Anthrax

Bacilus AnthracisBacilus Anthracis
AnthraxAnthrax
Sheref HassanSheref Hassan
Rachel HandwergerRachel Handwerger
Maria GarridoMaria Garrido

““The Agent”The Agent”
 CDC: Category “A” Biological AgentCDC: Category “A” Biological Agent
 Vegetative cell: large, gram-positive bacillusVegetative cell: large, gram-positive bacillus ((1.0-1.51.0-1.5 μμm by 3.0-5.0m by 3.0-5.0 μμm)m)
 Endospore: oval, central-to-sub terminal, does not usually swellEndospore: oval, central-to-sub terminal, does not usually swell
((1.0x1.51.0x1.5 μμmm));; COCO22 levels within the body inhibit sporulationlevels within the body inhibit sporulation
 Forms long chainsForms long chains in vitroin vitro; forms single cells or short chains in clinical; forms single cells or short chains in clinical
samplessamples
 Aerobic or facultative anaerobic; non-motile; catalase-positiveAerobic or facultative anaerobic; non-motile; catalase-positive
 Nonhemolytic on sheep blood agar; susceptible to lysis by gammaNonhemolytic on sheep blood agar; susceptible to lysis by gamma
phagephage
 Colonies: ground-glass appearance and consistency of beaten-eggColonies: ground-glass appearance and consistency of beaten-egg
whites and are 2-5 mm in diameter after 16-18 hours of incubationwhites and are 2-5 mm in diameter after 16-18 hours of incubation

““The Agent”The Agent” contd..contd..
● In environments rich in nutrients such asIn environments rich in nutrients such as
glucose, amino acids, and nucleosides, sporesglucose, amino acids, and nucleosides, spores
will germinate and form vegetative cells.will germinate and form vegetative cells.
Vegetative bacteria survive poorly outside ofVegetative bacteria survive poorly outside of
mammalian host.mammalian host.
● In a nutrient-deficient setting, vegetative cells willIn a nutrient-deficient setting, vegetative cells will
form spores.form spores.
● *Spores have been shown to survive in the*Spores have been shown to survive in the
environment for more than 40 years.environment for more than 40 years.

History of Anthrax:History of Anthrax:
Early HistoryEarly History
 The first recorded report of anthrax was during the fifthThe first recorded report of anthrax was during the fifth
Egyptian Plague in 1500 B.C.Egyptian Plague in 1500 B.C.  responsible for the deathresponsible for the death
of a large amount of livestock.of a large amount of livestock.
 The sixth Egyptian Plague, also known as the “plague ofThe sixth Egyptian Plague, also known as the “plague of
boils” was probably the first recorded instance ofboils” was probably the first recorded instance of
cutaneous anthrax and was responsible for many deaths.cutaneous anthrax and was responsible for many deaths.
 The “Black Bane” in Europe during the 1600s killed wellThe “Black Bane” in Europe during the 1600s killed well
over 60,000 cattle and typified anthrax symptoms.over 60,000 cattle and typified anthrax symptoms.
 1863: French biologist Casimir-Joseph Davaine makes1863: French biologist Casimir-Joseph Davaine makes
B.A.B.A. the first microorganism to be conculsively linked to athe first microorganism to be conculsively linked to a
disease.disease.
 1876:1876: B.A.B.A. isolated in pure culture for the first time byisolated in pure culture for the first time by
Robert KochRobert Koch
 Was also called: wool sorter’s disease, Bradford disease,Was also called: wool sorter’s disease, Bradford disease,
and rag picker’s disease.and rag picker’s disease.

Early HistoryEarly History
 1897: The Bradford Rules are passed as law1897: The Bradford Rules are passed as law
Anthrax Investigation Board.Anthrax Investigation Board.
 1919: The Anthrax Prevention Act passed in1919: The Anthrax Prevention Act passed in
EnglandEngland established a wool-disinfectingestablished a wool-disinfecting
complex in Liverpool.complex in Liverpool.
 Similar laws, gov’t regulations, better disinfectantSimilar laws, gov’t regulations, better disinfectant
methods and technology thereafter reducedmethods and technology thereafter reduced
Anthrax cases linked to occupational hazards,Anthrax cases linked to occupational hazards,
as well as making Anthrax viability andas well as making Anthrax viability and
outbreaks rare in the modern world.outbreaks rare in the modern world.

Natural OutbreaksNatural Outbreaks
Animal:Animal:
o 1945: Iran1945: Iran  outbreak causes 1outbreak causes 1
million sheep deathsmillion sheep deaths

Natural Outbreaks - cont...Natural Outbreaks - cont...
Inhalation:Inhalation:
o U.S.U.S.  1900-1978: only 18 reported cases1900-1978: only 18 reported cases
mostly among high risk groups such as goat millmostly among high risk groups such as goat mill
or goatskin workers and wool or tannery workersor goatskin workers and wool or tannery workers
(only 2 were laboratory associated)(only 2 were laboratory associated)
o New HampshireNew Hampshire  1957: 5 cases reported in1957: 5 cases reported in
goat-hair processing plantgoat-hair processing plant
o No Case of inhalation anthrax was reportedNo Case of inhalation anthrax was reported
since 1978 in the U.S. prior to post-Sept. 11since 1978 in the U.S. prior to post-Sept. 11thth
attacks.attacks.

CutaneousCutaneous (most common):(most common):
o U.S.U.S.  1944 -1994: 224 cases reported1944 -1994: 224 cases reported
o New HampshireNew Hampshire  1957: 4 cases reported in1957: 4 cases reported in
goat-hair processing plantgoat-hair processing plant
o ZimbabweZimbabwe  1979-1985: largest reported1979-1985: largest reported
epidemic with over 10,000 reported humanepidemic with over 10,000 reported human
cases and 182 deaths (nearly all cutaneous);cases and 182 deaths (nearly all cutaneous);
explained by spread via insect vectors orexplained by spread via insect vectors or
contaminated meat; however, biological warfarecontaminated meat; however, biological warfare
tactics have been also postulatedtactics have been also postulated

GastrointestinalGastrointestinal (uncommonly reported)(uncommonly reported)::
2 distinct syndromes: oral pharyngeal2 distinct syndromes: oral pharyngeal (unusual(unusual
manifestation of infection and is of more interest)manifestation of infection and is of more interest) & abdominal& abdominal
diseasedisease
o Since mid 1970’s: small outbreaks reported inSince mid 1970’s: small outbreaks reported in
Africa and AsiaAfrica and Asia
o Northern ThailandNorthern Thailand
 1982: 24 cases of oral pharyngeal reported due to the1982: 24 cases of oral pharyngeal reported due to the
consumption of contaminated buffalo meatconsumption of contaminated buffalo meat
 1987: 14 cases involving both oral pharyngeal and1987: 14 cases involving both oral pharyngeal and
abdominal diseaseabdominal disease

Statistics of Anthrax OccurrencesStatistics of Anthrax Occurrences
In the U.S.:In the U.S.:
• Approximately 130 cases occurred annually in the earlyApproximately 130 cases occurred annually in the early
1900’s1900’s
• This number decreased with less than 10 cases reportedThis number decreased with less than 10 cases reported
each year since the early 1960’s and no occurrences fromeach year since the early 1960’s and no occurrences from
1978 until after Sept. 111978 until after Sept. 11thth
20012001
• 95% of naturally occurring cases are cutaneous and 5% are95% of naturally occurring cases are cutaneous and 5% are
due to inhalation.due to inhalation. (cases of gastrointestinal anthrax have not been recognized yet in(cases of gastrointestinal anthrax have not been recognized yet in
the U.S.)the U.S.)
• Only 18 cases of naturally occurring inhalation cases haveOnly 18 cases of naturally occurring inhalation cases have
been reported during the 20been reported during the 20thth
century with the most recent incentury with the most recent in
19761976
• Since 1990, only 2 cases (1992 and 2000) of naturallySince 1990, only 2 cases (1992 and 2000) of naturally
occurring infection have been reported (both cutaneous).occurring infection have been reported (both cutaneous).

Statistics of Anthrax OccurrencesStatistics of Anthrax Occurrences
Globally:Globally:
• Approximately 2,000 - 20,000 cases of anthraxApproximately 2,000 - 20,000 cases of anthrax
occur each year.occur each year.
• Most cases are cutaneous with inhalation andMost cases are cutaneous with inhalation and
gastrointestinal being less frequent.gastrointestinal being less frequent.
• Human cases usually follow diseaseHuman cases usually follow disease
occurrences in ruminants and are mostoccurrences in ruminants and are most
prevalent in Africa, the Middle East, and parts ofprevalent in Africa, the Middle East, and parts of
Southeast Asia.Southeast Asia.

