bacillus anthracis hirotaka ishibashi jennifer jolivet sean patrick kelly

Bacillus anthracisBacillus anthracis

Hirotaka IshibashiJennifer Jolivet

Sean Patrick Kelly

Bacillus anthracisBacillus 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

EndosporeEndospore 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

GeneticsGenetics 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?Where is Anthrax?

http://www.vetmed.lsu.edu/whocc/mp_world.htm

AnthraxAnthrax From the Greek word From the Greek word anthrakosanthrakos for coal for coal Caused by sporesCaused by spores Primarily a disease of Primarily a disease of domesticated & wild domesticated & wild

animalsanimals– Herbivores such as sheep, cows, horses, goatsHerbivores such as sheep, cows, horses, goats

Natural reservoir is Natural reservoir is soilsoil– Does not depend on an animal reservoir Does not depend on an animal reservoir

making it hard to eradicatemaking it hard to eradicate– Cannot be regularly cultivated from soils Cannot be regularly cultivated from soils

where there is an absence of endemic anthrax where there is an absence of endemic anthrax – Occurs sporadically throughout US Occurs sporadically throughout US – South Dakota, Arkansas, Texas, Louisiana, South Dakota, Arkansas, Texas, Louisiana,

Mississippi, California recognized endemic Mississippi, California recognized endemic areasareas

Anthrax zonesAnthrax zones– Soil rich in organic matter (pH < 6.0) Soil rich in organic matter (pH < 6.0) – Dramatic changes in climateDramatic changes in climate

Anthrax Infection & SpreadAnthrax Infection & Spread May be spread by streams, insects, wild animals, May be spread by streams, insects, wild animals,

birds, contaminated wastesbirds, contaminated wastes

Animals infected by soilborne spores in food & water Animals infected by soilborne spores in food & water or bites from certain insectsor bites from certain insects

Humans can be infected when in contact with flesh, Humans can be infected when in contact with flesh, bones, hides, hair, & excrementbones, hides, hair, & excrement– nonindustrial or industrialnonindustrial or industrial– cutaneous & inhalational most commoncutaneous & inhalational most common

Risk of natural infection Risk of natural infection 1/100,0001/100,000– Outbreaks occur in endemic areas after outbreaks Outbreaks occur in endemic areas after outbreaks

in livestockin livestock

Three forms of AnthraxThree forms of Anthrax CutaneousCutaneous anthrax anthrax

– Skin Skin – Most commonMost common– Spores enter to skin through small lesionsSpores enter to skin through small lesions

InhalationInhalation anthraxanthrax– Spores are inhaledSpores are inhaled

GastrointestinalGastrointestinal (GI) (GI) anthraxanthrax– Spores are ingestedSpores are ingested– Oral-pharyngeal and abdominalOral-pharyngeal and abdominal

Milestones in Anthrax Milestones in Anthrax HistoryHistory

Early historyEarly history 1800s1800s 1900s1900s Recent yearsRecent years Outbreaks in Thailand and USOutbreaks in Thailand and US

History of Anthrax (Early History of Anthrax (Early history)history)

Although anthrax dates back more than 3,000 years, Although anthrax dates back more than 3,000 years, it was not recognized as a disease until the 18th it was not recognized as a disease until the 18th century.century.

1500 B.C - A “plague of boils” in Egypt affected the 1500 B.C - A “plague of boils” in Egypt affected the Pharaoh’s cattle. ‘Boils’ are symptomatic of anthrax.Pharaoh’s cattle. ‘Boils’ are symptomatic of anthrax.

1600s - The “Black Bane” thought to be anthrax, in 1600s - The “Black Bane” thought to be anthrax, in Europe kills over 60,000 cattle.Europe kills over 60,000 cattle.

1700s - There are some accounts of human cases.1700s - There are some accounts of human cases.

History (1800s)History (1800s)

Early 1800s - The first human cases of cutaneous Early 1800s - The first human cases of cutaneous anthrax in the US and UK were reported in men anthrax in the US and UK were reported in men who contracted the disease after having been in who contracted the disease after having been in contact with infected livestock.contact with infected livestock.

The disease was called Wool Sorter’s disease or The disease was called Wool Sorter’s disease or Rag Picker’s disease because it affected workers Rag Picker’s disease because it affected workers in those trades.in those trades.

1868 - Anthrax was observed under a microscope.1868 - Anthrax was observed under a microscope.

1876 - German bacteriologist Robert Koch 1876 - German bacteriologist Robert Koch confirmed bacterial origin of anthrax.confirmed bacterial origin of anthrax.

History (Early 1900s)History (Early 1900s)

1915 - German agents injected horses, mules, and 1915 - German agents injected horses, mules, and cattle with anthrax during WWI. This was the cattle with anthrax during WWI. This was the firstfirst recorded use of anthrax as a recorded use of anthrax as a biologicalbiological weaponweapon..

1937 - Japan started a biological warfare program in 1937 - Japan started a biological warfare program in Manchuria, including tests involving anthrax.Manchuria, including tests involving anthrax.

1942 - UK demonstrated experiments using anthrax at 1942 - UK demonstrated experiments using anthrax at Gruinard IslandGruinard Island off the coast of Scotland. off the coast of Scotland.

1943 - United States began developing anthrax 1943 - United States began developing anthrax weapons.weapons.

1945 - In 1945 - In Iran Iran an anthrax outbreak killed more than 1 an anthrax outbreak killed more than 1 million sheep.million sheep.

History (Late 1900s)History (Late 1900s) 1950s and 60s - U.S. biological warfare program 1950s and 60s - U.S. biological warfare program

continues after WWII at Fort Detrick, Marylandcontinues after WWII at Fort Detrick, Maryland

1969 - President Nixon ended United States' 1969 - President Nixon ended United States' offensive biological weapons program, but offensive biological weapons program, but defensive work still continues.defensive work still continues.

1970 - Anthrax vaccine for 1970 - Anthrax vaccine for humanshumans was approved was approved by U.S. FDA. by U.S. FDA.

1978-80 - The world's largest outbreak of human 1978-80 - The world's largest outbreak of human anthrax via insect vectors or contaminated meat anthrax via insect vectors or contaminated meat struck Zimbabwe, struck Zimbabwe, AfricaAfrica where more than 10,000 where more than 10,000 cases were recorded and over 180 people died.cases were recorded and over 180 people died.

1979 - In 1979 - In Soviet UnionSoviet Union, aerosolized anthrax , aerosolized anthrax spores were released accidentally at a military spores were released accidentally at a military facility, affecting 94 and killing 64 people.facility, affecting 94 and killing 64 people.

History (Recent years)History (Recent years) 1991 - About 150,000 U.S. troops were vaccinated 1991 - About 150,000 U.S. troops were vaccinated

for anthrax in preparation for for anthrax in preparation for Gulf WarGulf War..

1990-93 - The cult group, 1990-93 - The cult group, Aum ShinrikyoAum Shinrikyo, released , released anthrax spores in Tokyo, fortunately no one was anthrax spores in Tokyo, fortunately no one was injured. On February 27, 2004, the leader of this injured. On February 27, 2004, the leader of this group was given a sentence of death at a district group was given a sentence of death at a district court in Tokyo.court in Tokyo.

1995 - Iraq produced 8,500 liters of concentrated 1995 - Iraq produced 8,500 liters of concentrated anthrax as part of the biological weapon program anthrax as part of the biological weapon program under under Saddam Hussein’sSaddam Hussein’s administration. administration.

2001 - 2001 - LettersLetters containing anthrax spores were containing anthrax spores were mailed to many places in the US such as NBC, New mailed to many places in the US such as NBC, New York Times, and Media in Miami. In Florida, a man York Times, and Media in Miami. In Florida, a man died after inhaling anthrax at the office.died after inhaling anthrax at the office.

Outbreaks in ThailandOutbreaks in Thailand

This picture is 9 This picture is 9 days after the onset days after the onset of symptoms of oral-of symptoms of oral-pharyngeal anthrax.pharyngeal anthrax.

19821982 - In rural Northern - In rural Northern Thailand, an outbreak of 52 Thailand, an outbreak of 52 cases of cutaneous anthrax cases of cutaneous anthrax and 24 cases of oral-and 24 cases of oral-pharyngeal anthrax occurred.pharyngeal anthrax occurred.

Oral-pharyngeal anthrax: an Oral-pharyngeal anthrax: an unusual manifestation of unusual manifestation of humanhumaninfection with B. anthracis.infection with B. anthracis.

