Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

91
Bacilus Anthracis Bacilus Anthracis Anthrax Anthrax Sheref Hassan Sheref Hassan Rachel Handwerger Rachel Handwerger Maria Garrido Maria Garrido

Transcript of Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

Page 1: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

Bacilus AnthracisBacilus Anthracis

AnthraxAnthrax

Sheref HassanSheref Hassan

Rachel HandwergerRachel Handwerger

Maria GarridoMaria Garrido

Page 2: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria 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 ( (1.0-1.5 1.0-1.5 μμm by 3.0-5.0 m 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 ((1.0x1.5 1.0x1.5 μμmm));; COCO2 2 levels within the body inhibit sporulationlevels within the body inhibit sporulation

Forms long chains Forms long chains in vitroin vitro; forms single cells or short chains in clinical ; forms single cells or short chains in clinical samples samples

Aerobic or facultative anaerobic; non-motile; catalase-positiveAerobic or facultative anaerobic; non-motile; catalase-positive Nonhemolytic on sheep blood agar; susceptible to lysis by gamma Nonhemolytic on sheep blood agar; susceptible to lysis by gamma

phagephage Colonies: ground-glass appearance and consistency of beaten-egg Colonies: 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

Page 3: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

““The Agent” The Agent” contd..contd..

● In environments rich in nutrients such as In environments rich in nutrients such as glucose, amino acids, and nucleosides, spores glucose, amino acids, and nucleosides, spores will germinate and form vegetative cells. will germinate and form vegetative cells. Vegetative bacteria survive poorly outside of Vegetative bacteria survive poorly outside of mammalian host.mammalian host.

● In a nutrient-deficient setting, vegetative cells will In 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.

Page 4: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

History of Anthrax:History of Anthrax:Early HistoryEarly History

The first recorded report of anthrax was during the fifth The first recorded report of anthrax was during the fifth Egyptian Plague in 1500 B.C. Egyptian Plague in 1500 B.C. responsible for the death responsible for the death of a large amount of livestock.of a large amount of livestock.

The sixth Egyptian Plague, also known as the “plague of The sixth Egyptian Plague, also known as the “plague of boils” was probably the first recorded instance of boils” 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 well The “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 makes 1863: French biologist Casimir-Joseph Davaine makes B.A.B.A. the first microorganism to be conculsively linked to a the first microorganism to be conculsively linked to a disease.disease.

1876: 1876: B.A.B.A. isolated in pure culture for the first time by isolated in pure culture for the first time by Robert Koch Robert 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.

Page 5: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

History of Anthrax:History of Anthrax: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 in 1919: The Anthrax Prevention Act passed in EnglandEngland established a wool-disinfecting established a wool-disinfecting complex in Liverpool. complex in Liverpool.

Similar laws, gov’t regulations, better disinfectant Similar laws, gov’t regulations, better disinfectant methods and technology thereafter reduced methods and technology thereafter reduced Anthrax cases linked to occupational hazards, Anthrax cases linked to occupational hazards, as well as making Anthrax viability and as well as making Anthrax viability and outbreaks rare in the modern world.outbreaks rare in the modern world.

Page 6: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

History of Anthrax:History of Anthrax:Natural OutbreaksNatural Outbreaks

Animal:Animal:o 1945: Iran 1945: Iran outbreak causes 1 outbreak causes 1

million sheep deathsmillion sheep deaths

Page 7: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

History of Anthrax:History of Anthrax:Natural Outbreaks - cont...Natural Outbreaks - cont...

Inhalation:Inhalation:o U.S. U.S. 1900-1978: only 18 reported cases 1900-1978: only 18 reported cases

mostly among high risk groups such as goat mill mostly among high risk groups such as goat mill or goatskin workers and wool or tannery workers or goatskin workers and wool or tannery workers (only 2 were laboratory associated)(only 2 were laboratory associated)

o New Hampshire New Hampshire 1957: 5 cases reported in 1957: 5 cases reported in goat-hair processing plantgoat-hair processing plant

o No Case of inhalation anthrax was reported No 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. 11 thth attacks.attacks.

Page 8: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

History of Anthrax:History of Anthrax:Natural Outbreaks - cont...Natural Outbreaks - cont...

Cutaneous Cutaneous (most common):(most common):

o U.S. U.S. 1944 -1994: 224 cases reported 1944 -1994: 224 cases reportedo New Hampshire New Hampshire 1957: 4 cases reported in 1957: 4 cases reported in

goat-hair processing plantgoat-hair processing planto Zimbabwe Zimbabwe 1979-1985: largest reported 1979-1985: largest reported

epidemic with over 10,000 reported human epidemic 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 or explained by spread via insect vectors or contaminated meat; however, biological warfare contaminated meat; however, biological warfare tactics have been also postulatedtactics have been also postulated

Page 9: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

History of Anthrax:History of Anthrax:Natural Outbreaks - cont...Natural Outbreaks - cont...

Gastrointestinal Gastrointestinal (uncommonly reported)(uncommonly reported)::2 distinct syndromes: oral pharyngeal 2 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 in Since 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 the 1982: 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 and 1987: 14 cases involving both oral pharyngeal and

abdominal diseaseabdominal disease

Page 10: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

Statistics of Anthrax OccurrencesStatistics of Anthrax Occurrences

In the U.S.:In the U.S.:• Approximately 130 cases occurred annually in the early Approximately 130 cases occurred annually in the early

1900’s1900’s• This number decreased with less than 10 cases reported This number decreased with less than 10 cases reported

each year since the early 1960’s and no occurrences from each year since the early 1960’s and no occurrences from 1978 until after Sept. 111978 until after Sept. 11thth 2001 2001

• 95% of naturally occurring cases are cutaneous and 5% are 95% of naturally occurring cases are cutaneous and 5% are due to inhalation. due to inhalation. (cases of gastrointestinal anthrax have not been recognized yet (cases of gastrointestinal anthrax have not been recognized yet in the U.S.)in the U.S.)

