injection of even a minute amount of blood (0.00000001 ml) from an afflicted animal into an uninfected one produced the illness.6 Heating infected tissue or fluid to 55-60o C for five minutes killed the organism, which was presumably a bacterium because it did not pass through filters that viruses could transgress. Subcutaneous

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inoculation of guinea pigs with crushed squirrel fleas taken from squirrels or guinea pigs sick or dead from the disease reproduced the disorder. Infected animals were not contagious: illness did not spread directly between suffering and healthy animals when caged together, but

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indirect transmission occurred when fleas, fed on infected animals, bit unaffected ones. In an experiment, for example, healthy squirrels were housed with sick ones. In one group of cages, numerous squirrel fleas were also present, but in the other, they were absent. Even though the

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cages were adjacent, healthy animals developed tularemia only in those that had fleas. 6 In 1912, McCoy and Charles W. Chapin, another USPHS physician, announced that, using

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certain stains, they had detected the causative bacteria microscopically as round or rod-shaped microbes and had cultured them within 3-5 days on an egg-based medium.7 They named the

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organism Bacterium tularense, after Tulare County in California, where the disease was first

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discovered. They determined that the bacteria were Gram-negative and that their virulence diminished after several generations of serial cultures. Guinea pigs that survived cutaneous

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inoculation with a culture were resistant to infection when subsequently injected with spleen from an infected animal, suggesting that immunity had developed. McCoy and Chapin also devised complement fixation and agglutination tests to measure antibodies to the microbe. 8 They found that sheep, but not calves, swine or goats, sickened after receiving subcutaneous injections of spleen tissue from an infected guinea pig. Feeding contaminated food to guinea 5

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pigs caused disease, but an inoculation of fresh feces into guinea pigs did not, presumably because the bacterium was absent from the intestinal tract of infected animals.7 McCoy, who became the nation’s preeminent authority on leprosy, published nothing further on the topic of tularemia, but from 1915-37 he was director of the United States Hygienic Laboratory in

much important research on the disease.5(p36) DEER FLIES, RABBITS, AND HUMAN DISEASE

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Washington, D. C. (re-named the National Institute of Health in 1930), the subsequent site of

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Although six cases of presumed human tularemia were described in 1911,9 the first microbiologically proven human case occurred in Cincinnati, Ohio, in 1913 when a butcher developed ocular inflammation and regional lymphadenitis. When William B. Wherry, who had

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left California to join the University of Cincinnati Medical School,5(p60) injected conjunctival scrapings from the butcher into guinea pigs, the animals died and their tissues grew B.

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tularense when inoculated onto the medium that McCoy and Chapin had employed.10

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In 1919, at the behest of the Utah state health commissioner, the USPHS dispatched Edward Francis (1872-1957) from Washington D.C. to investigate a febrile disease affecting the

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rural population of Millard County in southwest Utah.11 Francis had received his MD from the

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University of Cincinnati in 1897 and joined the USPHS in 1900.5(p17) In “deer fly fever,” the disorder that he was sent to study, the location of the bite from the fly, Crysops discalis, became inflamed, as did the regional lymph nodes, which commonly suppurated and drained through the skin. Other features included fever for 3-6 weeks and marked prostration, often necessitating lengthy bed rest.11

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In August 1919, Francis examined a 52 year-old farmer ill with deer fly fever who had lymphadenitis behind his left ear. He collected both pus from the lymph node and blood from a peripheral vein, each of which, when inoculated into guinea pigs, produced a fatal disease resembling what McCoy had described. Material from the infected animals grew B. tularense.12

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Unfortunately, the patient died after a month-long illness, and at his autopsy Francis obtained tissue without wearing rubber gloves. Five days later, he had the abrupt onset of fatigue,

weakness, and fever, and spent two weeks in the hospital. His pyrexia abated after 24 days, but

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he languished for the next month, largely spent lying around his room. He slowly convalesced over the third month. He had no lymph node enlargement or identifiable entry site. He did not obtain cultures from himself, but antibodies detected months later confirmed a previous infection, presumably this illness. Nearly every week from May 1920 to 1936, he performed

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autopsies on affected animals without gloves because of “a satisfied feeling of immunity.”12 His

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sense of invulnerability was illusive: he incurred four more infections, consisting of finger ulcers and regional lymphadenitis, accompanied twice by short-lived fever and lassitude. He isolated

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B. tularense all three times that he cultured the digital lesions. The mildness of these

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subsequent attacks suggested that the original infection had provoked partial immunity. 12 Francis was not alone in having laboratory-acquired infections. During two years, all five

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of his assistants who dissected or handled infected rodents also became ill and had serologic evidence that B. tularense was the cause.13 One scientist had contact with sick animals in a field station in Utah, but the other four victims worked in the Hygienic Laboratory in Washington, D. C. At the time, that facility employed about 100 personnel, and all of them frequently passed by the area where the animals were inoculated, dissected, and handled after autopsy. Only 7

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those four people who had direct contact with the animals, however, became ill. None had an obvious entry site or lymph node enlargement, and the pattern of their disease was characteristic—sudden high fever and constitutional symptoms, often with musculoskeletal pain, headache, and weight loss. The illness gradually abated after several weeks. No one died,

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but convalescence was slow. Francis discovered that Chapin, who handled infected rodents while working with McCoy, had contracted a similar illness in 1912 that incapacitated him for three weeks. Shortly afterwards, he developed antibodies to B. tularense.13

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In 1921 Francis reported seven cases of deer fly fever from the previous year and

dubbed the disease “tularaemia,” a combination of tularense from the California county name and aemia, Greek for “blood,” to indicate bacteremia, detected in two patients.14 Because

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jackrabbits were concurrently dying in large numbers, Francis suspected that they too had the disease, and from May to June 1920, 556 were shot in the wild, with B. tularense isolated from

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17. He also captured female adult deer flies on horses near where human cases had earlier occurred.15 Using guinea pigs and rabbits shipped from Washington, D. C., he allowed the flies

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to feed on experimental animals infected with B. tularense that he had isolated from four

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human cases and then to feed on healthy animals. His studies showed that the flies could transmit the disease by a single bite, and death in the rabbits and guinea pigs occurred four to

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seven days later. The deer flies remained infective as long as two weeks after feeding on the sick animals.