19871987 - 14 cases of both oral- - 14 cases of both oral- pharyngeal and abdominal pharyngeal and abdominal anthrax occurred.anthrax occurred.

Caused by the consumption Caused by the consumption of contaminated water and of contaminated water and buffalo meat.buffalo meat.

Thira Sirisanthana, Arthuer Brown, Thira Sirisanthana, Arthuer Brown, Anthrax of Anthrax of the Gastrointestinal Tractthe Gastrointestinal Tract, Emerging Infectious , Emerging Infectious Diseases, Vol. 8, 7, July 2002Diseases, Vol. 8, 7, July 2002

Outbreaks in the USOutbreaks in the US

In the early 1900’s approximately 130 cases occurred In the early 1900’s approximately 130 cases occurred annually due to the following reasons.annually due to the following reasons.

1) Agricultural, farm workers exposed to infected 1) Agricultural, farm workers exposed to infected animalsanimals

2) Processors exposed to infected animal products 2) Processors exposed to infected animal products (hair, (hair,

leather, wool, bone)leather, wool, bone)

3) Laboratory workers contacted with anthrax spores3) Laboratory workers contacted with anthrax spores

4) Civilians exposed to contaminated imported animal 4) Civilians exposed to contaminated imported animal productsproducts

These four are rare today.These four are rare today.

Natural Outbreaks in the U.S. (1951–2003)Natural Outbreaks in the U.S. (1951–2003)

60

4745

22

3938

26

1612

23

149

3 5 7 5 2 3 4 2 5 2 2 2 2 2 06

0 1 0 0 0 1 0 0 0 2 0 0 0 1 0 0 0 0 0 0 0 1 0 0 00

10203040506070

Year

Hum

an c

ases

in 1957 animal vaccination startedin 1957 animal vaccination started

20032003

• N = 409N = 409• 391 cases were cutaneous anthrax.391 cases were cutaneous anthrax.• 18 cases were inhalation anthrax18 cases were inhalation anthrax• GI anthrax has not been recognized yetGI anthrax has not been recognized yet• Since 1990 only 2 cases of cutaneous anthrax of naturally Since 1990 only 2 cases of cutaneous anthrax of naturally occurring infection have been reported.occurring infection have been reported.

Anthrax Overview PPT, CDC, 2001 Anthrax Overview PPT, CDC, 2001 (http://www.cdc.gov/)(http://www.cdc.gov/)

in 1970 human vaccination startedin 1970 human vaccination started

Natural Outbreaks in North Natural Outbreaks in North DakotaDakota

The The highesthighest occurrence of occurrence of Anthrax outbreaks in the USAnthrax outbreaks in the US

1989-1999 - 26 cases of 1989-1999 - 26 cases of infected livestock were infected livestock were reported.reported.

2000 - 33 cases were 2000 - 33 cases were reported during July-reported during July-September.September.

Total of 180 animals (beef Total of 180 animals (beef cattle, horses, and bison) cattle, horses, and bison) died and one person was died and one person was infected with cutaneous infected with cutaneous anthrax.anthrax.

Roughly, cases appear Roughly, cases appear every every 2 years2 years in North Dakota in North Dakota

PathogenesisPathogenesis The infectious dose of The infectious dose of

B. anthracisB. anthracis in humans in humans by any route is not by any route is not precisely known. precisely known. – Rely on primate dataRely on primate data– Minimum infection Minimum infection

dose of ~ 1,000-dose of ~ 1,000-8,000 spores8,000 spores

– LDLD5050 of 8,000-10,000 of 8,000-10,000 spores for inhalationspores for inhalation

Virulence depends on Virulence depends on 2 2 factorsfactors– CapsuleCapsule– 3 toxins3 toxins

http://www.kvarkadabra.net/index.html?/biologija/teksti/biolosko_orozje.htm

CapsuleCapsule GlycocalyxGlycocalyx

– Sticky, gelatinous Sticky, gelatinous polymer external to cell polymer external to cell wallwall

pX02 plasmidpX02 plasmid

Made up of Made up of D-glutamic D-glutamic acidacid

Non-toxic on its ownNon-toxic on its own

Only encapsulated Only encapsulated B. B. anthracisanthracis virulent virulent

Most important role Most important role during during establishment of establishment of diseasedisease– Protects against Protects against

phagocytosis & lysis phagocytosis & lysis during vegetative stateduring vegetative state

http://textbookofbacteriology.net/BSRP.html

ToxinsToxins pX01 plasmidpX01 plasmid AB modelAB model

– BindingBinding– ActivatingActivating

Protective antigenProtective antigen (PA), (PA), edema factoredema factor (EF) & (EF) & lethal factorlethal factor (LF)(LF)– Make up 50% of Make up 50% of

proteins in the proteins in the organismorganism

Individually non-toxicIndividually non-toxic– PA+LF PA+LF lethal lethal

activityactivity– EF+PA EF+PA edema edema – EF+LF EF+LF inactive inactive– PA+LF+EF PA+LF+EF edema edema

& necrosis; lethal& necrosis; lethal

http://www.rcsb.org/pdb/molecules/pdb28_1.html

Toxins (2)Toxins (2) Protective antigen (PA, Protective antigen (PA,

83kDa)83kDa)– PagPag gene gene– Binds to receptor & Binds to receptor &

helps internalize other 2 helps internalize other 2 proteinsproteins

Edema factor (EF, 89 kDa)Edema factor (EF, 89 kDa)– CyaCya gene gene– Adenylate cyclaseAdenylate cyclase– Affects all cellsAffects all cells

Lethal factor (LF, 87 kDa)Lethal factor (LF, 87 kDa)– LefLef gene gene– More important More important

virulence factorvirulence factor– MetalloproteaseMetalloprotease– Cleaves mitogen Cleaves mitogen

activated protein kinase activated protein kinase kinsase (MAPKK)kinsase (MAPKK)

– Affects only Affects only macrophagesmacrophages

http://www.ericse.org/anthrax/anthraxmicrographs.html

Mechanism of Infection Mechanism of Infection Anthrax spores enter bodyAnthrax spores enter body Germinate & multiple in Germinate & multiple in

lymph nodeslymph nodes PA, EF, LF excreted from PA, EF, LF excreted from

bacteriabacteria PA binds to TEM8. PA binds to TEM8. PA nicked by protease PA nicked by protease

furinfurin– 20-kDa segment off 20-kDa segment off

leaving 63-kDa peptideleaving 63-kDa peptide– Heptamer formsHeptamer forms

EF and/or LF bindsEF and/or LF binds Complex internalized by Complex internalized by

endocytosisendocytosis Acidification of endosomeAcidification of endosome LF or EF crosses into LF or EF crosses into

cytosol via PA mediated cytosol via PA mediated ion-conductive channelsion-conductive channels

LF cleaves MAPKK 1 & 2LF cleaves MAPKK 1 & 2 EF stimulates cAMPEF stimulates cAMP

http://kugi.kribb.re.kr/KUGI/Pathways/BioCarta/anthraxPathway/

OutcomeOutcome Do not understand exactly how symptoms occurDo not understand exactly how symptoms occur

EF converts EF converts ATP to cAMPATP to cAMP– Increases cAMP levels over 1,000 foldIncreases cAMP levels over 1,000 fold– Impairs neutrophil functionImpairs neutrophil function– Alters water homeostasisAlters water homeostasis– EdemaEdema

LF cleaves MAPKKLF cleaves MAPKK at its N terminus at its N terminus– Disrupts pathways involved in cell growth & maturationDisrupts pathways involved in cell growth & maturation– Increased synthesis of tumor necrosis factor-Increased synthesis of tumor necrosis factor-αα & &

interleukin-1interleukin-1ββ– Macrophage lysisMacrophage lysis– More cells infected with bacteria & toxinMore cells infected with bacteria & toxin– Septic shock & deathSeptic shock & death

Death probably results from high levels of bacteria secreting Death probably results from high levels of bacteria secreting LF toxins in bloodLF toxins in blood– At death, blood contains as many as At death, blood contains as many as 109 bacilli/ml109 bacilli/ml

(depending on the species)(depending on the species)

RegulatorsRegulators

Bicarbonate or Bicarbonate or COCO22 stimulates capsule and PA stimulates capsule and PA formationformation

LF requires LF requires zinczinc ions ions

EF requires EF requires calmodulincalmodulin, a major intracellular calcium , a major intracellular calcium receptorreceptor

Transcriptional regulator Transcriptional regulator AcpAAcpA on pX02 controls on pX02 controls expression of capsuleexpression of capsule

atxAatxA on pX01 is a positive regulator necessary for on pX01 is a positive regulator necessary for transcription of all 3 toxin genestranscription of all 3 toxin genes

Clinical InformationClinical Information

InfectionInfection Symptoms (1Symptoms (1stst and 2 and 2ndnd phase) phase) Three forms of Anthrax infection Three forms of Anthrax infection

and their Pathologyand their Pathology DiagnosisDiagnosis

Infection of AnthraxInfection of Anthrax The estimated number of naturally occurring human The estimated number of naturally occurring human

cases of anthrax in the world is 20,000 to 100,000 per cases of anthrax in the world is 20,000 to 100,000 per year.year.