““Travelers Beware”Travelers Beware”
Anthrax in animals is hyper-endemic or endemic in:Anthrax in animals is hyper-endemic or endemic in:
(caution when eating meat in these areas)(caution when eating meat in these areas)
 Most areas of the Middle EastMost areas of the Middle East
 Most areas of equatorial AfricaMost areas of equatorial Africa
 Mexico and Central AmericaMexico and Central America
 Chile, Argentina, Peru, BoliviaChile, Argentina, Peru, Bolivia
 Certain Southeast Asian countries such as Myanam,Certain Southeast Asian countries such as Myanam,
Vietnam, Cambodia, & ThailandVietnam, Cambodia, & Thailand
 Papua New GuineaPapua New Guinea
 ChinaChina
 Some Mediterranean CountriesSome Mediterranean Countries
(for more information see WHOCC)(for more information see WHOCC)

Bacillus anthracisBacillus anthracis
 Review:Review:
Gram PositiveGram Positive
Rod shapedRod shaped
AerobicAerobic
Spore FormingSpore Forming
Non-motileNon-motile
A B
A1 A2 A3 A4 B1 B2

Bacillus anthracisBacillus anthracis exists in two forms:exists in two forms:
1.1. Inert spore formInert spore form
– 11 μmμm
– Rod shapedRod shaped
– Aerobic (free O2 present)Aerobic (free O2 present)
– Highly Resistant to extreme conditions (e.g.Highly Resistant to extreme conditions (e.g.
temperature, weather, radiation, etc.)temperature, weather, radiation, etc.)
– Spores have been known to last up to 40 yrsSpores have been known to last up to 40 yrs

2.2. Vegetative formVegetative form
– 1 – 1.51 – 1.5 μm by 5 – 8 μmμm by 5 – 8 μm
– Square-ended rodSquare-ended rod
– Anaerobic environment of hostAnaerobic environment of host
– Only multiply within host in the presence ofOnly multiply within host in the presence of
amino acids, nucleosides, and glucoseamino acids, nucleosides, and glucose

 Bacillus anthracisBacillus anthracis is composed of:is composed of:
A capsuleA capsule
A three part proteinA three part protein
Protective Antigen (PA)Protective Antigen (PA)
Edema Factor (EF)Edema Factor (EF)
Lethal Factor (LF)Lethal Factor (LF)
 In the bacteria there is one chromosomeIn the bacteria there is one chromosome
and 2 plasmids (pXO1 and pXO2)and 2 plasmids (pXO1 and pXO2)

 ChromosomeChromosome  not involved in virulencenot involved in virulence
 pXO1pXO1  codes for PA, EF, and LFcodes for PA, EF, and LF
 pXO2pXO2  codes for the capsulecodes for the capsule

pXO2/CapsulepXO2/Capsule
 pXO2 codes for the homopolymericpXO2 codes for the homopolymeric γγ--
linked poly-D-glutamic acid capsulelinked poly-D-glutamic acid capsule
 Contains three genes: capBCAContains three genes: capBCA
 96,231 bp, ~85 ORFs (16 sm. ones)96,231 bp, ~85 ORFs (16 sm. ones)
 Antiphagocytic functionAntiphagocytic function
 Is non-toxic by itselfIs non-toxic by itself
 Is important in the onset of the infectionIs important in the onset of the infection
 Poorly understoodPoorly understood

pXO1pXO1
 181,654 nucleotides, ~143 ORFs181,654 nucleotides, ~143 ORFs
 Codes for the three toxin genes whichCodes for the three toxin genes which
are transcribed simultaneously:are transcribed simultaneously:
pagApagA (for PA)(for PA)
cyacya (for EF)(for EF)
leflef (for LF)(for LF)
 Codes for regulatory proteins, from theCodes for regulatory proteins, from the
atxAatxA gene, which causes ~10x increasegene, which causes ~10x increase
in transcription of all three toxinsin transcription of all three toxins

PAPA
 Is non-toxic without EF and LFIs non-toxic without EF and LF
 Coded fromCoded from pagpag genegene
 A/T rich (69%)A/T rich (69%)
 Cysteine-freeCysteine-free
 83 kDa (735-amino acid)83 kDa (735-amino acid)
 Long, flat proteinLong, flat protein
 Function – to aid in the insertion of EFFunction – to aid in the insertion of EF
and LF toxins in the cytosoland LF toxins in the cytosol

PA StructurePA Structure
 Domain 1 (Domain 1 (Green and Blue)
 residues 1 – 249,residues 1 – 249, ββ-sandwich fold,-sandwich fold,
 2 Ca2 Ca2+2+
, forms cleavage site during, forms cleavage site during
proteolytic activationproteolytic activation
 Domain 2 (Domain 2 (Magenta)
 Residues 250 – 487,Residues 250 – 487, ββ-barrel core-barrel core
 Forms membrane inserted channelForms membrane inserted channel
/pore formation/pore formation
 Domain 3 (Domain 3 (Yellow)
 Residues 488 – 594,Residues 488 – 594,
4-stranded mixed4-stranded mixed ββ-sheet, 4 small helices-sheet, 4 small helices
 Helps to oligomerize(?)Helps to oligomerize(?)
 Domain 4 (Purple)Domain 4 (Purple)
 Residues 595 – 735,Residues 595 – 735, ββ-sandwich-sandwich
 Contains receptor binding domainContains receptor binding domain
Koehler, T.M. Anthrax. Springer. 2002.

PA MechanismPA Mechanism
Mechanism:Mechanism:
1.1. PA83 binds to cell receptorPA83 binds to cell receptor
2.2. Furin or a furin-like molecule cleaves PA83Furin or a furin-like molecule cleaves PA83
between aa 164-167 (Arg-Lys-Lys-Arg) to twobetween aa 164-167 (Arg-Lys-Lys-Arg) to two
fragments: PA20 (released) and PA63 (remainsfragments: PA20 (released) and PA63 (remains
attached to the receptor)attached to the receptor)
http://www.biotechjournal.com/Pathways/anthrax.htm

PA Mechanism (cont.)PA Mechanism (cont.)
3.3. PA63/receptor complexes join to form aPA63/receptor complexes join to form a
heptamer at reduced pHheptamer at reduced pH
4.4. The heptamer complex can then bind to oneThe heptamer complex can then bind to one
LF or EF molecule at each subunitLF or EF molecule at each subunit

PA Mechanism (cont.)PA Mechanism (cont.)
5.5. The heptamer is internalized via RMEThe heptamer is internalized via RME
6.6. Acidification of the vesicle causes theAcidification of the vesicle causes the
heptamer to form a pore in the membraneheptamer to form a pore in the membrane
7.7. EF and LF are released through the poreEF and LF are released through the pore
into the cytosolinto the cytosol

General MechanismGeneral Mechanism

EFEF
 Coded from cya geneCoded from cya gene
A/T rich (71%)A/T rich (71%)
Cysteine-freeCysteine-free
 89 kDa (767-amino acid)89 kDa (767-amino acid)
 MainlyMainly ββ strandsstrands
 N-terminal ~250 aa bind to PA63N-terminal ~250 aa bind to PA63
 Remaining molecule possesses catalyticRemaining molecule possesses catalytic
activity in substrate and calmodulin-activity in substrate and calmodulin-
binding subdomainsbinding subdomains
 A-B toxinA-B toxin

EF FunctionEF Function
 EF receptors are present on most cellsEF receptors are present on most cells
 EF is a calmodulin dependent adenylateEF is a calmodulin dependent adenylate
cyclasecyclase
Therefore only functions in eukaryotic cellsTherefore only functions in eukaryotic cells
 Catalyzes breakdown of ATP to cAMPCatalyzes breakdown of ATP to cAMP
~200 fold increase~200 fold increase
 Why Calmodulin?Why Calmodulin?
1% total cellular protein1% total cellular protein
Responsible for mediatingResponsible for mediating CaCa2+2+
signaling insignaling in
cellscells

EF MechanismEF Mechanism
 MechanismMechanism
1.1. EF enters the cell by binding to PA (calledEF enters the cell by binding to PA (called
EdTx)EdTx)
2.2. Inside, EF binds to calmodulin at the N-Inside, EF binds to calmodulin at the N-
terminal lowterminal low CaCa2+2+
affinity site in a very uniqueaffinity site in a very unique
way:way:
– One half grips lower lobe while the other grasps the upperOne half grips lower lobe while the other grasps the upper
lobe and twistslobe and twists
– The method of binding forms a contact region of 6,000The method of binding forms a contact region of 6,000 ÅÅ22
––
an extremely large binding region!an extremely large binding region!
– The conformational change experienced by both moleculesThe conformational change experienced by both molecules
inhibits Cainhibits Ca2+2+
from binding and inhibits the reverse reaction offrom binding and inhibits the reverse reaction of
cAMP to ATPcAMP to ATP

EF Mechanism (cont.)EF Mechanism (cont.)
2.2. The conformational change in EF allowsThe conformational change in EF allows
it to act as an adenylate cyclase andit to act as an adenylate cyclase and
bind to ATPbind to ATP
3.3. cAMP is formed due to the binding of EFcAMP is formed due to the binding of EF
to ATPto ATP
Liddington, R.C., A Molecular Full Nelson. Nature. 415: 373-374, 2002.