In November 1921 a meat merchant in Washington, D. C., developed pyrexia, an ulcerated finger, and axillary lymphadenitis.16 He self-diagnosed “rabbit fever,” a condition widely recognized in his trade, which included selling rabbit meat to the public. Francis 8

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serologically confirmed the illness as tularemia. In January 1923 the meat merchant extracted 914 livers from the wild cottontail rabbits that he sold and took them to Francis, who found tularemia in seven. Two animals had come from Tennessee, indicating that this disorder was present not only in the West and Midwest, but also in the Eastern United States. 16

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This was not the first reported case of human disease acquired from lagomorphs (hares and rabbits), however. In 1837, Homma Soken (1804-72), a Japanese court physician, described a similar illness that developed after eating hares and that caused chills, fever, and

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lymphadenitis.17 In 1924, Dr. Hachiro Ohara (1882-1943) diagnosed this disorder in a family several days after they had skinned, cooked, and eaten a dead hare. He autopsied other dead hares, which his brother had found nearby, and removed their hearts, which he rubbed on the left hand of his accommodating wife. Two days later, she developed a headache, and on day

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four, she had chills, fever, and the onset of gradually enlarging left axillary lymph nodes, which,

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when excised, grew an unidentified bacterium. Wondering if this disease was tularemia, in 1925 Ohara sent blood from five affected patients to Francis, who confirmed the diagnosis

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serologically.17 That year, Ohara also sent an excised lymph node, which, when Francis injected

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its contents into guinea pigs, killed the animals. Cultures from the dead animals yielded B. tularense.18 Francis called the disorder Ohara’s disease, while Ohara called it yato-byo, which

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means “wild rabbit disease” in Japanese.17 ANOTHER VECTOR In 1921, the USPHS hired Ralph Parker (1889-1949), an entomologist, to establish a field station in an abandoned schoolhouse in Hamilton, Montana, in order to study Rocky Mountain spotted fever (RMSF), a tick-borne 9

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infection causing serious, often lethal, illness among people in the Bitterroot Valley.5(p41) He and Roscoe Spencer (1888-1982), a USPHS physician who had graduated from Johns Hopkins Medical School in 1913,5(p51) routinely collected wood ticks (Dermacentor andersoni) by dragging a cloth made of outing-flannel (a soft fabric with thread projections on both sides)

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over vegetation in known infested areas and allowed them to feed on healthy guinea pigs in order to determine how often the ticks were infected with RMSF.19 In May 1923 they

unexpectedly discovered that some of the animals had developed tularemia instead and

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realized that ticks could be infected with B. tularense in their native habitat. When an engorged female tick deposits her eggs, the hatching larvae feed that season, transform into nymphs the next year, and into adults the following one. When tick larvae were allowed to feed on experimental animals with tularemia, the investigators found that the ticks remained infective

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during their next two stages and could transmit the disease both as nymphs and adults to

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healthy animals, including guinea pigs, snowshoe rabbits, and woodchucks. They suggested that the ticks might both maintain and transmit tularemia in their natural habitat because they

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normally feed on wild rabbits and woodchucks.19 They also hypothesized that bites by infected

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ticks might cause disease in humans, a conjecture that Francis confirmed serologically in a patient from Montana who sustained a tick bite in 1924.20

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Despite using careful technique, both investigators also contracted the illness in 1924.21 Spencer became sick on July 4 with malaise, weakness, and abdominal pain. Two days later, he developed pyrexia and headache that persisted for the next five days, after which he was hospitalized for suspected typhoid fever. Following several days of rest, he returned to work on July 28 and gradually convalesced. He felt that he had completely recovered by October, as 10

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evidenced by his winning a tennis tournament. Parker became ill on October 2 with headache, weakness, lumbar pains, chills, sweating, and fever. He remained febrile, but from October 1025 his worst symptoms, primarily musculoskeletal discomfort, occurred, especially in the back and in various joints, where he experienced migratory arthralgias. From October 10-15, he had

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a rash that first appeared behind his ears and spread to the back of his neck, face, forehead, and arms. It consisted of red papules, followed by peeling at the sites of the eruption. The published report includes three photographs of a balding and pudgy Parker lying in bed with

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the rash visible on both his face and, especially, his neck.21 He returned to part-time work on November 22, but, because he suffered from weakness and nervousness, he did not resume his full schedule until December 15. Spencer and Parker were not the only ones who became sick: four of their six laboratory assistants who handled infected ticks or helped perform autopsies

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on diseased guinea pigs and rabbits also developed tularemia. Based on strong circumstantial

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evidence, it seemed likely that three of the workers had acquired the infection by inhaling organisms from affected animals. As with Francis’ laboratory-acquired cases, neither the chief

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their illnesses.21

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investigators nor their assistants had any evident entry sites or enlarged lymph nodes during

CLINICAL FEATURES AND EPIDEMIOLOGY

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In 1928, Francis summarized 679 cases of tularemia, providing clinical details in 565.22 The major sources of infection were rabbits in over 400 patients and ticks in at least 70. The incubation period, clearly discernible in 259 victims, ranged from 1-10 days, with an average of 3.5. The onset was generally dramatic, with fever, chills, muscle aches, sweating, headache, vomiting, and prostration. Rashes, such as Parker had, were uncommon, occurring in about 5% 11