Humans are infected through contact with infected Humans are infected through contact with infected animalsanimals and their products because of human and their products because of human intervention.intervention.

Anthrax spores contaminate the ground when an affected Anthrax spores contaminate the ground when an affected animal dies and can live in the animal dies and can live in the soilsoil for many years. for many years.

Anthrax can also be spread by eating Anthrax can also be spread by eating undercooked meatundercooked meat from infected animals.from infected animals.

Anthrax is Anthrax is NOTNOT transmitted from person to person. transmitted from person to person.

Humans can be exposed but not be infected.Humans can be exposed but not be infected.

What are the symptoms for What are the symptoms for anthrax?anthrax?

There are two phases of symptom.There are two phases of symptom.

1) 1) Early phaseEarly phase - Many symptoms can occur within 7 - Many symptoms can occur within 7 days of days of

infectioninfection

2) 2) 2nd phase2nd phase - Will hit hard, and usually occurs - Will hit hard, and usually occurs within 2 or 3 within 2 or 3

days after the early phase.days after the early phase.

- Early Phase Symptoms -- Early Phase Symptoms - Fever (temperature > 100 degrees F)Fever (temperature > 100 degrees F)

Chills or night sweatsChills or night sweats

Headache, cough, chest discomfort, sore throatHeadache, cough, chest discomfort, sore throat

Joint stiffness, joint pain, muscle achesJoint stiffness, joint pain, muscle aches

Shortness of breathShortness of breath

Enlarged lymph nodes, nausea, loss of appetite, Enlarged lymph nodes, nausea, loss of appetite, abdominal distress, vomiting, diarrheaabdominal distress, vomiting, diarrhea

MeningitisMeningitis

- 2nd Phase Symptoms -- 2nd Phase Symptoms -

Breathing problems, pneumoniaBreathing problems, pneumonia

ShockShock

Swollen lymph glandsSwollen lymph glands

Profuse sweatingProfuse sweating

Cyanosis (skin turns blue) Cyanosis (skin turns blue)

DeathDeath

Three clinical forms of Three clinical forms of AnthraxAnthrax

3 types of anthrax infection occur in humans:3 types of anthrax infection occur in humans:

1) 1) CutaneousCutaneous

2) 2) InhalationInhalation

3) 3) GIGI

Cutaneous Anthrax Cutaneous Anthrax 95%95% of anthrax infections of anthrax infections

occur when the bacterium occur when the bacterium enters a cut or scratch on the enters a cut or scratch on the skin due to handling of skin due to handling of contaminated animal products contaminated animal products or infected animals.or infected animals.

May also be spread by biting May also be spread by biting insects that have fed on insects that have fed on infected hosts. infected hosts.

After the spore germinates in After the spore germinates in skin tissues, toxin production skin tissues, toxin production initially results in itchy bump initially results in itchy bump that develops into a vesicle that develops into a vesicle and then painless black ulcer.and then painless black ulcer.

http://science.howstuffworks.com/anthrax1.htm

Cutaneous Anthrax (2)Cutaneous Anthrax (2)

The most common naturally occurring form of The most common naturally occurring form of anthrax. anthrax.

Ulcers are usually 1-3 cm in diameter.Ulcers are usually 1-3 cm in diameter.

Incubation period:Incubation period:– Usually an immediate response up to 1 dayUsually an immediate response up to 1 day

Case fatality after 2 days of infection: Case fatality after 2 days of infection: – Untreated (20%)Untreated (20%)– With antimicrobial therapy (1%)With antimicrobial therapy (1%)

Cutaneous Anthrax (3)Cutaneous Anthrax (3)

CDC, Cutaneous Anthrax—Vesicle DevelopmentCDC, Cutaneous Anthrax—Vesicle Development

Inhalation Anthrax Inhalation Anthrax The infection begins with The infection begins with

the inhalation of the the inhalation of the anthrax spore.anthrax spore.

Spores need to be less than Spores need to be less than 5 microns (millionths of a 5 microns (millionths of a meter) to reach the meter) to reach the alveolus.alveolus.

Macrophages lyse and Macrophages lyse and destroy some of the spores.destroy some of the spores.

Survived sporesSurvived spores are are transported to lymph transported to lymph nodes.nodes.

At least At least 2,500 spores2,500 spores have have to be inhaled to cause an to be inhaled to cause an infection.infection.

Inhalation Anthrax, Introduction, DRP, Armed Forces Institute of PathologyInhalation Anthrax, Introduction, DRP, Armed Forces Institute of Pathology

Inhalation Anthrax (2)Inhalation Anthrax (2) Disease immediately follows Disease immediately follows

germination.germination.

Spores replicate in the Spores replicate in the lymph nodes.lymph nodes.

The two lungs are separated The two lungs are separated by a structure called the by a structure called the mediastinummediastinum, which , which contains the heart, trachea, contains the heart, trachea, esophagus, and blood esophagus, and blood vessels.vessels.

Bacterial toxins released Bacterial toxins released during replication result in during replication result in mediastinal widening and mediastinal widening and pleural effusions pleural effusions (accumulation of fluid in the (accumulation of fluid in the pleural space)pleural space)..


Inhalation Anthrax (3)Inhalation Anthrax (3) Death usually results 2-3 days after the onset of Death usually results 2-3 days after the onset of

symptoms.symptoms.

Natural infection is extremely rare (in the US, 20 Natural infection is extremely rare (in the US, 20 cases were reported in last century).cases were reported in last century).

Inhalation Anthrax is the Inhalation Anthrax is the most lethal typemost lethal type of of Anthrax.Anthrax.

Incubation period:Incubation period:– 1–7 days1–7 days– Possibly ranging up to 42 days (depending on how Possibly ranging up to 42 days (depending on how

many spores were inhaled).many spores were inhaled).

Case fatality after 2 days of infection: Case fatality after 2 days of infection: – Untreated (97%)Untreated (97%)– With antimicrobial therapy (75%)With antimicrobial therapy (75%)

Gastrointestinal Anthrax Gastrointestinal Anthrax

GI anthrax may follow GI anthrax may follow after the consumption after the consumption of contaminated, of contaminated, poorly cooked meat.poorly cooked meat.

There are 2 different There are 2 different forms of GI anthrax:forms of GI anthrax:1) Oral-pharyngeal1) Oral-pharyngeal2) Abdominal2) Abdominal

Abdominal anthrax is Abdominal anthrax is more common than more common than the oral-pharyngeal the oral-pharyngeal form.form.

http://science.howstuffworks.com/anthrax1.htmhttp://science.howstuffworks.com/anthrax1.htm

GI Anthrax (2)GI Anthrax (2)

Oral-pharyngeal formOral-pharyngeal form - results from the deposition - results from the deposition and germination of spores in the upper and germination of spores in the upper gastrointestinal tract.gastrointestinal tract.

Local lumphadenopathy (an infection of the lymph Local lumphadenopathy (an infection of the lymph glands and lymph channels), edema, sepsis glands and lymph channels), edema, sepsis develop after an oral or esophageal ulcer.develop after an oral or esophageal ulcer.

Abdominal formAbdominal form - develops from the deposition and - develops from the deposition and germination of spores in the lower gastrointestinal germination of spores in the lower gastrointestinal tract, which results in a primary intestinal lesion.tract, which results in a primary intestinal lesion.

Symptoms such as abdominal pain and vomiting Symptoms such as abdominal pain and vomiting appear within a few days after ingestion.appear within a few days after ingestion.

GI Infection (3)GI Infection (3) GI anthrax cases are GI anthrax cases are uncommonuncommon..

