Tularemia: The Rabbit

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Tularemia The Rabbits Revenge
Koushik Das Clara Lee Anusha Viswanathan
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Tularemia

• Zoonotic disease caused by bacterium Francisella
  tularensis
• Three sub-species biovar A (tularensis) biovar
  B (palaearctica), and biovar C (novicida)
• One of the most infectious pathogenic bacteria
  known

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Transmission
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Transmission

• F. tularensis infects through skin, mucous
  membranes, lungs, and GI tract.
• Target organs lymph nodes, lungs, spleen, liver,
  and kidney

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History

• I showed my hand to papa and he diagnosed it as
  blood poisoning and burned the thing out with
  nitric acidin the afternoon papa got out his
  lancet and after injecting cocaine sawed the
  place
• - 1904, excerpts from Letter to Myrtle

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History California

• 1906 Earthquake!
• Increase in bubonic plague in region
• 1908-11 War of extermination against rodents
• Plague-like disease in rodents discovered
• 1912 Dr. McCoy isolates Bacterium Tularense
  from infected squirrels in Tulare County,
  California

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History

• 1912 Wherry and Lamb identify first tularemia
  case in humans
• Dr. Francis main researcher to research the route
  of disease transmission
• 1922 publishes Tularemia, Francis, 1921 A New
  Disease of Man in JAMA
• The first American disease

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History Japan

• Early 1800s cases of disease transferred from
  rodents fatal to humans
• 1925 Oharas Disease studied in Japan
• Francis clinical and serological findings show
  that Oharas Disease is tularemia
• Ohara uses human volunteer (his wife) to study
  transmission of disease

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History Russia

• 1920s extensive outbreaks in Astrakhan and
  Riazan regions (over 900 people affected)
• Resulting from direct contact with European
  water vole
• Researchers infected during course of
  investigations

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Modern Outbreaks

• Vermont, 1968
• 47 cases, people handled muskrats four weeks
  before onset of illness
• No fatalities, but 14 patients had severe illness
  lasting around ten days
• Utah, 1971
• 39 cases, contracted from bite of an infected
  deerfly
• All patients recovered
• South Dakota, 1984
• 20 cases of glandular tularemia in children
• Illness mild
• Thought to be caused by type B

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Recent Outbreaks

• Marthas Vineyard, 2000

• 15 cases of tularemia
• 11 patients had primary pneumonic disease
• 1 fatality
• Caused by type A

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Recent Outbreaks

• Laboratory Workers (Marthas Vineyard, cont.)
• Began with a fatal case of pulmonary tularemia in
  a 43 year old man
• 13 people (including one pregnant woman) in the
  microbiology laboratory and autopsy services
  exposed despite adhering to established
  laboratory protocol
• Tularemia ranks second in the US and third
  worldwide as leading lab associated infections

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Recent Outbreaks

• August 2002 Prairie dogs at Texas pet
  distribution facility die unexpectedly
• Health officials determine that cause of death is
  tularemia
• Worse.potentially infected dogs were shipped to
  Ohio, West Virginia, Florida, Washington, as well
  as to Japan, the Czech Republic, the Netherlands,
  Belgium

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Recent International Outbreaks

• 1966-1967, Sweden 600 patients infected with
  strains of milder F. tularensis
• 1995, Moscow tainted milk and contaminated
  wells spur epidemic
• 2001, Germany two cases reported, when father
  and daughter eat rabbit run over by fathers car
  (!!)

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Outbreak!

