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Bacillus anthracis

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Title: Bacillus anthracis


1
Bacillus anthracis
  • Hirotaka Ishibashi
  • Jennifer Jolivet
  • Sean Patrick Kelly

2
Bacillus anthracis
  • Gram rod
  • Facultative anaerobe
  • 1 - 1.2µm in width x 3 - 5µm in length
  • Belongs to the B. cereus family
  • Thiamin growth requirement
  • Glutamyl-polypeptide capsule
  • Nonmotile
  • Forms oval, centrally located endospores

http//www.bact.wisc.edu/Bact330/lectureanthrax
3
Endospore
  • Oxygen required for sporulation
  • 1 spore per cell
  • dehydrated cells
  • Highly resistant to heat, cold, chemical
    disinfectants, dry periods
  • Protoplast carries the material for future
    vegetative cell
  • Cortex provides heat and radiation resistance
  • Spore wall provides protection from chemicals
    enzymes

http//www.gsbs.utmb.edu/microbook/ch015.htm
4
Genetics
  • 1 chromosome
  • 5.2 million bp
  • Ames strain sequenced
  • 2 plasmids
  • px01
  • 184 kbp
  • Pathogenicity island
  • pX02
  • 95.3 kbp
  • Capsule
  • Anthrax receptor
  • Occurs gt than ten thousendfold on macrophage cell
  • ATR/TEM8 gene
  • Chromosome 4

gib.genes.nig.ac.jp/single/ main.php?spidBant_AME

5
Where is Anthrax?
http//www.vetmed.lsu.edu/whocc/mp_world.htm
6
Anthrax
  • From the Greek word anthrakos for coal
  • Caused by spores
  • Primarily a disease of domesticated wild
    animals
  • Herbivores such as sheep, cows, horses, goats
  • Natural reservoir is soil
  • Does not depend on an animal reservoir making it
    hard to eradicate
  • Cannot be regularly cultivated from soils where
    there is an absence of endemic anthrax
  • Occurs sporadically throughout US
  • South Dakota, Arkansas, Texas, Louisiana,
    Mississippi, California recognized endemic areas
  • Anthrax zones
  • Soil rich in organic matter (pH lt 6.0)
  • Dramatic changes in climate

7
Anthrax Infection Spread
  • May be spread by streams, insects, wild animals,
    birds, contaminated wastes
  • Animals infected by soilborne spores in food
    water or bites from certain insects
  • Humans can be infected when in contact with
    flesh, bones, hides, hair, excrement
  • nonindustrial or industrial
  • cutaneous inhalational most common
  • Risk of natural infection 1/100,000
  • Outbreaks occur in endemic areas after outbreaks
    in livestock

8
Three forms of Anthrax
  • Cutaneous anthrax
  • Skin
  • Most common
  • Spores enter to skin through small lesions
  • Inhalation anthrax
  • Spores are inhaled
  • Gastrointestinal (GI) anthrax
  • Spores are ingested
  • Oral-pharyngeal and abdominal

9
Milestones in Anthrax History
  • Early history
  • 1800s
  • 1900s
  • Recent years
  • Outbreaks in Thailand and US

10
History of Anthrax (Early history)
  • Although anthrax dates back more than 3,000
    years, it was not recognized as a disease until
    the 18th century.
  • 1500 B.C - A plague of boils in Egypt affected
    the Pharaohs cattle. Boils are symptomatic of
    anthrax.
  • 1600s - The Black Bane thought to be anthrax,
    in Europe kills over 60,000 cattle.
  • 1700s - There are some accounts of human cases.

11
History (1800s)
  • Early 1800s - The first human cases of cutaneous
    anthrax in the US and UK were reported in men who
    contracted the disease after having been in
    contact with infected livestock.
  • The disease was called Wool Sorters disease or
    Rag Pickers disease because it affected workers
    in those trades.
  • 1868 - Anthrax was observed under a microscope.
  • 1876 - German bacteriologist Robert Koch
    confirmed bacterial origin of anthrax.

12
History (Early 1900s)
  • 1915 - German agents injected horses, mules, and
    cattle with anthrax during WWI. This was the
    first recorded use of anthrax as a biological
    weapon.
  • 1937 - Japan started a biological warfare program
    in Manchuria, including tests involving anthrax.
  • 1942 - UK demonstrated experiments using anthrax
    at Gruinard Island off the coast of Scotland.
  • 1943 - United States began developing anthrax
    weapons.
  • 1945 - In Iran an anthrax outbreak killed more
    than 1 million sheep.

13
History (Late 1900s)
  • 1950s and 60s - U.S. biological warfare program
    continues after WWII at Fort Detrick, Maryland
  • 1969 - President Nixon ended United States'
    offensive biological weapons program, but
    defensive work still continues.
  • 1970 - Anthrax vaccine for humans was approved by
    U.S. FDA.
  • 1978-80 - The world's largest outbreak of human
    anthrax via insect vectors or contaminated meat
    struck Zimbabwe, Africa where more than 10,000
    cases were recorded and over 180 people died.
  • 1979 - In Soviet Union, aerosolized anthrax
    spores were released accidentally at a military
    facility, affecting 94 and killing 64 people.

14
History (Recent years)
  • 1991 - About 150,000 U.S. troops were vaccinated
    for anthrax in preparation for Gulf War.
  • 1990-93 - The cult group, Aum Shinrikyo, released
    anthrax spores in Tokyo, fortunately no one was
    injured. On February 27, 2004, the leader of this
    group was given a sentence of death at a district
    court in Tokyo.
  • 1995 - Iraq produced 8,500 liters of concentrated
    anthrax as part of the biological weapon program
    under Saddam Husseins administration.
  • 2001 - Letters containing anthrax spores were
    mailed to many places in the US such as NBC, New
    York Times, and Media in Miami. In Florida, a man
    died after inhaling anthrax at the office.

