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Anthrax: Dr.T.V.Rao MD
Anthrax: Dr.T.V.Rao MD
Dr.T.V.Rao MD
Dr.T.V.Rao MD 1
General Characteristics of Bacillus
~ 60 species; Gram-positive or Gram-variable bacilli
Large (0.5 x 1.2 to 2.5 x 10 um)
Most are saprophytic contaminants or normal flora
Bacillus anthracis is most important member
Produce endospores
Aerobic or facultatively anaerobic
Dr.T.V.Rao MD 4
Bacillus anthrax
Several landmarks
Dr.T.V.Rao MD 5
20,000-100,000 cases estimated globally/year
http://www.vetmed.lsu.edu/whocc/mp_world.htm
Dr.T.V.Rao MD
Animal Transmission
Most commonly infected by ingestion from
contaminated soil or contaminated feed or bone
meal
Dr.T.V.Rao MD 7
Anthrax
An infectious,
Usually fatal disease
of warm-blooded
animals, especially of
cattle and sheep,
caused by the
bacterium Bacillus
anthracis.
Dr.T.V.Rao MD 8
B. anthracis
Gram-positive, spore-forming, non-motile bacillus
Dr.T.V.Rao MD 9
Anthrax Bacilli
Dr.T.V.Rao MD 10
Bacillus anthracis
General characteristics
Bacillus anthracis
Large, Gram positive, non-motile
rod
Vegetative form
and spores
Nearly worldwide distribution
Over 1,200 strains
Dr.T.V.Rao MD 11
Bacillus anthracis
Gram + rod
Facultative anaerobe
1 - 1.2m in width x 3 - 5m in
length
Dr.T.V.Rao MD 13
Endospore
Oxygen required for sporulation
1 spore per cell
dehydrated cells
Highly resistant to heat, cold,
chemical disinfectants, dry periods
Protoplast carries the material for
future vegetative cell
Cortex provides heat and radiation
resistance
Spore wall provides protection from
chemicals & enzymes
Dr.T.V.Rao MD 14
Gram Stain Morphology
of B. anthracis
Broad, gram-positive
rod: 11.5 x 35
Oval, central to
subterminal spores: 1 x
1.5 with no significant
swelling of cell
Spores usually NOT
present in clinical
specimens unless
exposed to atmospheric
O2
Dr.T.V.Rao MD 15
Mechanism of Infection
Anthrax spores enter body
Germinate & multiple in
lymph nodes
PA, EF, LF excreted from
bacteria
PA binds to TEM8.
EF and/or LF binds
Complex internalized by
endocytosis
Acidification of endosome
LF or EF crosses into cytosol
via PA mediated ion-
conductive channels
Dr.T.V.Rao MD 16
Cultural
characteristics
Aerobe, facultative anaerobe
Grows between 12 -45 c
2-3 mm colonies
Edge like matted hair
Medusa head appearance
Blood agar Hemolytic colonies
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Appearance of Anthrax
String of pearl
appearance with
Pencillin
Differentiates Anthrax
and Cereus
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SELECTIVE MEDIUM
PLET
Contain
1 polymyxins
2 Lysozyme
3 Ethylene dioxide
4 Tetra acetic acid
Contains EDTA
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Biochemical Reactions
Gelatin
Inverted fir
tree
appearance
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Biochemical
Reactions
Glucose, Maltose
and Sucrose
fermented with
acid but no gas
Catalase positive
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Sterilization of environments Floor
space/shed/vehicle
Preliminary disinfection using 10% formaldehyde; (1-1.5 It/
sq.m.) or 4%
Gluteraldehydes for at least 2 hours
Cleaning - by washing or scrubbing with hot water
Final disinfection by one of the following disinfectants
applied for at least 2 hours.