EFEF
What happens as a result of thisWhat happens as a result of this
activity?activity?
Causes edema by altering HCauses edema by altering H22O and ionO and ion
movementmovement
Inhibits immune response againstInhibits immune response against
http://www.biocarta.com/pathfiles/h_anthraxPathway.asp

EFEF
Inhibits activity of TF NF-Inhibits activity of TF NF-ĸB inĸB in
macrophagesmacrophages
StrainsStrains with only EF and PA (LF-) arewith only EF and PA (LF-) are
not likely to be lethalnot likely to be lethal

LFLF
 Coded from lef geneCoded from lef gene
 A/T rich (70%)A/T rich (70%)
 Cystenine-freeCystenine-free
 90.2-kDa (776-amino acid)90.2-kDa (776-amino acid)
 3 domains:3 domains:
 N-terminal ~250 aa competesN-terminal ~250 aa competes
with EF to bind PA63with EF to bind PA63
 5 imperfect repeats,5 imperfect repeats,
each 19 aa longeach 19 aa long
 Contains a zinc proteaseContains a zinc protease
active site (HEXXH helix)active site (HEXXH helix)
 A-B ToxinA-B Toxin Pannifer, A.D., Wong, T.Y., et al, Crystal Structure of the Anthrax Lethal Factor. Nature. 414: 229-233, 2001.

LF FunctionLF Function
 LF only binds to macrophagesLF only binds to macrophages
 LF is responsible for most of the toxicityLF is responsible for most of the toxicity
Can induce lysis in about 90-120 minutesCan induce lysis in about 90-120 minutes
 LF is a zinc dependent metalloproteaseLF is a zinc dependent metalloprotease
which cleaves MAPKKwhich cleaves MAPKK

LF Mechanism and FunctionLF Mechanism and Function
 LF is internalized via PA (called LeTx)LF is internalized via PA (called LeTx)
 Mechanism and Result of MAPKKMechanism and Result of MAPKK
cleavage:cleavage:
LF cleaves between 7-10 residues on theLF cleaves between 7-10 residues on the
N-terminus of the MAPKKs at a pair ofN-terminus of the MAPKKs at a pair of
proline residues (separtated by 0 – 4 aa)proline residues (separtated by 0 – 4 aa)
E.g. MAPKK1, 2, 3, 4, 6E.g. MAPKK1, 2, 3, 4, 6
Duesbery, N.S., Vande Woude, G.F., Anthrax Toxins. Cellular and Molecular Life Sciences. 55: 1599-1609, 1999.

LF Mechanism and Func. (cont)LF Mechanism and Func. (cont)
This cleavage serves to inactivate the MAPKKThis cleavage serves to inactivate the MAPKK
MAPKK cleavage leads to lysis of theMAPKK cleavage leads to lysis of the
macrophages, although the mechanism ismacrophages, although the mechanism is
unclearunclear

Some MAPKK aid in the activation of TF NF-Some MAPKK aid in the activation of TF NF-
ĸBĸB
This illustrates how EF and LF work together toThis illustrates how EF and LF work together to
promote macrophage lysispromote macrophage lysis

 At sublytic levels it stimulates production of IL-1At sublytic levels it stimulates production of IL-1ββ
and TNFαand TNFα
 At lytic levels the mechanism is unclear, but it isAt lytic levels the mechanism is unclear, but it is
possible that in the early stages they inhibit thepossible that in the early stages they inhibit the
immune response to allow the bacteria to proliferate,immune response to allow the bacteria to proliferate,
and at later stages it stimulates them to cause lysis ofand at later stages it stimulates them to cause lysis of
cellscells
 Causes leakage in cell membrane without ATPCauses leakage in cell membrane without ATP
 increased Caincreased Ca2+2+
conc. in cellconc. in cell
 Strains with only LF and PA (EF-) are likely to beStrains with only LF and PA (EF-) are likely to be
lethal, although to a lesser degreelethal, although to a lesser degree

ReviewReview
 EFEF
 Increased productionIncreased production
of cAMPof cAMP
 Causes swellingCauses swelling
 Inhibits immuneInhibits immune
responseresponse
 Allows for invasion!Allows for invasion!
 LFLF
 Cleaves MAPKKsCleaves MAPKKs
 Inhibits immuneInhibits immune
responseresponse
 DestroysDestroys
macrophagesmacrophages
 Cells can’t fight offCells can’t fight off
infectioninfection  they Die!they Die!

Overall Mechanism ReviewOverall Mechanism Review
Duesbery, N.S., Vande Woude, G.F., Anthrax Toxins. Cellular and Molecular Life Sciences. 55: 1599-1609, 1999.

Clinical Features of B. AnthracisClinical Features of B. Anthracis
 Anthrax can manifest itself in 3 forms:Anthrax can manifest itself in 3 forms:
cutaneous, gastrointestinal, & inhalationalcutaneous, gastrointestinal, & inhalational
(pulmonary).(pulmonary).
 The type contracted depends on the modes ofThe type contracted depends on the modes of
transmission, which include:transmission, which include:
 Contact with infected tissues of dead animals (eg, butchering,Contact with infected tissues of dead animals (eg, butchering,
preparing contaminated meat)preparing contaminated meat)  cutaneouscutaneous
 Consumption of contaminated undercooked meatConsumption of contaminated undercooked meat
gastrointestinalgastrointestinal
 Contact with contaminated hair, wool, or hides (duringContact with contaminated hair, wool, or hides (during
processing) or contact with products made from themprocessing) or contact with products made from them
inhalational &/or gastrointestinalinhalational &/or gastrointestinal
 biological warfarebiological warfare mainly inhalational &/or gastrointestinalmainly inhalational &/or gastrointestinal

PathologyPathology
Inhalational Anthrax:Inhalational Anthrax:
 Endospores are introduced into the body and alveoli via inhalation.Endospores are introduced into the body and alveoli via inhalation.
(small size of spores permits this)(small size of spores permits this)
 Macrophages phagocytose and lyse endospores, and then travelMacrophages phagocytose and lyse endospores, and then travel
to regional lymph nodes.to regional lymph nodes.
 Spores germinate to become vegetative cells within macrophages;Spores germinate to become vegetative cells within macrophages;
they then leave macrophages and multiply within the lymphaticthey then leave macrophages and multiply within the lymphatic
system. (1-43 days)system. (1-43 days)
 Bacteria enter the bloodstream and can lead to septic shock andBacteria enter the bloodstream and can lead to septic shock and
toxemia with system wide edema and necrosis; hematogenoustoxemia with system wide edema and necrosis; hematogenous
spread can lead to hemorrhagic meningitis.spread can lead to hemorrhagic meningitis.
 True pneumonia is rare w/ inhalational anthrax but focal,True pneumonia is rare w/ inhalational anthrax but focal,
hemorrhagic, necrotizing pneumonic lesions may be observed.hemorrhagic, necrotizing pneumonic lesions may be observed.
 Major causes of death is compression of lungs and septic shock.Major causes of death is compression of lungs and septic shock.

