BW-ch08.pdf

Tularemia

Chapter 8

TULAREMIA

MATTHEW J. HEPBURN, MD * ; ARTHUR M. FRIEDLANDER, MD † ; and ZYGMUNT F. DEMBEK, P h D, MS, MPH ‡

INTRODUCTION

INFECTIOUS AGENT

CLINICAL DISEASE

Epidemiology

Pathogenesis

Clinical Manifestations

Diagnosis

Treatment

PROPHYLAXIS

Postexposure Prophylaxis

Vaccination with Live Vaccine Strain

ISSUES FOR LABORATORY WORKERS

USE OF TULAREMIA AS A BIOLOGICAL WEAPON

SUMMARY

* Major,MedicalCorps,USArmy;InfectiousDiseasesPhysician,DivisionofMedicine,USArmyMedicalResearchInstituteofInfectiousDiseases,

1425PorterStreet,FortDetrick,Maryland21702

† Colonel,MedicalCorps,USArmy(Ret);SeniorScientist,DivisionofBacteriology,USArmyMedicalResearchInstituteofInfectiousDiseases,1425

PorterStreet,FortDetrick,Maryland21702;andAdjunctProfessorofMedicine,UniformedServicesUniversityoftheHealthSciences,4301Jones

BridgeRoad,Bethesda,Maryland20814

‡ Lieutenant Colonel, Medical Service Corps, US Army Reserve; Chief, Biodefense Epidemiology and Training & Education Programs, Operational

MedicineDepartment,DivisionofMedicine,USArmyMedicalResearchInstituteofInfectiousDiseases,1425PorterStreet,FortDetrick,Maryland

21702

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MedicalAspectsofBiologicalWarfare

INTRODUCTION

Francisellatularensis poses a substantial threat as a bio-

logical weapon, and it is viewed by most experts as a dan-

gerous pathogen if weaponized. Both the United States

and the former Soviet Union developed weaponized F

tularensis during the Cold War. 1,2 It is unclear whether

tularemia has ever been used deliberately as a biological

weapon. The Japanese experimented with Ftularensis as

a biological weapon, but there is no documentation of its

use in military operations. 3 There is also speculation that

the former Soviet Union used Ftularensis as a weapon

against German troops in the Battle of Stalingrad during

World War II. 2 Despite the tularemia outbreak among

soldiers of both armies during this battle, some authors

suggest that natural causes, as opposed to an intentional

release, were responsible for the epidemic. 4 There was

also speculation that Ftularensis was used as a biological

weapon by Serbia in the Kosovo conflict, although the

subsequent investigation suggested the observed cases

were not caused by an intentional release. 5,6

Ftularensis has been included in the list of Centers

for Disease Control and Prevention Category A threat

organisms because of the infectivity with exposure to

low numbers of organisms, the ease of administration,

and the serious consequences of infection. 1 Tularemia’s

effectiveness as a biological weapon includes a nonspe-

cific disease presentation, high morbidity, significant

mortality if untreated, and the limited ability to obtain

a rapid diagnosis. Although tularemia responds to

antibiotics, the use of an antibiotic-resistant strain can

make these countermeasures ineffective.

INFECTIOUS AGENT

Tularemia was named after Tulare County, Califor-

nia, where an epidemic disease outbreak resembling

plague occurred in ground squirrels in 1911. McCoy

and Chapin successfully cultured the causative agent

and named it Bacteriumtularense . 7 Wherry and Lamb

subsequently identified the pathogen as the cause of

conjunctival ulcers in a 22-year-old man. 8 Edward

Francis made significant scientific contributions to the

understanding of the disease in the early 20th century,

including naming it “tularemia.” 9

Ftularensis is an aerobic, gram-negative coccobacilli.

