Anthrax

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

Bacillus spp. are ubiquitous

• Soil, water, and airborne dust
• Thermophilic (< 75°C) and psychrophilic (>5-8°C)
• Can flourish at extremes of acidity & alkalinity (pH 2 to
10)
Dr.T.V.Rao MD 2
 Anthrax
• From the Greek word anthrakos for coal
• Caused by spores
• Primarily a disease of domesticated & wild animals
– Herbivores such as sheep, cows, horses, goats
• Natural reservoir is soil
– Does not depend on an animal reservoir making it
hard to eradicate
– Cannot be regularly cultivated from soils where there
is an absence of endemic anthrax
– Occurs sporadically throughout US
– South Dakota, Arkansas, Texas, Louisiana, Mississippi,
California recognized endemic areas
Dr.T.V.Rao MD 3
 A Closer Look at Anthrax
• Anthrax is a disease of cattle, goats, and sheep
caused by a bacterium, Bacillus anthracis. It is rare
for humans to be infected. Most infections that do
occur are localized to small cuts in the skin whose
edges turn black (hence the name “anthracis”,
after anthracite coal). The disease is deadly for
humans because B. anthracis produces lethal
toxins.

Dr.T.V.Rao MD 4
 Bacillus anthrax
Several landmarks

• 1st to Observe under

Microscope
• 1st to communicable
disease.
• 1st observe the spores (
Robert Koch )
• 1st to prepare for
attenuated vaccine.
( Louis Pasteur)

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 - 1.2µm in width x 3 - 5µm in

length

• Belongs to the B. cereus family

– Thiamin growth requirement
– Glutamyl-polypeptide
capsule
– Nonmotile

• Forms oval, centrally located

endospores
Dr.T.V.Rao MD 12
 The Spore
• Sporulation requires
– Poor nutrient conditions
– Presence of oxygen
• Spores
– Very resistant to extremes
– Survive for decades
– Taken up by host and
germinate
• Lethal dose 2,500 to
55,000 spores

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: 1–1.5 x 3–5 µ
• Oval, central to
subterminal spores: 1 x
1.5 µ 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

Dr.T.V.Rao MD 17
 Appearance of Anthrax

• String of pearl
appearance with
Pencillin
• Differentiates Anthrax
and Cereus

Dr.T.V.Rao MD 18
 SELECTIVE MEDIUM
PLET
• Contain
• 1 polymyxins
• 2 Lysozyme
• 3 Ethylene dioxide
• 4 Tetra acetic acid
Contains EDTA

Dr.T.V.Rao MD 19
 Biochemical Reactions

• Gelatin –
Inverted fir
tree
appearance

Dr.T.V.Rao MD 20
 Biochemical
Reactions

• Glucose, Maltose
and Sucrose
fermented with
acid but no gas
• Catalase positive

Dr.T.V.Rao MD 21
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

Dr.T.V.Rao MD 26
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

Dr.T.V.Rao MD 28
 Anthrax:
Clinical Presentation
Cutaneous
Inhalational
Gastrointestinal

Dr.T.V.Rao MD 29
Epidemiology
of Anthrax in
Animal and
Human Hosts

Dr.T.V.Rao MD 30
 Three forms of Anthrax
• Cutaneous anthrax
– Skin
– Most common
– Spores enter to skin through small lesions

• Inhalation anthrax
– Spores are inhaled

• Gastrointestinal (GI) anthrax

– Spores are ingested
– Oral-pharyngeal and abdominal
Dr.T.V.Rao MD 31
 Cutaneous Anthrax
• 95% of all cases globally
• Incubation: 2-3 days (up to 12 days)
• Spores enter skin through open wound or abrasion
• Papule progresses to black Escher
• Severe edema
• Fever and malaise

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: 1–12 days
 Case-fatality:
 Without antibiotic treatment—20%
 With antibiotic treatment—1%

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

• Made up of D-glutamic acid

• Non-toxic on its own

• Only encapsulated B. anthracis

virulent

• Most important role during

establishment of disease
– Protects against phagocytosis &
lysis during vegetative state

Dr.T.V.Rao MD 35
 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
Dr.T.V.Rao MD 37
 How Toxicity Manifests

• They are not toxic indivually but whole complex

produces local edema and generalisaed shock.
• PA I which is the fraction which binds to the target
cell surface and in turn provides attachment sites
for OF or LF facilitating .their entry into the cell

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

CDC, Cutaneous Anthrax—Vesicle Development

Dr.T.V.Rao MD 42
 Anthrax: Cutaneous

Left, Forearm lesion on day 7—vesiculation and ulceration of initial macular

or papular anthrax skin lesion. Right, Eschar of the neck on day 15 of
illness, typical of the last stage of the lesion. From Binford CH, Connor DH,
eds. Pathology of Tropical and Extraordinary Diseases. Vol 1. Washington,
DC: AFIP; 1976:119. AFIP negative 71-1290–2.
Dr.T.V.Rao MD 43
Anthrax: Cutaneous

Healing after treatment

Dr.T.V.Rao MD 44
 Inhalation Anthrax
• The infection begins with the inhalation
of the anthrax spore.