15
Outbreaks in Thailand
  • 1982 - In rural Northern Thailand, an outbreak of
    52 cases of cutaneous anthrax and 24 cases of
    oral-pharyngeal anthrax occurred.
  • Oral-pharyngeal anthrax an unusual manifestation
    of humaninfection with B. anthracis.
  • 1987 - 14 cases of both oral- pharyngeal and
    abdominal anthrax occurred.
  • Caused by the consumption of contaminated water
    and buffalo meat.
  • This picture is 9 days after the onset of
    symptoms of oral-pharyngeal anthrax.

Thira Sirisanthana, Arthuer Brown, Anthrax of the
Gastrointestinal Tract, Emerging Infectious
Diseases, Vol. 8, 7, July 2002
16
Outbreaks in the US
  • In the early 1900s approximately 130 cases
    occurred annually due to the following reasons.
  • 1) Agricultural, farm workers exposed to
    infected animals
  • 2) Processors exposed to infected animal
    products (hair,
  • leather, wool, bone)
  • 3) Laboratory workers contacted with anthrax
    spores
  • 4) Civilians exposed to contaminated imported
    animal products
  • These four are rare today.

17
  • Natural Outbreaks in the U.S. (19512003)

in 1957 animal vaccination started
in 1970 human vaccination started
2003
  • N 409
  • 391 cases were cutaneous anthrax.
  • 18 cases were inhalation anthrax
  • GI anthrax has not been recognized yet
  • Since 1990 only 2 cases of cutaneous anthrax of
    naturally
  • occurring infection have been reported.

Anthrax Overview PPT, CDC, 2001
(http//www.cdc.gov/)
18
Natural Outbreaks in North Dakota
  • The highest occurrence of Anthrax outbreaks in
    the US
  • 1989-1999 - 26 cases of infected livestock were
    reported.
  • 2000 - 33 cases were reported during
    July-September.
  • Total of 180 animals (beef cattle, horses, and
    bison) died and one person was infected with
    cutaneous anthrax.
  • Roughly, cases appear every 2 years in North
    Dakota

19
Pathogenesis
  • The infectious dose of B. anthracis in humans by
    any route is not precisely known.
  • Rely on primate data
  • Minimum infection dose of 1,000-8,000 spores
  • LD50 of 8,000-10,000 spores for inhalation
  • Virulence depends on 2 factors
  • Capsule
  • 3 toxins

http//www.kvarkadabra.net/index.html?/biologija/t
eksti/biolosko_orozje.htm
20
Capsule
  • Glycocalyx
  • Sticky, gelatinous polymer external to cell wall
  • pX02 plasmid
  • Made up of D-glutamic acid
  • Non-toxic on its own
  • Only encapsulated B. anthracis virulent
  • Most important role during establishment of
    disease
  • Protects against phagocytosis lysis during
    vegetative state

http//textbookofbacteriology.net/BSRP.html
21
Toxins
  • pX01 plasmid
  • AB model
  • Binding
  • Activating
  • Protective antigen (PA), edema factor (EF)
    lethal factor (LF)
  • Make up 50 of proteins in the organism
  • Individually non-toxic
  • PALF ? lethal activity
  • EFPA ? edema
  • EFLF ? inactive
  • PALFEF ? edema necrosis lethal

http//www.rcsb.org/pdb/molecules/pdb28_1.html
22
Toxins (2)
  • Protective antigen (PA, 83kDa)
  • Pag gene
  • Binds to receptor helps internalize other 2
    proteins
  • Edema factor (EF, 89 kDa)
  • Cya gene
  • Adenylate cyclase
  • Affects all cells
  • Lethal factor (LF, 87 kDa)
  • Lef gene
  • More important virulence factor
  • Metalloprotease
  • Cleaves mitogen activated protein kinase kinsase
    (MAPKK)
  • Affects only macrophages

http//www.ericse.org/anthrax/anthraxmicrographs.h
tml
23
Mechanism of Infection
  • Anthrax spores enter body
  • Germinate multiple in lymph nodes
  • PA, EF, LF excreted from bacteria
  • PA binds to TEM8.
  • PA nicked by protease furin
  • 20-kDa segment off leaving 63-kDa peptide
  • Heptamer forms
  • EF and/or LF binds
  • Complex internalized by endocytosis
  • Acidification of endosome
  • LF or EF crosses into cytosol via PA mediated
    ion-conductive channels
  • LF cleaves MAPKK 1 2
  • EF stimulates cAMP

http//kugi.kribb.re.kr/KUGI/Pathways/BioCarta/ant
hraxPathway/
24
Outcome
  • Do not understand exactly how symptoms occur
  • EF converts ATP to cAMP
  • Increases cAMP levels over 1,000 fold
  • Impairs neutrophil function
  • Alters water homeostasis
  • Edema
  • LF cleaves MAPKK at its N terminus
  • Disrupts pathways involved in cell growth
    maturation
  • Increased synthesis of tumor necrosis factor-a
    interleukin-1ß
  • Macrophage lysis
  • More cells infected with bacteria toxin
  • Septic shock death
  • Death probably results from high levels of
    bacteria secreting LF toxins in blood
  • At death, blood contains as many as 109
    bacilli/ml (depending on the species)