Ftularensis is not motile, and appears as small (approxi-

mately 0.2–0.5 µm by 0.7–1.0 µm), 10 faintly staining

gram-negative bacteria on Gram’s stain (Figure 8-1). F

tularensis was formerly included in the Pasteurella and

the Brucella genera. Eventually a new genus was cre-

ated, and the name Francisella was proposed in tribute

to Edward Francis. 11 A closely related species, Fran-

cisellaphilomiragia , has also been described as a human

pathogen. 12,13 F tularensis is considered to have four

subspecies: (1) tularensis , (2) holarctica , (3) mediasiatica,

and (4) novicida. 14 Ftularensis subspecies tularensis , also

known as Type A (or biovar A), occurs predominantly

in North America and is the most virulent subspecies

in both animals and humans. This subspecies was

recently divided into A.I. and A.II. subpopulations.

Subpopulation A.I. causes disease in the central United

States, and subpopulation A.II. is found mostly in the

western United States. 15 Ftularensis subspecies holarc-

tica (formerly described as palearctica ), also known as

Type B (or biovar B), is found in Europe and Asia, but

also occurs in North America. Ftularensis subspecies

holarctica causes a less virulent form of disease than

subspecies tularensis , but has been documented to

cause bacteremia in immunocompetent individuals. 16,17

Before antibiotics, F tularensis subspecies tularensis

resulted in 5% to 57% mortality, yet Ftularensis sub-

species holarctica was rarely fatal. 18 Unlike these other

subspecies, Fnovicida rarely causes human disease. 12

F tularensis subspecies mediasiatica has been isolated

in the central Asian republics of the former Soviet

Union, and it appears to be substantially less virulent

in a rabbit model compared to Ftularensis subspecies

tularensis . 19,20 The four subspecies can be distinguished

with biochemical tests and genetic analysis.

Fig. 8-1 . Gram’s stain of Francisellatularensis .

Photograph: Courtesy of Dr Larry Stauffer, Oregon State

Public Health Laboratories, Centers for Disease Control and

Prevention, Atlanta, Georgia, Public Health Image Library,

#1904.

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Tularemia

THE CLINICAL DISEASE

Tularemia is an infection with protean clinical mani-

festations. Healthcare providers need to understand

the range of possible presentations of tularemia to use

diagnostic testing and antibiotic therapy appropriately

for these infections. Most cases of naturally occurring

tularemia are ulceroglandular disease, involving an ul-

cer at the inoculation site and regional lymphadenopa-

thy. Variations of ulceroglandular disease associated

with different inoculation sites include ocular (oculo-

glandular) and oropharyngeal disease. Occasionally

patients with tularemia present with a nonspecific

febrile systemic illness (typhoidal tularemia) without

evidence of a primary inoculation site. Pulmonary

disease from F tularensis can occur naturally (pneu-

monic tularemia), but is uncommon and should raise

suspicion of a biological attack, particularly if signifi-

cant numbers of cases are diagnosed. Because of the

threat of this microorganism as a biological weapon,

clusters of cases in a population or geographic area

not accustomed to tularemia outbreaks should trigger

consideration for further investigation. 21 Rotz et al

provide criteria for determining the likelihood that a

tularemia outbreak is caused by intentional use of tu-

laremia as a biological weapon. 21 A tularemia outbreak

in US military personnel deployed to a nonendemic

environment would be one example of an incident

that should be investigated. The investigation should

yield the likely cause of the outbreak, which could be

varied (exposure to infected animals, arthropod-borne,

etc). By determining the cause of tularemia, it may be

possible to implement control measures, such as water

treatment or use of an alternative water supply if the

outbreak is traced to a waterborne source.

an aerosol or contaminated dust.