• 1942-1943, Battle of Stalingrad
• - tens of thousands of soldiers and civilians
  affected
• - possible cause destroyed infrastructure,
  rampant rodents, contaminated food and water

Or..Soviet conspiracy theory? - Biohazard by
Ken Alibek - deliberate release of airborne
tularemia on German troops during turning point
of WWII?
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History as Bioweapon

• WWII Japans Unit 731 used bacterium in
  experiments on prisoners of war in Manchuria
  during the Japanese occupation of China
• Cold War Both the USSR and the American-British
  biological weapon researchers worked to
  aerosolize tularemia as a bioweapon
• - 1958 tularemia became the American weapon of
  choice for biological retaliation.
• - American development of tularemia ceased in
  1966, Soviet development continued through the
  early 1990s

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Microbiology
Classification
Cellular Physiology of the Bacterium
Bacterial Infection
Immune System
Recovery
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Francisella Tularemia
Coccobacilli
Nonmotile
Gram negative
Size 0.2 x 0.2-0.7 um
Aerobe
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Grow Your Own F. tularensis
Iron
Cysteine
24-39ºC Range
Minimal medium Broth, Blood or Chocolate Agar
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Problem
Highly Infectious
Not popular bacterium to research
Labworkers dont have a death wish
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Two Biovariants
Biovar tularemia palearctica
Synonym Type A holarctica
Virulence High Lower
Capsule Density High Low
Cysteine uridase activity Yes No
Geographic Distribution North America Europe and Asia
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Virulence
No toxin secreted
No toxic endotoxin
Virulence caused by
Capsule
LPS
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The Immune System

• Non Specific

• Specific

Humoral
Inflammation
B cells - Antibodies
Macrophages
Cell-Mediated
Neutrophils
T cells
cytotoxic
Natural Killer Cells
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HOT
RED
IL-1
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IL-1
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Fibrin
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SWELLING
PAIN
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http//www.cat.cc.md.us/courses/bio141/lecguide/un
it1/bacpath/lpsan.html
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http//www.cat.cc.md.us/courses/bio141/lecguide/un
it1/bacpath/macro.html
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GRANULOMA
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Areas of F. Tularensis Infection
Point of Entry - Ulcers and Granulomas
Lungs
Blood
Lymph Nodes
Kidney and Spleen
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Needed for Full Recovery
Only Slows Growth
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Epidemiology

• Geographic Distribution (US World)
• Human Exposure Risk
• Incidence

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It Goes By Many Names

• United States
• Rabbit fever
• Deer-fly fever
• Market men's disease
• Japan
• Wild hare disease (yato-byo)
• Ohara's disease
• Russia
• Water-rat trappers' disease in Russia.

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Geographic Distribution

• Disease of the Northern hemisphere, between 30º
  71º N Latitude.
• No cases in UK, Africa, South America, Australia
• Bacterial strain differs in each continent
• Type B associated with Eastern Europe and
  Scandinavia
• Type A associated with United States
• Clinical distribution of disease varies from
  country to country

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Geographic Distribution

• Primarily affects rural areas because of its
  vector based transmission.
• Vector of transmission is specific to the ecology
  of region
• Endemic in Eurasia (Scandanavia, Eastern Europe,
  Former USSR) as well as Japan.

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Geographic Distribution (US)

• Every state, except Hawaii, has reported cases of
  Tularemia.
• Missouri, Arkansas, Oklahoma account for 53 of
  reported cases.

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Incidence

• Very common pre WW I several 1000s of cases
  reported every year
• Declined to 0.15 case/100,000 people since 1965
• Declined to 0.04 case/100,000 people (about
  100-200 nationally per year) during the 1990s.
• Still small, annual mortality rate.

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Incidence

• Between 1985 1992
• 1409 cases
• 20 deaths (mostly elderly and children)
• 1.4 fatality
• On the whole, pre-antibiotic era mortality was as
  high as 30-60, though today it has fallen to
  approximately gt 3 with effective treatment.

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Human Exposure Risks
Animal Host Bites (A huge number of possible
vectors depending on location, including voles,
mice, water rats, squirrels, rabbits, ticks,
flies, mosquitoes etc.)
Environmental Contamination (Exposure from
tainted food, water, soil, vegetation,
bio-terrorist aerosol)
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Human Exposure

• Peak diagnosis occurs from June September with
  a peak in December
• Summer months correspond to tick season, as
  tularemia is the 3rd most common tick transmitted
  disease in the US.
• December peak corresponds to hunters eating
  poorly cooked game meats.