10% formaldehyde
4% Gluteraldehydes
3% hydrogen peroxide or
1% per acetic acid
Dr.T.V.Rao MD 22
Wool and Hair
By duckering process
(five stages) i.e.
lmmersion in 0.25-0.3%
soda liquor
Immersion in soap
liquor;
Two immersions in 2%
formaldehyde solution;
and
Rinsing in water
Dr.T.V.Rao MD 23
Antibiotics
Pencillin
Erythrocin
Tetracycline
Chloramphenicol
Occasional strains
resistant to penicillin
are encountered
Dr.T.V.Rao MD 24
Transmitted
The disease can be
transmitted to humans
through contact with
contaminated animal
substances, such as hair,
feces, or hides, and is
characterized by ulcerative
skin lesions
Dr.T.V.Rao MD 25
Criteria in Transmission
Skin: direct skin contact with spores; in nature, contact
with infected animals or animal products (usually related
to occupational exposure)
Respiratory tract: inhalation of aerosolized spores
GI: consumption of undercooked or raw meat products or
dairy products from infected animals
NO person-to-person transmission of inhalation or GI
anthrax
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Anthrax Cycle
Dr.T.V.Rao MD 27
Pathogenesis
The infectious dose of B.
anthracis in humans by any
route is not precisely known.
Rely on primate data
Minimum infection dose of
~ 1,000-8,000 spores
LD50 of 8,000-10,000
spores for inhalation
Virulence depends on 2
factors
Capsule
3 toxins http://www.kvarkadabra.net/index.html?/biologija/teksti/biolosko_orozje.htm
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Anthrax:
Clinical Presentation
Cutaneous
Inhalational
Gastrointestinal
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Epidemiology
of Anthrax in
Animal and
Human Hosts
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Three forms of Anthrax
Cutaneous anthrax
Skin
Most common
Spores enter to skin through small lesions
Inhalation anthrax
Spores are inhaled
Dr.T.V.Rao MD 32
Anthrax:
Cutaneous
Begins as a papule, progresses through a
vesicular stage to a depressed black necrotic
ulcer (Escher)
Edema, redness, and/or necrosis without
ulceration may occur
Form most commonly encountered in naturally
occurring cases
Incubation period: 112 days
Case-fatality:
Without antibiotic treatment20%
With antibiotic treatment1%
Dr.T.V.Rao MD 33
Anthrax:
Inhalational
A brief prodromal resembling a viral-like
illness, characterized by myalgia, fatigue,
fever, with or without respiratory
symptoms, followed by hypoxia and
dyspnea, often with radiographic evidence
of mediastinal widening.
Meningitis in 50% of patients
Rhinorrhea (rare)
Dr.T.V.Rao MD 34
Glycocalyx
Capsule
Sticky, gelatinous polymer
external to cell wall
pX02 plasmid
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Virulence Factors
1 Capsular polypeptide
Anthrax Toxin
Both are coded by separate plasmid
The capsular polypeptide aids virulence by
inhibiting phagocytosis, loss of plasmid loss of
virulence
How the live attenuated anthrax spore vaccine (
Sterne strain )
Dr.T.V.Rao MD 36
Anthrax Toxin
The toxin is a three factions
The edema factor, (OF Factor I )
The protective antigen factor ( PA or
Factor II )
The lethal factor ( LF or Factor III )
Individually they are not toxic
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How Toxicity Manifests
Dr.T.V.Rao MD 38
TOXIGENICITY
OF island adenyl cyclase which is activated only inside
the target cells leading to the intracellular
accumulation of cyclic AMP
Responsible for edema and other biological effects of
toxin.
Entry of LF toxin into the target cell causes cell death.
Loss of plasmid which encodes anthrax toxin renders
the strain avirulant.
Sterne vaccine strain devoid of Plasmid coding for the
capsule polysaccharide.
Dr.T.V.Rao MD 39
Pathogenesis
Anthrax spores enter body
Germinate & multiple in lymph nodes
PA, EF, LF excreted from bacteria
PA binds to TEM8.