Various epidemiological categories of tularemia

have been suggested, often dependent on the infective

vector, mode of infection, or occupation of the infected

individuals. 18

Direct Contact

In 1914 a meat cutter with oculoglandular disease,

manifested by conjunctival ulcers and preauricular

lymphadenopathy, had the first microbiologically

proven human tularemia case reported. 8 An early re-

view of tularemia established that a majority of human

cases (368 of 488, or 75%) in North America resulted

from dressing and eating wild rabbits. 9 Other wild

mammals may potentially serve as sources for tula-

remia transmission from direct contact, such as wild

prairie dogs that are captured and sold as pets. 24

Food and Water Ingestion

Tularemia can also be contracted by eating meat

from infected animals 9 or food contaminated by in-

fected animals. 25 Water can also become contaminated

from animals infected with tularemia and cause hu-

man infection. During March through April 1982, 49

cases of oropharyngeal tularemia were identified in

Sansepolcro, Italy. 26 The case distribution in this city

suggested that a water system was the source. The

infected individuals had consumed unchlorinated

water, and a dead rabbit from which Ftularensis was

isolated was found nearby. 26 Waterborne transmission

of ulceroglandular tularemia also occurred during a

Spanish outbreak among 19 persons who had contact

with river-caught crayfish. 27 Contaminated water may

have contributed to recent outbreaks of oropharyngeal

tularemia in Turkey 28 and Bulgaria. 25 It is unclear how

Ftularensis survives in water, but it may be linked to

its ability to survive in certain protozoa species such

as Acanthamoebacastellanii . 29

Epidemiology

Ftularensis subspecies tularensis (Type A) is the most

common Ftularensis subspecies causing clinical tulare-

mia in North America. 10 Type A was once thought not

to occur in Europe, but a type A strain has recently been

isolated from flea and mite parasites of small rodents

trapped in Slovakia. 22 Ftularensis subspecies holarctica

(type B), found throughout the Northern Hemisphere,

is less pathogenic. 1 In the United States an average of

124 tularemia cases per year were reported from 1990

through 2000. 23 Over half of all cases reported came

from Arkansas, Missouri, South Dakota, and Okla-

homa, where the foci of infection are well-established.

Tularemia can be transmitted by direct contact with

infected animals or their tissues, ingestion of under-

cooked infected meat or contaminated water, animal

bites or scratches, arthropod bites, and inhalation of

Mammalian Bites and Arthropod Vectors

Mammalian bites are another source of tularemia

transmission to humans. Instances of human transmis-

sion from the bites or scratch of a cat, coyote, ground

squirrel, and a hog were documented over 80 years ago. 9

In April 2004 a 3-year-old boy from Denver, Colorado,

contracted tularemia from a hamster bite, providing

evidence of disease transmission from these pets. 30

Transmission of tularemia by the bites of ticks and

flies is also well-documented. 10 Dermacentor species

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MedicalAspectsofBiologicalWarfare

ticks (dog ticks) are important vectors in areas where

enzootic transmission occurs in North America 31 and

Europe. 32 Ixodes species ticks may also contribute to F

tularensis transmission. 33 In Utah during the summer

of 1971, 28 of 39 tularemia cases were contracted from

deerfly ( Chrysopsdiscalis ) bites. 34 An epidemic of 121

tularemia cases (115 ulceroglandular) in Siberia from

July through August 1941 may have resulted from

transmission of F tularensis by mosquitoes, midges

( Chironomidae ), and small flies ( Similia ). 35

Tularemia in an Unusual Setting

Some tularemia cases have occurred in geographic

areas where the disease has never been reported. An

orienteering contest on an isolated Swedish island in

2000 resulted in two cases of ulceroglandular tulare-

mia. 42 These cases were theorized to have occurred

from contact with migratory birds carrying the micro-

organism. The social disruption caused by war also

has been linked to tularemia outbreaks. During World

War II, an outbreak of over 100,000 tularemia cases oc-

curred in the former Soviet Union, 4 and outbreaks with

hundreds of cases after the war occurred in Austria

and France. 43 Outbreaks of zoonoses during war since

that time have led to speculation that these epidemics

were purposefully caused. For example, no tularemia

cases had been reported from Kosovo between 1974

and 1999, and tularemia was not previously recognized

endemically or enzootically in the Balkan countries. 5

However, after a decade of warfare, an outbreak of over

900 suspected tularemia cases occurred in Kosovo dur-

ing 1999 and 2000, leading researchers to investigate

claims of use of this agent as a biological weapon by the

Serbs against the Albanian inhabitants of the country. 5,6

The Kosovo outbreak and subsequent investigation

are described in detail in chapter 3, Epidemiology of

Biowarfare and Bioterrorism.