There have been reported outbreaks in Zimbabwe, There have been reported outbreaks in Zimbabwe, Africa and northern Thailand in the world.Africa and northern Thailand in the world.

GI anthrax has not been reported in the US. GI anthrax has not been reported in the US.

Incubation period:Incubation period:– 1-7 days1-7 days

Case fatality at 2 days of infection: Case fatality at 2 days of infection: – Untreated (25-60%)Untreated (25-60%)– With antimicrobial therapy (undefined) due to the With antimicrobial therapy (undefined) due to the

rarityrarity

How is anthrax diagnosed?How is anthrax diagnosed?

Gram stainGram stain

Culture of Culture of B. anthracisB. anthracis from the blood, skin lesions, from the blood, skin lesions, vesicular fluid, or respiratory secretionsvesicular fluid, or respiratory secretions

X-ray and Computed Tomography (CT) scanX-ray and Computed Tomography (CT) scan

Rapid detection methodsRapid detection methods- PCR for detection of nucleic acid- PCR for detection of nucleic acid- ELISA assay for antigen detection- ELISA assay for antigen detection- Other immunohistochemical and - Other immunohistochemical and immunoflourescence immunoflourescence examinations examinations - These are available only at certain labs- These are available only at certain labs

Gram Stain AnalysisGram Stain Analysis

Useful for cutaneous Useful for cutaneous and inhalation anthrax.and inhalation anthrax.

A blood sample or skin A blood sample or skin lesion is taken from the lesion is taken from the patient and cultured for patient and cultured for 6 to 24 hours6 to 24 hours. .

Gram stain takes about Gram stain takes about 10 to 15 minutes10 to 15 minutes..

Identify whether the Identify whether the bacteria come from the bacteria come from the anthrax category.anthrax category.

Bacillus anthracisBacillus anthracis in Gram in Gram stainstain

Chest X-rayChest X-ray Useful for inhalation and GI Useful for inhalation and GI

anthrax anthrax

Chest X-rays is advised as Chest X-rays is advised as an initial method of an initial method of inhalation anthrax inhalation anthrax detection, but it is detection, but it is sometimessometimes not useful for not useful for patients without symptoms.patients without symptoms.

Find Find a widened a widened mediastinum and pleural mediastinum and pleural effusion.effusion.

Picture shows Picture shows widened widened mediastinummediastinum caused by caused by B. B. anthracis anthracis infectioninfection, , resulting less available resulting less available space in lungsspace in lungs


At day 1At day 1 At day 3At day 3

CT scanCT scan

Useful for inhalation and GI anthraxUseful for inhalation and GI anthrax Even when X-rays are negative, CT scans may provide Even when X-rays are negative, CT scans may provide

more precise information.more precise information. Chest CT (Right) shows the increase in the size of the Chest CT (Right) shows the increase in the size of the

pleural effusionspleural effusions (accumulation of fluid in the pleural (accumulation of fluid in the pleural space)space)..


PCR PCR Assay Assay PCR is a target amplification method of nucleic acid PCR is a target amplification method of nucleic acid

based B. anthracis detection.based B. anthracis detection.

Used for the detection of anthrax toxin genes.Used for the detection of anthrax toxin genes.ex) rpoB gene - used as a specific chromosomal ex) rpoB gene - used as a specific chromosomal marker for marker for RT-PCR detection. RT-PCR detection.

The rpoB gene was sequenced from 36 Bacillus The rpoB gene was sequenced from 36 Bacillus strainsstrains

The assay was specific for 144 Bacillus anthracis The assay was specific for 144 Bacillus anthracis strains from different geographical locations.strains from different geographical locations.

Provided Provided 100%100% sensitivity and specificity sensitivity and specificity

PCR PCR Assay (2)Assay (2) Detection time:Detection time:

- PCR only takes - PCR only takes severalseveral hours hours ex) Rapid-cycle RT-PCR can be finished within ex) Rapid-cycle RT-PCR can be finished within 1-2 1-2 hourshours

Can start Can start early treatment early treatment of Anthraxof Anthrax

There are many different types of PCR assays for There are many different types of PCR assays for the detection of Anthrax such as multiplex PCR, the detection of Anthrax such as multiplex PCR, enterobacterial repetitive intergenic consensus-PCR enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR), and long-range repetitive element (ERIC-PCR), and long-range repetitive element polymorphism-PCR.polymorphism-PCR.

Rapid diagnostic methods provide answers in Rapid diagnostic methods provide answers in minutes or hours instead of days.minutes or hours instead of days.

Distinguishing inhalation Anthrax Distinguishing inhalation Anthrax from cold or influenzafrom cold or influenza

Anthrax, cold, and influenza patients have similar Anthrax, cold, and influenza patients have similar symptoms at early phase such as flu-like symptoms (fever, symptoms at early phase such as flu-like symptoms (fever, chills, cough, and muscle aches etc.)chills, cough, and muscle aches etc.)

Symptoms of Anthrax do not include a Symptoms of Anthrax do not include a runny noserunny nose, which is , which is common in cold and influenza .common in cold and influenza .

Anthrax involves severe breathing problems and more Anthrax involves severe breathing problems and more vomiting. These symptoms are not very common in cold or vomiting. These symptoms are not very common in cold or influenza. influenza.

Anthrax have high white blood cell counts and no increase Anthrax have high white blood cell counts and no increase in the number of lymphocytes.in the number of lymphocytes.

Flu usually have low white blood cell counts and an Flu usually have low white blood cell counts and an increase in the number of lymphocytes. increase in the number of lymphocytes.

Inhalation anthrax has abnormality in X-ray or CT scanInhalation anthrax has abnormality in X-ray or CT scan

TreatmentTreatment 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

VaccineVaccine BioThrax/Anthrax vaccine absorbedBioThrax/Anthrax vaccine absorbed

– Made by BioportMade by Bioport– Route of exposure not importantRoute of exposure not important

Administered subcutaneously Administered subcutaneously

– .5mL at 0, 2, and 4 weeks, and at 6, 12, & 18 months, & .5mL at 0, 2, and 4 weeks, and at 6, 12, & 18 months, & booster doses at 1 yr intervalsbooster doses at 1 yr intervals

PA from PA from attenuated, nonencapsulatedattenuated, nonencapsulated Sterne strain Sterne strain absorbed onto aluminum hydroxideabsorbed onto aluminum hydroxide– Contains no dead or live bacteria in the preparation Contains no dead or live bacteria in the preparation – Antibodies to PA prevent binding to the target cell & Antibodies to PA prevent binding to the target cell &

confer protection from anthrax. confer protection from anthrax.

95% of vaccinated Rhesus monkeys survived lethal doses 95% of vaccinated Rhesus monkeys survived lethal doses of inhaled anthrax of inhaled anthrax

A December 22, 2003 ruling temporarily halted the A December 22, 2003 ruling temporarily halted the Department of Defense’s anthrax vaccination program Department of Defense’s anthrax vaccination program – Lifting of that injunction on January 7, 2004Lifting of that injunction on January 7, 2004

Who gets it?Who gets it?

People who work directly with it in the labPeople who work directly with it in the lab

People who work with imported animal hides or furs People who work with imported animal hides or furs in areas where standards are insufficient to prevent in areas where standards are insufficient to prevent exposure to anthrax spores.exposure to anthrax spores.

People who handle potentially infected animal People who handle potentially infected animal products in high-incidence areasproducts in high-incidence areas

Military personnel deployed to areas with high risk Military personnel deployed to areas with high risk for exposure to the organism. for exposure to the organism.

Continuing researchContinuing research

Anthrax Immunity Gene in Mice Anthrax Immunity Gene in Mice – Kif1C Kif1C – Four varieties (two resistant & two susceptible) Four varieties (two resistant & two susceptible)

Hepatitis drugHepatitis drug– Hepsera Hepsera – Blocks the toxic edema factorBlocks the toxic edema factor

Monoclonal antibodiesMonoclonal antibodies– ABthrax from Human Genome ScienceABthrax from Human Genome Science– Phase 1 clinical trialsPhase 1 clinical trials– Neutralizes protective antigenNeutralizes protective antigen

Identify other strains for improved, faster diagnosisIdentify other strains for improved, faster diagnosis

Weaponization & Weaponization & Bacillus Bacillus AnthracisAnthracis::

Why is this Agent Considered to be the Department of Why is this Agent Considered to be the Department of Defense’s Number-One/Two Biological Threat?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 Why are Biological Agents Attractive Weapons?Attractive Weapons?