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of cases. Francis classified tularemia into four clinical types. The most frequent was ulceroglandular, present in 480 cases (85%), in which a papule appeared at the exposure site and then ulcerated. Regional lymph nodes (“glands”) enlarged, became tender, and about half of them ruptured through the skin, draining pus. Sometimes, linear nodules appeared along

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proximal lymphatics (“nodular lymphangitis”). The second type, occurring in 32 patients (6%), was oculoglandular, in which the entry site was the eye. Lids swelled, conjunctival ulcers

formed, and regional lymph nodes became inflamed. A third type was glandular, present in 25

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cases (4%) and characterized by lymphadenitis, but without cutaneous lesions. The fourth was typhoidal, present in 28 patients (5%), in which general symptoms, such as fever, chills, and myalgias, occurred in the absence of cutaneous, ocular, or lymphatic abnormalities. Because of Francis’ extensive investigations of tularemia, a proposal in 1959 recommended that the

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causative organism, initially labeled by McCoy and Chapin as Bacterium tularense, but later

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classified as Pasteurella tularensis, be renamed Francisella tularensis.23 Microbiologists subsequently adopted this suggestion, and that nomenclature persists.

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Francis’ classification of clinical tularemia also persists, but with two later additions.24

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Oropharyngeal tularemia comprises throat pain and ulceration, combined with cervical lymphadenitis. It occurs from consuming contaminated water or food of by inhaling aerosols

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from infected animals or tainted water. The final type is pneumonia, the primary form of which develops from inspiring the organism directly into the lungs from infected mammals or from environments, such as hay, soil, or water, contaminated by affected animals. “Secondary pneumonia” occurs when bacteria originating in a focal site of infection enter the circulation and travel to the lungs. 12

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When Francis wrote his 1928 review article, tularemia had been found only in the United States and Japan, but it was reported in Russia that year, in Norway in 1929, in Canada in 1930, in Sweden in 1931, and in Austria in 1935.25 Retrospective studies, however, suggest that what was probably tularemia occurred in Russians in the 18 th Century,26 and in lemmings in

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Norway in 1653.27 Since then, reports have documented tularemia in many countries, almost exclusively in the Northern Hemisphere, and F. tularensis has been isolated from numerous species of mammals, birds, reptiles, amphibians, fish, and invertebrates.28

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Tularemia can increase in times of armed conflict. Large outbreaks occurred in the Soviet Union during World War II, when uncultivated lands, delayed harvests, and destruction of grain storage facilities led to a marked increase in rodents. The high incidence in Red Army soldiers during the Battle of Stalingrad from 1942-3 apparently arose when these animals

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contaminated food, drinking water and hay used for bedding.29

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In Francis’ review of 679 cases in 1928, the mortality rate was only about 4%, but the morbidity was substantial.22 In general, fever lasted for a few weeks, patients were weak during

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the second month, and they slowly recuperated during the third month. No effective therapy

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was available until 1932, when Lee Foshay, a Cincinnati physician, introduced an antiserum. 30 By 1940 he had treated 600 patients with this product, elicited by repeated subcutaneous

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inoculations of formaldehyde-killed organisms into a horse or goat. Systemic symptoms and lymphadenitis subsided much more rapidly than in untreated patients, but about 50% developed serum sickness, severe in 16%, with symptoms similar to, and sometimes worse than, tularemia itself, including fever, headache, backache, and lymphadenitis.31

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Streptomycin, discovered in 1943, was first used in tularemia in 1944. 32 Treated patients had a dramatic and rapid response, with marked improvement within 24 hours. The course of therapy was 7-10 days of twice-daily intramuscular injections. In 1949, investigators found that an oral antibiotic, tetracycline, was effective, but subsequent experience indicated

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that it required a more protracted regimen than streptomycin to avert relapses. 33 Several newer antimicrobial agents are also efficacious in treating tularemia.24 EXPERIMENTS IN VOLUNTEERS

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The cases of laboratory-acquired tularemia suggested that only a small number of organisms was necessary to cause infection. Human studies confirmed this hypothesis in

untreated control subjects participating in trials funded by the United States Army, which was seeking a vaccine to defend combatants from potential exposure to F. tularensis during

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biological warfare. In 1956, investigators produced ulceroglandular tularemia by injecting just

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10 bacteria into the forearms of inmate volunteers from the Ohio State Penitentiary. 34 When re-challenged with the same inoculum two or eight months later, all developed skin ulcers and

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lymph node enlargement, but few had fever and other systemic symptoms, confirming Francis’

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hypothesis that a primary infection could induce partial immunity. In 1958, the same researchers, again using prisoners, demonstrated that fever, headache, myalgias, and chest

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pain developed 4-7 days after inhaling 10-52 bacteria. Illness was usually typhoidal, but pneumonia could occur.35 Other studies, enlisting volunteers from the Maryland House of Correction or soldiers who were conscientious objectors, demonstrated that inhaling anywhere from 200 to 10 8 bacteria generally caused typhoidal tularemia, but sometimes pneumonia.36,37 Ingesting 14

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encapsulated organisms, however, required 108 microbes to induce disease, again usually typhoidal.38 In these studies, ill subjects received antibiotic therapy, and investigators reported no deaths or serious long-term complications. The soldiers, whose religious beliefs prohibited combat, were participating in “Project Whitecoat,” a program from 1954-73 in which about

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2300 conscientious objectors, more than 90% of them Seventh Day Adventists, worked in

various positions, such as laboratory technicians, hospital corpsmen, and administrators, at Fort Detrick, Maryland.39 Periodically, they were invited to participate in studies involving organisms

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that had the potential for biological warfare, including those causing Eastern, Western, and Venezuelan equine encephalitis, typhoid fever, Q fever, RMSF, Rift Valley fever, and tularemia.40 Fortunately, they seemed to suffer no long-term health effects, based on a retrospective study in 2005.39