Pathology cont…Pathology cont…
 Development of symptoms and pathogenesis occurs in 2Development of symptoms and pathogenesis occurs in 2
stages:stages:
1)1) Exhibition of cold and flu like symptomsExhibition of cold and flu like symptoms malaise, fever,malaise, fever,
prominent cough, nausea, vomiting, drenching sweats, dyspnea,prominent cough, nausea, vomiting, drenching sweats, dyspnea,
chest pain, and headacheschest pain, and headaches
2)2) Develops rapidly (hours) and is characterized byDevelops rapidly (hours) and is characterized by acute dyspnea,acute dyspnea,
subsequent cyanosis, pleural effusion, widened mediastinum,subsequent cyanosis, pleural effusion, widened mediastinum,
pulmonary edema, hemorrhagic symptoms of stage 1 becomepulmonary edema, hemorrhagic symptoms of stage 1 become
severe (>24hrs and leads to death)severe (>24hrs and leads to death)
 IDID5050 (infective dosage that will infect 50% of exposed) is(infective dosage that will infect 50% of exposed) is
8,000-50,000 spores.8,000-50,000 spores.
 LDLD5050 (Lethal dosage that will kill 50% of exposed) is 2,500-(Lethal dosage that will kill 50% of exposed) is 2,500-
55,000 spores.55,000 spores.
 UntreatedUntreated 80% mortality rate80% mortality rate

Cutaneous Anthrax:Cutaneous Anthrax:
 Endospores enter body through the skin usually viaEndospores enter body through the skin usually via
preexisting skin lesions, abrasions, and skin mucuspreexisting skin lesions, abrasions, and skin mucus
membranes.membranes.
 Low-level germination and toxin production occurs atLow-level germination and toxin production occurs at
entrance site leading to localized necrosis with escharentrance site leading to localized necrosis with eschar
formation and edemaformation and edema (massive in some cases)(massive in some cases)
 Eschar formation:Eschar formation:
• Day 1Day 1 pruritic red papule appears at the initial site of infectionpruritic red papule appears at the initial site of infection
• Day 2Day 2 papule becomes round ulcerpapule becomes round ulcer
• Day 3Day 3 plaque develops around edema site and central papuleplaque develops around edema site and central papule
necrotizes, ulcerates, and forms a normally painless black escharnecrotizes, ulcerates, and forms a normally painless black eschar
 After 1-2 weeks eschar falls off and leaves ulcer that can heal by week 3.After 1-2 weeks eschar falls off and leaves ulcer that can heal by week 3.

Anthrax eschar, neckAnthrax eschar, neck
CDC Public Health Image Library,CDC Public Health Image Library,
number 1933number 1933
Edema around anthrax lesionEdema around anthrax lesion
AB Christie, LiverpoolAB Christie, Liverpool
Anthrax eschar, neckAnthrax eschar, neck
CDC Public Health Image Library,CDC Public Health Image Library,
number 1934number 1934

Cutaneous Anthrax:Cutaneous Anthrax:
 Endospores are often phagocytosed by macrophages andEndospores are often phagocytosed by macrophages and
carried to local lymph nodes, which can result in painfulcarried to local lymph nodes, which can result in painful
lymphadenopathy and lymphangitislymphadenopathy and lymphangitis
 Hematogenous spread with resultant toxemia can occur,Hematogenous spread with resultant toxemia can occur,
with symptoms such as headache and fevers (up to 102with symptoms such as headache and fevers (up to 102
°F)°F) although such spread is not common with appropriatealthough such spread is not common with appropriate
antibiotic therapyantibiotic therapy
 On very rare occasions system wide infection can occurOn very rare occasions system wide infection can occur
and results in death within days.and results in death within days.
 IDID5050 & LD& LD5050 are unknown for cutaneous anthraxare unknown for cutaneous anthrax
 UntreatedUntreated mortality rate is 20%mortality rate is 20%
 With antibiotic treatment death is very rare.With antibiotic treatment death is very rare.

Gastrointestinal Anthrax:Gastrointestinal Anthrax:
 Pathogenesis and statistics are unclear due to rarity ofPathogenesis and statistics are unclear due to rarity of
this formthis form
 Vegetative cells from uncooked meat rather than sporesVegetative cells from uncooked meat rather than spores
(germination unnecessary)(germination unnecessary)
 In oral pharyngeal anthrax, bacteria enter via the oral orIn oral pharyngeal anthrax, bacteria enter via the oral or
pharyngeal mucosapharyngeal mucosa mucosal ulcers can occur initially,mucosal ulcers can occur initially,
followed by local/regional lymphadenopathy andfollowed by local/regional lymphadenopathy and
localized edema.localized edema.
 In abdominal anthrax, entrance occurs @ the ileum orIn abdominal anthrax, entrance occurs @ the ileum or
cecumcecum Intestinal lesions, regional lymphadenopathy,Intestinal lesions, regional lymphadenopathy,
and edema of the bowel wall and ascitesand edema of the bowel wall and ascites initiallyinitially
causescauses nausea, vomiting, and malaise progressing intonausea, vomiting, and malaise progressing into
bloody diarrhea, acute abdomen, or sepsis.bloody diarrhea, acute abdomen, or sepsis.
 Mortality may drop with antibiotic treatmentMortality may drop with antibiotic treatment (inconclusive)(inconclusive)

Summary of PathologySummary of Pathology

DiagnosisDiagnosis
 Because of rarity of gastrointestinal cases diagnosisBecause of rarity of gastrointestinal cases diagnosis
focus is limited. Onset of characteristic symptomsfocus is limited. Onset of characteristic symptoms
should signal patient to consult a physicianshould signal patient to consult a physician
immediately for antibiotic treatment.immediately for antibiotic treatment.
 Cutaneous anthrax can be distinguished by 2 keyCutaneous anthrax can be distinguished by 2 key
features: painlessness of lesions and relatively largefeatures: painlessness of lesions and relatively large
extent of associated edema (presence of eschars)extent of associated edema (presence of eschars)
 Distinguishing features of inhalational anthrax:Distinguishing features of inhalational anthrax:
presence of widened mediastinum and pleuralpresence of widened mediastinum and pleural
effusions on chest radiograph or CT (chesteffusions on chest radiograph or CT (chest
compound tomographic) scan with minimumcompound tomographic) scan with minimum
evidence of pneumonia.evidence of pneumonia.

Diagnosis cont…Diagnosis cont…
 Easier to diagnose when occurs in several suspicious cases than singleEasier to diagnose when occurs in several suspicious cases than single
case (or at high risk situationscase (or at high risk situations postal employee)postal employee)
 chest x-ray showing a widened mediastinum, infiltrates, pleural effusion,chest x-ray showing a widened mediastinum, infiltrates, pleural effusion,
etc. = likely anthrax infectionetc. = likely anthrax infection
 CT scan showing hyperdense hilar and mediastinal nodesCT scan showing hyperdense hilar and mediastinal nodes (white arrow, middle(white arrow, middle
diagram)diagram) + extensive mediastinal edema = anthrax infection+ extensive mediastinal edema = anthrax infection
 Molecular Biology tests only available at Laboratory Response NetworkMolecular Biology tests only available at Laboratory Response Network
(LRN) labs but highly reliable(LRN) labs but highly reliable
Pulmonary anthrax chest X-rayPulmonary anthrax chest X-ray
showing widened mediastinumshowing widened mediastinum
Dept of Radiological PathologyDept of Radiological Pathology
Armed Forces Institute ofArmed Forces Institute of
PathologyPathology

Diagnosis cont…Diagnosis cont…
Distinguish Anthrax from Influenza-Like IllnessDistinguish Anthrax from Influenza-Like Illness
(ILI):(ILI):
 Early symptoms (fever, chills, myalgias, fatigue,Early symptoms (fever, chills, myalgias, fatigue,
malaise, and nonproductive cough) are similar.malaise, and nonproductive cough) are similar.
 ILI patients have rhinorrhea and this is uncommonILI patients have rhinorrhea and this is uncommon
in Anthrax patients.in Anthrax patients.
 Anthrax patients have shortness of breath, anAnthrax patients have shortness of breath, an
uncommon trait of ILI patients.uncommon trait of ILI patients.
 Nausea and vomiting are more common in anthraxNausea and vomiting are more common in anthrax
infectioninfection
 Abnormal chest radiographs and CT scansAbnormal chest radiographs and CT scans 
Anthrax infectionAnthrax infection

Post-exposure Prophylaxis:Post-exposure Prophylaxis:
Treatment and TherapyTreatment and Therapy
 Limited number of cases and data for treatmentLimited number of cases and data for treatment
 Must be administered before critical toxin levelsMust be administered before critical toxin levels
are reachedare reached
 Early attemptsEarly attempts (cutaneous)(cutaneous) surgery, but fear ofsurgery, but fear of
septicemiasepticemia (early 20(early 20thth
century)century)
 Pasteur, Jourbert, & FortineauPasteur, Jourbert, & Fortineau antibioticsantibiotics (focus(focus
of modern treatment now)of modern treatment now)
 Early therapy utilized Penicillin G procaine butEarly therapy utilized Penicillin G procaine but
has been shifted to other antibiotics due tohas been shifted to other antibiotics due to
Penicillin-resistant strain appearancePenicillin-resistant strain appearance