Aerosol Transmission

The largest recorded pneumonic tularemia outbreak

occurred in Sweden during the winter of 1966 through

1967, when 676 cases were reported. 36 Most of the cases

occurred among the farming population, 71% among

adults older than 45 years and 63% among men. The

hundreds of pneumonic cases likely resulted from

contact with hay and dust contaminated by voles

infected with tularemia. Ftularensis was later isolated

from the dead rodents found in barns, as well as from

vole feces and hay.

In the summer of 2000, an outbreak of primary

pneumonic tularemia occurred in Martha’s Vineyard,

Massachusetts. 37 Fifteen confirmed tularemia cases were

identified, 11 of which were the pneumonic form of tula-

remia. One 43-year-old man died of primary pneumonic

tularemia. Epidemiological analysis revealed that using

a lawn mower or brush cutter was significantly associ-

ated with illness in the 2 weeks before presentation of

this case. 38 Feldman et al proposed that in Martha’s Vine-

yard, Ftularensis was shed in animal excreta, persisted in

the environment, and was transmitted to humans after

mechanical aerosolization by mower or brush cutter

and subsequent inhalation. 38 The strong epidemiological

link with grass cutting adds plausibility to this expla-

nation. 39 A seroprevalence survey conducted in 2001

in Martha’s Vineyard demonstrated that landscapers

were more likely to have antibodies to Ftularensis than

nonlandscapers, suggesting an increased occupational

risk for tularemia. 38

The only other previously reported outbreak of

pneumonic tularemia in the United States occurred at

Martha’s Vineyard during the summer of 1978. 40 In a

single week, seven persons who stayed together in a

vacation cottage eventually developed typhoidal tula-

remia. A search for additional cases on the island uncov-

ered six other tularemia cases (five typhoidal and one

ulceroglandular). No confirmed source for the disease

exposure was discovered. Tularemia had been reported

sporadically since the introduction of rabbits to Martha’s

Vineyard in the 1930s, 40 and pneumonic tularemia was

initially reported in Massachusetts in 1947. 41

Laboratory-acquired Tularemia

Soon after the discovery of Ftularensis as a pathogen,

cases of laboratory-acquired infection were recognized.

Edward Francis observed that many laboratory per-

sonnel working with the pathogen, including himself,

became infected. 9 Six tularemia cases occurred during

US Public Health Service laboratory investigations of

tularemia outbreaks from 1919 through 1921. 44 Tulare-

mia is the third most commonly acquired laboratory

infection, 45 and recent laboratory-acquired infections

of tularemia emphasize the laboratory hazard that this

organism presents. 46 Because of the extreme infectivity

of this microorganism, investigators of a 2000 outbreak

in Kosovo chose not to culture the organisms from pa-

tients, but instead relied on empirical clinical evidence

of tularemia cases.

Pathogenesis

For infection to occur, bacterial pathogens must tra-

verse the normal skin and mucosal barriers that typi-

cally prevent microorganisms from entering the body.

Breaks in the skin from lacerations or abrasions provide

opportunity for Ftularensis transmission and infection.

Arthropod vectors can bypass the skin defenses with

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Tularemia

a bite, thus inoculating the organism directly into the

host. However, the portal of entry can also be mucous

membranes in the respiratory tract, ocular membranes,

or the gastrointestinal tract.

One of the remarkable attributes of F tularensis is

the low infectious dose. As few as 10 organisms can

produce an infection when injected subcutaneously

into human volunteers, and only 10 to 50 organisms

are required when administered to human volunteers

by aerosol. 47,48 Recent investigations have attempted

to elucidate the unique characteristics that allow F

tularensis to cause infection at such a low number of

organisms. As an intracellular pathogen, Ftularensis

has developed the means to survive in the typically

hostile environment inside macrophages by interfer-

ing with multiple aspects of macrophage function.