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EXPERIMENTS WITH TULAREMIA AS A BIOLOGICAL WEAPON

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The U. S. Army had established its center for biological warfare research in Fort Detrick in 1943, where it began testing F. tularensis, among other organisms.41 From 1944-59

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tularemia inadvertently developed in 104 vaccinated laboratory personnel, many caused by

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strains engineered to make them streptomycin-resistant.42,43 Human experimentation began there in 1955 and entailed exposing civilian and military volunteers to aerosolized F. tularensis

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in a spherical chamber known as the “eight ball,”44 where bombs containing organisms were detonated and their contents dispersed by fans to distribute them evenly into the ambient air. A 1958 report indicated that a wet suspension of F. tularensis was a feasible retaliatory weapon if the United States became a target of germ warfare, and the following year another study

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indicated that aerial spraying of this material would cause more than 50% casualties over an area of 16 square miles.45 In 1948, British investigators dispersed F. tularensis above the sea near the Caribbean

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islands of Antigua, Nevis, and St. Kitts to determine if it could infect experimental animals at various distances from the dissemination site.40(pp120-6) In 1964, Americans conducted similar tests off Johnston Atoll, about 800 miles south of Honolulu, where aircraft traveling at nearly supersonic speed released F. tularensis. In the largest investigation, a 32-mile swath of bacteria

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traveled over 60 miles downwind before becoming non-infective.40(pp202-4) In 1969 and 1970, President Richard Nixon’s executive orders unilaterally prohibited biological weapons, confining future research to biological defense, such as vaccines.40(pp207-8) In 1972 more than 100

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countries signed a treaty that banned the development, possessing, and stockpiling of

peaceful purposes.”44

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pathogens in quantities that could not be justified for “prophylactic, protective or other

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Scientists in the Soviet Union, a treaty signatory, allegedly violated it. They produced an aerosolized, vaccine-resistant F. tularensis, mixed it with additives to maintain virulence, and

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packed it into small metal balls designed to explode several miles upwind from a target.

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Outdoor studies conducted from 1982-3 on an island in the Aral Sea showed that the vaccineresistant preparation was very effective in killing caged, vaccinated monkeys.46

Earlier, Japan had used F. tularensis against soldiers and citizens. Although the 1925 Geneva Protocol had forbidden biological weapons, many of its signatories, including Japan,

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conducted secret research. Shiro Ishii (1892-1959), a physician and microbiologist, felt that biological warfare could help Japan achieve its territorial ambitions.47 Under Ishii’s supervision, the Japanese established centers in Manchuria, elsewhere in China, and in other conquered Asian countries to conduct studies on chemical and biological warfare, which involved several

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organisms, including F. tularensis. One component of his investigations was to infect military, civilian, and political prisoners—mostly Chinese, Koreans, Russians, and Mongolians—with these agents to determine their lethality and the prophylactic benefit of 18 investigational

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vaccines. One trial involved subcutaneous injection of F. tularensis.48

Prisoners who survived these experiments were usually executed. Investigators vivisected some, often without anesthesia, to study their organs as the infections progressed. The largest, most notorious research site was Unit 731, officially named the Army Epidemic

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Prevention Research Laboratory and established in 1932 near the city of Harbin in central

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Manchuria. It employed over 1000 Japanese personnel, many of them prominent physicians and scientists from Japan’s finest universities. The facility held up to 400 prisoners, sometimes

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abducted from the streets of nearby towns to provide the necessary experimental subjects. In

gases.47

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a field north of Harbin, prisoners tied to posts were exposed to aerosolized germs or poisonous

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Investigators also targeted towns and cities to determine the best methods of dispersing

pathogens. Plague-infected fleas, for example, were dropped in ceramic bombs that easily cracked on impact, releasing the fleas, along with rice and wheat, included to attract rats on which the fleas could feed and then spread infection to humans. After these experiments, large plague epidemics occurred in several cities in 1940. An estimated 580,000 people died from 17

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Japanese biological warfare, with about 20,000 perishing from laboratory experiments. More than 20,000 physicians and scientists participated in this research.49 How often F. tularensis was employed and its efficacy as a biological weapon are unknown because the Japanese destroyed most of their records when World War II ended. In a 1970 publication, however, a World Health

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Organization expert committee calculated its potential in warfare by estimating that 50

kilograms of dried F. tularensis released over a city of 5 million inhabitants in an economically developed country would cause disease in about 250,000 people, 50,000 would require

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hospitalization, and approximately 4500 would die.50 SERENDIPITY IN THE HISTORY OF TULAREMIA

One of the salient features of the early history of tularemia is the importance of serendipity in scientific discovery. The word “serendipity” is a neologism of Horace Walpole

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(1717-97), Whig politician, man of letters, art historian, author of the first Gothic novel (The

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Castle of Otranto) in 1764, and son of the first British Prime Minister, Robert Walpole. In a letter dated January 28, 1754 and addressed to his friend Horace Mann (1706-86), British

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minister to the Court of Florence, he confirmed the arrival of the portrait of Grand Duchess

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Bianca Capello by Giorgio Vasari (1511-74) that Mann had sent to him.51 Walpole described how he unexpectedly found “a critical discovery” about the Capello coats of arms in an old

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Venetian book on heraldry. He said that he had a propensity for such lucky revelations, one kind of which he labeled “serendipity.” He derived the word from a “silly fairy tale, called The Three Princes of Serendip” (Serendip was the name of the country later named Ceylon and now called Sri Lanka). As the princes traveled, “they were always making discoveries, by accidents and sagacity, of things which they were not in quest of.” Serendipity is an “accidental 18

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sagacity,” he explained: “no discovery of a thing you are looking for comes under this description.”51 In 18th Century England, “sagacity” meant “an acute sense of smell” as well as an “acuteness of discovery,” according to Samuel Johnson’s Dictionary, published in 1755, 52 suggesting that Walpole’s definition emphasized not only an unexpected revelation, but also

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the ability to discern its significance. Robert K. Merton (1910-2003), a sociologist who traced the history and use of the word, asserted that serendipity in science “refers to the fairly

common experience of observing an unanticipated, anomalous and strategic datum which

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becomes the occasion for developing a new theory or extending an existing theory.”53 By “strategic” he meant the observer’s ability to “detect the universal in the particular.”