PA nicked by protease furin
20-kDa segment off leaving 63-kDa peptide
Heptamer forms
EF and/or LF binds
Complex internalized by endocytosis
Acidification of endosome
LF or EF crosses into cytosol via PA mediated ion-conductive
channels
LF cleaves MAPKK 1 & 2
EF stimulates cAMP
Dr.T.V.Rao MD 40
Clinical Presentation of Anthrax
Cutaneous Anthrax
95% human cases are cutaneous infections
1 to 5 days after contact
Small, pruritic, non-painful papule at inoculation site
Papule develops into hemorrhagic vesicle & ruptures
Slow-healing painless ulcer covered with black Escher
surrounded by edema
Infection may spread to lymphatic's w/ local adenopathy
Septicemia may develop
20% mortality in untreated cutaneous anthrax
Dr.T.V.Rao MD 41
Cutaneous Anthrax
Dr.T.V.Rao MD 42
Anthrax: Cutaneous
Mediastinal widening
JAMA 1999;281:17351745
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Mediastinal Widening and Pleural
Effusion on Chest X-Ray in Inhalational
Anthrax
Dr.T.V.Rao MD 48
Gastrointestinal Anthrax
GI anthrax may follow after the
consumption of contaminated,
poorly cooked meat.
http://science.howstuffworks.com/anthrax1.htm Dr.T.V.Rao MD 49
Clinical Presentation of Anthrax
Inhalation Anthrax
Virtually 100% fatal (pneumonic)
Meningitis may complicate cutaneous
and inhalation forms of disease
Pharyngeal anthrax
Fever
Pharyngitis
Neck swelling
Dr.T.V.Rao MD 50
Clinical Presentation of Anthrax
Gastrointestinal (Ingestion) Anthrax
Dr.T.V.Rao MD 53
Diagnosis in Humans
Anthrax quick ELISA test
New test approved by FDA on June 7th, 2004.
Detects antibodies produced during infection with
Bacillus anthracis
Quicker and easier to interpret than previous antibody
testing methods
Results in less than ONE hour
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Clues to diagnosis
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Laboratory Criteria for
Identification of B. anthracis
From clinical samples, such as blood,
cerebrospinal fluid (CSF), skin lesion
(eschar), or oropharyngeal ulcer
Encapsulated gram-positive rods on Gram
stain
From growth on sheep blood agar:
Large gram-positive rods
Nonmotile
Nonhemolytic
Dr.T.V.Rao MD 56
Anthrax:
Diagnosis
Inhalational
Chest X-raywidened mediastinum,
pleural effusions, infiltrates, pulmonary
congestion
Affected tissue biopsy for
immunohistochemistry
Any available sterile site fluid for Gram
stain, PCR, or culture
Pleural fluid cell block for
immunohistochemistry
Dr.T.V.Rao MD 57
B. anthracis:
Confirmatory Identification
Isolate
Capsule DFA
Phage
lysis Capsule antigen
Horse Bicarbonate
Cell wall
blood media
(MFadyean (MFadyean stain
Stain) India ink stain)
Dr.T.V.Rao MD 58
B. anthracis:
Presumptive Identification
Dr.T.V.Rao MD 59
Laboratory Criteria for
Identification of B. anthracis
From clinical samples, such as blood,
cerebrospinal fluid (CSF), skin lesion
(eschar), or oropharyngeal ulcer
Encapsulated gram-positive rods on Gram
stain
From growth on sheep blood agar:
Large gram-positive rods
Nonmotile
Nonhemolytic
Dr.T.V.Rao MD 60
Laboratory Criteria for
Identification of B. anthracis
Rapid screening assay (PCR- and antigen-
detection based) for use on cultures and
directly on clinical specimens
Confirmatory criteria for identification of
B. anthracis
Capsule production
Lysis by gamma-phage
Direct fluorescent antibody assay (DFA)
Dr.T.V.Rao MD 61
PCR Assay
Detection time:
- PCR only takes several hours
ex) Rapid-cycle RT-PCR can be finished within 1-2 hours
There are many different types of PCR assays for the detection of
Anthrax such as multiplex PCR, enter bacterial repetitive
intragenic consensus-PCR (ERIC-PCR), and long-range repetitive
element polymorphism-PCR.