GenerallyGenerally Less Expensive than Other WMDLess Expensive than Other WMD– The “Poor Man’s Nuke”The “Poor Man’s Nuke”– Equivalent lethal effect from nuclear weapon Equivalent lethal effect from nuclear weapon

would cost approximately 800 times as much would cost approximately 800 times as much ($800 invested in NW for every $1 put toward BW)($800 invested in NW for every $1 put toward BW)

Dual UseDual Use - Is the Biological Material a - Is the Biological Material a Vaccine or Weapon?Vaccine or Weapon?– Same equipment often used to produce both legal Same equipment often used to produce both legal

vaccines/ pharmaceuticals and BWvaccines/ pharmaceuticals and BW– Perpetrator can deny agents were intended for Perpetrator can deny agents were intended for

use as BW use as BW – Helps to lower cost of BW facility if also involved Helps to lower cost of BW facility if also involved

in legal activityin legal activity– Staff of trained professionals likely always Staff of trained professionals likely always

availableavailable

Why are Biological Agents Why are Biological Agents Attractive Weapons? (2)Attractive Weapons? (2)

Silent, Unnoticeable Silent, Unnoticeable AttacksAttacks– Bombs & bullets are Bombs & bullets are

loud and there effects loud and there effects often dramatic and often dramatic and widely evident - not the widely evident - not the case with BWcase with BW

– BW can be tasteless, BW can be tasteless, odorless, colorless and odorless, colorless and unnoticeable unnoticeable

– Allows for more facile Allows for more facile attacks on large attacks on large populationspopulations

– People could be People could be inflicted and not inflicted and not immediately realize it - immediately realize it - time lagtime lag


Plausible DeniabilityPlausible Deniability– State or terrorist group can easily deny State or terrorist group can easily deny

deliverancedeliverance– Proof difficult to come by - 2001 anthrax Proof difficult to come by - 2001 anthrax

terrorists still not found & likely never will be terrorists still not found & likely never will be (very little evidence) (very little evidence)

– DNA sequencing of biological agent and DNA sequencing of biological agent and matching it with an agent in the assumed matching it with an agent in the assumed perpetrator’s possession likely the only way to perpetrator’s possession likely the only way to locate attacker - even sequencing is locate attacker - even sequencing is speculativespeculative

VS.


Lengthy IncubationLengthy Incubation PeriodPeriod– Most weapons act immediately, not Most weapons act immediately, not

usually the case with BWusually the case with BW– Various BW agents have incubation Various BW agents have incubation

periods of 1-60 days on averageperiods of 1-60 days on average– Can surprise opposition/victims and Can surprise opposition/victims and

put them in “survival” mode rather than put them in “survival” mode rather than defense mode ==> greater vulnerabilitydefense mode ==> greater vulnerability

– No way of knowing where to heighten No way of knowing where to heighten security, often impossible to trace BW originsecurity, often impossible to trace BW origin

Specific Benefits of Using Specific Benefits of Using Anthrax as a Biological Anthrax as a Biological

WeaponWeapon Highly Lethal (Inhalational Anthrax)Highly Lethal (Inhalational Anthrax)

– Virtually 100% of exposed personnel will die Virtually 100% of exposed personnel will die from one breath of air with a high anthrax from one breath of air with a high anthrax concentration (LD50 determined to be about 8,000-concentration (LD50 determined to be about 8,000-10,000 spores or .08-.5 micrograms)10,000 spores or .08-.5 micrograms)

– Inhalation of about 1,000 spores (.01Inhalation of about 1,000 spores (.01g) can g) can cause pulmonary anthraxcause pulmonary anthrax

– 100,000 times deadlier than the deadliest 100,000 times deadlier than the deadliest chemical warfare agentchemical warfare agent

– If treatment begins 48 hrs after symptoms, If treatment begins 48 hrs after symptoms, mortality still ~95%mortality still ~95%


Weapon (2)Weapon (2) Non-contagiousNon-contagious

– Eliminates concern of spread from one Eliminates concern of spread from one person to another (sheep different)person to another (sheep different)

– Allows for anthrax to be targeted at specific Allows for anthrax to be targeted at specific populations w/o worrypopulations w/o worry

– In contrast, Smallpox and pneumonic In contrast, Smallpox and pneumonic plague are communicableplague are communicable

Easy to Protect with Advance PreparationEasy to Protect with Advance Preparation– Enemy could vaccinate troops prior to an Enemy could vaccinate troops prior to an

attack and/or antibiotics could be provided in attack and/or antibiotics could be provided in order to mitigate disease’s effectsorder to mitigate disease’s effects

– Physical and psychological advantage for Physical and psychological advantage for attacker - no harm in entering contaminated zoneattacker - no harm in entering contaminated zone


Weapon (3)Weapon (3) Long Shelf LifeLong Shelf Life

– Anthrax spores decay at a rate of less than Anthrax spores decay at a rate of less than one tenth of a percent per minute (very slow for one tenth of a percent per minute (very slow for an organism)an organism)

– During WW II, Britain detonated During WW II, Britain detonated experimental anthrax bombs on Gruinard Island - experimental anthrax bombs on Gruinard Island - anthrax spores remained viable in top 15-20cm of anthrax spores remained viable in top 15-20cm of soil for ~40 yrs until fully decontaminated in 1986soil for ~40 yrs until fully decontaminated in 1986

Anthrax is Stable in Many Various Types of Weapons Anthrax is Stable in Many Various Types of Weapons SystemsSystems– Withstands the turbulence experienced Withstands the turbulence experienced

from being sprayed/detonatedfrom being sprayed/detonated– Can be loaded in munitions (freeze-dried Can be loaded in munitions (freeze-dried

condition) , disseminated as an aerosol with crude condition) , disseminated as an aerosol with crude sprayers or even packaged in milled or un-milled sprayers or even packaged in milled or un-milled powder form (ex. envelopes through mail)powder form (ex. envelopes through mail)


Weapon (4)Weapon (4) Short Incubation Period (Relative to Most Short Incubation Period (Relative to Most

Other BW)Other BW)– Lag-time between attack and the first Lag-time between attack and the first

symptoms is only 1-6 dayssymptoms is only 1-6 days– Prediction of intended effect is much more Prediction of intended effect is much more

facile to estimatefacile to estimate– In contrast, bacterial agent brucellosis has an In contrast, bacterial agent brucellosis has an

incubation of 5-60 daysincubation of 5-60 days

UV ResistantUV Resistant– One of only two bacterial agents that is One of only two bacterial agents that is

considered resistant to sunlight (the other being considered resistant to sunlight (the other being Coxiella)Coxiella)


Weapon (5)Weapon (5) Widely AvailableWidely Available

– Animal disease - soil samples from all over the Animal disease - soil samples from all over the world contain anthraxworld contain anthrax

– Approx. 1500 microbiologic repositories across Approx. 1500 microbiologic repositories across the globe sell cultures to laboratories, vaccine the globe sell cultures to laboratories, vaccine companies and other entities (diagnostic/treatment)companies and other entities (diagnostic/treatment)

Facile to Produce in Basic FormFacile to Produce in Basic Form– Knowledge/technology available in open Knowledge/technology available in open

market with few controlsmarket with few controls– Cost is low (approximately $50 per kilogram in Cost is low (approximately $50 per kilogram in

basic from) basic from) – One test tube of feed stock (samples of One test tube of feed stock (samples of

anthrax) in a fermenter can produce a kg of anthrax anthrax) in a fermenter can produce a kg of anthrax in about 96 hoursin about 96 hours

– Any country with basic Any country with basic healthcare/pharmaceutical industry can producehealthcare/pharmaceutical industry can produce


Weapon (6)Weapon (6) Spores Naturally Occurring at 1-5 Spores Naturally Occurring at 1-5 mm

– Optimal size for BW agent b/c it is right Optimal size for BW agent b/c it is right diameter to get to the bottom of the alveoli in the diameter to get to the bottom of the alveoli in the lungs - if too big, spores will stick to top of lung and lungs - if too big, spores will stick to top of lung and will likely get blown back outwill likely get blown back out

Anthrax Dangerous as Both a Powder and Anthrax Dangerous as Both a Powder and LiquidLiquid– Enhances perpetrator’s delivery optionsEnhances perpetrator’s delivery options