George McCoy found a new disease while searching for plague in squirrels. He might have disregarded his unexpected findings, dismissed them as “atypical plague” in which the

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causative bacillus was not visible or viable, or relegated the task of investigating this issue to

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other researchers because his designated responsibility was to detect plague in squirrels. Yet, he discerned the importance of his discovery and had the curiosity and determination to pursue

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its elucidation. Edward Francis detected the same illness when investigating an apparently

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unrelated infection, deer-fly fever, and possessed the insight to hypothesize that concurrent deaths in jackrabbits were a related phenomenon, rather than a coincidence. Spencer and

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Parker unintentionally found the disease in ticks while looking for RMSF. All these investigators had the wisdom to recognize the potential importance of their unanticipated revelations, diligently conducted extensive laboratory and animal studies to understand their findings, and, consequently, developed new theories of the disease, as Merton’s definition of scientific

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serendipity requires. THE USPHS INVOLVEMENT IN TULAREMIA RESEARCH The early history of tularemia is also a tribute to the courage of its investigators.

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Because of the high infectivity of F. tularensis, their research entailed the substantial personal risk of acquiring tularemia, which, though seldom lethal, was a lengthy, debilitating illness, without any safe and effective treatment until 1944. Unfortunately, one scientist in the USPHS died of laboratory-acquired tularemia.54

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Indeed, most of the early investigators of tularemia were members of the USPHS.

Founded in 1798 through a bill signed by President John Adams, its initial responsibility was to care for sick or disabled seaman in hospitals or other institutions.54(p14) Originally called the

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Marine Hospital Service, its duties broadened over the years to include quarantining sick travelers, screening immigrants for medical conditions, collaborating with state health

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departments, investigating epidemics, and manufacturing vaccines and antitoxins. Its name

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changed to the United States Public Health Service in 1912.54(p58) Its Hygienic Laboratory opened on Staten Island in 1887 and in 1891 moved to Washington D. C., with new quarters

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established in 1904. Its founder and director, Joseph Kinyoun (1860-1919), had been sent to

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San Francisco in 1899 in anticipation of the arrival of bubonic plague from Asia, 54(p35-40) and George McCoy became head of the US Plague Laboratory there in 1908.5(p31) William B. Wherry, who discovered plague in California ground squirrels, had been temporarily assigned to the USPHS as a bacteriologist in Oakland.5(p60) Edward Francis did his research in the Hygienic Laboratory, where his modest quarters became the major center for tularemia studies. When he began his work, 14 human cases were known, but by four years later 800 had been 20

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diagnosed, most of which he had confirmed himself.18 He became the pre-eminent expert on tularemia, and a later investigator stated that “the one man more responsible for unraveling the intricacies and many manifestations of tularemia than any and all others combined was Dr. Edward Francis.”18 Roscoe Spencer and Ralph Parker were also members of the USPHS. They

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worked together in the Hamilton, Montana facility, which later became the Rocky Mountain Laboratory, now operated by the National Institutes of Health and devoted to the investigation of infectious diseases. Parker stayed there until his death in 1949.5(p41) Spencer left in 1928, but

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remained a government scientist and became assistant chief of the National Cancer Institute in 1937, then chief from 1943 until his retirement in 1947.5(p51) Thus, the early history of tularemia is a tribute to the USPHS specifically and more generally to the contributions of the United States government in facilitating important scientific discoveries.

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ETHICAL ISSUES WITH TULAREMIA RESEARCH

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The saga of tularemia also highlights the vacillations in ethical perspectives. Medical experimentation in American prisons dates back at least to the beginning of the 20 th Century

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and included, among many others, the famous investigations of Joseph Goldberger (1874-

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1929), another member of the USPHS, who demonstrated the cause of pellagra by feeding a nutritionally deficient diet to inmates who volunteered to participate in exchange for receiving

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pardons of their crimes.55 Studies on prison volunteers became more common during World War II and included experiments with sleeping sickness, malaria, gonorrhea, and gas gangrene. Despite the Nuremberg Code that emerged from trials of Nazi doctors in 1947 and dealt with the ethics of human experimentation, studies on prisoners remained largely acceptable in the 1950s and 1960s and included testing of novel pharmaceuticals. Experiments on incarcerated 21

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subjects, however, began to receive widespread condemnation in the 1970s, and in 1980 the Food and Drug Administration (FDA) banned clinical phase I studies of new drugs in prisons. 56 The underlying ethical rationale for discontinuing experiments on inmates was that completely informed consent is impossible to obtain and participation is inherently coercive. Some recent

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guidelines, however, have supported such trials, albeit cautiously and with many constraints, partly based on the concept that the prisoners might benefit directly or indirectly as individuals or as a group by studies on medical problems common in their population.56

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TULAREMIA IN BIOLOGICAL WARFARE

Another issue that emerged from the early history of tularemia was the potential use of F. tularensis as a weapon. Biological warfare goes back at least to 300 BCE when Greeks polluted their enemies’ wells and drinking water supplies with the bodies of dead animals.41

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Later, Romans and Persians employed the same tactic. In 1155 at a battle in Tortona, Italy, the

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Holy Roman Emperor Frederick I, also known as Frederick Barbarossa, used the bodies of both dead soldiers and dead animals to pollute the wells of their Norman enemies.41 In 1346, during

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the second pandemic of bubonic plague, a Mongol army hurled corpses of their plague-stricken