25
Regulators
  • Bicarbonate or CO2 stimulates capsule and PA
    formation
  • LF requires zinc ions
  • EF requires calmodulin, a major intracellular
    calcium receptor
  • Transcriptional regulator AcpA on pX02 controls
    expression of capsule
  • atxA on pX01 is a positive regulator necessary
    for transcription of all 3 toxin genes

26
Clinical Information
  • Infection
  • Symptoms (1st and 2nd phase)
  • Three forms of Anthrax infection and their
    Pathology
  • Diagnosis

27
Infection of Anthrax
  • The estimated number of naturally occurring human
    cases of anthrax in the world is 20,000 to
    100,000 per year.
  • Humans are infected through contact with infected
    animals and their products because of human
    intervention.
  • Anthrax spores contaminate the ground when an
    affected animal dies and can live in the soil for
    many years.
  • Anthrax can also be spread by eating undercooked
    meat from infected animals.
  • Anthrax is NOT transmitted from person to person.
  • Humans can be exposed but not be infected.

28
What are the symptoms for anthrax?
  • There are two phases of symptom.
  • 1) Early phase - Many symptoms can occur within 7
    days of
  • infection
  • 2) 2nd phase - Will hit hard, and usually occurs
    within 2 or 3
  • days after the early phase.

29
- Early Phase Symptoms -
  • Fever (temperature gt 100 degrees F)
  • Chills or night sweats
  • Headache, cough, chest discomfort, sore throat
  • Joint stiffness, joint pain, muscle aches
  • Shortness of breath
  • Enlarged lymph nodes, nausea, loss of appetite,
    abdominal distress, vomiting, diarrhea
  • Meningitis

30
- 2nd Phase Symptoms -
  • Breathing problems, pneumonia
  • Shock
  • Swollen lymph glands
  • Profuse sweating
  • Cyanosis (skin turns blue)
  • Death

31
Three clinical forms of Anthrax
  • 3 types of anthrax infection occur in humans
  • 1) Cutaneous
  • 2) Inhalation
  • 3) GI

32
Cutaneous Anthrax
  • 95 of anthrax infections occur when the
    bacterium enters a cut or scratch on the skin due
    to handling of contaminated animal products or
    infected animals.
  • May also be spread by biting insects that have
    fed on infected hosts.
  • After the spore germinates in skin tissues, toxin
    production initially results in itchy bump that
    develops into a vesicle and then painless black
    ulcer.

http//science.howstuffworks.com/anthrax1.htm
33
Cutaneous Anthrax (2)
  • The most common naturally occurring form of
    anthrax.
  • Ulcers are usually 1-3 cm in diameter.
  • Incubation period
  • Usually an immediate response up to 1 day
  • Case fatality after 2 days of infection
  • Untreated (20)
  • With antimicrobial therapy (1)

34
Cutaneous Anthrax (3)
CDC, Cutaneous AnthraxVesicle Development
35
Inhalation Anthrax
  • The infection begins with the inhalation of the
    anthrax spore.
  • Spores need to be less than 5 microns (millionths
    of a meter) to reach the alveolus.
  • Macrophages lyse and destroy some of the spores.
  • Survived spores are transported to lymph nodes.
  • At least 2,500 spores have to be inhaled to cause
    an infection.

Inhalation Anthrax, Introduction, DRP, Armed
Forces Institute of Pathology
36
Inhalation Anthrax (2)
  • Disease immediately follows germination.
  • Spores replicate in the lymph nodes.
  • The two lungs are separated by a structure called
    the mediastinum, which contains the heart,
    trachea, esophagus, and blood vessels.
  • Bacterial toxins released during replication
    result in mediastinal widening and pleural
    effusions (accumulation of fluid in the pleural
    space).

Inhalation Anthrax, Introduction, DRP, Armed
Forces Institute of Pathology
37
Inhalation Anthrax (3)
  • Death usually results 2-3 days after the onset of
    symptoms.
  • Natural infection is extremely rare (in the US,
    20 cases were reported in last century).
  • Inhalation Anthrax is the most lethal type of
    Anthrax.
  • Incubation period
  • 17 days
  • Possibly ranging up to 42 days (depending on how
    many spores were inhaled).
  • Case fatality after 2 days of infection
  • Untreated (97)
  • With antimicrobial therapy (75)

38
Gastrointestinal Anthrax
  • GI anthrax may follow after the consumption of
    contaminated, poorly cooked meat.
  • There are 2 different forms of GI anthrax
  • 1) Oral-pharyngeal
  • 2) Abdominal
  • Abdominal anthrax is more common than the
    oral-pharyngeal form.

http//science.howstuffworks.com/anthrax1.htm
39
GI Anthrax (2)
  • Oral-pharyngeal form - results from the
    deposition and germination of spores in the upper
    gastrointestinal tract.
  • Local lumphadenopathy (an infection of the lymph
    glands and lymph channels), edema, sepsis develop
    after an oral or esophageal ulcer.
  • Abdominal form - develops from the deposition and
    germination of spores in the lower
    gastrointestinal tract, which results in a
    primary intestinal lesion.
  • Symptoms such as abdominal pain and vomiting
    appear within a few days after ingestion.

