Bacillus anthracis
Hirotaka Ishibashi
Jennifer Jolivet
Sean Patrick Kelly
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
Endospore



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

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
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 > than ten
thousendfold on
macrophage cell
– ATR/TEM8 gene
 Chromosome 4
gib.genes.nig.ac.jp/single/ main.php?spid=Bant_AME
Where is Anthrax?
http://www.vetmed.lsu.edu/whocc/mp_world.htm
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 < 6.0)
– Dramatic changes in climate
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
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
Milestones in Anthrax History





Early history
1800s
1900s
Recent years
Outbreaks in Thailand and US
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
Pharaoh’s 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.
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 Sorter’s disease or Rag
Picker’s 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.
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.
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.
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
Hussein’s 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.
Outbreaks in Thailand

This picture is 9 days
after the onset of
symptoms of oralpharyngeal anthrax.
Thira Sirisanthana, Arthuer Brown, Anthrax of the
Gastrointestinal Tract, Emerging Infectious Diseases,
Vol. 8, 7, July 2002

1982 - In rural Northern
Thailand, an outbreak of 52
cases of cutaneous anthrax
and 24 cases of oralpharyngeal anthrax occurred.

Oral-pharyngeal anthrax: an
unusual manifestation of
human
infection with B. anthracis.

1987 - 14 cases of both oralpharyngeal and abdominal
anthrax occurred.

Caused by the consumption of
contaminated water and
buffalo meat.
Outbreaks in the US

In the early 1900’s 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.
Natural Outbreaks in the U.S. (1951–2003)
60
in 1970 human vaccination started
4745
3938
2003
23
9
7
6
3 5 5 2 3 4 2 5 2 2 2 2 2
0 0 1 0 0 0 1 0 0 0 2 0 0 0 1 0 0 0 0 0 0 0 1 0 0 0
20
03
16
12 14
19
95
22
26
19
75
70
60
50
40
30
20
10
0
19
51
19
55
Human cases
in 1957 animal vaccination started
• N = 409
Year
• 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/)
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
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/teksti/biolosko_orozje.htm
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
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
– PA+LF  lethal
activity
– EF+PA  edema
– EF+LF  inactive
– PA+LF+EF  edema &
necrosis; lethal
http://www.rcsb.org/pdb/molecules/pdb28_1.html
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.html
Mechanism of Infection

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
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
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 ionconductive channels
LF cleaves MAPKK 1 & 2
EF stimulates cAMP
http://kugi.kribb.re.kr/KUGI/Pathways/BioCarta/anthraxPathway/
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-α & interleukin-1β
– 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)
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
Clinical Information
Infection
 Symptoms (1st and 2nd phase)
 Three forms of Anthrax infection and
their Pathology
 Diagnosis

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.
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.
- Early Phase Symptoms 
Fever (temperature > 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
- 2nd Phase Symptoms 
Breathing problems, pneumonia

Shock

Swollen lymph glands

Profuse sweating

Cyanosis (skin turns blue)

Death
Three clinical forms of Anthrax

3 types of anthrax infection occur in humans:
1) Cutaneous
2) Inhalation
3) GI
Cutaneous Anthrax
http://science.howstuffworks.com/anthrax1.htm

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.
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%)
Cutaneous Anthrax (3)
CDC, Cutaneous Anthrax—Vesicle Development
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
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
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:
– 1–7 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%)
Gastrointestinal Anthrax
http://science.howstuffworks.com/anthrax1.htm

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.
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.
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
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
Gram Stain Analysis
Bacillus anthracis in Gram stain

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.
Chest X-ray
At day 1

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 3
Inhalation Anthrax, Introduction, DRP, Armed Forces Institute of Pathology
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
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
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 longrange repetitive element polymorphism-PCR.

Rapid diagnostic methods provide answers in minutes or
hours instead of days.
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
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
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
Defense’s anthrax vaccination program
– Lifting of that injunction on January 7, 2004
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.
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
Weaponization & Bacillus
Anthracis:
Why is this Agent Considered to be the Department of
Defense’s Number-One/Two Biological Threat?
A sample of anthrax bacteria at the National
School of Biological Sciences, Mexico City
Why are Biological Agents
Attractive Weapons?

Generally Less Expensive than Other WMD
– The “Poor Man’s 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
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
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 perpetrator’s
possession likely the only way to locate attacker even sequencing is speculative
VS.
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
==> greater vulnerability
– No way of knowing where to heighten
security, often impossible to trace BW origin
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%
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
disease’s effects
– Physical and psychological advantage for attacker - no
harm in entering contaminated zone
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)
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)
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
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 perpetrator’s 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
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
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 capability”1
– 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 Reform’s Subcommittee on
National Security,” 1, 2; and “Information about the Anthrax Vaccine,” 2.
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 Union’s
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): 68–71.
2 - Atlas, 160; and Raymond A. Zilinskas, “Verifying Compliance to the Biological and Toxin Weapons Convention,”
Critical Issues in Microbiology 24, no. 3 (1998): 195–218.
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-inlaw & 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,” 195–218; and Rolf Ekéus, “UN
biological Inspections in Iraq,” in The New Terror: Facing the Threat of
Biological and Chemical Weapons, 246–47.
Locating the Threat of Anthrax:
Who Has Weapons? (5)
BW Programs by Country & Sources of Information
Country
ACDA
DOD
( 1995-97) (1996-98)
Bulgaria
China
Cuba
Egypt
India
Iran
Iraq
Israel
Laos
Libya
North Korea
Russia/Soviet Union
South Africa
Syria
Taiwan
Vietnam
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
FIS
(1993)
X
X
X
X
X
DOD Open Sources
(1988-90) (Pre-1993)
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
ACDA = Arms Control & Disarmament Agency DOD = Department of Defense
= Foreign Intelligence Service of the Russian Federation
W. Seth Carus, “Biological Warfare Threats in Perspective,” Critical Issues in Microbiology 24, no. 3 (1998): 154.
FIS
Source:
Weaponizing Anthrax:
How is it made?