Only Need a Small Amount for a Mass EffectOnly Need a Small Amount for a Mass Effect– 1,763 lbs of nerve gas sarin, .2 lbs of Type A 1,763 lbs of nerve gas sarin, .2 lbs of Type A

botulinum toxin or only .02 lbs of anthrax spores botulinum toxin or only .02 lbs of anthrax spores produce the same lethal effectproduce the same lethal effect

Locating the Threat of Locating the Threat of Anthrax: Anthrax:

Who Has Weapons? Who Has Weapons? Exact # of Countries & Terrorist Exact # of Countries & Terrorist

Groups UnknownGroups Unknown– Intuitively, it would seem that any Intuitively, it would seem that any

country/terrorist group that has an country/terrorist group that has an offensive BW program also has anthraxoffensive BW program also has anthrax

– Any country/terrorist group with Any country/terrorist group with biotech/pharmaceutical corporations biotech/pharmaceutical corporations and/or facilities could easily make and/or facilities could easily make anthrax anthrax

– United States and Russia are only United States and Russia are only countries confirmed to currently be in countries confirmed to currently be in possession of weaponized anthrax possession of weaponized anthrax

– Many other nations and terrorist groups Many other nations and terrorist groups believed to have anthraxbelieved to have anthrax

Locating the Threat of Locating the Threat of Anthrax: Anthrax: Who Has Weapons? (2) Who Has Weapons? (2)

Determining the Most Significant ThreatsDetermining the Most Significant Threats– US Department of Defense: “More than seven US Department of Defense: “More than seven

countries including Iraq, Iran, Syria & Russia countries including Iraq, Iran, Syria & Russia have or are suspected of developing have or are suspected of developing (anthrax) biological warfare capability”(anthrax) biological warfare capability”11

– US, Britain, Iraq, Germany, the USSR, Japan, US, Britain, Iraq, Germany, the USSR, Japan, South Africa & Aum Shinrikyo (Japanese South Africa & Aum Shinrikyo (Japanese terrorist group) have used/tested anthrax as terrorist group) have used/tested anthrax as a weapon in the past - what state/group was a weapon in the past - what state/group was responsible for 2001 attacks?responsible for 2001 attacks?

– US Department of State has identified seven US Department of State has identified seven states as sponsors of international terrorism: states as sponsors of international terrorism: Iran, Iraq, Syria, Libya, Cuba, Sudan & North Iran, Iraq, Syria, Libya, Cuba, Sudan & North KoreaKorea

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.


Determining the Most Significant Determining the Most Significant Threats (continued)Threats (continued)– Secretary of the Air Force F. Whitten Secretary of the Air Force F. Whitten

Peters to Senate Armed Services Peters to Senate Armed Services Committee on 07/21/99: “[Anthrax] has Committee on 07/21/99: “[Anthrax] has been weapon-ized and we know it is been weapon-ized and we know it is deployed in about 10 countries around the deployed in about 10 countries around the world.” world.” 11

– During the 1980s, some of the Soviet During the 1980s, some of the Soviet Union’s intercontinental ballistic missiles Union’s intercontinental ballistic missiles (ICBM) reportedly were loaded with (ICBM) reportedly were loaded with “cocktails” of BW agents (including “cocktails” of BW agents (including anthrax) and targeted at major US cities anthrax) and targeted at major US cities (One ICBM could carry enough anthrax to (One ICBM could carry enough anthrax to wipe out the population of NYC). wipe out the population of NYC). 22

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.


Determining the Most Significant Threats Determining the Most Significant Threats (continued)(continued)– 1991/92 UN Special Commission (UNSCOM) 1991/92 UN Special Commission (UNSCOM)

inspection: Iraq definitely has BW, including anthraxinspection: Iraq definitely has BW, including anthrax– In 1995, Lt General Hussein Kamal (Saddam's Son-In 1995, Lt General Hussein Kamal (Saddam's Son-

in-law & former head of Iraqi BW program) told UN in-law & former head of Iraqi BW program) told UN that: that: Iraq indeed had large stores of weaponizable anthrax Iraq indeed had large stores of weaponizable anthrax

and many weapons loaded with anthrax and many weapons loaded with anthrax (bombs, Scuds, Al Hussayn (bombs, Scuds, Al Hussayn warheads, 122 mm rockets, artillery shells, spray tanks warheads, 122 mm rockets, artillery shells, spray tanks for fighters and for fighters and remotely piloted aircraft) remotely piloted aircraft) 11

– Iraq was able to hide much of its BW program in Iraq was able to hide much of its BW program in spite of the intense UNSCOM inspections - could spite of the intense UNSCOM inspections - could other countries as well?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 Locating the Threat of Anthrax: Anthrax:

Who Has Weapons? Who Has Weapons? (5)(5)

Country ACDA DOD FIS DOD Open Sources ( 1995-97) (1996-98) (1993) (1988-90) (Pre-1993)Bulgaria XChina X X X XCuba X XEgypt X X XIndia X Iran X X X X XIraq X X X X XIsrael X XLaos XLibya X X X X XNorth Korea X X X XRussia/Soviet Union X X X XSouth Africa XSyria X X X XTaiwan X X XVietnam X

BW Programs by Country & Sources of InformationBW Programs by Country & Sources of Information

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.

Weaponizing Anthrax: Weaponizing Anthrax: How is it How is it

made?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: Best Type of Anthrax for Use as Weapon:

INHALATIONALINHALATIONAL


made? (2)made? (2) Simplistic Approach:Simplistic Approach:

– Grow bacteria culture (germination => Grow bacteria culture (germination => vegetation, in vitro @37º C)vegetation, in vitro @37º C)

– Allow bacteria to sporulate, separate by Allow bacteria to sporulate, separate by filtration or centrifugationfiltration or centrifugation

– Weaponize - what type of dispersal?Weaponize - what type of dispersal? Wet dispersalWet dispersal

– Spray liquid solution (droplets) over enemySpray liquid solution (droplets) over enemy Dry dispersalDry dispersal

– Dry with drying agent (ex. Magnesium Sulfate) = Dry with drying agent (ex. Magnesium Sulfate) = powderpowder

– Aerosolize and spray over targeted populationAerosolize and spray over targeted population– Disperse in mail or by some other meansDisperse in mail or by some other means

But is it this easy? Answer: NOBut is it this easy? Answer: NO


made? (3)made? (3) Wet DispersalWet Dispersal

– Very difficult to formulate for effective useVery difficult to formulate for effective use When solution is sprayed, droplets tend to be large When solution is sprayed, droplets tend to be large

(ex. Windex forms droplets ~100(ex. Windex forms droplets ~100m in m in size)size)

Droplets do not stay in air for longDroplets do not stay in air for long Would need jet aircraft flying at 600mph or Would need jet aircraft flying at 600mph or

special high pressure sprayers that cost ~$100,000 special high pressure sprayers that cost ~$100,000 and would have to be mounted on a truckand would have to be mounted on a truck

Not practicalNot practical

Solution: Use Dry PowderSolution: Use Dry Powder


made? (4)made? (4) Dry DispersalDry Dispersal

– More facile, but not necessarily a walk in More facile, but not necessarily a walk in the park…the park… After spore formation & filtration/centrifugation, After spore formation & filtration/centrifugation,

spores & remaining cells will form sticky paste spores & remaining cells will form sticky paste (consistency of peanut butter)(consistency of peanut butter)

Paste dried down ==> forms brick (or freeze Paste dried down ==> forms brick (or freeze dried)dried)

Brick needs to be ground into fine (1 Brick needs to be ground into fine (1 m) powder m) powder ==> spores will have surface charge ==> ==> spores will have surface charge ==> clumpingclumping

Need to neutralize “static cling” (similar to Need to neutralize “static cling” (similar to putting a sheet of Bounce in dryer)putting a sheet of Bounce in dryer)


made? (5)made? (5)

Neutralizing “Static Cling” of SporesNeutralizing “Static Cling” of Spores– Exact formulations and recipes are classifiedExact formulations and recipes are classified

Basic approach is to coat spores with a fine silica or Basic approach is to coat spores with a fine silica or alumina clay (Iraqi’s use the chemical Bentonite)alumina clay (Iraqi’s use the chemical Bentonite)

Spores no longer clump, actually want to stay apart, Spores no longer clump, actually want to stay apart, repel each other ==> do not stick to surfacesrepel each other ==> do not stick to surfaces

Without surface charges, spores can easily re-Without surface charges, spores can easily re-aerosolize after coming in contact with aerosolize after coming in contact with objects/ground objects/ground

Treated area can be infectious for a long time after Treated area can be infectious for a long time after dispersal (Increases danger and lethality of BW)dispersal (Increases danger and lethality of BW)


made? (6)made? (6) How to Aerosolize?How to Aerosolize?