40
GI Infection (3)
  • GI anthrax cases are uncommon.
  • There have been reported outbreaks in Zimbabwe,
    Africa and northern Thailand in the world.
  • GI anthrax has not been reported in the US.
  • Incubation period
  • 1-7 days
  • Case fatality at 2 days of infection
  • Untreated (25-60)
  • With antimicrobial therapy (undefined) due to the
    rarity

41
How is anthrax diagnosed?
  • Gram stain
  • Culture of B. anthracis from the blood, skin
    lesions, vesicular fluid, or respiratory
    secretions
  • X-ray and Computed Tomography (CT) scan
  • Rapid detection methods
  • - PCR for detection of nucleic acid
  • - ELISA assay for antigen detection
  • - Other immunohistochemical and
    immunoflourescence
  • examinations
  • - These are available only at certain labs

42
Gram Stain Analysis
  • Useful for cutaneous and inhalation anthrax.
  • A blood sample or skin lesion is taken from the
    patient and cultured for 6 to 24 hours.
  • Gram stain takes about 10 to 15 minutes.
  • Identify whether the bacteria come from the
    anthrax category.

Bacillus anthracis in Gram stain
43
Chest X-ray
  • Useful for inhalation and GI anthrax
  • Chest X-rays is advised as an initial method of
    inhalation anthrax detection, but it is sometimes
    not useful for patients without symptoms.
  • Find a widened mediastinum and pleural effusion.
  • Picture shows widened mediastinum caused by B.
    anthracis infection, resulting less available
    space in lungs

At day 1
At day 3
Inhalation Anthrax, Introduction, DRP, Armed
Forces Institute of Pathology
44
CT scan
  • Useful for inhalation and GI anthrax
  • Even when X-rays are negative, CT scans may
    provide more precise information.
  • Chest CT (Right) shows the increase in the size
    of the pleural effusions (accumulation of fluid
    in the pleural space).

Inhalation Anthrax, Introduction, DRP, Armed
Forces Institute of Pathology
45
PCR Assay
  • PCR is a target amplification method of nucleic
    acid based B. anthracis detection.
  • Used for the detection of anthrax toxin genes.
  • ex) rpoB gene - used as a specific chromosomal
    marker for
  • RT-PCR detection.
  • The rpoB gene was sequenced from 36 Bacillus
    strains
  • The assay was specific for 144 Bacillus anthracis
    strains from different geographical locations.
  • Provided 100 sensitivity and specificity

46
PCR Assay (2)
  • Detection time
  • - PCR only takes several hours
  • ex) Rapid-cycle RT-PCR can be finished within
    1-2 hours
  • Can start early treatment of Anthrax
  • There are many different types of PCR assays for
    the detection of Anthrax such as multiplex PCR,
    enterobacterial repetitive intergenic
    consensus-PCR (ERIC-PCR), and long-range
    repetitive element polymorphism-PCR.
  • Rapid diagnostic methods provide answers in
    minutes or hours instead of days.

47
Distinguishing inhalation Anthrax from cold or
influenza
  • Anthrax, cold, and influenza patients have
    similar symptoms at early phase such as flu-like
    symptoms (fever, chills, cough, and muscle aches
    etc.)
  • Symptoms of Anthrax do not include a runny nose,
    which is common in cold and influenza .
  • Anthrax involves severe breathing problems and
    more vomiting. These symptoms are not very common
    in cold or influenza.
  • Anthrax have high white blood cell counts and no
    increase in the number of lymphocytes.
  • Flu usually have low white blood cell counts and
    an increase in the number of lymphocytes.
  • Inhalation anthrax has abnormality in X-ray or CT
    scan

48
Treatment
  • Before 2001, 1st line of treatment was penicillin
    G
  • Stopped for fear of genetically engineered
    resistant strains
  • 60 day course of antibiotics
  • Ciprofloxacin
  • fluoroquinolone
  • 500 mg tablet every 12h or 400 mg IV every 12h
  • Inhibits DNA synthesis
  • Doxycycline
  • 6-deoxy-tetracycline
  • 100 mg tablet every 12h or 100 mg IV every 12h
  • Inhibits protein synthesis
  • For inhalational, need another antimicrobial
    agent
  • clindamycin
  • rifampin
  • chloramphenico

http//nmhm.washingtondc.museum/news/anthrax.html
49
Vaccine
  • BioThrax/Anthrax vaccine absorbed
  • Made by Bioport
  • Route of exposure not important
  • Administered subcutaneously
  • .5mL at 0, 2, and 4 weeks, and at 6, 12, 18
    months, booster doses at 1 yr intervals
  • PA from attenuated, nonencapsulated Sterne strain
    absorbed onto aluminum hydroxide
  • Contains no dead or live bacteria in the
    preparation
  • Antibodies to PA prevent binding to the target
    cell confer protection from anthrax.
  • 95 of vaccinated Rhesus monkeys survived lethal
    doses of inhaled anthrax
  • A December 22, 2003 ruling temporarily halted the
    Department of Defenses anthrax vaccination
    program
  • Lifting of that injunction on January 7, 2004

50
Who gets it?
  • People who work directly with it in the lab
  • People who work with imported animal hides or
    furs in areas where standards are insufficient to
    prevent exposure to anthrax spores.
  • People who handle potentially infected animal
    products in high-incidence areas
  • Military personnel deployed to areas with high
    risk for exposure to the organism.

