Bacterial agents of bioterroism

Laboratory network for biological terrorism

Bacillus anthracis

Anthrax

Anthrax: Overview • Primarily disease of

herbivores

• Humans usually

infected by contact with infected animals or contaminated animal products

• Soil reservoir • Woolsorter’s disease

(inhalation anthrax)

• No person-to-person

transmission of inhalational anthrax

CDC

ANTHRAX • Three forms of human anthrax occur: 1. Cutaneous 2. Gastrointestinal • Oropharyngeal • Abdominal 3. Inhalation (Woolsorter’s Disease)

Cutaneous anthrax

Vesicle development, day 2 Eschar formation, day 4

Inhalation Anthrax • Infective dose = 8,000 - 15,000 spores • Incubation period = 1-6 days • Duration of illness = 3-5 days • Fever, malaise, and fatigue • Short period of improvement = up to 2 days • Abrupt respiratory distress…death <24hrs • Person to person transmission = no

Anthrax: Specimen Selection • Inhalation: Sputum and Blood • Cutaneous: Vesicles and Eschar • Gastrointestinal: Stool and Blood

Bacillus anthracis Key Sentinel Lab Tests • Gram stain • Growth characteristics on agar • Sporulation, in air • Motility • Capsule by India Ink

Bacillus anthracis Gram Stain Morphology

• Broad gram-positive rod: 1-1.5 X 3-5 µ • Oval, central - subterminal spores: 1 X 1.5 µ

with no significant swelling of cell

• Spores are NOT usually present in clinical

specimens unless exposed to atmospheric O 2

B. anthracis,

Gram stain demonstrating spores

B. anthracis Colonial Morphology

• Colonial morphology of 18-24hr @ 35 C: – Well isolated colonies are 2-5 mm in diameter – Flat or slightly convex, irregularly round – Edges: slightly undulate, often curly tailing

edges

– Ground glass appearance – “Sticky” consistency….stands up like beaten

egg whites

B. anthracis

, colony on SBA

“STICKY” consistency of anthracis’ colony on SBA B.

Bacillus anthracis Presumptive Identification

• Gram-positive, broad rod, catalase-

positive, spore-positive, aerobe: Bacillus sp.

• Spores are oval and nonswelling with

ground glass colony appearance: Bacillus morphology group 1, includes B. anthracis, B. cereus, B cereus var mycoides, and B. thuringiensis

Bacillus anthracis Presumptive Identification, con’t

• Nonmotile:

B anthracis and B cereus var mycoides ( megaterium) and B.

• Nonhemolytic, forms capsule:

Presumptive B. anthracis

• Refer to state lab for testing

Yersinia pestis

Plague

Plague: Overview

• Natural vector - Rodent flea • Mammalian hosts – rats, squirrels,

chipmunks, rabbits, and carnivores

• Enzootic or Epizootic

Plague Epidemiology • U.S. averages 13 cases/yr • 30% of cases are in Native Americans in the

Southwest. 15% case fatality rate

• Most cases occur in summer and near the

patient’s residence

– bubonic (infected lymph nodes) – septicemic (blood-borne organisms) – pneumonic (transmissible by aerosol;

deadliest)

Yersinia pestis

Specimen Selection • Specimen selection is important – Bubo - lymph node aspirate – Blood - organisms may be intermittent. Take

three specimens 10-30 minutes apart

– Pneumonic • Sputum/throat - use Wayson stain • Bronchial washings - Wayson stain • Inoculate routine plating media

Sentinel Lab Procedures

Yersinia pestis

• Gram stain • Wayson stain • Growth characteristics on agar • Growth characteristics in broth

Yersinia pestis

Gram stain • Small, gram-negative coccobacilli

Yersinia pestis

Wayson Stain • Used for rapid assessment – when it is a part of the identification process • Best with tissue, sputum, blood • Stains of pure culture isolates tend to lose bipolarity • Pink-blue cells with polar granules (safety pin appearance)

Yersinia pestis

Wayson Stain • Pink-blue cells with a closed safety pin look

Wayson stain alone is not diagnostic

Y.pestis

48 h culture on SBA 72 h culture on SBA

Yersinia pestis Technical Hints

• Small Gram-negative, poorly staining rods

from blood, lymph node aspirate, or respiratory specimens

• Safety pin appearance in Gram, Wright,

Giemsa, or Wayson stain

• More than one patient in a short, specified

period with fever, lymphadenopathy

• Refer to state lab

Francisella tularensis Tularemia

Tularemia: Overview • Disease of Northern Hemisphere • In U.S., most cases associated with rabbits/hares and ticks • About 200 cases/year in U.S.

– most in South central and Western states – majority of cases in summer, some in winter

Reported Cases of Tularemia 1990-1998

Tularemia: Overview (cont’d) • Several forms of human tularemia exist: - Ulceroglandular, glandular, oculoglandular, oropharyngeal, intestinal, pneumonic, and typhoidal • Low infectious dose –1 to 10 organisms by aerosol or intradermal

route

• No person-to-person transmission

Tularemia: Specimen Selection • Serum - acute and convalescent • Blood cultures • Sputum • Swab – ulcer or eye

Sentinel Lab Procedures

Francisella tularensis

• This is a dangerous, highly virulent organism and it should not be manipulated at the bench. Laboratory-acquired infections can occur easily .