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Human Exposure

• No inherent susceptibility among any race, sex,
  or age group however 75 of diagnosed patients
  are men and 33 are children
• More commonly associated with outdoor activities.
• Occupations at risk include
• Lab workers
• Farmers
• Veternarian
• Sheep Workers
• Hunters
• Cooks

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Human Exposure

• No documented person to person transmission of
  the disease
• EXTREMELY potent and pathogenic
• Intradermaly or subcutaneously, only 10-50
  organisms necessary for infection
• Ingestion requires 108 organisms for infection
• Infection occurs in families during outbreaks
  mainly because of shared activities.

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Human Exposure - Vectors

• One of the difficulties in diagnosis and
  treatment is the HUGE variety of vectors (over
  200 known)
• Numerous species of ticks or any blood-feeding
  organism, mosquitoes, a variety of lagomorphs
  (rabbits), biting flies, aquatic rodents,
  muskrats, beavers, voles, mice, prairie dogs,
  sheep, cats.
• Vectors are specific to the ecology of the region
• Ticks common in Rocky Mountains
• Biting Flies common in CA, NV, UT
• Mosquitoes common in Sweden, Finland, Russia

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Human Exposure
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Human Exposure - Vectors

• Type A biovar (F. tularensis) more virulent.
• Almost exclusively in North America
• Transmitted primarily via cotton tail rabbits,
  amblyomma americanum (lone star tick),
  dermacentor variabilis (dog tick), dermacentor
  andersoni (wood tick)
• Type B biovar (F. tularensis palaearctica) less
  virulent.
• Eastern Europe other endemic areas
• Prone to Typhoidal type clinical manifestation
• Transmitted via aquatic rodents like muskrats,
  beavers, ground voles (all infected by
  mosquitoes).
• Also poorly cooked game meats

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Pathogenesis

• Infection can occur through any open mucous
  membrane, eyes, broken skin, inhalation, GI
  tract.
• Major targets include lymph nodes, lungs, pleura,
  spleen, liver, and kidney, causing failure in all
  of these organs in final stages.

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Pathogenesis

• Spreads from mucous membrane to proximal lymph
  node and then throughout the body.
• Ranges from asymptomatic to rapid death.
• Incubation avg. 3-5 days, but ranges 1-21 days.
• Generally, within 3-5 days of infection, reaction
  is focal to site of inoculation, with a papule
  forming, suppurative necrosis, and ulceration.
• Marked by accumulated polymorphonuclear
  leukocytes macrophage invasion, epitheliod
  cells, and lymphocytes.

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Pathogenesis

• Suppurative lesions become granulomatous with
  central, necrotic eschar.
• Histologically, includes epithelioid cells,
  multinucleated giant cells, fibroblasts typical
  of tuberculosis or sarcoidosis.

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Clinical Manifestations

• Greatest difficulty of managing tularemia is
  difficulty in diagnosis due to an immensely broad
  set of symptoms of disease and seven distinct
  presentations.
• Vary in severity according to
• Virulence
• Portal of entry
• Extent of systemic involvement
• Immune status of host

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The Seven States of Tularemia

• Ulceroglandular
• Glandular
• Typhoidal
• Oculoglandular
• Oropharyngeal
• Pneumonic
• Septic

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Clinical Ulceroglandular

• Accounts for 21-87 of total cases.
• Method of Infection
• Typical from handling contaminated carcass, but
  more typically from arthropod bite.
• Clinical Presentation
• Local cutaneous papule reflecting point of
  infection, that becomes pustular ulcerates.
• One or more regional afferent lymph nodes becomes
  tender enlarged.
• Even with antiobiotics, nodes may become
  fluctuant rupture.