51
Continuing research
  • Anthrax Immunity Gene in Mice
  • Kif1C
  • Four varieties (two resistant two susceptible)
  • Hepatitis drug
  • Hepsera
  • Blocks the toxic edema factor
  • Monoclonal antibodies
  • ABthrax from Human Genome Science
  • Phase 1 clinical trials
  • Neutralizes protective antigen
  • Identify other strains for improved, faster
    diagnosis

52
Weaponization Bacillus Anthracis Why is this
Agent Considered to be the Department of
Defenses Number-One/Two Biological Threat?
A sample of anthrax bacteria at the National
School of Biological Sciences, Mexico City
53
Why are Biological Agents Attractive Weapons?
  • Generally Less Expensive than Other WMD
  • The Poor Mans Nuke
  • Equivalent lethal effect from nuclear weapon
    would cost approximately 800 times as much (800
    invested in NW for every 1 put toward BW)
  • Dual Use - Is the Biological Material a Vaccine
    or Weapon?
  • Same equipment often used to produce both legal
    vaccines/ pharmaceuticals and BW
  • Perpetrator can deny agents were intended for use
    as BW
  • Helps to lower cost of BW facility if also
    involved in legal activity
  • Staff of trained professionals likely always
    available

54
Why are Biological Agents Attractive Weapons? (2)
  • Silent, Unnoticeable Attacks
  • Bombs bullets are loud and there effects often
    dramatic and widely evident - not the case with
    BW
  • BW can be tasteless, odorless, colorless and
    unnoticeable
  • Allows for more facile attacks on large
    populations
  • People could be inflicted and not immediately
    realize it - time lag

55
Why are Biological Agents Attractive Weapons? (3)
  • Plausible Deniability
  • State or terrorist group can easily deny
    deliverance
  • Proof difficult to come by - 2001 anthrax
    terrorists still not found likely never will be
    (very little evidence)
  • DNA sequencing of biological agent and matching
    it with an agent in the assumed perpetrators
    possession likely the only way to locate attacker
    - even sequencing is speculative

VS.
56
Why are Biological Agents Attractive Weapons? (4)
  • Lengthy Incubation Period
  • Most weapons act immediately, not usually the
    case with BW
  • Various BW agents have incubation periods of
    1-60 days on average
  • Can surprise opposition/victims and put them in
    survival mode rather than defense mode gt
    greater vulnerability
  • No way of knowing where to heighten security,
    often impossible to trace BW origin

57
Specific Benefits of Using Anthrax as a
Biological Weapon
  • Highly Lethal (Inhalational Anthrax)
  • Virtually 100 of exposed personnel will die
    from one breath of air with a high anthrax
    concentration (LD50 determined to be about
    8,000-10,000 spores or .08-.5 micrograms)
  • Inhalation of about 1,000 spores (.01?g) can
    cause pulmonary anthrax
  • 100,000 times deadlier than the deadliest
    chemical warfare agent
  • If treatment begins 48 hrs after symptoms,
    mortality still 95

58
Specific Benefits of Using Anthrax as a
Biological Weapon (2)
  • Non-contagious
  • Eliminates concern of spread from one person to
    another (sheep different)
  • Allows for anthrax to be targeted at specific
    populations w/o worry
  • In contrast, Smallpox and pneumonic plague are
    communicable
  • Easy to Protect with Advance Preparation
  • Enemy could vaccinate troops prior to an attack
    and/or antibiotics could be provided in order to
    mitigate diseases effects
  • Physical and psychological advantage for
    attacker - no harm in entering contaminated zone

59
Specific Benefits of Using Anthrax as a
Biological Weapon (3)
  • Long Shelf Life
  • Anthrax spores decay at a rate of less than one
    tenth of a percent per minute (very slow for an
    organism)
  • During WW II, Britain detonated experimental
    anthrax bombs on Gruinard Island - anthrax spores
    remained viable in top 15-20cm of soil for 40
    yrs until fully decontaminated in 1986
  • Anthrax is Stable in Many Various Types of
    Weapons Systems
  • Withstands the turbulence experienced from being
    sprayed/detonated
  • Can be loaded in munitions (freeze-dried
    condition) , disseminated as an aerosol with
    crude sprayers or even packaged in milled or
    un-milled powder form (ex. envelopes through
    mail)

60
Specific Benefits of Using Anthrax as a
Biological Weapon (4)
  • Short Incubation Period (Relative to Most Other
    BW)
  • Lag-time between attack and the first symptoms
    is only 1-6 days
  • Prediction of intended effect is much more
    facile to estimate
  • In contrast, bacterial agent brucellosis has an
    incubation of 5-60 days
  • UV Resistant
  • One of only two bacterial agents that is
    considered resistant to sunlight (the other being
    Coxiella)

61
Specific Benefits of Using Anthrax as a
Biological Weapon (5)
  • Widely Available
  • Animal disease - soil samples from all over the
    world contain anthrax
  • Approx. 1500 microbiologic repositories across
    the globe sell cultures to laboratories, vaccine
    companies and other entities (diagnostic/treatment
    )
  • Facile to Produce in Basic Form
  • Knowledge/technology available in open market
    with few controls
  • Cost is low (approximately 50 per kilogram in
    basic from)
  • One test tube of feed stock (samples of anthrax)
    in a fermenter can produce a kg of anthrax in
    about 96 hours
  • Any country with basic healthcare/pharmaceutical
    industry can produce

62
Specific Benefits of Using Anthrax as a
Biological Weapon (6)
  • Spores Naturally Occurring at 1-5 ?m
  • Optimal size for BW agent b/c it is right
    diameter to get to the bottom of the alveoli in
    the lungs - if too big, spores will stick to top
    of lung and will likely get blown back out
  • Anthrax Dangerous as Both a Powder and Liquid
  • Enhances perpetrators delivery options
  • Only Need a Small Amount for a Mass Effect
  • 1,763 lbs of nerve gas sarin, .2 lbs of Type A
    botulinum toxin or only .02 lbs of anthrax spores
    produce the same lethal effect