• Only 18 cases of naturally occurring inhalation cases have Only 18 cases of naturally occurring inhalation cases have been reported during the 20been reported during the 20thth century with the most recent in century with the most recent in 19761976

• Since 1990, only 2 cases (1992 and 2000) of naturally Since 1990, only 2 cases (1992 and 2000) of naturally occurring infection have been reported (both cutaneous). occurring infection have been reported (both cutaneous).

Page 11: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

Statistics of Anthrax OccurrencesStatistics of Anthrax Occurrences

Globally:Globally:• Approximately 2,000 - 20,000 cases of anthrax Approximately 2,000 - 20,000 cases of anthrax

occur each year.occur each year.• Most cases are cutaneous with inhalation and Most cases are cutaneous with inhalation and

gastrointestinal being less frequent.gastrointestinal being less frequent.• Human cases usually follow disease Human cases usually follow disease

occurrences in ruminants and are most occurrences in ruminants and are most prevalent in Africa, the Middle East, and parts of prevalent in Africa, the Middle East, and parts of Southeast Asia.Southeast Asia.

Page 12: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

““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)

Page 13: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

Bacillus anthracisBacillus anthracis

Review:Review:Gram PositiveGram PositiveRod shapedRod shapedAerobicAerobicSpore FormingSpore FormingNon-motileNon-motile

A B

A1 A2 A3 A4 B1 B2

Page 14: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

Bacillus anthracisBacillus anthracis

Bacillus anthracis Bacillus anthracis exists in two forms:exists in two forms:1.1. Inert spore formInert spore form

– 1 1 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

Page 15: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

Bacillus anthracisBacillus anthracis

2.2. Vegetative formVegetative form– 1 – 1.5 1 – 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 of Only multiply within host in the presence of

amino acids, nucleosides, and glucoseamino acids, nucleosides, and glucose

Page 16: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

Bacillus anthracisBacillus anthracis

Bacillus anthracisBacillus anthracis is composed of: is composed of:A capsuleA capsuleA 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 chromosome In the bacteria there is one chromosome and 2 plasmids (pXO1 and pXO2)and 2 plasmids (pXO1 and pXO2)

Page 17: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

Bacillus anthracisBacillus anthracis

Chromosome Chromosome not involved in virulence not involved in virulencepXO1 pXO1 codes for PA, EF, and LF codes for PA, EF, and LFpXO2 pXO2 codes for the capsule codes for the capsule

Page 18: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

pXO2/CapsulepXO2/Capsule

pXO2 codes for the homopolymeric pXO2 codes for the homopolymeric --linked poly-D-glutamic acid capsulelinked poly-D-glutamic acid capsule

Contains three genes: capBCAContains three genes: capBCA96,231 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 infectionPoorly understoodPoorly understood

Page 19: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

pXO1pXO1

181,654 nucleotides, ~143 ORFs181,654 nucleotides, ~143 ORFsCodes for the three toxin genes which Codes 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 the Codes for regulatory proteins, from the atxAatxA gene, which causes ~10x increase gene, which causes ~10x increase in transcription of all three toxinsin transcription of all three toxins

Page 20: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

PAPA

Is non-toxic without EF and LFIs non-toxic without EF and LF Coded from Coded from pagpag gene gene

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 EF Function – to aid in the insertion of EF and LF toxins in the cytosoland LF toxins in the cytosol

Page 21: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

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 mixed 4-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.

Page 22: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

PA MechanismPA MechanismMechanism:Mechanism:

1.1. PA83 binds to cell receptorPA83 binds to cell receptor

2.2. Furin or a furin-like molecule cleaves PA83 Furin or a furin-like molecule cleaves PA83 between aa 164-167 (Arg-Lys-Lys-Arg) to two between aa 164-167 (Arg-Lys-Lys-Arg) to two fragments: PA20 (released) and PA63 (remains fragments: PA20 (released) and PA63 (remains attached to the receptor)attached to the receptor)

http://www.biotechjournal.com/Pathways/anthrax.htm

Page 23: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

PA Mechanism (cont.)PA Mechanism (cont.)

3.3. PA63/receptor complexes join to form a PA63/receptor complexes join to form a heptamer at reduced pHheptamer at reduced pH

4.4. The heptamer complex can then bind to one The heptamer complex can then bind to one LF or EF molecule at each subunitLF or EF molecule at each subunit

http://www.biotechjournal.com/Pathways/anthrax.htm

Page 24: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

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 the Acidification 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 pore EF and LF are released through the pore into the cytosol into the cytosol

Koehler, T.M. Anthrax. Springer. 2002.

Page 25: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

General MechanismGeneral Mechanism

Koehler, T.M. Anthrax. Springer. 2002.

Page 26: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

EFEF

Coded from cya geneCoded from cya geneA/T rich (71%)A/T rich (71%)Cysteine-freeCysteine-free

89 kDa (767-amino acid)89 kDa (767-amino acid)Mainly Mainly ββ strands strandsN-terminal ~250 aa bind to PA63N-terminal ~250 aa bind to PA63Remaining molecule possesses catalytic Remaining molecule possesses catalytic

activity in substrate and calmodulin-activity in substrate and calmodulin-binding subdomainsbinding subdomains

A-B toxinA-B toxin

Page 27: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

EF FunctionEF Function

EF receptors are present on most cellsEF receptors are present on most cells EF is a calmodulin dependent adenylate EF is a calmodulin dependent adenylate

cyclasecyclaseTherefore 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 proteinResponsible for mediating Responsible for mediating CaCa2+ 2+ signaling in signaling in

cellscells

Page 28: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

EF MechanismEF Mechanism

MechanismMechanism1.1. EF enters the cell by binding to PA (called EF 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 low terminal low CaCa2+ 2+ affinity site in a very unique affinity site in a very unique way:way:

– One half grips lower lobe while the other grasps the upper One half grips lower lobe while the other grasps the upper lobe and twistslobe and twists

– The method of binding forms a contact region of 6,000 The method of binding forms a contact region of 6,000 ÅÅ2 2 – – an extremely large binding region! an extremely large binding region!