– Once anthrax is weapons-grade Once anthrax is weapons-grade (appropriate size and w/o surface (appropriate size and w/o surface charges), can simply be dispersed by charges), can simply be dispersed by basic spray methods (such as a crop-basic spray methods (such as a crop-duster plane)duster plane)

The True Danger: The True Danger: Possible Possible Effects Anthrax AttackEffects Anthrax Attack

World Health Organization (WHO)World Health Organization (WHO)– Estimates that 250,000 people would Estimates that 250,000 people would

develop disease and 100,000 would die if develop disease and 100,000 would die if 50kg of aerosolized anthrax was released 50kg of aerosolized anthrax was released from an aircraft over a developed urban from an aircraft over a developed urban population of five million (w/o treatment).population of five million (w/o treatment).11

US Congressional Office of Technology US Congressional Office of Technology AssessmentAssessment– Estimates that between 130,000 and 3 Estimates that between 130,000 and 3

million deaths could follow the aerosolized million deaths could follow the aerosolized release of 100 kg of anthrax spores release of 100 kg of anthrax spores upwind of the Washington, DC, area—upwind of the Washington, DC, area—lethality matching or exceeding that of a lethality matching or exceeding that of a hydrogen bomb.hydrogen bomb.22

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: The True Danger: Possible Possible

Effects Anthrax Attack (2)Effects Anthrax Attack (2) Centers for Disease Control and Prevention Centers for Disease Control and Prevention

(CDC)(CDC)– Produced an economic model that suggested a Produced an economic model that suggested a

cost of $26.2 billion per 100,000 persons exposed cost of $26.2 billion per 100,000 persons exposed to inhalational anthrax to inhalational anthrax 11

Decontamination of Gruinard Island in the Decontamination of Gruinard Island in the UKUK– Decontamination took place from 1979 to 1987Decontamination took place from 1979 to 1987– Total cost is unpublished (assumed to be Total cost is unpublished (assumed to be

~500,000 British pounds), but materials required ~500,000 British pounds), but materials required included 280 tons of formaldehyde & 2000 tons of included 280 tons of formaldehyde & 2000 tons of seawater seawater 22

– Decontamination of buildings/other areas equally Decontamination of buildings/other areas equally as costly/difficultas 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: The True Danger: Possible Effects Possible Effects

Anthrax Attack (3)Anthrax Attack (3) Sverdlovsk Accident (Russia BW Plant)Sverdlovsk Accident (Russia BW Plant)

- New cases of inhalational anthrax developed as late as 43 days - New cases of inhalational anthrax developed as late as 43 days after the presumed date of release (lengthy period of lethality)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: The True Danger: Possible Effects Anthrax Possible Effects Anthrax

Attack (4)Attack (4)

Peters, C.J., D.M. Hartley. Anthrax Inhalation and Lethal Human Infection. Lancet. 359 (9307): 710-711.Peters, C.J., D.M. Hartley. Anthrax Inhalation and Lethal Human Infection. Lancet. 359 (9307): 710-711.

Analysis of the 2001 US Analysis of the 2001 US Anthrax AttacksAnthrax Attacks

Period From 10/02/01 - 11/20/01Period From 10/02/01 - 11/20/01– 22 cases of anthrax infection, 11 22 cases of anthrax infection, 11

inhalational (all confirmed) and 11 inhalational (all confirmed) and 11 cutaneous (4 suspected & 7 confirmed)cutaneous (4 suspected & 7 confirmed)

– 7 states : CT(1), FL(2), MD(3), NJ(5), 7 states : CT(1), FL(2), MD(3), NJ(5), NYC(8), PA(1) & VA(2)NYC(8), PA(1) & VA(2)

– 5 of 11 inhalational infections resulted in 5 of 11 inhalational infections resulted in death (~45% mortality rate)death (~45% mortality rate)

– All persons received immediate treatment All persons received immediate treatment upon onset of symptomsupon onset of symptoms

– Mean duration between exposure and Mean duration between exposure and onset of symptoms: 4.5 days (estimate)onset of symptoms: 4.5 days (estimate)

Analysis of the 2001 US Analysis of the 2001 US Anthrax Attacks (2)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


http://www.anthraxinvestigation.com/


Anthrax in EnvelopesAnthrax in Envelopes– Concentration of about 1 trillion spores per Concentration of about 1 trillion spores per

gramgram– 2 grams anthrax per envelope2 grams anthrax per envelope– Each letter contained ~200 million times Each letter contained ~200 million times

average LDaverage LD5050

– All anthrax was unmilled, contained a certain All anthrax was unmilled, contained a certain type of silica to reduce electrostatic charges type of silica to reduce electrostatic charges and was of the Ames strain and was of the Ames strain

– all characteristic of US weapons-grade anthraxall characteristic of US weapons-grade anthrax


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 FacilitiesFacilities - NYC & Trenton– Both facilities tested positive for bacillus anthracis as

well as at least 5 other facilities associated with Trenton facility


What Did These Attacks Exemplify?What Did These Attacks Exemplify?– Lethality of anthraxLethality of anthrax: 5 of 11 inhalatory victims : 5 of 11 inhalatory victims

died (though provided with best possible died (though provided with best possible treatment), less than 2 grams of anthrax per treatment), less than 2 grams of anthrax per envelopeenvelope

– Facile spread of weaponized anthraxFacile spread of weaponized anthrax: 7 states : 7 states affected and only 4 letters found, rapid spread in affected and only 4 letters found, rapid spread in buildingsbuildings

– Long shelf-life of anthraxLong shelf-life of anthrax: some people in NYC : some people in NYC were affected weeks after others (varying were affected weeks after others (varying incubation periods?)incubation periods?)

– Incredibly high cost of clean-up/decontamination: Incredibly high cost of clean-up/decontamination: millions of $millions of $

– Difficulty of finding perpetratorDifficulty of finding perpetrator: still not found, : still not found, likely never will belikely never will be


Concerns Resulting from 2001 AttacksConcerns Resulting from 2001 Attacks– What would have happened if more What would have happened if more

people were affected? Would the mortality people were affected? Would the mortality rate have been higher?rate have been higher?

– Who created this weapons-grade Who created this weapons-grade anthrax? Terrorist group thought to be anthrax? Terrorist group thought to be unlikely, but then what state sponsor? Did unlikely, but then what state sponsor? Did US lab employee(s) or government US lab employee(s) or government official(s) lend a hand to perpetrators?official(s) lend a hand to perpetrators?

– How can the US prevent other similar How can the US prevent other similar attacks?attacks?

– What would have happened if anthrax What would have happened if anthrax would have been aerosolized?would have been aerosolized?

Defense: Protection Defense: Protection Against AnthraxAgainst Anthrax

VaccinationVaccination– Very Effective: 2 dose efficacy against up to 1,000 Very Effective: 2 dose efficacy against up to 1,000

LDLD5050 in monkeys (human response believed to be in monkeys (human response believed to be very similar)very similar)

Early DetectionEarly Detection– Extremely important: How can the US learn about Extremely important: How can the US learn about

an anthrax attack before the appearance of an anthrax attack before the appearance of symptoms? (would eliminate much of danger)symptoms? (would eliminate much of danger)

– Time lag between exposure and symptoms is Time lag between exposure and symptoms is primary reason for the high mortality rate primary reason for the high mortality rate experienced with anthrax infectionsexperienced with anthrax infections

– A device similar to a smoke alarm or carbon A device similar to a smoke alarm or carbon monoxide detector?monoxide detector?

– Currently nothing on the market that could Currently nothing on the market that could serve such a purpose, little research in this serve such a purpose, little research in this particular areaparticular area

Defense: Protection Defense: Protection Against Anthrax (2)Against Anthrax (2)

Air Cleansers/FiltersAir Cleansers/Filters– Simply a reduction of risk, by no means Simply a reduction of risk, by no means

an eliminationan elimination– Appropriate for mailrooms, wool-sorting Appropriate for mailrooms, wool-sorting

facilities, etc.facilities, etc. Irradiation of Susceptible MaterialsIrradiation of Susceptible Materials

– Virtually eliminates threat of anthrax in Virtually eliminates threat of anthrax in mailmail

– But is it worth it? Do the disadvantages But is it worth it? Do the disadvantages outweigh the benefits?outweigh the benefits?