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warriors over the walls of Kaffa, a seaport on the Crimean coast.57 During the battle of Carolstein in 1422, Lithuanian troops catapulted the bodies of their slain soldiers plus 200

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cartloads of excrement over the castle walls onto the ranks of the defenders, in whom deadly fevers soon developed. In 1495, Cesalpino, an Italian physician, gave wine contaminated with the blood of lepers to French forces during the Naples Campaign.58 In 1763 during the French and Indian War, the British commander Jeffrey Amherst suggested giving blankets contaminated with smallpox to “Inoculate the Indians…to Extirpate this Execrable Race.” 41 22

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These examples indicate the longstanding predilection of warring armies to employ infections as weapons during combat, sometimes with the complicity of physicians. Given the high infectivity of F. tularensis, it is unsurprising that Japanese, Russian, British and American scientists studied it for its potential in biological warfare.

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Concern about biological weapons intensified during the early 21 st Century with the anthrax attacks in 2001, when several news media outlets and two Democratic Senators

received spores of Bacillus anthracis in postal letters. That year, a Working Group on Civilian

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Biodefense, which had formed two years earlier, published a paper on the use of tularemia as a biological weapon, much of it based on insights developed from the studies discussed above.59 It suggested that aerosol release of F. tularensis in a densely populated area would cause the acute onset of a febrile illness 3-5 days later, with lung involvement developing in many cases.

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Because person-person transmission of the disease has not been reported, isolation of infected

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people would be unnecessary. Furthermore, tularemia would have a slower progression and a lower fatality rate than inhalational plague or anthrax, two other potential biological weapons.

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Although F. tularensis can survive for weeks at low temperatures in water, moist soil, hay,

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straw, and decaying animal carcasses, the likelihood of secondary dispersal seemed low because desiccation, solar radiation, oxidation, and other environmental factors would

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substantially reduce its viability. CONCLUSION

This paper documents how an infection came to be discovered and characterized.

George McCoy’s investigation into the prevalence in bubonic plague in ground squirrels led to the serendipitous detection of a new disease. Edward Francis unexpectedly found that “deer 23

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fly fever” in Utah was the same infection, and Spencer and Parker, studying RSMF in Montana, surprisingly detected the pathogen in ticks, identifying another vector of disease transmission. These researchers were indeed lucky, but they explored their unanticipated discoveries methodically and meticulously. What deserves special emphasis is their heavy dependence on

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the use of experimental animals, primarily guinea pigs, which were extraordinarily susceptible to this infection and usually died from it. The guinea pigs had their skin and peritoneum

injected and their cutaneous surfaces abraded to determine whether tissue and fluid from

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ground squirrels, deer flies, rabbits, woodchucks, and ticks contained the organism. Fleas, deer flies, and ticks fed on them to assess whether these were vectors of disease transmission. To determine what other animals were susceptible, investigators inoculated them with tissue or body fluids from sick or dead guinea pigs. McCoy and Chapin determined the thermal death

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point of the organism by injecting material heated at increasing temperatures into guinea pigs

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until they found the level at which the infected tissue no longer caused disease. All the investigators kept the microbe viable for subsequent experimentation by inoculating material

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from ill guinea pigs into successive groups of healthy ones, which then became ill and were the

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source of material to inject into the next generation. The scientists were systematic, determined, and persistent in other ways, as well.

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McCoy and Chapin tried to isolate the organism on numerous culture media before attaining success with an egg-based one. They also developed the serologic tests that later investigators adopted and were critical in detecting the infection and determining the immunologic response to it. Francis collected the available information from diagnosed cases to define the clinical characteristics of the disorder, including such important issues as the incubation period, risk 24

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factors, clinical manifestations, categories of disease, course of illness, and the mortality rate. He performed serologic tests on suspected cases, studied the frequency of tularemia in rabbits sold in a Washington, D. C., meat market, and tested various culture media on which to grow the organism.

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Many of those who discovered other infections during the late 19th and in early 20th Century used a similar paradigm to elucidate the disorders. It consisted of some or all of these elements: 1) animal experimentation to demonstrate the organism’s pathogenicity, establish an

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animal model, and delineate the extent of the infection’s presence in nature, its transmissibility to other animals, the method of its acquisition, and the vectors that spread it; 2) injection of successive groups of animals with infected material to keep the microbe viable for several generations and available for further experimentation; 3) techniques to visualize and culture

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the organism; 4) observations of patients to detail the spectrum of its clinical manifestations;

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and 5) serologic studies to simplify the diagnosis and to determine immune responses to the pathogen. For example, in 1892 an Argentine medical student, Alejandro Posadas, found

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organisms that resembled protozoa in a biopsy from a man with disfiguring skin lesions and

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reproduced the disease by injecting the tissue into several mammals, including a dog, cat, and monkey.60 Later investigators also employed inoculations of this pathogen into experimental

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animals, which determined that it was a fungus with two forms, depending upon the environment in which it grew. It was isolated onto culture media, and it was named Coccidioides (“resembling coccidia,” a protozoan) immitis (“merciless,” because the initial cases were lethal). Systematic studies by Charles Smith in the 1930s in the San Joaquin Valley of California defined the epidemiologic, serologic, and clinical characteristics of the infection. 25

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Another example occurred in 1935 when an outbreak of a febrile disease among abattoir workers in Brisbane, Australia, led E. H. Derrick to inoculate the urine and blood of the sick patients into guinea pigs, rats, rabbits, and mice, which successfully transmitted the infection, usually without killing the animals.61 He showed that they developed immunity after

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being infected, for they did not get ill when re-inoculated weeks later with the material that had previously sickened them. He tried to discover the natural mode of transmission by

feeding healthy animals with tissue from infected animals, rubbing it onto intact and abraded

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skin, and instilling it into the conjunctival sac, all without success. He also showed that guinea pig lice did not transmit the infection. Later studies demonstrated that the main mode of acquisition was through inhaling the organism.62 Derrick named the infection Q fever (the Q for “query” because he did not know the cause), and, using the information from nine patients, he

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deduced its incubation period, disease course, and epidemiology. His Australian colleague, F.