– The conformational change experienced by both molecules The conformational change experienced by both molecules inhibits Cainhibits Ca2+ 2+ from binding and inhibits the reverse reaction from binding and inhibits the reverse reaction of cAMP to ATP of cAMP to ATP

Page 29: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

EF Mechanism (cont.)EF Mechanism (cont.)2.2. The conformational change in EF allows The conformational change in EF allows

it to act as an adenylate cyclase and bind it to act as an adenylate cyclase and bind to ATPto ATP

3.3. cAMP is formed due to the binding of EF cAMP is formed due to the binding of EF to ATPto ATP

Liddington, R.C., A Molecular Full Nelson. Nature. 415: 373-374, 2002.

Page 30: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

EFEFWhat happens as a result of this What happens as a result of this

activity?activity?Causes edema by altering HCauses edema by altering H22O and ion O and ion

movementmovementInhibits immune response against Inhibits immune response against

Bacillus anthracisBacillus anthracis

http://www.biocarta.com/pathfiles/h_anthraxPathway.asp

Page 31: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

EFEF

Inhibits activity of TF NF-Inhibits activity of TF NF-ĸB in ĸB in macrophagesmacrophages

Strains Strains with only EF and PA (LF-) are with only EF and PA (LF-) are not likely to be lethalnot likely to be lethal

Page 32: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

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 competeswith EF to bind PA63with EF to bind PA63 5 imperfect repeats, 5 imperfect repeats, each 19 aa longeach 19 aa long Contains a zinc protease Contains a zinc protease active site (HEXXH helix)active site (HEXXH helix)

A-B ToxinA-B ToxinPannifer, A.D., Wong, T.Y., et al, Crystal Structure of the Anthrax Lethal Factor. Nature. 414: 229-233, 2001.

Page 33: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

LF FunctionLF Function

LF only binds to macrophagesLF only binds to macrophagesLF 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 minutesLF is a zinc dependent metalloprotease LF is a zinc dependent metalloprotease

which cleaves MAPKKwhich cleaves MAPKK

Page 34: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

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 MAPKK Mechanism and Result of MAPKK

cleavage:cleavage:LF cleaves between 7-10 residues on the LF cleaves between 7-10 residues on the

N-terminus of the MAPKKs at a pair of N-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.

Page 35: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

LF Mechanism and Func. (cont)LF Mechanism and Func. (cont)

This cleavage serves to inactivate the MAPKKThis cleavage serves to inactivate the MAPKKMAPKK cleavage leads to lysis of the MAPKK cleavage leads to lysis of the

macrophages, although the mechanism is macrophages, although the mechanism is unclearunclear

http://www.biotechjournal.com/Pathways/anthrax.htm

Page 36: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

LF Mechanism and Func. (cont)LF Mechanism and Func. (cont)

Some MAPKK aid in the activation of TF NF-Some MAPKK aid in the activation of TF NF-ĸBĸBThis illustrates how EF and LF work together to This illustrates how EF and LF work together to

promote macrophage lysispromote macrophage lysis

Page 37: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

LF Mechanism and Func. (cont)LF Mechanism and Func. (cont)

At sublytic levels it stimulates production of IL-1At sublytic levels it stimulates production of IL-1β β and TNFαand TNFα At lytic levels the mechanism is unclear, but it is At lytic levels the mechanism is unclear, but it is

possible that in the early stages they inhibit the possible 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 of and at later stages it stimulates them to cause lysis of cellscells

Causes leakage in cell membrane without ATP Causes leakage in cell membrane without ATP increased Ca increased Ca2+2+ conc. in cell conc. in cell

Strains with only LF and PA (EF-) are likely to be Strains with only LF and PA (EF-) are likely to be lethal, although to a lesser degreelethal, although to a lesser degree

Page 38: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

ReviewReview

EFEF Increased production Increased production

of cAMPof cAMP Causes swellingCauses swelling Inhibits immune Inhibits immune

responseresponse Allows for invasion!Allows for invasion!

LFLF Cleaves MAPKKsCleaves MAPKKs Inhibits immune Inhibits immune

responseresponse Destroys Destroys

macrophagesmacrophages Cells can’t fight off Cells can’t fight off

infection infection they Die! they Die!

Page 39: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

Overall Mechanism ReviewOverall Mechanism Review

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

Page 40: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

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, & inhalational cutaneous, gastrointestinal, & inhalational (pulmonary).(pulmonary).

The type contracted depends on the modes of The 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) cutaneous cutaneous Consumption of contaminated undercooked meatConsumption of contaminated undercooked meat

gastrointestinalgastrointestinal Contact with contaminated hair, wool, or hides (during Contact 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 gastrointestinal mainly inhalational &/or gastrointestinal

Page 41: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

PathologyPathologyInhalational Anthrax:Inhalational Anthrax: Endospores are introduced into the body and alveoli via Endospores are introduced into the body and alveoli via

inhalation. (small size of spores permits this)inhalation. (small size of spores permits this) Macrophages phagocytose and lyse endospores, and then travel Macrophages 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 lymphatic they 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 and Bacteria enter the bloodstream and can lead to septic shock and toxemia with system wide edema and necrosis; hematogenous toxemia 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.