Treatment and Therapy cont…Treatment and Therapy cont…
 Fluoroquinone class antibiotics are now usedFluoroquinone class antibiotics are now used
Ciprofloxacin in conjunction w/ Doxycycline as firstCiprofloxacin in conjunction w/ Doxycycline as first
lineline
 Usual dosages:Usual dosages:
 Cutaneous: adults & pregnant womenCutaneous: adults & pregnant women 500mg500mg
Ciprofloxacin and 100mg Doxycycline twice daily;Ciprofloxacin and 100mg Doxycycline twice daily;
childrenchildren varies, based on age and weight (All for atvaries, based on age and weight (All for at
least 60 days)least 60 days)
 Inhalational & Gastrointestinal via IV:Inhalational & Gastrointestinal via IV: adults and pregnantadults and pregnant
womenwomen 400mg Ciprofloxacin400mg Ciprofloxacin OROR 100mg Doxycycline100mg Doxycycline
every 12hrsevery 12hrs ANDAND 2 additional antimicrobials (rifampin,2 additional antimicrobials (rifampin,
vancomycin, penicilin, ampicillin, chloramphenicol,vancomycin, penicilin, ampicillin, chloramphenicol,
imipenem, clindamycin, & clarithromycin);imipenem, clindamycin, & clarithromycin); childrenchildren
CiprofloxacinCiprofloxacin OROR Doxycycline varies, based on age &Doxycycline varies, based on age &
weightweight ANDAND one or two of the previously mentionedone or two of the previously mentioned
antimicrobials. (Therapy switched to oral treatment whenantimicrobials. (Therapy switched to oral treatment when
clinically appropriate and lasts for at least 60 days)clinically appropriate and lasts for at least 60 days)

CategoryCategory Initial IV TherapyInitial IV Therapy DurationDuration
AdultsAdults Ciprofloxacin 400 mg everyCiprofloxacin 400 mg every
12 hr OR12 hr OR
Doxycycline 100mg everyDoxycycline 100mg every
12 hr12 hr
ANDAND
Additional 1 or 2 antibioticsAdditional 1 or 2 antibiotics
When clinically appropriateWhen clinically appropriate
switch to oral therapy:switch to oral therapy:
Ciprofloxacin 500mg 2xCiprofloxacin 500mg 2x
daily ORdaily OR
Doxycycline 100 mg 2xDoxycycline 100 mg 2x
dailydaily
Continue oral or IV therapyContinue oral or IV therapy
for 60 daysfor 60 days
Children*Children* Ciprofloxacin 10-15 mg/kgCiprofloxacin 10-15 mg/kg
ever 12 hr ORever 12 hr OR
Doxycycline:Doxycycline:
>8y and > 45kg: 100mg>8y and > 45kg: 100mg
every 12 hrevery 12 hr
>8y and>8y and << 45kg: 2.245kg: 2.2
mg/kg every 12 hrmg/kg every 12 hr
<8y 2.2mg/kg every 12<8y 2.2mg/kg every 12
hrhr
AND 1 or 2 additionalAND 1 or 2 additional
antibioticsantibiotics
Switch to oral whenSwitch to oral when
clinically appropriateclinically appropriate
Ciprofloxacin 10-15 mg/kgCiprofloxacin 10-15 mg/kg
every 12 hr ORevery 12 hr OR
Doxycycline:Doxycycline:
>8y and > 45kg: 100mg>8y and > 45kg: 100mg
2x daily2x daily
>8y and>8y and << 45kg: 2.245kg: 2.2
mg/kg 2x dailymg/kg 2x daily
<8y 2.2mg/kg 2x daily<8y 2.2mg/kg 2x daily
Continue oral or IV for 60Continue oral or IV for 60
daysdays
Pregnant Women*Pregnant Women* Same for non-pregnantSame for non-pregnant
adultsadults
Same for non-pregnantSame for non-pregnant
adultsadults
*Although ciprofloxacin and certain tetracyclines are not recommended for children and/or pregnant women, in life
threatening circumstances their use would be warranted. Adapted from CDC.

Pre-exposure Prophylaxis: AnthraxPre-exposure Prophylaxis: Anthrax
VaccineVaccine
 1881: first developed by Louis Pasteur1881: first developed by Louis Pasteur
 1937: vaccine modified to use attenuated1937: vaccine modified to use attenuated
non pathogenic strain ofnon pathogenic strain of B. anthracisB. anthracis w/w/
dormant sporesdormant spores (other than former Soviet Union(other than former Soviet Union
and associate countries, this vaccine is preferred overand associate countries, this vaccine is preferred over
live attenuated vaccine)live attenuated vaccine)
 U.S. currently uses AVAU.S. currently uses AVA (Anthrax Vaccine(Anthrax Vaccine
Adsorbed)Adsorbed) aka BioThrax™ licensed in 1970aka BioThrax™ licensed in 1970
and produced by Bioport Corp.and produced by Bioport Corp.

Anthrax Vaccine cont…Anthrax Vaccine cont…
 AVA: made from cell-free infiltrates of microaerophilic cultures of anAVA: made from cell-free infiltrates of microaerophilic cultures of an
avirulent, nonencapsulated strain ofavirulent, nonencapsulated strain of B anthracis concentrated with PAB anthracis concentrated with PA
(protective antigen)(protective antigen) final no live or dead bacteria are in final productfinal no live or dead bacteria are in final product
 Final product contains: 1.2 mg/mL aluminum, added as aluminumFinal product contains: 1.2 mg/mL aluminum, added as aluminum
hydroxide in 0.85% sodium chloridehydroxide in 0.85% sodium chloride (Al = adjuvant that increase immune(Al = adjuvant that increase immune
response)response); & 25 mcg/mL benzethonium chloride and 100 mcg/mL; & 25 mcg/mL benzethonium chloride and 100 mcg/mL
formaldehyde, added as preservativeformaldehyde, added as preservative
 How efficacious? : several animal studies & 1 controlled human trialHow efficacious? : several animal studies & 1 controlled human trial
induced immune response in 83% of adults vaccinated with one dose andinduced immune response in 83% of adults vaccinated with one dose and
91% for 2 or more doses.91% for 2 or more doses.
 ** Correlation between antibody titer and protection against infection not** Correlation between antibody titer and protection against infection not
yet defined.yet defined.
 Efficacy duration unknown, but animal tests suggest 1-2 years after 2Efficacy duration unknown, but animal tests suggest 1-2 years after 2
doses.doses.
 Vaccine not licensed for children due to lack of any pediatric testing, butVaccine not licensed for children due to lack of any pediatric testing, but
likely to be safe based on results with other inactive vaccines.likely to be safe based on results with other inactive vaccines.
 1998: Department of Defense makes vaccination for anthrax mandatory1998: Department of Defense makes vaccination for anthrax mandatory
for all troops (AVIP)for all troops (AVIP) over 400 quit or court marshaled due to fear ofover 400 quit or court marshaled due to fear of
adverse side effects.adverse side effects.

 Dosage:Dosage:
 In U.S.: 6 doses of 0.5 mL at weeks 0, 2, & 4 ANDIn U.S.: 6 doses of 0.5 mL at weeks 0, 2, & 4 AND
months 6, 12, & 18, as well as annual boostersmonths 6, 12, & 18, as well as annual boosters
 In U.K.: 6 doses of 0.5 mL at weeksIn U.K.: 6 doses of 0.5 mL at weeks 0, 3, 6, & 32 with0, 3, 6, & 32 with
annual boosters.annual boosters.
 Post-exposure vaccination:Post-exposure vaccination:
 Vaccine can be used with antibiotics after inhalationVaccine can be used with antibiotics after inhalation
exposureexposure
 3-dose regime @ weeks 0, 2,& 4 in combination with3-dose regime @ weeks 0, 2,& 4 in combination with
at least 30-days of antimicrobial therapy (w/outat least 30-days of antimicrobial therapy (w/out
vaccine 60-day antimicrobial therapy is needed)vaccine 60-day antimicrobial therapy is needed)
 Vaccine not licensed for post-exposure therapy.Vaccine not licensed for post-exposure therapy.
Therefore, it is administered as an investigational newTherefore, it is administered as an investigational new
drug (IND) under FDA regulations.drug (IND) under FDA regulations.

 Adverse effects:Adverse effects:
 USAMRIID data: inflammatory reaction greaterUSAMRIID data: inflammatory reaction greater
than 5 cm in diameter around the site of injectionthan 5 cm in diameter around the site of injection
found in about 2.4 - 3.9% of casesfound in about 2.4 - 3.9% of cases
 1990-200: Vaccine Adverse Event reporting1990-200: Vaccine Adverse Event reporting
System (VAERS) receives 1,544 reports ofSystem (VAERS) receives 1,544 reports of
adverse events after vaccinationadverse events after vaccination mostmost
frequent included:frequent included: injection site hypersensitivityinjection site hypersensitivity
(334), injection site edema (283), injection site(334), injection site edema (283), injection site
pain (247), headache (239), arthralgia (232),pain (247), headache (239), arthralgia (232),
asthenia (215), and pruritus (212).asthenia (215), and pruritus (212).