63
Locating the Threat of Anthrax
Who Has Weapons?
  • Exact of Countries Terrorist Groups Unknown
  • Intuitively, it would seem that any
    country/terrorist group that has an offensive BW
    program also has anthrax
  • Any country/terrorist group with
    biotech/pharmaceutical corporations and/or
    facilities could easily make anthrax
  • United States and Russia are only countries
    confirmed to currently be in possession of
    weaponized anthrax
  • Many other nations and terrorist groups believed
    to have anthrax

64
Locating the Threat of Anthrax
Who Has Weapons? (2)
  • Determining the Most Significant Threats
  • US Department of Defense More than seven
    countries including Iraq, Iran, Syria Russia
    have or are suspected of developing (anthrax)
    biological warfare capability1
  • US, Britain, Iraq, Germany, the USSR, Japan,
    South Africa Aum Shinrikyo (Japanese terrorist
    group) have used/tested anthrax as a weapon in
    the past - what state/group was responsible for
    2001 attacks?
  • US Department of State has identified seven
    states as sponsors of international terrorism
    Iran, Iraq, Syria, Libya, Cuba, Sudan North
    Korea

1 - DoD Response to the Staff Report of the
House Government Reforms Subcommittee on
National Security, 1, 2 and Information about
the Anthrax Vaccine, 2.
65
Locating the Threat of Anthrax
Who Has Weapons? (3)
  • Determining the Most Significant Threats
    (continued)
  • Secretary of the Air Force F. Whitten Peters to
    Senate Armed Services Committee on 07/21/99
    Anthrax has been weapon-ized and we know it is
    deployed in about 10 countries around the world.
    1
  • During the 1980s, some of the Soviet Unions
    intercontinental ballistic missiles (ICBM)
    reportedly were loaded with cocktails of BW
    agents (including anthrax) and targeted at major
    US cities (One ICBM could carry enough anthrax
    to wipe out the population of NYC). 2

1 - Peter Grier, Up in the Air about Anthrax,
Air Force Magazine 82, no. 10 (October 1999)
6871.

2 - Atlas, 160 and Raymond A.
Zilinskas, Verifying Compliance to the
Biological and Toxin Weapons Convention,
Critical Issues in Microbiology 24, no. 3 (1998)
195218.
66
Locating the Threat of Anthrax
Who Has Weapons? (4)
  • Determining the Most Significant Threats
    (continued)
  • 1991/92 UN Special Commission (UNSCOM)
    inspection Iraq definitely has BW, including
    anthrax
  • In 1995, Lt General Hussein Kamal (Saddam's
    Son-in-law former head of Iraqi BW program)
    told UN that
  • Iraq indeed had large stores of weaponizable
    anthrax and many weapons loaded with anthrax

    (bombs, Scuds, Al Hussayn warheads, 122 mm
    rockets, artillery shells, spray tanks for
    fighters
    and remotely piloted
    aircraft) 1
  • Iraq was able to hide much of its BW program in
    spite of the intense UNSCOM inspections - could
    other countries as well?

1 - Zilinskas, Verifying Compliance, 195218
and Rolf Ekéus, UN biological Inspections in
Iraq, in The New Terror Facing the Threat of
Biological and Chemical Weapons, 24647.
67
  • Locating the Threat of Anthrax
    Who Has Weapons? (5)

BW Programs by Country Sources of Information
Country ACDA DOD
FIS DOD Open Sources
( 1995-97) (1996-98) (1993)
(1988-90) (Pre-1993) Bulgaria         X China X
X   X X Cuba   X     X Egypt X   X   X India  
  X     Iran X X X X X Iraq X X X X X Israel    
X   X Laos         X Libya X X X X X North
Korea   X X X X Russia/Soviet Union X X   X X Sout
h Africa         X Syria X X   X X Taiwan X    
X X Vietnam         X
ACDA Arms Control Disarmament Agency DOD
Department of Defense
FIS Foreign
Intelligence Service of the Russian Federation

Source
W. Seth Carus, Biological Warfare Threats in
Perspective, Critical Issues in Microbiology 24,
no. 3 (1998) 154.
68
Weaponizing Anthrax
How is it made?
  • What Type of Anthrax to Use?
  • Inhalational (lungs)
  • Incredibly Lethal (untreated death rate gt90)
  • Facile attack methods (silent, flu-like, spray
    dispersible, etc.)
  • Cutaneous (skin)
  • Not near as lethal (untreated death rate 20)
  • More difficult to administer (need cut or
    abrasion)
  • Gastrointestinal (intestines)
  • Somewhat lethal (untreated death rate 25-60)
  • More difficult to administer (one has to consume
    anthrax)
  • Best Type of Anthrax for Use as Weapon
    INHALATIONAL

69
Weaponizing Anthrax
How is it made? (2)
  • Simplistic Approach
  • Grow bacteria culture (germination gt vegetation,
    in vitro _at_37º C)
  • Allow bacteria to sporulate, separate by
    filtration or centrifugation
  • Weaponize - what type of dispersal?
  • Wet dispersal
  • Spray liquid solution (droplets) over enemy
  • Dry dispersal
  • Dry with drying agent (ex. Magnesium Sulfate)
    powder
  • Aerosolize and spray over targeted population
  • Disperse in mail or by some other means
  • But is it this easy? Answer NO

70
Weaponizing Anthrax
How is it made? (3)
  • Wet Dispersal
  • Very difficult to formulate for effective use
  • When solution is sprayed, droplets tend to be
    large (ex. Windex forms droplets 100?m in size)
  • Droplets do not stay in air for long
  • Would need jet aircraft flying at 600mph or
    special high pressure sprayers that cost
    100,000 and would have to be mounted on a truck
  • Not practical
  • Solution Use Dry Powder