Page 42: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

Pathology cont…Pathology cont…

Inhalational Anthrax:Inhalational Anthrax: Development of symptoms and pathogenesis occurs in 2 Development 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 become pulmonary edema, hemorrhagic symptoms of stage 1 become severe (>24hrs and leads to death)severe (>24hrs and leads to death)

IDID50 50 (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 rate 80% mortality rate

Page 43: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

Pathology cont…Pathology cont…Cutaneous Anthrax:Cutaneous Anthrax: Endospores enter body through the skin usually via Endospores enter body through the skin usually via

preexisting skin lesions, abrasions, and skin mucus preexisting skin lesions, abrasions, and skin mucus membranes.membranes.

Low-level germination and toxin production occurs at Low-level germination and toxin production occurs at entrance site leading to localized necrosis with eschar entrance site leading to localized necrosis with eschar formation and edema formation 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 infection pruritic red papule appears at the initial site of infection

• Day 2Day 2 papule becomes round ulcer papule becomes round ulcer • Day 3Day 3 plaque develops around edema site and central papule plaque develops around edema site and central papule

necrotizes, ulcerates, and forms a normally painless black eschar necrotizes, 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.

Page 44: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

Pathology cont…Pathology cont…

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

Page 45: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

Pathology cont…Pathology cont…

Cutaneous Anthrax:Cutaneous Anthrax: Endospores are often phagocytosed by macrophages and Endospores are often phagocytosed by macrophages and

carried to local lymph nodes, which can result in painful carried 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 102 with symptoms such as headache and fevers (up to 102 °F) °F) although such spread is not common with appropriate although such spread is not common with appropriate antibiotic therapy antibiotic therapy

On very rare occasions system wide infection can occur On 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 anthrax are 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.

Page 46: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

Pathology cont…Pathology cont…Gastrointestinal Anthrax:Gastrointestinal Anthrax: Pathogenesis and statistics are unclear due to rarity of Pathogenesis and statistics are unclear due to rarity of

this formthis form Vegetative cells from uncooked meat rather than spores Vegetative cells from uncooked meat rather than spores

(germination unnecessary)(germination unnecessary) In oral pharyngeal anthrax, bacteria enter via the oral or In 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 and followed by local/regional lymphadenopathy and localized edema. localized edema.

In abdominal anthrax, entrance occurs @ the ileum or In 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 initially initially causes causes nausea, vomiting, and malaise progressing into nausea, vomiting, and malaise progressing into bloody diarrhea, acute abdomen, or sepsis.bloody diarrhea, acute abdomen, or sepsis.

Mortality may drop with antibiotic treatment Mortality may drop with antibiotic treatment (inconclusive)(inconclusive)

Page 47: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

Summary of PathologySummary of Pathology

Page 48: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

DiagnosisDiagnosis Because of rarity of gastrointestinal cases diagnosis Because of rarity of gastrointestinal cases diagnosis

focus is limited. Onset of characteristic symptoms focus is limited. Onset of characteristic symptoms should signal patient to consult a physician should signal patient to consult a physician immediately for antibiotic treatment.immediately for antibiotic treatment.

Cutaneous anthrax can be distinguished by 2 key Cutaneous anthrax can be distinguished by 2 key features: painlessness of lesions and relatively features: painlessness of lesions and relatively large extent of associated edema (presence of large extent of associated edema (presence of eschars)eschars)

Distinguishing features of inhalational anthrax: Distinguishing features of inhalational anthrax: presence of widened mediastinum and pleural presence of widened mediastinum and pleural effusions on chest radiograph or CT (chest effusions on chest radiograph or CT (chest compound tomographic) scan with minimum compound tomographic) scan with minimum evidence of pneumonia.evidence of pneumonia.

Page 49: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

Diagnosis cont…Diagnosis cont…

Inhalational Anthrax:Inhalational Anthrax: Easier to diagnose when occurs in several suspicious cases than single case (or at Easier to diagnose when occurs in several suspicious cases than single case (or at

high risk situationshigh risk situations postal employee) postal employee) chest x-ray showing a widened mediastinum, infiltrates, pleural effusion, etc. = chest x-ray showing a widened mediastinum, infiltrates, pleural effusion, etc. =

likely anthrax infection likely anthrax infection CT scan showing hyperdense hilar and mediastinal nodes CT scan showing hyperdense hilar and mediastinal nodes (white arrow, middle diagram)(white arrow, middle diagram) + +

extensive mediastinal edema = anthrax infectionextensive mediastinal edema = anthrax infection Molecular Biology tests only available at Laboratory Response Network (LRN) labs Molecular Biology tests only available at Laboratory Response Network (LRN) labs

but highly reliablebut highly reliable

Pulmonary anthrax chest X-rayPulmonary anthrax chest X-rayshowing widened mediastinumshowing widened mediastinum

Dept of Radiological PathologyDept of Radiological PathologyArmed Forces Institute of Armed Forces Institute of PathologyPathology

Page 50: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

Diagnosis cont…Diagnosis cont…

Distinguish Anthrax from Influenza-Like Illness Distinguish 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 uncommon ILI patients have rhinorrhea and this is uncommon in Anthrax patients.in Anthrax patients.

Anthrax patients have shortness of breath, an Anthrax patients have shortness of breath, an uncommon trait of ILI patients.uncommon trait of ILI patients.