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Macfarlane Burnet, visualized the organism as coccobacilli in mice inoculated with material that Derrick had sent to him,63 and the microbe, later identified as a bacterium, was eventually

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named Coxiella burnetii.64

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The history of the discovery and subsequent exploration of tularemia, as with that of C. immitis and C. burnetii, illustrates how industrious, ingenious and lucky researchers can be in

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their investigative enterprises. Unfortunately, however, the potential use of F. tularensis in warfare lured physicians and other scientists to conduct involuntary human experimentation, test tularemia as a biological weapon, and employ it against defenseless citizens. Thus, the history of this disease displays both the grandeur and the potential debasement of science: in the discovery and elucidation of this infection, scientists were at their very best, but in the 26

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depraved Japanese human experimentation on involuntary subjects they were at their very

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worst.

REFERENCES

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1. Zietz BP, Dunkelberg H. The history of the plague and the research on the causative agent Yersinia pestis. Int J Environ Health 2004;207:165-178. 2. Echenberg M. Plague ports. The global impact of bubonic plague, 1894-1901. New York: New

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York University Press, 2007: 5. 3. McCoy GW. Plague among ground squirrels in America. J Hyg 1910;10: 589-601.

4. Wherry WB. Plague among ground squirrels of California, J Infect Dis 1908; 5:485-506.

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5. Barry J. Notable contributions to medical research by Public Health Service scientists. A biobibliography to 1940, Washington, D. C.: Department of Health, Education and Welfare. Public Health Service, Public Health Service Publication No. 752, 1960; 36, 60.

M

6. McCoy GW. A plague-like disease of rodents, Public Health Bull 1911; 45: 53-71.

ED

7. McCoy GW, Chapin CW. Further observations on a plague-like disease of rodents with a preliminary note on the causative agent, Bacterium tularense, J Infect Dis 1912;10: 61-72.

PT

8. McCoy GW, Chapin CW. V. Bacterium tularense the cause of a plague-like disease of rodents.

CE

Public Health Bull 1912;53: 17-23.

AC

9. Francis E. A summary of the present knowledge of tularaemia, Medicine 1928;7: 411-432. 10. Wherry WB, Lamb BH. Infection of man with Bacterium tularense, J Infect Dis 1914; 15: 331-340.

11. Francis E. Deer-fly fever, or Pahvant Valley plague, Public Health Rep 1919; 34: 2061-2062. 12. Francis E. Immunity in tularemia, Trans Assoc Amer Physicians 1936; 51: 394-398. 28

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13. Francis E. Tularaemia. Francis 1921. VII. Six cases of tularaemia occurring in laboratory workers, Public Health Rep 1922;37: 392-413. 14. Francis E. Tularaemia Francis 1921. I. The occurrence of tularaemia in nature as a disease of

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man, Public Health Rep 1921;36: 1731-1738. 15. Francis E, Mayne B. Tularaemia. Francis 1921. II. Experimental transmission of tularaemia by flies of the species Chrysops discalis, Public Health Rep 1921;36: 1738-1753.

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16. Francis E. Tularaemia. IX. Tularemia in the Washington (D.C.) Market, Public Health Rep 1923;38: 1391-1404.

17. Ohara S, Studies on Yato-byo (Ohara’s Disease, Tularemia in Japan), Report 1,” Jpn J Exp

M

Med 1954;24:69-79.

18. Jellison W. Tularemia. Dr. Edward Francis and his first 23 isolates of Francisella tularensis,

ED

Bull Hist Med 1972;46: 477-485.

PT

19. Parker RR, Spencer RR, Francis E. Tularaemia. XI. Tularaemia infection in ticks of the species

1073.

CE

Dermacentor andersoni Stiles in the Bitterroot Valley, Mont. Public Health Rep 1924;39: 1057-

AC

20. Francis E. Tularemia. JAMA 1925;84: 1243-1250. 21. Parker RR, Spencer RR. Six additional cases of laboratory infection of tularaemia in Man. Public Health Rep 1926;41: 1341-1355. 22. Francis E. Symptoms, diagnosis and pathology of tularemia, JAMA 1928;91: 1151-1161.

29

ACCEPTED MANUSCRIPT

23. Evans ME, Gregory DW, Shaffner W, McGee Z. Tularemia: A 30-year experience with 88 cases. Medicine 1985;64: 251-269. 24. Thomas LD, Shaffner W. Tularemia pneumonia. Infect Dis Clin North Amer 2010;24: 43-55.

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25. Francis E. Sources of infection and seasonal incidence of tularaemia in man. Public Health Rep 1937;52: 103-113.

26. Pollitzer R. History and incidence of tularemia in the Soviet Union. A review. Bronx, NY:

AN US

Fordham University Press, 1967, p. 2.

27. Sjöstedt A. Tularemia: History, epidemiology, pathogen, physiology, and clinical manifestations. Ann NY Acad Sci 2007;1105: 1-29.

M

28. Keim P, Johansson A, Wagner DM. Molecular epidemiology, evolution, and ecology of

ED

Francisella. Ann NY Acad Sci 2007;1105: 30-66.

29. Croddy E, Krcalova S. Tularemia, biological warfare, and the Battle for Stalingrad (1942-

PT

1943). Milit Med 2001;166: 837-838.