On initial entry into the macrophage, Ftularensis uses

a bacterial acid phosphatase, AcpA, to inhibit the

bactericidal respiratory burst response of the macro-

phage. 49,50 Additionally, both Ftularensis Type A and B

can inhibit acidification of the phagosome after entry

into the macrophage, escape from the phagosome,

and reside in the macrophage cytoplasm. 51,52 Another

survival mechanism of Ftularensis is the interference

with the normal macrophage response by inhibiting

Toll-like receptor signaling and cytokine secretion,

as demonstrated in experiments with murine macro-

phages and the live vaccine strain (referred to as LVS,

which is subspecies holarctica or a Type B strain) of F

tularensis. 53 An absence of Toll-like receptor signaling

inhibits the typical robust innate immune response

that could eliminate the bacteria. Replication of the

organism in the macrophage begins slowly, but even-

tually large numbers of organisms can be found in a

single macrophage. 52,54,55 Although F tularensis may

initially delay apoptosis (programmed cell death) of

the macrophage, the organism eventually induces

apoptosis through mechanisms similar to intrinsic cel-

lular signals. 56 Researchers have identified only some

of the factors required by Ftularensis for survival in

macrophages, including IglC , a 23-kDa protein that

most likely affects Toll-like receptor-4 signal transduc-

tion, 53,57 and the MglAB operon that regulates transcrip-

tion of virulence factors. 58 The MinD protein functions

as a pump for substances containing free radicals such

as hydrogen peroxide, allowing the organism to resist

oxidative killing. 59

The early innate immune response to F tularensis

involves intracellular killing of the pathogen by the

macrophages and proinflammatory cytokine secre-

tion. Murine experiments have demonstrated the

importance of an effective early cytokine response.

Interferon-g-deficient mice die from sublethal doses of

LVS 60 and tumor necrosis factor-a is at least as impor-

tant as interferon-g for control of Ftularensis infection. 61,

62 The host defense within macrophages appears to be

crucial at controlling infection by Ftularensis . In human

monocytes/macrophages, LVS strain and F novicida

induced the processing and release of interleukin

(IL)-1b, an essential component of the inflammatory

immune response. 63 However, killed bacteria did not

induce this response, but did induce the early phases

required for IL-1b, such as mRNA transcription. The

results suggest that only live Francisella can escape

from the phagosome, and thus trigger the function of

caspase-1, which converts the precursor of IL-1b to its

active form. In mice deficient in caspase-1 as well as

ASC, an adaptor protein involved in host cell death,

substantially higher bacterial loads were observed, as

well as early mortality, compared to normal mice. 64

Neutrophils perform an important function in limiting

the spread of F tularensis after inoculation. Experi-

ments have demonstrated that neutrophils can kill F

tularensis , 65 and mice depleted of neutrophils appear

susceptible to infection with Ftularensis LVS. 66

The late adaptive immune response to Ftularensis

requires an intact cell-mediated immune system,

particularly in resolving the initial infection and in

producing long-term immunity. 67 There is no clear

immunodominant epitope on any one Ftularensis viru-

lence protein that stimulates the required cell-mediated

response; however, studies have demonstrated that

multiple protein/peptides are required. 68 Vaccination

with Ftularensis LVS appears to produce a long-term

memory T-cell response (as measured by lymphocyte

stimulation), 69 but it is unclear what degree of long-

term protection is conferred by this response. Both

CD4 + and CD8 + lymphocytes are required for an ef-

fective cell-mediated response to F tularensis. 60 The

protective memory response is dependent on a robust

proinflammatory cellular response, because admin-

istration of anti-interferon-g and anti-tumor necrosis

factor-a antibodies to previously vaccinated mice

dramatically lowers the lethal infective intradermal

dose of Ftularensis. 62 This response initially appears 2

to 4 weeks after initial infection, 70-72 and it can remain

detectable for many years. 69,73

The importance of humoral immunity in the defense

against tularemia is not completely understood, but it

appears that the humoral response by itself provides

little or no value in protecting the host. 74 When labo-

ratory workers received a formalin-killed whole-cell

vaccine developed by Foshay et al, 75 a strong humoral

response was elicited but was not protective against

cutaneous 48 or respiratory 47 challenge. The failure of

this vaccine suggested that the formalin inactivation

procedures destroyed some of the essential protec-

tive antigens or that these protective antigens were

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