Nausea and vomiting are more common in anthrax Nausea and vomiting are more common in anthrax infectioninfection

Abnormal chest radiographs and CT scans Abnormal chest radiographs and CT scans Anthrax infectionAnthrax infection

Page 51: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

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 levels Must be administered before critical toxin levels

are reachedare reached Early attempts Early attempts (cutaneous)(cutaneous) surgery, but fear of surgery, but fear of

septicemia septicemia (early 20(early 20thth century) century)

Pasteur, Jourbert, & FortineauPasteur, Jourbert, & Fortineau antibiotics antibiotics (focus (focus of modern treatment now)of modern treatment now)

Early therapy utilized Penicillin G procaine but Early therapy utilized Penicillin G procaine but has been shifted to other antibiotics due to has been shifted to other antibiotics due to Penicillin-resistant strain appearance Penicillin-resistant strain appearance

Page 52: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

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 first Ciprofloxacin in conjunction w/ Doxycycline as first lineline

Usual dosages:Usual dosages: Cutaneous: adults & pregnant womenCutaneous: adults & pregnant women 500mg 500mg

Ciprofloxacin and 100mg Doxycycline twice daily; Ciprofloxacin and 100mg Doxycycline twice daily; childrenchildren varies, based on age and weight (All for at varies, 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 Ciprofloxacin 400mg Ciprofloxacin OROR 100mg Doxycycline 100mg Doxycycline every 12hrs every 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 Ciprofloxacin Ciprofloxacin OR OR Doxycycline varies, based on age & Doxycycline varies, based on age & weight weight ANDAND one or two of the previously mentioned one or two of the previously mentioned antimicrobials. (Therapy switched to oral treatment when antimicrobials. (Therapy switched to oral treatment when clinically appropriate and lasts for at least 60 days)clinically appropriate and lasts for at least 60 days)

Page 53: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

CategoryCategory Initial IV TherapyInitial IV Therapy Duration Duration

AdultsAdults Ciprofloxacin 400 mg every Ciprofloxacin 400 mg every 12 hr OR12 hr OR

Doxycycline 100mg every Doxycycline 100mg every 12 hr12 hr

ANDAND

Additional 1 or 2 antibiotics Additional 1 or 2 antibiotics

When clinically appropriate When clinically appropriate switch to oral therapy: switch to oral therapy:

Ciprofloxacin 500mg 2x Ciprofloxacin 500mg 2x daily ORdaily OR

Doxycycline 100 mg 2x Doxycycline 100 mg 2x dailydaily

Continue oral or IV therapy Continue oral or IV therapy for 60 days for 60 days

Children*Children* Ciprofloxacin 10-15 mg/kg Ciprofloxacin 10-15 mg/kg ever 12 hr ORever 12 hr OR

Doxycycline:Doxycycline:

>8y and > 45kg: 100mg >8y and > 45kg: 100mg every 12 hr every 12 hr

>8y and >8y and << 45kg: 2.2 45kg: 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 additional AND 1 or 2 additional antibiotics antibiotics

Switch to oral when Switch to oral when clinically appropriateclinically appropriate

Ciprofloxacin 10-15 mg/kg Ciprofloxacin 10-15 mg/kg every 12 hr ORevery 12 hr ORDoxycycline:Doxycycline:

>8y and > 45kg: 100mg >8y and > 45kg: 100mg 2x daily 2x daily

>8y and >8y and << 45kg: 2.2 45kg: 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 60 Continue oral or IV for 60 days days

Pregnant Women*Pregnant Women* Same for non-pregnant Same for non-pregnant adults adults

Same for non-pregnant Same for non-pregnant adults adults

*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.

Page 54: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

Pre-exposure Prophylaxis: Anthrax Pre-exposure Prophylaxis: Anthrax VaccineVaccine

1881: first developed by Louis Pasteur1881: first developed by Louis Pasteur 1937: vaccine modified to use attenuated 1937: vaccine modified to use attenuated

non pathogenic strain of non pathogenic strain of B. anthracisB. anthracis w/ w/ dormant spores dormant spores (other than former Soviet Union (other than former Soviet Union and associate countries, this vaccine is preferred over and 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 1970 aka BioThrax™ licensed in 1970 and produced by Bioport Corp. and produced by Bioport Corp.

Page 55: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

Anthrax Vaccine cont…Anthrax Vaccine cont… AVA: made from cell-free infiltrates of microaerophilic cultures of an AVA: made from cell-free infiltrates of microaerophilic cultures of an

avirulent, nonencapsulated strain of avirulent, nonencapsulated strain of B anthracis concentrated with PA B anthracis concentrated with PA (protective antigen)(protective antigen) final no live or dead bacteria are in final product final no live or dead bacteria are in final product

Final product contains: 1.2 mg/mL aluminum, added as aluminum Final product contains: 1.2 mg/mL aluminum, added as aluminum hydroxide in 0.85% sodium chloride hydroxide 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 preservative formaldehyde, 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 and induced 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 2 Efficacy 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, but Vaccine 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 mandatory 1998: 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 of over 400 quit or court marshaled due to fear of adverse side effects.adverse side effects.

Page 56: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

Anthrax Vaccine cont…Anthrax Vaccine cont…

Dosage:Dosage: In U.S.: 6 doses of 0.5 mL at weeks 0, 2, & 4 AND In 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 weeks In U.K.: 6 doses of 0.5 mL at weeks 0, 3, 6, & 32 with 0, 3, 6, & 32 with

annual boosters.annual boosters. Post-exposure vaccination:Post-exposure vaccination:

Vaccine can be used with antibiotics after inhalation Vaccine can be used with antibiotics after inhalation exposureexposure

3-dose regime @ weeks 0, 2,& 4 in combination with 3-dose regime @ weeks 0, 2,& 4 in combination with at least 30-days of antimicrobial therapy (w/out at 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 new Therefore, it is administered as an investigational new drug (IND) under FDA regulations.drug (IND) under FDA regulations.