71
Weaponizing Anthrax
How is it made? (4)
  • Dry Dispersal
  • More facile, but not necessarily a walk in the
    park
  • After spore formation filtration/centrifugation,
    spores remaining cells will form sticky paste
    (consistency of peanut butter)
  • Paste dried down gt forms brick (or freeze
    dried)
  • Brick needs to be ground into fine (1 ?m) powder
    gt spores will have surface charge gt clumping
  • Need to neutralize static cling (similar to
    putting a sheet of Bounce in dryer)

72
Weaponizing Anthrax
How is it made? (5)
  • Neutralizing Static Cling of Spores
  • Exact formulations and recipes are classified
  • Basic approach is to coat spores with a fine
    silica or alumina clay (Iraqis use the chemical
    Bentonite)
  • Spores no longer clump, actually want to stay
    apart, repel each other gt do not stick to
    surfaces
  • Without surface charges, spores can easily
    re-aerosolize after coming in contact with
    objects/ground
  • Treated area can be infectious for a long time
    after dispersal (Increases danger and lethality
    of BW)

73
Weaponizing Anthrax
How is it made? (6)
  • How to Aerosolize?
  • Once anthrax is weapons-grade (appropriate size
    and w/o surface charges), can simply be dispersed
    by basic spray methods (such as a crop-duster
    plane)

74
The True Danger
Possible Effects Anthrax Attack
  • World Health Organization (WHO)
  • Estimates that 250,000 people would develop
    disease and 100,000 would die if 50kg of
    aerosolized anthrax was released from an aircraft
    over a developed urban population of five million
    (w/o treatment).1
  • US Congressional Office of Technology Assessment
  • Estimates that between 130,000 and 3 million
    deaths could follow the aerosolized release of
    100 kg of anthrax spores upwind of the
    Washington, DC, arealethality matching or
    exceeding that of a hydrogen bomb.2

1 - World Health Organization. Health Aspects of
Chemical and Biological Weapons. Geneva,
Switzerland World Health Organization
197098-99. 2 - Office of Technology Assessment,
US Congress. Proliferation of Weapons of Mass
Destruction. Washington, DC US Government
Printing Office 199353-55. Publication
OTA-ISC-559.
75
The True Danger
Possible Effects Anthrax Attack (2)
  • Centers for Disease Control and Prevention (CDC)
  • Produced an economic model that suggested a cost
    of 26.2 billion per 100,000 persons exposed to
    inhalational anthrax 1
  • Decontamination of Gruinard Island in the UK
  • Decontamination took place from 1979 to 1987
  • Total cost is unpublished (assumed to be 500,000
    British pounds), but materials required included
    280 tons of formaldehyde 2000 tons of seawater
    2
  • Decontamination of buildings/other areas equally
    as costly/difficult
  • 1 - Kaufmann AF, Meltzer MI, Schmid GP. The
    economic impact of a bioterrorist attack. Emerg
    Infect Dis. 1997383-94.
  • 2 - Titball RW, Turnbull PC, Hutson RA. The
    monitoring and detection of Bacillus anthracis in
    the environment. J Appl Bacteriol.
    199170(suppl)9S-18S.

76
  • The True Danger
    Possible Effects Anthrax Attack (3)
  • Sverdlovsk Accident (Russia BW Plant)
  • - New cases of inhalational anthrax developed as
    late as 43 days after the presumed date of
    release (lengthy period of lethality)
  • Guillermin J. Anthrax The Investigation of a
    Lethal Outbreak. Berkeley University of
    California Press. In press.

77
  • The True Danger
    Possible Effects Anthrax Attack (4)

Peters, C.J., D.M. Hartley. Anthrax Inhalation
and Lethal Human Infection. Lancet. 359 (9307)
710-711.
78
Analysis of the 2001 US Anthrax Attacks
  • Period From 10/02/01 - 11/20/01
  • 22 cases of anthrax infection, 11 inhalational
    (all confirmed) and 11 cutaneous (4 suspected 7
    confirmed)
  • 7 states CT(1), FL(2), MD(3), NJ(5), NYC(8),
    PA(1) VA(2)
  • 5 of 11 inhalational infections resulted in death
    (45 mortality rate)
  • All persons received immediate treatment upon
    onset of symptoms
  • Mean duration between exposure and onset of
    symptoms 4.5 days (estimate)

79
  • Analysis of the 2001 US Anthrax Attacks (2)
  • Above anthrax-containing envelopes postmarked
    September 18th, 2001
  • Above anthrax-containing envelopes postmarked
    October 9, 2001

Also believed to be three or more other
envelopes that were never found
80
  • Analysis of the 2001 US Anthrax Attacks (3)

http//www.anthraxinvestigation.com/
81
Analysis of the 2001 US Anthrax Attacks (4)
  • Anthrax in Envelopes
  • Concentration of about 1 trillion spores per gram
  • 2 grams anthrax per envelope
  • Each letter contained 200 million times average
    LD50
  • All anthrax was unmilled, contained a certain
    type of silica to reduce electrostatic charges
    and was of the Ames strain
  • all characteristic of US weapons-grade anthrax