What Type of Anthrax to Use?
– Inhalational (lungs)
 Incredibly Lethal (untreated death rate >90%)
 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
Weaponizing Anthrax:
How is it made? (2)

Simplistic Approach:
– Grow bacteria culture (germination =>
vegetation, in vitro @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
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
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 ==> forms brick (or freeze dried)
 Brick needs to be ground into fine (1 m) powder
==> spores will have surface charge ==> clumping
 Need to neutralize “static cling” (similar to putting a
sheet of Bounce in dryer)
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 (Iraqi’s use the chemical Bentonite)
 Spores no longer clump, actually want to stay apart,
repel each other ==> do not stick to surfaces
 Without surface charges, spores can easily reaerosolize after coming in contact with
objects/ground
 Treated area can be infectious for a long time after
dispersal (Increases danger and lethality of BW)
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)
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, area—lethality 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; 1970:98-99.
2 - Office of Technology Assessment, US Congress. Proliferation of Weapons of Mass Destruction. Washington, DC:
US Government Printing Office; 1993:53-55. Publication OTA-ISC-559.
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. 1997;3:83-94.
2 - Titball RW, Turnbull PC, Hutson RA. The monitoring and detection of Bacillus anthracis in the
environment. J Appl Bacteriol. 1991;70(suppl):9S-18S.
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.
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.
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)
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
Analysis of the 2001 US Anthrax
Attacks (3)
http://www.anthraxinvestigation.com/
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
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
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
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?
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
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?
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
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 doesn’t
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
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/Gov’t 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
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
– Don’t 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
Anthrax vs. Other BW Agents:
Brief Overview
Disease
Inhalation
anthrax
Brucellosis
Trans mit
Man to Man
No
No
Infective Dose
(Aerosol)
8,000-50,000
spores
10 -100
organisms
Incubation
Period
1-6 days
Duration of Illness
Per sistence of Organism
Vaccine Efficacy
(ae rosol e xpos ure)
2 dose ef ficacy against up
to 1,000 LD50 in monkeys
No vaccine
<5% untreated
Very stable - spores remain
viable for > 40 years in soil
Very stable
> 1 week
Low with treatment,
high without
Unstable in aerosols & f resh
water; stable in salt water
No data on aerosol
Death in 7-10 days
in septicemic f orm
1-6 days
(usually f atal)
> 50%
Very stable
No vaccine
High unless treated
within 12-24 hours
For up to 1 year in soil; 270 days
in live tissue
2-10 days
(average 3-5)
10-40 days
> 2 weeks
2-14 days
Moderate if
untreated
Very low
For months in moist soil or other
media
For months on wood and sand
3 doses not protective
against 118 LD50 in
monkeys
80% protection against
1-10 LD50
94% protection against
3,500 LD50 in guinea pigs
Vaccine protects against
large doses in primates
5-60 days
(usually 1-2
months)
4 hours 5 days (usually
2-3 days)
10-14 days via
aerosol
2-3 days
3-5 days (usually
fatal if untreated)
Weeks to months
Le thality (approx.
cas e fatality rates)
High
Chole ra
Rare
10-500 organisms
Glanders
Low
Assumed low
Pneumonic
Plague
High
100-500
organisms
Tulare mia
No
10-50 organisms
Q Fe ver
Rare
1-10 organisms
Smallpox
High
7-17 days
(average 12)
4 weeks
High to moderate
Very stable
Vene zue lan
Equine
Ence phalitis
Viral
Hemorrhagic
Fevers
Botulism
Low
Assumed low
(10-100
organisms)
10-100 organisms
2-6 days
Days to weeks
Low
Relatively unstable
TC 83 protects against 30500 LD50 in hamsters
Moderate
1-10 organisms
4-21 days
Death between 7-16
days
Relatively unstable - depends on
agent
No vaccine
No
0.001 g/kg is
LD50 for type A
1-5 days
For weeks in nonmoving water
and food
Staph
Enterotoxin B
Ricin
No
0.03 g/person
incapacitation
3-5 g/kg is L D50
in mice
3-12 hours after
inhalation
18-24 hours
Death in 24-72
hours; lasts months
if not lethal
Hours
High for Zaire
strain, moderate
with Sudan
High without
respiratory support
< 1%
Resistant to f reezing
3 dose ef ficacy 100%
against 25-250 LD50 in
primates
No vaccine
High
Stable
No vaccine
T-2
Mycotoxins
No
Moderate
2-4 hours
Moderate
For years at room temperature
No vaccine
No
Days - death within
10-12 days for
ingestion
Days to months
www.nbc-med.org/SiteContent/HomePage/ WhatsNew/MedManual/Feb01/AppxC.doc -

http://www.sumanasinc.com/webcontent/
anisamples/ani_anthrax.html

http://www.bt.cdc.gov/training/historyofbt
/index.asp