• Gram stain • Growth characteristics in broth • Growth characteristics in agar

Francisella tularensis

• Poorly staining, tiny Gram-negative coccobacilli

Francisella tularensis

Growth Characteristics • Fastidious, requires cysteine for robust growth: Cysteine Heart Agar (CHA) is ideal – Enriched chocolate agar + 9% sheep blood + cysteine – Not part of Sentinel Lab routine procedures – BCYE (for

Legionella ) also works

• Will grow initially on sheep and chocolate blood agar and Thayer-Martin agar, but poorly or not at all on passage • Grows slowly at 35 o C, poorly at 28 o C

Francisella tularensis

Growth Characteristics • 24 hours – gray-white, translucent colonies – usually too small to be seen individually • 48 hours – Sheep Blood Agar - <1 mm, gray-white, opaque, no

hemolysis

Francisella tularensis

Sheep blood agar Chocolate agar Cysteine heart agar

Francisella tularensis

Technical Hints

If you see:

• Tiny, Gram-negative coccobacilli from

blood, lymph node aspirate, or respiratory specimens

• Blood isolates that will grow slowly on

chocolate agar but poorly or not at all on blood agar in 24 hours

• Faint growth in thio; requires cysteine in

other broth

• Refer to state lab

Brucella spp.

Brucellosis

BRUCELLOSIS • A zoonotic disease caused by any of 4

Brucella and canis sp.: abortus, melitensis, suis,

• A systemic infection characterized by

an undulant fever pattern

• But relatively rare in the U.S. with

approximately 100 cases/yr

BRUCELLOSIS: TRANSMISSION • Unpasteurized dairy products – The most common mode of transmission • Direct skin contact – Occupational hazard for farmers,

butchers, veterinarians, and laboratory personnel

• Aerosols – Highly infectious

BRUCELLOSIS

•Infective dose = 10 -100 organisms •Incubation period = 5 days - > 6 months •Duration of illness = weeks to months •Fever, profuse sweating, malaise, headache and muscle/back pain. •Person to person transmission = no •Mortality = <5% •Persistence of organism = very stable

Brucella

spp.

Specimen Selection • Serum – The diagnosis of brucellosis is frequently

achieved by serology. An acute & convalescent phase specimen should be collected (21d apart)

• Blood or bone marrow – Sources from which Brucellae are most

often isolated

• Tissue (spleen, liver) – Brucellae occasionally isolated

Brucella spp.

Biosafety Alert

•

Brucellosis

is THE most commonly

reported laboratory-associated bacterial infection.

• Cases have occurred in clinical

laboratory settings by “sniffing” cultures, direct skin contact with cultures, and aerosol generating procedures

Sentinel Lab Tests Brucella spp.

• Colonial morphology on SBA • Gram stain morphology • Oxidase positive • Urea hydrolysis positive

Brucella spp.

Key Sentinel Lab Tests

Colonial morphology on SBA

–Fastidious –Visible growth may take 48 - 72 hrs –Small (0.5-1.0mm), convex, glistening – Non-hemolytic and non-pigmented

B. melitensis on sheep blood agar

Brucella spp.

Key Sentinel Lab Tests

Gram Stain Morphology

–Tiny (very) –Faintly staining –Gram-negative coccobacilli –0.5 - 0.7

 x 0.6 - 1.5



Brucella spp.

Review of Key Tests

• Tiny, faintly staining, gram-negative coccobacilli from blood or bone marrow • Slow growth on Sheep Blood Agar, 2-3 days for colony appearance • Oxidase + • Urease + • Handle plates with care • Refer to state lab

Clostridium botulinum

Botulism

Botulism: Overview

• Caused by toxin from – toxin types A, B, E, most commonly associated with

human disease

– most potent lethal substance known to man (lethal

dose 1ng/kg)

Clostridium botulinum • C. botulinum spores found in soil worldwide • Approximately 100 reported cases/year in the U.S.

– infant most common (72%) – food borne not common • No person-to-person transmission

FOODBORNE BOTULISM

• Infective dose: 0.001  g/kg • Incubation period: 18 - 36 hr (6hr to 10 d) • Dry mouth, double vision, droopy eyelids, dilated pupils • Generalized, progressive descending bilateral muscle weakness & paralysis • Respiratory failure and death • Mortality usually 5 – 10%

FOODBORNE BOTULISM

• Among 309 persons with clinically diagnosed botulism reported to CDC from 1975 to 1988: – Stool cultures for

C. botulinum : 51% +

– Serum botulinum toxin testing: 37% + – Stool botulinum toxin testing: 23% + • Overall, at least one of the above tests was positive for 65% of all patients • Diagnosis is primarily clinical

Sentinel Lab Procedures for Botulism Event

• Properly collected specimens are to be referred to designated testing laboratories • Prior to the shipment of any botulism associated specimen, testing must be arranged with MDCH laboratory

Sentinel Lab Procedures for Botulism Event Clinical specimens to be collected:

1. Serum 2. Feces 3. Food samples

Autopsy specimens:

1. Serum 2. Gastric and intestinal contents

Botulism Biosafety Alert

Materials suspected of containing botulism toxin must be handled: –Biological Safety Cabinet (Class II) –Laboratory Coats –Disposable surgical gloves –Face shield (as needed)

BOTULISM

• The diagnosis of botulism is made

clinically, i.e., based on the patient’s case history and physical findings

• Health care providers suspecting

botulism should contact the Michigan Department of Community Health

Botulism Referral Lab Procedures • Mouse bioassay • Isolation of C. botulinum