Page 57: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

Anthrax Vaccine cont…Anthrax Vaccine cont…

Adverse effects:Adverse effects: USAMRIID data: inflammatory reaction greater USAMRIID data: inflammatory reaction greater

than 5 cm in diameter around the site of injection than 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 reporting 1990-200: Vaccine Adverse Event reporting System (VAERS) receives 1,544 reports of System (VAERS) receives 1,544 reports of adverse events after vaccinationadverse events after vaccination most frequent most frequent included: included: injection site hypersensitivity (334), injection site hypersensitivity (334), injection site edema (283), injection site pain injection site edema (283), injection site pain (247), headache (239), arthralgia (232), asthenia (247), headache (239), arthralgia (232), asthenia (215), and pruritus (212).(215), and pruritus (212).

Page 58: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

Anthrax Vaccine cont…Anthrax Vaccine cont… 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 as such as B. subtilis, and creatingB. subtilis, and creating recombinant vaccines recombinant vaccines

Creating mutant strains of vaccines that utilize non-human Creating mutant strains of vaccines that utilize non-human aromatic compounds for virulencearomatic compounds for virulence may establish better may establish better immunicity (minimizes self-cell recognition problems) w/out immunicity (minimizes self-cell recognition problems) w/out risk of deadly infectionrisk of deadly infection

Experimenting with purified PA preparations with different Experimenting with purified PA preparations with different combinations of adjuvant molecules (similar to AVA combinations 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 chain no 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 in expression of the cereolysine AB gene in B. anthracisB. anthracis gives gives hemolytic properties to the bacteria; conferred immunity against hemolytic properties to the bacteria; conferred immunity against both H-7 strain wildtype and H-7 AB recombinant when immunized both 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 immunized 100% 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

Page 59: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

““Safety First”Safety First”

Pre-Anthrax testing/handling Post-Anthrax handling: Decontamination

Page 60: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

ANTHRAX AS A BIOLOGICAL ANTHRAX AS A BIOLOGICAL WEAPONWEAPON

WHY USE WHY USE ANTHRAX? ANTHRAX?

Relatively easy to Relatively easy to acquire, in vitro acquire, in vitro growth (any lab growth (any lab media @ 37media @ 37°C) °C) and induction of and induction of sporulation is sporulation is easy. easy.

Page 61: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

WHY USE ANTHRAX?WHY USE ANTHRAX?

Most likely agent for large-scale biological Most likely agent for large-scale biological attack.attack.

Inhalational anthrax is the most potent of the Inhalational anthrax is the most potent of the three disease forms. ~80% mortality and with three disease forms. ~80% mortality and with prompt treatment ~50% mortality.prompt treatment ~50% mortality.

Anthrax spores can be easily spread in the Anthrax spores can be easily spread in the environment.Ultimate reservoir is soil where they environment.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.

Page 62: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

WHY USE ANTHRAX? – cont.WHY USE ANTHRAX? – cont.

Spore size of 3-6 Spore size of 3-6 μm suitable for aerosolization μm suitable for aerosolization and ideal for human alveolar deposition. and ideal for human alveolar deposition.

Early differentiation of inhalational anthrax from Early differentiation of inhalational anthrax from common infections is difficult.common infections is difficult.

Anthrax spores decay at the rate of less than 0.1 Anthrax spores decay at the rate of less than 0.1 per cent a minute, which is very slow for an per cent a minute, which is very slow for an organism. organism.

Page 63: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

WHY USE ANTHRAX? – cont.WHY USE ANTHRAX? – cont.

USDD reported that 3 defense employees with USDD reported that 3 defense employees with some technical skills but without expertise on some technical skills but without expertise on bioweapons could manufacture a simulant of BA bioweapons 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 have spores are odorless and invisible and have potential to travel many kms before dissipating.potential to travel many kms before dissipating.

Page 64: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

Some data on offensive Some 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 10,000 anthracis during rush hour would result in 10,000 deaths.In this test more than 1M NY were exposed deaths.In this test more than 1M NY were exposed to the Bacillus.to the Bacillus.

Aerial spray of BA along 100km line under ideal Aerial spray of BA along 100km line under ideal meteorological cond could produce 50%lethality meteorological 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 a US study near Johnston Atoll in SP reported that a plane “sprayed a 32mile long line of agent that plane “sprayed a 32mile long line of agent that traveled for more than 60 miles before it lost its traveled for more than 60 miles before it lost its infectiousness”.infectiousness”.

Page 65: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

Some data on offensive Some data on offensive capacity of anthrax spores –capacity of anthrax spores –

cont.cont.

WHO estimated that 50kg of BA released in a WHO 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 deaths 50 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 an 100 kg = bw 130,000 and 3M deaths over an area size of W-DC. This lethality matches that of area size of W-DC. This lethality matches that of a hydrogen bomb.a hydrogen bomb.

Page 66: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

                                                                   

Page 67: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

Some data on offensive capacity Some data on offensive capacity of anthrax spores –cont.of anthrax spores –cont.

CDC suggested a cost of $26.2B per 100,000 CDC suggested a cost of $26.2B per 100,000 persons exposed.persons exposed.

Human LD50 is ~2,500 to 55,000 inhaled BA Human LD50 is ~2,500 to 55,000 inhaled BA spores but studies on primates suggest that as spores but studies on primates suggest that as few as 1-3 spores may be sufficient to cause few as 1-3 spores may be sufficient to cause infection.infection.

Little information of possible risks of direct Little information of possible risks of direct contamination of food or water with anthrax contamination of food or water with anthrax spores.spores.

Page 68: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

So, who (possibly) has anthrax So, who (possibly) has anthrax spores ready?spores ready?