Dr.T.V.Rao MD 63
Treatment & Prophylaxis
Treatment
Penicillin is drug of choice
Erythromycin, chloramphenicol acceptable alternatives
Doxycycline now commonly recognized as prophylactic
Vaccine (controversial)
Laboratory workers
Employees of mills handling goat hair
Active duty military members
Potentially entire populace of U.S. for herd immunity
Dr.T.V.Rao MD 64
Treatment
Penicillin
Has been the drug of choice
Some strains resistant to penicillin and doxycycline
Ciprofloxacin
Chosen as treatment of choice in 2001
No strains known to be resistant
Doxycycline may be preferable
Dr.T.V.Rao MD 65
Treatment
Before 2001, 1st line of treatment was
penicillin G
Stopped for fear of genetically
engineered resistant strains
60 day course of antibiotics
Ciprofloxacin
fluoroquinolone
500 mg tablet every 12h or 400 mg IV
every 12h
Inhibits DNA synthesis
Doxycycline
6-deoxy-tetracycline
100 mg tablet every 12h or 100 mg IV
every 12h
Inhibits protein synthesis
For inhalational, need another
antimicrobial agent
clindamycin
rifampin hrax.html
chloramphenico
Dr.T.V.Rao MD 66
Trends on Vaccine
BioThrax/Anthrax vaccine absorbed
Made by Bioport
Route of exposure not important
Administered subcutaneously
.5mL at 0, 2, and 4 weeks, and at 6, 12, & 18 months, & booster doses at 1 yr
intervals
A December 22, 2003 ruling temporarily halted the Department of Defenses anthrax
vaccination program
Lifting of that injunction on January 7, 2004
Dr.T.V.Rao MD 67
Immune Protection Against Anthrax
Live cellular vaccines
"Sterne" type live spore (toxigenic, noncapsulating)
Former USSR STI live spore (toxigenic, non-
capsulating)
"Pasteur" type (mixed culture, reduced virulence)
Sterile, acellular vaccines
US "anthrax vaccine adsorbed" (AVA)not licensed
for use in civilian populations
UK "anthrax vaccine precipitated" (AVP)
Recombinant PA research vaccines
AI3+; Freunds; Saponin, Monophosphoryl lipid A; Ribi
Dr.T.V.Rao MD 68
Vaccination
Cell-free filtrate
Licensed in 1970
At risk
Wool mill workers
Veterinarians
Lab workers
Livestock handlers
Military personnel
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Vaccine Schedule
3 injections at two-week intervals
3 injections 6 months apart
Annual booster
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Recommended Post exposure Prophylaxis to
Prevent Inhalational Anthrax
Initial Therapy Duration
Adults Ciprofloxacin 60 days
(including pregnant 500 mg PO BID
women and OR
immunocompromised) Doxycycline
100 mg PO BID
Children Ciprofloxacin* 60 days
1015 mg/kg PO Q 12 hrs. Change to
OR amoxicillin
Doxycycline: if susceptible
>8 yrs. and >45 kg: 100 mg PO BID
>8 yrs. and <45 kg: 2.2 mg/kg PO BID
<8 yrs.: 2.2 mg/kg PO BID
*Ciprofloxacin not to exceed 1 gram daily in children
Patient information sheets at www.bt.cdc.gov
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Precautions to Health care Workers
Standard contact
precautions. Avoid
direct contact
with wound or
wound drainage.
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Anthrax Has Been Used As a
Bioweapon
Because it is deadly, noncontagious, and dispersed
by spores, anthrax has always been considered a
good candidate for a bioweapon (table 3). Late in
2001, this possibility became a reality. Letters
containing anthrax spores were sent to several
news reporters and two United States Senators.
Five people died of inhalational anthrax as a result
of exposure to these spores.
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Weaponization & Bacillus Anthracis:
Why is this Agent Considered to be the Department of Defenses
Number-One/Two Biological Threat?
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Analysis of the 2001 US Anthrax
Attacks
*Also believed to be three or more other envelopes that were never found
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Anthrax Cases, 2001
22 cases
11 cutaneous
11 inhalational
5 deaths (all inhalational)
Index case in Florida
2 postal workers in Maryland
Hospital supply worker in NYC
Elderly farm woman in Connecticut
Dr.T.V.Rao MD 76
Analysis of the 2001 US Anthrax Attacks
Anthrax in Envelopes
Concentration of about 1 trillion spores per gram
2 grams anthrax per envelope
Each letter contained ~200 million times average LD50
All anthrax was unmilled, contained a certain type of
silica to reduce electrostatic charges and was of the
Ames strain
all characteristic of US weapons-grade anthrax
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Created by Dr.T.V.Rao MD for e
learning resources in Developing
World
Email
doctortvrao@gmail.com
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