• Spores need to be less than 5 microns

(millionths of a meter) to reach the
alveolus.

• Macrophages lyse and destroy some of

the spores.

• Survived spores are transported to

lymph nodes.

• At least 2,500 spores have to be inhaled

to cause an infection.

Inhalation Anthrax, Introduction, DRP, Armed Forces

Dr.T.V.Rao MD Institute of Pathology 45
 Inhalation Anthrax
• Disease immediately follows germination.

• Spores replicate in the lymph nodes.

• The two lungs are separated by a structure

called the mediastinum, which contains
the heart, trachea, esophagus, and blood
vessels.

• Bacterial toxins released during replication

result in mediastinal widening and pleural
effusions (accumulation of fluid in the
pleural space).

Inhalation Anthrax, Introduction, DRP, Armed Forces

Dr.T.V.Rao MD Institute of Pathology 46
Anthrax: Inhalational

Mediastinal widening
JAMA 1999;281:1735–1745
Dr.T.V.Rao MD 47
 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.

• There are 2 different forms of GI

anthrax:
1) Oral-pharyngeal
2) Abdominal

• Abdominal anthrax is more

common than the oral-pharyngeal
form.

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

Virtually 100% fatal

Abdominal pain

Hemorrhagic ascites

Paracentesis fluid may reveal
gram-positive rods
Dr.T.V.Rao MD 51
 Anthrax:
Diagnosis
Cutaneous
 Gram stain, polymerase chain reaction
(PCR), or culture of vesicular fluid,
exudate, or eschar
 Blood culture if systemic symptoms
present
 Biopsy for immunohistochemistry,
especially if person taking
antimicrobials
Dr.T.V.Rao MD 52
 Diagnosis in Humans
• Isolation of B. anthracis
– Blood, skin
– Respiratory secretions
• Serology
• ELISA
• Nasal swabs
– Screening tool

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

Dr.T.V.Rao MD 54
 Clues to diagnosis

• Aerobic blood culture

growth of large, gram-
positive bacilli
provides preliminary
identification of
Bacillus species

Dr.T.V.Rao MD 55
 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-ray—widened 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
(M’Fadyean (M’Fadyean stain
Stain) India ink stain)

Dr.T.V.Rao MD 58
 B. anthracis:
Presumptive Identification

Clinical specimen (blood, CSF, etc.)

Gram stain Isolate on SBA

Capsule production
Colony morphology
Hemolysis
Motility
Spores Gram stain
Malachite green

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

• Can start early treatment of Anthrax

• 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.

• Rapid diagnostic methods provide answers in minutes or hours

instead of days.
Dr.T.V.Rao MD 62
 Cautions on Treatment
• Obtain specimens for
culture BEFORE initiating
antimicrobial therapy.
• Do NOT use extended-
spectrum cephalosporins
or
trimethoprim/sulfametho
xazole because anthrax
may be resistant to these
drugs.

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

• PA from attenuated, nonencapsulated Sterne strain absorbed onto aluminum hydroxide

– Contains no dead or live bacteria in the preparation
– Antibodies to PA prevent binding to the target cell & confer protection from anthrax.

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

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

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+; Freund’s; 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

Dr.T.V.Rao MD 69
 Vaccine Schedule
• 3 injections at two-week intervals
• 3 injections 6 months apart
• Annual booster

Dr.T.V.Rao MD 70
 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
10–15 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
Dr.T.V.Rao MD 71
 Precautions to Health care Workers
• Standard contact
precautions. Avoid
direct contact
with wound or
wound drainage.

Dr.T.V.Rao MD 72
 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.

Dr.T.V.Rao MD 73
 Weaponization & Bacillus Anthracis:
Why is this Agent Considered to be the Department of Defense’s
Number-One/Two Biological Threat?

A sample of anthrax bacteria at the National School

of Biological Sciences, Mexico City

Dr.T.V.Rao MD 74
Analysis of the 2001 US Anthrax
Attacks

 Above anthrax-containing envelopes Above anthrax-containing envelopes

postmarked September 18th, 2001 postmarked October 9, 2001

*Also believed to be three or more other envelopes that were never found
Dr.T.V.Rao MD 75
 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

Dr.T.V.Rao MD 77
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Dr.T.V.Rao MD 78