 Possible future Improvements on vaccine:Possible future Improvements on vaccine:
 Cloning the PA gene into organisms of low pathogenicity,Cloning the PA gene into organisms of low pathogenicity,
such assuch as B. subtilis, and creatingB. subtilis, and creating recombinant vaccinesrecombinant vaccines
 Creating mutant strains of vaccines that utilize non-humanCreating mutant strains of vaccines that utilize non-human
aromatic compounds for virulencearomatic compounds for virulence may establish bettermay establish better
immunicity (minimizes self-cell recognition problems) w/outimmunicity (minimizes self-cell recognition problems) w/out
risk of deadly infectionrisk of deadly infection
 Experimenting with purified PA preparations with differentExperimenting with purified PA preparations with different
combinations of adjuvant molecules (similar to AVAcombinations of adjuvant molecules (similar to AVA
approach)approach)
1)1) PA with monophosphoryl lipid A (MPL)PA with monophosphoryl lipid A (MPL) no cold storage chainno cold storage chain
required and possibly more efficacious in conferring immunityrequired and possibly more efficacious in conferring immunity
2)2) expression of the cereolysine AB gene inexpression of the cereolysine AB gene in B. anthracisB. anthracis givesgives
hemolytic properties to the bacteria; conferred immunity againsthemolytic properties to the bacteria; conferred immunity against
both H-7 strain wildtype and H-7 AB recombinant when immunizedboth H-7 strain wildtype and H-7 AB recombinant when immunized
by recombinant STI-1 strain containing the cereolysine AB geneby recombinant STI-1 strain containing the cereolysine AB gene
3)3) AVA may be more effectiveAVA may be more effective 100% immunity in 2-dose immunized100% immunity in 2-dose immunized
rhesus monkeys when compared to 100% mortality for non-rhesus monkeys when compared to 100% mortality for non-
immunized monkeysimmunized monkeys

““Safety First”Safety First”
Pre-Anthrax testing/handling
Post-Anthrax handling: Decontamination

ANTHRAX AS A BIOLOGICALANTHRAX AS A BIOLOGICAL
WEAPONWEAPON
WHY USEWHY USE
ANTHRAX?ANTHRAX?
 Relatively easyRelatively easy
to acquire, into acquire, in
vitro growthvitro growth
(any lab media(any lab media
@ 37@ 37°C)°C) andand
induction ofinduction of
sporulation issporulation is
easy.easy.

WHY USE ANTHRAX?WHY USE ANTHRAX?
 Most likely agent for large-scale biologicalMost likely agent for large-scale biological
attack.attack.
 Inhalational anthrax is the most potent of theInhalational anthrax is the most potent of the
three disease forms. ~80% mortality and withthree disease forms. ~80% mortality and with
prompt treatment ~50% mortality.prompt treatment ~50% mortality.
 Anthrax spores can be easily spread in theAnthrax spores can be easily spread in the
environment.Ultimate reservoir is soil where theyenvironment.Ultimate reservoir is soil where they
can survive for decades.can survive for decades.
 Spores are stable to drying, heat, UV light,Spores are stable to drying, heat, UV light,
gamma radiation, and many disinfectants.gamma radiation, and many disinfectants.

WHY USE ANTHRAX? – cont.WHY USE ANTHRAX? – cont.
 Spore size of 3-6Spore size of 3-6 μm suitable for aerosolizationμm suitable for aerosolization
and ideal for human alveolar deposition.and ideal for human alveolar deposition.
 Early differentiation of inhalational anthrax fromEarly differentiation of inhalational anthrax from
common infections is difficult.common infections is difficult.
 Anthrax spores decay at the rate of less than 0.1Anthrax spores decay at the rate of less than 0.1
per cent a minute, which is very slow for anper cent a minute, which is very slow for an
organism.organism.

WHY USE ANTHRAX? – cont.WHY USE ANTHRAX? – cont.
 USDD reported that 3 defense employees withUSDD reported that 3 defense employees with
some technical skills but without expertise onsome technical skills but without expertise on
bioweapons could manufacture a simulant of BAbioweapons could manufacture a simulant of BA
in less than a month and for $1M.in less than a month and for $1M.
 Aerosol release would be difficult to establish,Aerosol release would be difficult to establish,
spores are odorless and invisible and havespores are odorless and invisible and have
potential to travel many kms before dissipating.potential to travel many kms before dissipating.

Some data on offensiveSome data on offensive
capacity of anthrax sporescapacity of anthrax spores
 (1960) Experiments w/ simulant B. globigii in NYC(1960) Experiments w/ simulant B. globigii in NYC
subway system: release of similar amount of B.subway system: release of similar amount of B.
anthracis during rush hour would result in 10,000anthracis during rush hour would result in 10,000
deaths.In this test more than 1M NY were exposeddeaths.In this test more than 1M NY were exposed
to the Bacillus.to the Bacillus.
 Aerial spray of BA along 100km line under idealAerial spray of BA along 100km line under ideal
meteorological cond could produce 50%lethalitymeteorological cond could produce 50%lethality
rates as far as 160 km downwind.rates as far as 160 km downwind.
 US study near Johnston Atoll in SP reported that aUS study near Johnston Atoll in SP reported that a
plane “sprayed a 32mile long line of agent thatplane “sprayed a 32mile long line of agent that
traveled for more than 60 miles before it lost itstraveled for more than 60 miles before it lost its
infectiousness”.infectiousness”.

Some data on offensiveSome data on offensive
capacity of anthrax spores –capacity of anthrax spores –
cont.cont.
 WHO estimated that 50kg of BA released in aWHO estimated that 50kg of BA released in a
pop of 500,000 would result in 95,000 fatalities +pop of 500,000 would result in 95,000 fatalities +
125,000 incapacitated.125,000 incapacitated.
 50 kg over urban pop of 5M = 100,000 deaths50 kg over urban pop of 5M = 100,000 deaths
and 250,000 sick.and 250,000 sick.
 100 kg = bw 130,000 and 3M deaths over an100 kg = bw 130,000 and 3M deaths over an
area size of W-DC. This lethality matches that ofarea size of W-DC. This lethality matches that of
a hydrogen bomb.a hydrogen bomb.

Some data on offensive capacitySome data on offensive capacity
of anthrax spores –cont.of anthrax spores –cont.
 CDC suggested a cost of $26.2B per 100,000CDC suggested a cost of $26.2B per 100,000
persons exposed.persons exposed.
 Human LD50 is ~2,500 to 55,000 inhaled BAHuman LD50 is ~2,500 to 55,000 inhaled BA
spores but studies on primates suggest that asspores but studies on primates suggest that as
few as 1-3 spores may be sufficient to causefew as 1-3 spores may be sufficient to cause
infection.infection.
 Little information of possible risks of directLittle information of possible risks of direct
contamination of food or water with anthraxcontamination of food or water with anthrax
spores.spores.

So, who (possibly) has anthraxSo, who (possibly) has anthrax
spores ready?spores ready?
 1995 Iraq acknowledged producing and1995 Iraq acknowledged producing and
weaponizing BA to the UN special commission.weaponizing BA to the UN special commission.
 Former Soviet Union also known to have a BAFormer Soviet Union also known to have a BA
production program.production program.
 Existence of offensive biological weaponsExistence of offensive biological weapons
programs in at least 13-17 countries includingprograms in at least 13-17 countries including
Syria, Iran, Libya, China, SK, NK, Taiwan andSyria, Iran, Libya, China, SK, NK, Taiwan and
Israel. (US Dep of Def)Israel. (US Dep of Def)

Britain – Gruinard islandBritain – Gruinard island
19421942
 Britain conducted research during 1942 atBritain conducted research during 1942 at
Gruinard Island (Scotland).Gruinard Island (Scotland).
 Sheep were taken to an open field, secured inSheep were taken to an open field, secured in
wooden frames, and exposed to a bomb thatwooden frames, and exposed to a bomb that
scattered the spores. The sheep started dyingscattered the spores. The sheep started dying
three days later.three days later.
 An infected sheep's carcass washed ashoreAn infected sheep's carcass washed ashore
from the island led to the deaths of sevenfrom the island led to the deaths of seven
cattle, two horses, three cats and up to 50cattle, two horses, three cats and up to 50
sheep in a nearby village. They had probablysheep in a nearby village. They had probably
been infected by a dog which had itself beenbeen infected by a dog which had itself been
infected by the carcass.infected by the carcass.