82
Analysis of the 2001 US Anthrax Attacks (5)
  • Anthrax Found at American Media Inc. Building
    (Florida)
  • No letters found, but anthrax isolated in 90
    different locations in the building
  • Estimated cost of cleanup 7 million
  • Anthrax Found at Hart Senate Office (Washington
    D.C.)
  • 628 persons tested for bacillus anthracis, 28
    found positive
  • Cleanup took three months and cost an estimated
    23 million (with chlorine dioxide liquid)
  • 4 Envelopes Processed at Two Facilities - NYC
    Trenton
  • Both facilities tested positive for bacillus
    anthracis as well as at least 5 other facilities
    associated with Trenton facility

83
Analysis of the 2001 US Anthrax Attacks (6)
  • What Did These Attacks Exemplify?
  • Lethality of anthrax 5 of 11 inhalatory victims
    died (though provided with best possible
    treatment), less than 2 grams of anthrax per
    envelope
  • Facile spread of weaponized anthrax 7 states
    affected and only 4 letters found, rapid spread
    in buildings
  • Long shelf-life of anthrax some people in NYC
    were affected weeks after others (varying
    incubation periods?)
  • Incredibly high cost of clean-up/decontamination
    millions of
  • Difficulty of finding perpetrator still not
    found, likely never will be

84
Analysis of the 2001 US Anthrax Attacks (7)
  • Concerns Resulting from 2001 Attacks
  • What would have happened if more people were
    affected? Would the mortality rate have been
    higher?
  • Who created this weapons-grade anthrax?
    Terrorist group thought to be unlikely, but then
    what state sponsor? Did US lab employee(s) or
    government official(s) lend a hand to
    perpetrators?
  • How can the US prevent other similar attacks?
  • What would have happened if anthrax would have
    been aerosolized?

85
Defense Protection Against Anthrax
  • Vaccination
  • Very Effective 2 dose efficacy against up to
    1,000 LD50 in monkeys (human response believed to
    be very similar)
  • Early Detection
  • Extremely important How can the US learn about
    an anthrax attack before the appearance of
    symptoms? (would eliminate much of danger)
  • Time lag between exposure and symptoms is primary
    reason for the high mortality rate experienced
    with anthrax infections
  • A device similar to a smoke alarm or carbon
    monoxide detector?
  • Currently nothing on the market that could serve
    such a purpose, little research in this
    particular area

86
Defense Protection Against Anthrax (2)
  • Air Cleansers/Filters
  • Simply a reduction of risk, by no means an
    elimination
  • Appropriate for mailrooms, wool-sorting
    facilities, etc.
  • Irradiation of Susceptible Materials
  • Virtually eliminates threat of anthrax in mail
  • But is it worth it? Do the disadvantages
    outweigh the benefits?
  • Important Question To what point are we willing
    to protect ourselves from anthrax threat? What
    are we willing to sacrifice?

87
Defense Protection Against Anthrax (3)
  • Pre/Post Exposure Antibiotic Treatment
  • Isolates from the 2001 US attacks were sensitive
    to fluoroquinolones
  • Quite effective in mitigating effects of anthrax
    if caught at early stage
  • Decontamination of Exposed Areas
  • Often costly timely, but can be done rather
    successfully (using liquid chlorine dioxide or
    some other disinfectant)
  • Use of Protective Clothing Equipment
  • US military M17 M40 gas masks provide good
    protection against 1-5 ?m particles (cost 325)
  • Protective suits can be worn to easily eliminate
    cutaneous threat

88
Defense Protection Against Anthrax (4)
  • Only Reactionary, Post-Exposure Protection
    Methods Exist!
  • Besides vaccination/antibiotic treatment, there
    is no other preventative methods of protecting
    against anthrax
  • Most methods of protection are reactionary, this
    doesnt help to solve the main problem anthrax
    infections need to be determined earlier, before
    onset of symptoms
  • How can we currently protect ourselves from a
    massive bioterrorist attack? How can we make it
    more facile to distinguish btw. common maladies
    and anthrax?
  • PRIMARY OBJECTIVE NEED DEVICE/METHOD THAT WILL
    HELP TO PROTECT LARGE POPULATION FROM LARGE-SCALE
    ANTHRAX ATTACK

89
Anthrax Why it May Not be as Big of a Threat as
People Think
  • LD50 for Anthrax is Quite High
  • Smallpox 10-100 organisms, Q Fever
    1-10organisms, etc.
  • Very Difficult to Weaponize (Need State/Govt
    Support)
  • Aum Shinrikyo has failed to aerosolize anthrax on
    several occasions
  • Need sufficient nanotechnology engineering to be
    able to keep spores from clumping and sticking to
    surfaces (Army scientists could not weaponize
    anthrax when given the equipment a BW terrorist
    would likely have in his/her home)
  • Even if have Technology, Weapons-Grade Anthrax
    Very Costly
  • Only US Russia known to definitely have
    weaponized anthrax, large-scale production
    requires multi-million dollar investment

90
Anthrax Why it May Not be as Big of a Threat as
People Think (2)
  • Anthrax Strains are Highly Specific
  • Only certain strains are effective against
    humans, some are harmless
  • Lengthy Incubation
  • While this is also an advantage, it can serve as
    a disadvantage
  • Dont immediately know if weapon has struck
    target
  • Difficult to Use Anthrax Against Precise Targets
  • 2001 US attacks directed against high-status
    people, never reached targets
  • Aerosolization is most deadly, but also the most
    difficult to direct as a result of environmental
    conditions

91
  • Anthrax vs. Other BW Agents Brief
    Overview


www.nbc-med.org/SiteContent/HomePage/
WhatsNew/MedManual/Feb01/AppxC.doc -
92
  • http//www.sumanasinc.com/webcontent/anisamples/an
    i_anthrax.html
  • http//www.bt.cdc.gov/training/historyofbt/index.a
    sp
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