CE

30. Foshay L. Serum treatment of tularemia. JAMA 1932;98: 552. 31. Foshay L. Tularemia: A summary of certain aspects of the disease including methods of early

AC

diagnosis and the results of serum treatment in 600 patients,” Medicine 1949;19: 1-83. 32. Foshay L, Pasternack AB. Streptomycin treatment of tularemia. JAMA 1946;130: 393-398. 33. Woodward T, Raby WT, Eppes W, Hightower JA. Aureomycin in treatment of experimental and human tularemia. JAMA 1949;139: 830-832.

30

ACCEPTED MANUSCRIPT

34. Saslaw S, Eigelsbach HT, Wilson HE, Prior JA, Carhart S. Tularemia Vaccine Study. I. Intracutaneous challenge. Arch Internal Med 1961;107: 689-701. 35. Saslaw S, Eigelsbach H, Prior JA, Wilson HE, Carhart S. Tularemia Vaccine Study. II.

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Respiratory challenge. Arch Internal Med 1961;107: 702-714. 36. Sawyer W, Dangerfield HG, Hogge AL, Crozier D. Antibiotic prophylaxis and therapy of airborne tularemia. Bacteriol Rev 1966;30: 542-548.

AN US

37. McCrumb Jr FR. Aerosol infection of man with Pasteurella tularensis. Bacteriol Rev 1961;25: 262-267.

38. Hornick RB, Dawkins AT, Eigelsbach HT, Tulis JJ. Oral tularemia vaccine in man. Antimicrob

M

Agents Chemother (Bethesda). 1966;6: 11-14.

39. Pittman PR, Norris SL, Coonan KM, McKee Jr KT. An assessment of health status among

ED

medical research volunteers who served in the Project Whitecoat Program at Fort Detrick,

PT

Maryland. Milit Med 2005;170: 183-187.

CE

40. Regis E. The biology of doom. New York: Henry Holt and Co. 1999: 168. 41. Poupard JA, Miller LA. History of biological warfare: Catapults to capsomeres. Ann NY Acad

AC

Sci 1992;666: 9-20.

42. Van Metre TE, Kadull PJ. Laboratory-acquired tularemia in vaccinated individuals: A report of 62 cases. Ann Internal Med 1959;50: 621-632.

31

ACCEPTED MANUSCRIPT

43. Overholt EL, Tigertt WD, Kadull PJ, Ward MK, Charkes ND, Rene RM, Salzman TE, Stephens M. An analysis of forty-two cases of laboratory-acquired tularemia. Treatment with broad spectrum antibiotics. Am J Med 1961;30: 785-806.

CR IP T

44. Christopher GW, Cieslak TJ, Pavlin JA, Eitzen EM. Biological warfare. A historical perspective. JAMA 1997;278: 412-17.

45. Hay A. Simulants, stimulants and diseases: The evolution of the United States Biological

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Warfare Programme, 1945-60. Med Confl Surviv 1999;15: 198-214. 46. Alibek K. Biohazard. New York: Random House, 1999: 26-28.

47. Harris S. Japanese biological warfare research on humans: A case study of microbiology and

M

ethics. Ann NY Acad Sci 1992;666: 21-52.

48. Harris SH. Factories of death. Japanese biological warfare, 1932-45, and the American

ED

cover-up. Revised edition. New York: Routledge, 2002: 85.

PT

49. Barenblatt D. A plague upon humanity. The secret genocide of Axis Japan’s germ warfare

CE

operation. New York: HarperCollins, 2004: xii-xiii. 50. Health aspects of chemical and biological weapons. Report of a WHO group of consultants.

AC

Geneva: World Health Organization, 1970: 94-107. 51. Lewis WS, Smith WH, Lam GL, eds. Horace Walpole’s correspondence with Sir Horace Mann. IV. New Haven: Yale University Press, 1960: 407-408. 52. Johnson S. A dictionary of the English language in which the words are deduced from the originals, and illustrated in their different significations by examples from the best writers to 32

ACCEPTED MANUSCRIPT

which are prefixed, a history of the language and an English grammar. London: W. Stahan 1755, volume II: 1739. 53. Merton RK, Barber E. The travels and adventures of serendipity. A study in the sociological

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semantics and sociology of science. Princeton: Princeton University Press, 2004: 240-241. 54. Mullan F. Plagues and politics. The story of the United States Public Health Service. New York: Basic Books, 1989: 89.

AN US

55. Hornblum AM. They were cheap and available: Prisoners as research subjects in Twentieth Century America. BMJ 1997;315: 1437-1441

56. Pont J. Ethics in research involving prisoners. Int J. Prison Health 2008);4: 184-197.

M

57. Barras V, Greub G. History of biological warfare and bioterrorism. Clin Microbiol Infect Dis

ED

2014;20: 497-502.

58. Lesho E, Dorsey D, Bunner D. Feces, dead horses, and fleas. Evolution of the hostile use of

PT

biological agents. West J Med 1998;168:512-516.

CE

59. Dennis DT, Ingelsby TV, Henderson DA, et al. Tularemia as a biological weapon. Medical and public health management. JAMA 2001;285: 2763-2773.

AC

60. Hirschmann JV. The early history of coccidioidomycosis: 1892-1945. Clin Infect Dis 2007;44: 1202-1207.

61. Derrick EH. ’Q’ Fever, a new fever entity: Clinical features, diagnosis, and laboratory investigation. Med J Austral 1937;2: 281-299.

33

ACCEPTED MANUSCRIPT

62. Eldin C, Melenotte D, Mediannikv O, Ghigo E, Million M, Edouard S, et al. From Q fever to Coxiella burnetii infection: a paradigm change. Clin Microbiol Rev 2017;30: 115-190. 63. Burnet FM, Freeman M. Experimental studies on the virus of ‘Q’ Fever. Med J Austral

CR IP T

1937;2:299-305. 64. Waag DM. Coxiella burnetii: Host and bacterial responses to infection. Vaccine 2007;25:

AC

CE

PT

ED

M

AN US

7288-7295.

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