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Clinical - Ulceroglandular
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Clinical - Ulceroglandular
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Clinical Ulceroglandular Glandular
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Clinical - Glandular

• Accounts for approximately 3-20 of US cases but
  62 of Japanese cases
• Method of Infection
• Typical from handling contaminated carcass, but
  more typically from arthropod bite.
• Clinical Presentation
• Same process as ulceroglandular main difference
  is there is minimal skin ulceration and primarily
  only adenopathy.
• Febrile period may have long passed
• Nodes may suppurate and persist for months

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Clinical Ulceroglandular Glandular
Determining vector in ulceroglandular and
glandular tularemia based on point of inoculation.
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Clinical Ulceroglandular Glandular
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Clinical Ulceroglandular Glandular

• Differential diagnosis of these two forms of
  tularemia is difficult because it is confusable
  with
• Bacterial infection
• Syphillis
• Chancroid
• Lymphogranuloma venereum
• Tuberculosis
• Toxoplasmosis
• Sporotrichosis
• Antrhax
• Plague
• Herpes simplex
• And many, many more.

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Clinical Typhoid

• Accounts for 5-30 of total cases.
• Method of Infection
• Possible from many causes, including contaminated
  food, or an unapparent bite.
• Clinical Presentation
• Presents with fever, diarrhea, nausea, pain,
  dehydration without cutaneous or mucosal membrane
  lesions or any regional lymphadenitis.
• Appears just like a normal influenza but if
  untreated symptoms goes on for weeks or months
  and can spread to pneumonia or even sepsis
• Some GI manifestations possible like diarrhea or
  necrotic colon lesions.
• VERY CONFUSING DIAGNOSIS
• May cause rapid death in some and protracted
  fever in others
• Common secondary pleuropulmonary involvement (95)

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Clinical - Oculoglandular

• Accounts for 5 of total cases.
• Method of Infection
• Typically from direct contamination of eye by
  contaminated fingers.
• Clinical Presentation
• Presents with photophobia, ulceration of
  conjunctiva (membrane around outer eye)
• Chemosis (swelling around iris), vasculitis
  (inflammation of vessel), lymphadenitis may
  also occur in addition to usual febrile state
  with chills, nausea, vomiting, etc.
• Rule out with distal enlarged lymph note
  (cervical, preauriculam, or submandibular)
• Confusable with mumps, syphillis, herpes simplex

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Clinical - Oculoglandular
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Clinical - Oropharyngeal

• Accounts for 0-12 of total cases.
• Method of Infection
• Ingestion of contaminated water, food, or
  sometimes droplets of aerosols.
• Clinical Presentation
• Presents with stomatis (swelling of mouth
  organs), exudative pharyngitis, or tonsilitis
  sometimes with ulceration.
• Intense throat pain unresponsive to penicillin.
• Pronounced cervical or retropharyngeal
  lymphadenopathy which may form an abscess.
• Confusable with mononucleosis, diptheria, etc.

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Cervical - Oropharyngeal
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Clinical - Pneumonia

• Accounts for 7-20 of total cases.
• Method of Infection
• Infection directly from inhalation of aerosols or
  often a secondary infection from hematogenous
  spread from a distal infection.
• Clinical Presentation
• Symptoms include pharyngitis, bronchiolitis,
  pleuropneumonitis (inflammation of lung
  pleura), hilar lymphadenitis (medial base of lung
  lymph adnopathy)
• Peribronchial infilitrates (leukocytes mainly)
  and bronchopneumonia in multiple lobes with
  collection of fluid in pleura (b/w the lung and
  chest wall).
• Plerual fluid is gram negative, mostly lymph
  cells and mimics Tuberculosis.

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Clinical - Pneumonia

• Scattered granulomatous lesions can occur
• Only 25-50 show positive chest X-Rays.
• Respirator necessary in many cases may rapidly
  progress to failure and death

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Clinical Sepsis Rashes

• Sepsis
• Can occur in cases of nonspecific findings and
  secondary infection (fever, pain, vomiting, coma,
  etc.)
• Septic shock may emerge
• Rash 35 of all patients develop it

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Diagnosis

• Widespread, rapid, fool proof diagnostic testing
  is not widely available.
• Several Techniques available
• Readily Available
• Culture
• Antibody Agglutination
• Fluorescent Antibody/Immunoperoxidase
• More Experimental
• RNA hybridization
• PCR
• ELISA

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Diagnosis - Staining

• Collect specimen of respiratory secretion
  (pleural effusions, etc.), verify by antibody
  staining.
• Viewable via light microscope (0.2µm x
  0.2-0.7µm), pleomorphic (in many stages of life
  cycle)
• Differential staining from plague (bipolar) and
  anthrax (gram )
• Staining tests are relatively quick but not
  definitive.