1995 Iraq acknowledged producing and 1995 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 BA Former Soviet Union also known to have a BA production program.production program.

Existence of offensive biological weapons Existence of offensive biological weapons programs in at least 13-17 countries including programs in at least 13-17 countries including Syria, Iran, Libya, China, SK, NK, Taiwan and Syria, Iran, Libya, China, SK, NK, Taiwan and Israel. (US Dep of Def)Israel. (US Dep of Def)

Page 69: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

Britain – Gruinard island Britain – Gruinard island 19421942

Britain conducted research during 1942 at Britain conducted research during 1942 at Gruinard Island (Scotland).Gruinard Island (Scotland).

Sheep were taken to an open field, secured in Sheep were taken to an open field, secured in wooden frames, and exposed to a bomb that wooden frames, and exposed to a bomb that scattered the spores. The sheep started dying scattered the spores. The sheep started dying three days later. three days later.

An infected sheep's carcass washed ashore from An infected sheep's carcass washed ashore from the island led to the deaths of seven cattle, two the island led to the deaths of seven cattle, two horses, three cats and up to 50 sheep in a nearby horses, three cats and up to 50 sheep in a nearby village. They had probably been infected by a dog village. They had probably been infected by a dog which had itself been infected by the carcass. which had itself been infected by the carcass.

Page 70: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

Britain – Gruinard island 1942Britain – Gruinard island 1942 Island was so Island was so

contaminated that the contaminated that the island was kept in island was kept in quarantine for 48 years.quarantine for 48 years.

In 1986 an English In 1986 an English company was paid company was paid ₤500,000 to ₤500,000 to decontaminate the 520-decontaminate the 520-acre island by soaking acre island by soaking the ground in 280 tons the ground in 280 tons of formaldehyde diluted of formaldehyde diluted in 2000 tons of in 2000 tons of seawater.seawater.

Page 71: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

Sverdlovsk case – Former Sverdlovsk case – Former Soviet Union – April 2, 1979Soviet Union – April 2, 1979

Odorless, colorless, unnoticeable aerosol was Odorless, colorless, unnoticeable aerosol was accidentally released from a secret Soviet accidentally released from a secret Soviet biological weapons facility in Sverdlovsk.biological weapons facility in Sverdlovsk.

96 people within a short distance fell sick; 66 96 people within a short distance fell sick; 66 died (68.75%MR). All where inhalational died (68.75%MR). All where inhalational anthrax, not single case was cutaneous. Animals anthrax, 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 after New cases developed as late as 43 days after the presumed date of released. None during the presumed date of released. None during months or years afterward. months or years afterward.

Page 72: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

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 meatAutopsy findings of hemorrhagic mediastinitisInfection later linked to an aerosol leak from a nearby biologic weapons facility operated by the Soviet army

Meselson et al, Science, 1994

Page 73: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

Sverdlovsk: Case onsets & DeathsSverdlovsk: Case onsets & Deaths

0

1

2

3

4

5

6

7

8

All case onsets All deaths

Page 74: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

Gulf war - 1991Gulf war - 1991

1990 Iraq had 50 R400 bombs filled with 1990 Iraq had 50 R400 bombs filled with anthrax.anthrax.

8,500L of anthrax.8,500L of anthrax.Bombs were not used due to a supposed Bombs were not used due to a supposed

fear by Iraq that winds and meteorological fear by Iraq that winds and meteorological conditions could make them also a target conditions could make them also a target for their own bombs.for their own bombs.

Page 75: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

Aum Shinrikyo failed attack -Aum Shinrikyo failed attack -19951995

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

Page 76: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

US Anthrax attacks - 2001US Anthrax attacks - 2001

Robert Stevens, 63, Robert Stevens, 63, photo editor at the photo editor at the supermarket tabloid supermarket tabloid The Sun, published The Sun, published by American Media by American Media Inc., died on October Inc., died on October 5, 2001 from 5, 2001 from contracting pulmonary contracting pulmonary anthrax from an anthrax from an envelope.envelope.

Page 77: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

US Anthrax attacks – 2001 - US Anthrax attacks – 2001 - contcont

Oct,2 – Nov, 20 22 cases of anthrax were Oct,2 – Nov, 20 22 cases of anthrax were confirmed: 11 inhalational anthrax and 11 (7 confirmed: 11 inhalational anthrax and 11 (7 confirmed and 4 suspected) of cutaneous confirmed 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 of Mean duration bw exposure and onset of

symptoms: 4.5 days.symptoms: 4.5 days.

Page 78: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

US Anthrax attacks – 2001 - US Anthrax attacks – 2001 - contcont

4 BA + powder-4 BA + powder-containing envelopes containing envelopes were recovered.were recovered.

2 mailed around 2 mailed around Trenton, NJ and Trenton, NJ and postmarked Sep postmarked Sep 18,2001.18,2001.

Page 79: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

US Anthrax attacks – 2001 - US Anthrax attacks – 2001 - contcont

Envelopes processed at US PS Trenton Mail Envelopes processed at US PS Trenton Mail Processing Ctr and Morgan Central Postal Processing Ctr and Morgan Central Postal Facility (NYC). Both facilities and at least 5 more Facility (NYC). Both facilities and at least 5 more affiliated with NJ facility had environmental affiliated with NJ facility had environmental samples positive for BA.samples positive for BA.

No envelopes found in FL but BA was isolated No envelopes found in FL but BA was isolated from environmental sampling at the AMI building from environmental sampling at the AMI building (were 2 cases from FL worked).(were 2 cases from FL worked).

Page 80: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

US Anthrax attacks – 2001 - US Anthrax attacks – 2001 - contcont

Oct 9 envelopes sent from NJ to Washington DC Oct 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 of 15. Prompt recognition of powder led to rapid initiation of post-exposure prophylaxis.post-exposure prophylaxis.