– Important Question: To what point are we Important Question: To what point are we willing to protect ourselves from anthrax willing to protect ourselves from anthrax threat? What are we willing to sacrifice?threat? What are we willing to sacrifice?


Pre/Post Exposure Antibiotic TreatmentPre/Post Exposure Antibiotic Treatment– Isolates from the 2001 US attacks were sensitive Isolates from the 2001 US attacks were sensitive

to fluoroquinolonesto fluoroquinolones– Quite effective in mitigating effects of anthrax if Quite effective in mitigating effects of anthrax if

caught at early stagecaught at early stage Decontamination of Exposed AreasDecontamination of Exposed Areas

– Often costly & timely, but can be done rather Often costly & timely, but can be done rather successfully (using liquid chlorine dioxide or some successfully (using liquid chlorine dioxide or some other disinfectant) other disinfectant)

Use of Protective Clothing & EquipmentUse of Protective Clothing & Equipment– US military M17 & M40 gas masks provide US military M17 & M40 gas masks provide

good protection against 1-5 good protection against 1-5 m particles (cost m particles (cost ~$325)~$325)

– Protective suits can be worn to easily Protective suits can be worn to easily eliminate cutaneous threateliminate cutaneous threat


Only Reactionary, Post-Exposure Protection Only Reactionary, Post-Exposure Protection Methods Exist!Methods Exist!– Besides vaccination/antibiotic treatment, there is Besides vaccination/antibiotic treatment, there is

no other preventative methods of protecting no other preventative methods of protecting against anthrax against anthrax

– Most methods of protection are reactionary, this Most methods of protection are reactionary, this doesn’t help to solve the main problem: anthrax doesn’t help to solve the main problem: anthrax infections need to be determined earlier, before infections need to be determined earlier, before onset of symptomsonset of symptoms

– How can we currently protect ourselves from a How can we currently protect ourselves from a massive bioterrorist attack? How can we make it massive bioterrorist attack? How can we make it more facile to distinguish btw. common maladies more facile to distinguish btw. common maladies and anthrax?and anthrax?

PRIMARY OBJECTIVE: NEED DEVICE/METHOD PRIMARY OBJECTIVE: NEED DEVICE/METHOD THAT WILL HELP TO PROTECT LARGE THAT WILL HELP TO PROTECT LARGE POPULATION FROM LARGE-SCALE ANTHRAX POPULATION FROM LARGE-SCALE ANTHRAX ATTACKATTACK

Anthrax: Why it May Not be Anthrax: Why it May Not be as Big of a Threat as People as Big of a Threat as People

ThinkThink LDLD5050 for Anthrax is Quite High for Anthrax is Quite High

– Smallpox: 10-100 organisms, Q Fever: 1-Smallpox: 10-100 organisms, Q Fever: 1-10organisms, etc. 10organisms, etc.

Very Difficult to Weaponize (Need State/Gov’t Very Difficult to Weaponize (Need State/Gov’t Support)Support)– Aum Shinrikyo has failed to aerosolize anthrax on Aum Shinrikyo has failed to aerosolize anthrax on

several occasionsseveral occasions– Need sufficient nanotechnology engineering to be Need sufficient nanotechnology engineering to be

able to keep spores from clumping and sticking to able to keep spores from clumping and sticking to surfaces (Army scientists could not weaponize surfaces (Army scientists could not weaponize anthrax when given the equipment a BW terrorist anthrax when given the equipment a BW terrorist would likely have in his/her home)would likely have in his/her home)

Even if have Technology, Weapons-Grade Anthrax Even if have Technology, Weapons-Grade Anthrax Very CostlyVery Costly– Only US & Russia known to definitely have Only US & Russia known to definitely have

weaponized anthrax, large-scale production weaponized anthrax, large-scale production requires multi-million dollar investmentrequires multi-million dollar investment

Anthrax: Why it May Not be Anthrax: Why it May Not be as Big of a Threat as People as Big of a Threat as People

Think (2)Think (2) Anthrax Strains are Highly SpecificAnthrax Strains are Highly Specific

– Only certain strains are effective against humans, Only certain strains are effective against humans, some are harmlesssome are harmless

Lengthy IncubationLengthy Incubation– While this is also an advantage, it can serve as While this is also an advantage, it can serve as

a disadvantagea disadvantage– Don’t immediately know if weapon has “struck Don’t immediately know if weapon has “struck

target”target” Difficult to Use Anthrax Against Precise TargetsDifficult to Use Anthrax Against Precise Targets

– 2001 US attacks: directed against high-status people, 2001 US attacks: directed against high-status people, never reached targetsnever reached targets

– Aerosolization is most deadly, but also the most Aerosolization is most deadly, but also the most difficult to direct as a result of environmental difficult to direct as a result of environmental conditionsconditions

Anthrax vs. Other BW Agents: Anthrax vs. Other BW Agents: Brief OverviewBrief Overview

www.nbc-med.org/SiteContent/HomePage/ WhatsNew/MedManual/Feb01/AppxC.doc -

Disease TransmitMan to Man

Infective Dose(Aerosol)

IncubationPeriod

Duration of Illness Lethality (approx.case fatality rates)

Persistence of Organism Vaccine Efficacy(aerosol exposure)

Inhalationanthrax

No 8,000-50,000spores

1-6 days 3-5 days (usuallyfatal if untreated)

High Very stable - spores remainviable for > 40 years in soil

2 dose efficacy against upto 1,000 LD50 in monkeys

Brucellosis No 10 -100organisms

5-60 days(usually 1-2months)

Weeks to months <5% untreated Very stable No vaccine

Cholera Rare 10-500 organisms 4 hours -5 days (usually2-3 days)

> 1 week Low with treatment,high without

Unstable in aerosols & freshwater; stable in salt water

No data on aerosol

Glanders Low Assumed low 10-14 days viaaerosol

Death in 7-10 daysin septicemic form

> 50% Very stable No vaccine

PneumonicPlague

High 100-500organisms

2-3 days 1-6 days(usually fatal)

High unless treatedwithin 12-24 hours

For up to 1 year in soil; 270 daysin live tissue

3 doses not protectiveagainst 118 LD50 inmonkeys

Tularemia No 10-50 organisms 2-10 days(average 3-5)

> 2 weeks Moderate ifuntreated

For months in moist soil or othermedia

80% protection against1-10 LD50

Q Fever Rare 1-10 organisms 10-40 days 2-14 days Very low For months on wood and sand 94% protection against3,500 LD50 in guinea pigs

Smallpox High Assumed low(10-100organisms)

7-17 days(average 12)

4 weeks High to moderate Very stable Vaccine protects againstlarge doses in primates

VenezuelanEquineEncephalitis

Low 10-100 organisms 2-6 days Days to weeks Low Relatively unstable TC 83 protects against 30-500 LD50 in hamsters

ViralHemorrhagicFevers

Moderate 1-10 organisms 4-21 days Death between 7-16days

High for Zairestrain, moderatewith Sudan

Relatively unstable - depends onagent

No vaccine

Botulism No 0.001 g/kg isLD50 for type A

1-5 days Death in 24-72hours; lasts monthsif not lethal

High withoutrespiratory support

For weeks in nonmoving waterand food

3 dose efficacy 100%against 25-250 LD50 inprimates

StaphEnterotoxin B

No 0.03 g/personincapacitation

3-12 hours afterinhalation

Hours < 1% Resistant to freezing No vaccine

Ricin No 3-5 g/kg is LD50

in mice18-24 hours Days - death within

10-12 days foringestion

High Stable No vaccine

T-2Mycotoxins

No Moderate 2-4 hours Days to months Moderate For years at room temperature No vaccine

http://http://www.sumanasinc.com/webcontent/awww.sumanasinc.com/webcontent/anisamples/ani_anthrax.htmlnisamples/ani_anthrax.html

http://http://www.bt.cdc.gov/training/historyofbt/iwww.bt.cdc.gov/training/historyofbt/index.aspndex.asp

bacillus anthracis hirotaka ishibashi jennifer jolivet sean patrick kelly

Documents

anthrax infection

anthrax dates

absence of endemic anthrax

us slide

kbp capsule anthrax

climate slide

abdominal slide

ame slide