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Diagnosis - Culture

• Growing Tularensis in culture is definitive but
  is much more difficult and dangerous, and
  requires a later stage patient.
• Take sample from pharyngeal washings, sputum, or
  gastric aspirates and grow in cysteine enriched
  broth or several other established culture
  mediums.
• Difficult procedure can take up to 10 days for
  culture (usually within 96 hours).
• Culture requires a BSL level 2 or level 3
  environment potentially dangerous for lab
  technicians

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Diagnosis

• Serum Agglutination
• Often successfully done with serum and
  commercially available antigens. A 4x change in
  titers is diagnostic.
• BUT, not detectable until the end of the 2nd week
  of infection.
• PCR
• Very promising, and very effective
• Highest efficiency at 73 effective
• RNA Hybridization
• This and other future techniques should also
  match the efficacy of PCR as well as detect
  differences in strains of bacteria.

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Treatment

• Three classes of antibiotics provide very
  effective management for Tularemia
• Streptomycin aminoglycosides (Gentamicin
  alternatively)
• Tetracyclines Chloramphenicol
• Fluoroquinolones including Ciprofloxacin

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Antibiotic Efficacy
Drug Cure / Relapse Pros Cons
Streptomycin (Aminoglycocside) 97 / 0 Highly effective Long term IV, High toxicity, Streptomycin resistant
Tetracycline / Chloramphenicol 88 / 12 77 / 21 Works well, Oral administration Relapse Rate
Fluoroquinolones Unknown Ubiquitous, Mass casualty drug of choice Not widely tested
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Antibiotic Efficacy

• If antibiotics given during incubation period,
  study showed that patients were fully protected
  from symptoms 24hrs after challenge, ONLY if long
  course (14-28 days) used. Short course (5 days)
  were symptomatic
• Very relevant for potential terrorist attack
• If successfully identified as a bioterror threat,
  antibiotics can be used prophylacticaly to those
  with fever of unknown origin.
• Difficulty in procuring so much antibiotic.

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Vaccine

• Two vaccines previously available from killed F.
  tularensis and a live vaccine version (LVS) from
  the USSR.
• Killed vaccine is ineffective because only induce
  an antibody response.
• LVS is attenuated live tularensis strain in two
  phenotypes and induces protective immunity.
• A third, new vaccine has recently (8/2003) been
  commissioned by the US Army

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Vaccine

• LVS was used extensively in USSR on 10s of
  millions in endemic areas.
• It is an incomplete protection against
  inhalational tularemia
• Study compared pre-vaccine with post-vaccine lab
  workers in army and showed that incidence of
  accidental acute inhalational tularemia
• 5.7 cases/1000-man years of risk pre-vaccine
• 0.27 cases/1000-man years of risk post-vaccine
• But, vaccine was in ineffective ulceroglandular
  tularemia and was in general incomplete
• Requires 2 weeks for protective immunity, so not
  recommended for post-exposure prophylaxis.

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Vaccine
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Francisella tularensis as a Weapon
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Category A Agents

• can be easily disseminated or transmitted from
  person to person cause high mortality, with
  potential for major public health impact might
  cause public panic and social disruption and
  require special action for public health
  preparedness
• - Centers for Disease Control and Prevention

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Category A Agents

• Anthrax
• Plague
• Tularemia
• Botulism
• Smallpox
• Ebola and Marburg Hemorrhagic Fevers

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What makes F. tularensis special?

• Hardy, non-spore forming organism
• Soil
• Moist hay
• Straw
• Decaying animal carcasses
• - Dies quickly upon exposure to high temperatures
  and sunlight
• - No documented person to person transmission

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What makes F. tularensis special?