Britain – Gruinard island 1942Britain – Gruinard island 1942
 Island was soIsland was so
contaminated thatcontaminated that
the island was kept inthe island was kept in
quarantine for 48quarantine for 48
years.years.
 In 1986 an EnglishIn 1986 an English
company was paidcompany was paid
₤500,000 to₤500,000 to
decontaminate thedecontaminate the
520-acre island by520-acre island by
soaking the ground insoaking the ground in
280 tons of280 tons of
formaldehyde dilutedformaldehyde diluted
in 2000 tons ofin 2000 tons of
seawater.seawater.

Sverdlovsk case – FormerSverdlovsk case – Former
Soviet Union – April 2, 1979Soviet Union – April 2, 1979
 Odorless, colorless, unnoticeable aerosol wasOdorless, colorless, unnoticeable aerosol was
accidentally released from a secret Sovietaccidentally released from a secret Soviet
biological weapons facility in Sverdlovsk.biological weapons facility in Sverdlovsk.
 96 people within a short distance fell sick; 6696 people within a short distance fell sick; 66
died (68.75%MR). All where inhalationaldied (68.75%MR). All where inhalational
anthrax, not single case was cutaneous. Animalsanthrax, not single case was cutaneous. Animals
found dead 50 km southeast of Sverdlovsk.found dead 50 km southeast of Sverdlovsk.
 New cases developed as late as 43 days afterNew cases developed as late as 43 days after
the presumed date of released. None duringthe presumed date of released. None during
months or years afterward.months or years afterward.

Sverdlovsk incident (1979)Sverdlovsk incident (1979)
120 to >400 became ill, 75
died (at least, probably ~ 90)
Soviet officials initially
reported that outbreak was due
to contaminated meat
Autopsy findings of
hemorrhagic mediastinitis
Infection later linked to an
aerosol leak from a nearby
biologic weapons facility
operated by the Soviet army
Meselson et al, Science, 1994

Sverdlovsk: Case onsets & DeathsSverdlovsk: Case onsets & Deaths
0
1
2
3
4
5
6
7
8
4/1/1979
4/8/19794/15/1979
4/22/19794/29/1979
5/6/1979
5/13/19795/20/19795/27/1979
6/3/19796/10/1979
All case onsets All deaths

Gulf war - 1991Gulf war - 1991
 1990 Iraq had 50 R400 bombs filled with1990 Iraq had 50 R400 bombs filled with
anthrax.anthrax.
 8,500L of anthrax.8,500L of anthrax.
 Bombs were not used due to a supposedBombs were not used due to a supposed
fear by Iraq that winds and meteorologicalfear by Iraq that winds and meteorological
conditions could make them also a targetconditions could make them also a target
for their own bombs.for their own bombs.

Aum Shinrikyo failed attackAum Shinrikyo failed attack
-1995-1995
 Terrorist group, Aum Shinrikyo,Terrorist group, Aum Shinrikyo,
responsible for the release of Sarin in aresponsible for the release of Sarin in a
Tokyo subway on 1995, dispersedTokyo subway on 1995, dispersed
aerosols of anthrax and botulismaerosols of anthrax and botulism
throughout Tokyo on at least 8throughout Tokyo on at least 8
occasions. Attacks failed because strainoccasions. Attacks failed because strain
released closely matched the Sternereleased closely matched the Sterne
34F2 strain used for animal vaccination34F2 strain used for animal vaccination
programs and is not a significant risk forprograms and is not a significant risk for
humans.humans.

US Anthrax attacks - 2001US Anthrax attacks - 2001
 Robert Stevens, 63,Robert Stevens, 63,
photo editor at thephoto editor at the
supermarket tabloidsupermarket tabloid
The Sun, publishedThe Sun, published
by American Mediaby American Media
Inc., died on OctoberInc., died on October
5, 2001 from5, 2001 from
contracting pulmonarycontracting pulmonary
anthrax from ananthrax from an
envelope.envelope.

US Anthrax attacks – 2001 -US Anthrax attacks – 2001 -
contcont
 Oct,2 – Nov, 20 22 cases of anthrax wereOct,2 – Nov, 20 22 cases of anthrax were
confirmed: 11 inhalational anthrax and 11 (7confirmed: 11 inhalational anthrax and 11 (7
confirmed and 4 suspected) of cutaneousconfirmed and 4 suspected) of cutaneous
anthrax.anthrax.
 7 states along east coast of US: CT (1), FL (2),7 states along east coast of US: CT (1), FL (2),
MD (3), NJ (5), NYC (8), PA (1),VA (2).MD (3), NJ (5), NYC (8), PA (1),VA (2).
 Case-fatality ratio for inhalational anthrax: 45%.Case-fatality ratio for inhalational anthrax: 45%.
 Mean duration bw exposure and onset ofMean duration bw exposure and onset of
symptoms: 4.5 days.symptoms: 4.5 days.

contcont
 4 BA + powder-4 BA + powder-
containing envelopescontaining envelopes
were recovered.were recovered.
 2 mailed around2 mailed around
Trenton, NJ andTrenton, NJ and
postmarked Seppostmarked Sep
18,2001.18,2001.

contcont
 Envelopes processed at US PS Trenton MailEnvelopes processed at US PS Trenton Mail
Processing Ctr and Morgan Central PostalProcessing Ctr and Morgan Central Postal
Facility (NYC). Both facilities and at least 5 moreFacility (NYC). Both facilities and at least 5 more
affiliated with NJ facility had environmentalaffiliated with NJ facility had environmental
samples positive for BA.samples positive for BA.
 No envelopes found in FL but BA was isolatedNo envelopes found in FL but BA was isolated
from environmental sampling at the AMI buildingfrom environmental sampling at the AMI building
(were 2 cases from FL worked).(were 2 cases from FL worked).

contcont
 Oct 9 envelopes sent from NJ to Washington DCOct 9 envelopes sent from NJ to Washington DC
(congress: Hart Building).(congress: Hart Building).
 Sen. Daschle’s letter was opened by office staff on Oct,Sen. Daschle’s letter was opened by office staff on Oct,
15. Prompt recognition of powder led to rapid initiation of15. Prompt recognition of powder led to rapid initiation of
post-exposure prophylaxis.post-exposure prophylaxis.
 Scientists estimate that the letter sent to Sen. DaschleScientists estimate that the letter sent to Sen. Daschle
originally contained about 2 grams of anthrax.originally contained about 2 grams of anthrax.

contcont
 Concentration - in the range of 1 trillion spores per gram -Concentration - in the range of 1 trillion spores per gram -
meant that the letter could have contained 200 million timesmeant that the letter could have contained 200 million times
the average dose necessary to kill a person.the average dose necessary to kill a person.
 625 persons from Hart Building were tested (nasal swaps)625 persons from Hart Building were tested (nasal swaps)
and 28 found positive for BA.and 28 found positive for BA.
 Environmental sampling of Building showed heavilyEnvironmental sampling of Building showed heavily
contamination. Building was closed for decontamination oncontamination. Building was closed for decontamination on
Oct 17,2001.Oct 17,2001.

contcont
 Letter to Sen. LeahyLetter to Sen. Leahy
was in 1/280 barrels ofwas in 1/280 barrels of
unopened mailunopened mail
collected from Capitolcollected from Capitol
Hill after Sen. Daschle’sHill after Sen. Daschle’s
letter was discovered.letter was discovered.

contcont
 An estimated 32,000 persons initiatedAn estimated 32,000 persons initiated
antimicrobial prophylaxis but completion of theantimicrobial prophylaxis but completion of the
60 day course was recommended for 10,30060 day course was recommended for 10,300
persons.persons.
 Cross-contamination of mail occurred but riskCross-contamination of mail occurred but risk
was low considering the high amount of mailwas low considering the high amount of mail
processed in NJ and NYC.processed in NJ and NYC.
 2 patients w/ inh. anthrax had no exposure to2 patients w/ inh. anthrax had no exposure to
media or government facilities or possiblemedia or government facilities or possible
sources of naturally occurring anthrax yet bothsources of naturally occurring anthrax yet both
were infected w/ BA isolates identical towere infected w/ BA isolates identical to
outbreak strain.outbreak strain.

contcont
 All of the anthrax spores in the mail were foundAll of the anthrax spores in the mail were found
to be of an identical strain (Ames strain). Thisto be of an identical strain (Ames strain). This
strain is one that the U.S. military used for studystrain is one that the U.S. military used for study
at USAMRIID and distributed to otherat USAMRIID and distributed to other
government and university labs as well as togovernment and university labs as well as to
other governments including Great Britain.other governments including Great Britain.
 Decontamination of Hart Building took threeDecontamination of Hart Building took three
months and cost was estimated at $23M.months and cost was estimated at $23M.