Scientists estimate that the letter sent to Sen. Daschle Scientists estimate that the letter sent to Sen. Daschle originally contained about 2 grams of anthrax.originally contained about 2 grams of anthrax.

Page 81: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

US Anthrax attacks – 2001 - US Anthrax attacks – 2001 - 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 times the meant that the letter could have contained 200 million times the average dose necessary to kill a person.average dose necessary to kill a person.

625 persons from Hart Building were tested (nasal swaps) and 625 persons from Hart Building were tested (nasal swaps) and 28 found positive for BA.28 found positive for BA.

Environmental sampling of Building showed heavily Environmental sampling of Building showed heavily contamination. Building was closed for decontamination on Oct contamination. Building was closed for decontamination on Oct 17,2001.17,2001.

Page 82: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

US Anthrax attacks – 2001 - US Anthrax attacks – 2001 - contcont

Letter to Sen. Leahy Letter to Sen. Leahy was in 1/280 barrels of was in 1/280 barrels of unopened mail unopened mail collected from Capitol collected from Capitol Hill after Sen. Daschle’s Hill after Sen. Daschle’s letter was discovered.letter was discovered.

Page 83: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

US Anthrax attacks – 2001 - US Anthrax attacks – 2001 - contcont

An estimated 32,000 persons initiated An estimated 32,000 persons initiated antimicrobial prophylaxis but completion of the 60 antimicrobial prophylaxis but completion of the 60 day course was recommended for 10,300 day course was recommended for 10,300 persons.persons.

Cross-contamination of mail occurred but risk was Cross-contamination of mail occurred but risk was low considering the high amount of mail processed low considering the high amount of mail processed in NJ and NYC.in NJ and NYC.

2 patients w/ inh. anthrax had no exposure to 2 patients w/ inh. anthrax had no exposure to media or government facilities or possible sources media or government facilities or possible sources of naturally occurring anthrax yet both were of naturally occurring anthrax yet both were infected w/ BA isolates identical to outbreak strain.infected w/ BA isolates identical to outbreak strain.

Page 84: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

US Anthrax attacks – 2001 - US Anthrax attacks – 2001 - contcont

All of the anthrax spores in the mail were found All of the anthrax spores in the mail were found to be of an identical strain (Ames strain). This to be of an identical strain (Ames strain). This strain is one that the U.S. military used for study strain is one that the U.S. military used for study at USAMRIID and distributed to other at USAMRIID and distributed to other government and university labs as well as to government and university labs as well as to other governments including Great Britain.other governments including Great Britain.

Decontamination of Hart Building took three Decontamination of Hart Building took three months and cost was estimated at $23M.months and cost was estimated at $23M.

Page 85: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

New FindingsNew Findings

Secondary aerosolization of viable BA spores Secondary aerosolization of viable BA spores under common office activities was tested and under common office activities was tested and found to be possible. Test was perform almost a found 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 in completed sequencing the anthrax bacillus in late November 2001, but has decided to delay late November 2001, but has decided to delay its publication and access to research its publication and access to research community for security reasons.community for security reasons.

Page 86: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

What to do in case of an attack?What to do in case of an attack?

US military M17 and M40 gas masks US military M17 and M40 gas masks provide excellent protection against 1-5 provide excellent protection against 1-5 μm μm particles ($324). Other articles include a portable particles ($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-exposure Use of pre-exposure and post-exposure antibiotics (US attack isolates were sensitive to antibiotics (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.

Page 87: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

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-flow chemical amplification continuous-flow PCR on a chip. Problem: still slow and PCR 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 sent Burke lab: PCR product could be sent directly onto miniature gel: 30 min. directly onto miniature gel: 30 min.

Phillip Belgrader: PCR in microfuge Phillip Belgrader: PCR in microfuge tubes: 7 min.tubes: 7 min.

Page 88: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

ReferencesReferencesMICROBIOLOGY REFERENCES:

1.1. Drum, C.L., Yan, S.Z., et al. Structural Basis for the Drum, C.L., Yan, S.Z., et al. Structural Basis for the Activation of Anthrax Adenylyl Cyclase Exotoxin Activation 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., Anthrax Duesbery, 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.

Page 89: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

References (cont.)References (cont.)7.7. Pannifer, A.D., Wong, T.Y., et al, Crystal Structure Pannifer, 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 Toxin Patocka, 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. Bacillus Bacillus anthracisanthracis and Other and Other BacillusBacillus Species. Academic Species. 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

Page 90: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

References (cont.)References (cont.)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. New Dixon, 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. Clinical Franz, D.R., P.B. Jahrling, A.M. Friedlander, et al. Clinical Recognition and Management of Patients Exposed to Biological Recognition 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 Biological Greenfield, Ronald A., et al. Bacterial Pathogens as Biological Weapons and Agents of Bioterrorism. The American Journal of Weapons 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: Medical Inglesby, MD, et. al. Anthrax as a Biological Weapon: Medical and Public Health Management. JAMA. Vol 281, No.18. May and Public Health Management. JAMA. Vol 281, No.18. May 12,1999.12,1999.

Page 91: Bacilus Anthracis Anthrax Sheref Hassan Rachel Handwerger Maria Garrido.

References (cont.)References (cont.)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 Lethal Peters, 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 Other and 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/anthraxfactsheet.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 Preventative US Army Center for Health Promotion and Preventative Medicine: Medicine: http://chppm-www.apgea.army.mil/HomelandSecurity/anthraxvihttp://chppm-www.apgea.army.mil/HomelandSecurity/anthraxvideo/default.aspdeo/default.asp