• Extreme infectivity
• inhalation of fewer than 10 organisms can
  result in infection

• Subway Test
• Light bulbs filled with bacteria dropped on
  subway predict causalities upon attack
• Underground release would lower decay rate to 2

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Weapon of Mass Disruption

• Aerosol release will result in a febrile illness
  in 3-5 days followed by pleuropneumonitis and
  systemic infection slower progression and
  fatality rate than anthrax or plague but illness
  would be expected to persist for several weeks
  with relapses
• Anthrax vs. Tularemia
• 12,000 fatalities predicted with anthrax
• Only 10,000 fatalities (or 5) with most severe
  strain of tularemia (and antibiotic treatment),
  but 190,000 heavily debilitated
• Aerosol dispersal of 50 kg F. tularensis over a
  metropolitan area with 5 million people can
  result in 250,000 incapacitating casualties,
  including 19,000 deaths (WHO)

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Cost of a Bioterrorist Attack

• CDC Estimate
• If 100,000 people were exposed to a "tularemic
  cloud," 82,500 cases (an 82.5 attack rate) with
  6188 deaths (6.2 death rate) would be expected.
• The medical costs of tularemia from this
  bioterrorist attack would be between 456 million
  and 561.8 million.

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Indications of Intentional Release of F.
tularensis

• Clustering of illness (temporal or spatial)
• Waves of exposure, with quickest onset occurring
  at source
• Abrupt, severe onset and a single peak of cases
• Rapid progression from upper respiratory symptoms
  to life threatening pneumonia
• Attack rates would be similar across age and sex
  groups
• An outbreak of inhalational tularemia in
  unexpected (example, urban) setting

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Treatment

• Case fatality rate for F. tularensis (type A)
• without antibiotics
• overall 5-15
• severe forms 30-60
• with antibiotics
• overall lt 2 (in USA)

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BUT resistance to antibiotics can be potentially
enhanced to transform the bacteria into a more
lethal agent - F. tularensis with resistance to
antibiotics (tetracycline and chloraphenicol)
already developed
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Mass Casualty Treatment

• Vaccine as post exposure prophylaxis ineffective
• does not provide complete protection against
  inhalational forms of tularemia
• immunity takes around two weeks to develop, but
  incubation period of disease is only 3 to 6 days
• If exposed persons identified during the
  incubation period, treat with post-exposure
  prophylaxis

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Mass Casualty Treatment

• If only identified after onset of illness, place
  on fever watch.
• If persons develop an unexplained fever or
  flu-like illness within 14 days of the possible
  exposure start antibiotic therapy
• Ciproflaxin fluoroquinones favored

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What Needs to Be Done

• Reliable diagnostic test to identify F.
  tularensis in people
• Procedures to quickly detect F. tularensis in
  environment
• Ways to monitor appearance of antibiotic
  resistant strains
• Effective antibiotics to target strains resistant
  to current antibiotics

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Whos Doing It?

• USAMRIID defensive research
• CDC
• FDA
• International Dialogue
• WHO
• 2001 Pentagon funded program cooperation
  between Russian and American scientists
• 4th International Conference on Tularemia (2003)

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What Can You Do?
Carrot? What Carrot

• Avoid infected animals
• dull, dopey, glass-eyed with rough, ragged
  hairor brought home by the children or family
  dog Scientific Monthly, 1928
• Wash your hands with soap
• Notice any changes in pets and livestock
• Use insect repellent
• Cook food completely
• Make sure water is safe

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Prevention

• If suspected of contamination (live in cold moist
  environments) then you can kill with 10 bleach
  70 ethanol.
• Standard Cl in drinking water should
  decontaminate.

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And Finally.
If you have been doing any of the the donts
and a few days later, out of a clear sky,
suddenly develop a frightful headache, are racked
with pains in every bone, your temperature is
high, with frequent chills, there is a huge
swelling under an armpit, and you feel sick and
miserable all over, go to bed and send for the
doctor. Ten to one he will find you have
tularemia. - Rabbit Fever or Tularemia,
Scientific Monthly, 1928
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Acknowledgements

• Dr. Geoffrey Zubay
• Mrs. Kathleen Kehoe
• Dr. Judith Gibber