New FindingsNew Findings
 Secondary aerosolization of viable BA sporesSecondary aerosolization of viable BA spores
under common office activities was tested andunder common office activities was tested and
found to be possible. Test was perform almost afound to be possible. Test was perform almost a
month after the incident.month after the incident.
 Institute for Genomic Research (Rockville, MD)Institute for Genomic Research (Rockville, MD)
completed sequencing the anthrax bacillus incompleted sequencing the anthrax bacillus in
late November 2001, but has decided to delaylate November 2001, but has decided to delay
its publication and access to researchits publication and access to research
community for security reasons.community for security reasons.

What to do in case of an attack?What to do in case of an attack?
 US military M17 and M40 gas masksUS military M17 and M40 gas masks
provide excellent protection against 1-5provide excellent protection against 1-5 μmμm
particles ($324). Other articles include a portableparticles ($324). Other articles include a portable
mask ($79.95) and anthrax test-kit ($24.95)mask ($79.95) and anthrax test-kit ($24.95)
 Use of pre-exposure and post-exposureUse of pre-exposure and post-exposure
antibiotics (US attack isolates were sensitive toantibiotics (US attack isolates were sensitive to
fluoroquinolones, .fluoroquinolones, .
 Pre-exposure and post-exposure vaccination??Pre-exposure and post-exposure vaccination??
 Decontamination of any exposed spaces.Decontamination of any exposed spaces.

How to determine it is anthrax?How to determine it is anthrax?
 Anthrax test-kit ($24.95)Anthrax test-kit ($24.95)
 1998-Manz lab- new method for PCR:1998-Manz lab- new method for PCR:
chemical amplification continuous-flowchemical amplification continuous-flow
PCR on a chip. Problem: still slow andPCR on a chip. Problem: still slow and
too much equipment for remote site use.too much equipment for remote site use.
 Burke lab: PCR product could be sentBurke lab: PCR product could be sent
directly onto miniature gel: 30 min.directly onto miniature gel: 30 min.
 Phillip Belgrader: PCR in microfugePhillip Belgrader: PCR in microfuge
tubes: 7 min.tubes: 7 min.

ReferencesReferences
MICROBIOLOGY REFERENCES:
1.1. Drum, C.L., Yan, S.Z., et al. Structural Basis for theDrum, C.L., Yan, S.Z., et al. Structural Basis for the
Activation of Anthrax Adenylyl Cyclase ExotoxinActivation of Anthrax Adenylyl Cyclase Exotoxin
by Calmodium. Nature. 415: 396-402, 2002.by Calmodium. Nature. 415: 396-402, 2002.
2.2. Duesbery, N.S., Vande Woude, G.F., AnthraxDuesbery, N.S., Vande Woude, G.F., Anthrax
Toxins. Cellular and Molecular Life Sciences. 55:Toxins. Cellular and Molecular Life Sciences. 55:
1599-1609, 1999.1599-1609, 1999.
3.3. Koehler, T.M. Anthrax. Springer. 2002.Koehler, T.M. Anthrax. Springer. 2002.
4.4. Liddington, R.C., A Molecular Full Nelson. Nature.Liddington, R.C., A Molecular Full Nelson. Nature.
415: 373-374, 2002.415: 373-374, 2002.
5.5. Mehta, A., Zubay, G., et al. Anthrax Chapter. 2003.Mehta, A., Zubay, G., et al. Anthrax Chapter. 2003.

References (cont.)References (cont.)
7.7. Pannifer, A.D., Wong, T.Y., et al, Crystal StructurePannifer, A.D., Wong, T.Y., et al, Crystal Structure
of the Anthrax Lethal Factor. Nature. 414: 229-233,of the Anthrax Lethal Factor. Nature. 414: 229-233,
2001.2001.
8.8. Patocka, J., Splino, M. Anthrax ToxinPatocka, J., Splino, M. Anthrax Toxin
Characterization. Acta Medica. 45: 3-6, 2002.Characterization. Acta Medica. 45: 3-6, 2002.
9.9. Turnbull, P.C.B., Quinn, C.P., et al.Turnbull, P.C.B., Quinn, C.P., et al. BacillusBacillus
anthracisanthracis and Otherand Other BacillusBacillus Species. AcademicSpecies. Academic
Press. 2001.Press. 2001.
10.10. http://http://
www.biocarta.com/pathfiles/h_anthraxPathway.aspwww.biocarta.com/pathfiles/h_anthraxPathway.asp
11.11. http://http://
www.biotechjournal.com/Pathways/anthrax.htmwww.biotechjournal.com/Pathways/anthrax.htm
12.12. http://http://
www.es.dis.titech.ac.jp/~park/anthrax/anthrax.htmlwww.es.dis.titech.ac.jp/~park/anthrax/anthrax.html

History & Clinical features references:History & Clinical features references:
1.1. Brook, Itzhak. The Prophylaxis and Treatment of Anthrax.Brook, Itzhak. The Prophylaxis and Treatment of Anthrax.
International Journal of Antimicrobial Agents. 20: 320-325,International Journal of Antimicrobial Agents. 20: 320-325,
2002.2002.
2.2. Dixon, T.C., M. Meselson, J. Guillemin, et al. Anthrax. NewDixon, T.C., M. Meselson, J. Guillemin, et al. Anthrax. New
England Journal of Medicine. 341: 815-826, 1999.England Journal of Medicine. 341: 815-826, 1999.
3.3. Franz, D.R., P.B. Jahrling, A.M. Friedlander, et al. ClinicalFranz, D.R., P.B. Jahrling, A.M. Friedlander, et al. Clinical
Recognition and Management of Patients Exposed to BiologicalRecognition and Management of Patients Exposed to Biological
Warfare Agents. JAMA. 278 (5): 399-411. 1997.Warfare Agents. JAMA. 278 (5): 399-411. 1997.
4.4. Greenfield, Ronald A., et al. Bacterial Pathogens as BiologicalGreenfield, Ronald A., et al. Bacterial Pathogens as Biological
Weapons and Agents of Bioterrorism. The American Journal ofWeapons and Agents of Bioterrorism. The American Journal of
The Medical Sciences. Volume 323, Number 6. June 2002.The Medical Sciences. Volume 323, Number 6. June 2002.
5.5. Inglesby, MD, et. al. Anthrax as a Biological Weapon: MedicalInglesby, MD, et. al. Anthrax as a Biological Weapon: Medical
and Public Health Management. JAMA. Vol 281, No.18. Mayand Public Health Management. JAMA. Vol 281, No.18. May
12,1999.12,1999.

6.6. IOM. The Anthrax Vaccine: Is it safe? Does it work?IOM. The Anthrax Vaccine: Is it safe? Does it work?
Washington, D.C.: National Academy Press. March 2002.Washington, D.C.: National Academy Press. March 2002.
7.7. Peters, C.J., D.M. Hartley. Anthrax Inhalation and LethalPeters, C.J., D.M. Hartley. Anthrax Inhalation and Lethal
Human Infection. Lancet. 359 (9307): 710-711.Human Infection. Lancet. 359 (9307): 710-711.
8.8. Turnbull, P.C.B., Quinn, C.P., et al.Turnbull, P.C.B., Quinn, C.P., et al. Bacillus anthracisBacillus anthracis and Otherand Other
BacillusBacillus Species. Academic Press. 2001.Species. Academic Press. 2001.
9.9. Center for Infectious Disease Research & Policy:Center for Infectious Disease Research & Policy:
http://www.cidrap.umn.edu/cidrap/content/bt/anthrax/biofacts/anhttp://www.cidrap.umn.edu/cidrap/content/bt/anthrax/biofacts/an
thraxfactsheet.htmlthraxfactsheet.html
10.10. Nas, Meryl M.D. Anthrax Epizootic in Zimbabwe, 1978-1980:Nas, Meryl M.D. Anthrax Epizootic in Zimbabwe, 1978-1980:
Due to Deliberate Spread?:Due to Deliberate Spread?:
http://www.anthraxvaccine.org/zimbabwe.htmlhttp://www.anthraxvaccine.org/zimbabwe.html
11.11. US Army Center for Health Promotion and PreventativeUS Army Center for Health Promotion and Preventative
Medicine:Medicine:
http://chppm-www.apgea.army.mil/HomelandSecurity/anthraxvideohttp://chppm-www.apgea.army.mil/HomelandSecurity/anthraxvideo

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