Detection of Anthrax Spores in Mail

Florine C. Cleary 2002

Anthrax
• Rare disease in humans (1/100,000 in natural environment) • Three types of anthrax in humans:
– cutaneous (infection of skin, eyes and/or mucous membranes) – gastrointestinal (infection of intestinal mucosa) – inhalation (infection of lungs – obtained by inhaling spores)

• Detection of anthrax spores has recently become a concern for us all.

These are the three letters sent containing Anthrax

“This is next

Take penacilin now Death to America Death to Israel Allah is Great”

“You can not stop us

We have anthrax You die now. Are you afraid? Death to America Death to Israel Allah is great”

“This is next

Take penacilin now Death to America Death to Israel Allah is Great”

Background
• Five people died in America last fall as a result of handling anthrax-tainted mail. • Two of the people killed were postal employees who worked at the Brentwood facility, which processes mail for the federal government • This led to a massive campaign by the U.S. Postal Service to sanitize mail, especially that destined for politicians on Capitol Hill.

Potential Health Risks Involved With Irradiation
• “Following the ‘clean up’ and new methods implemented by the US postal service, at least 73 Senate employees reported symptoms such as headaches, bloody noses, skin rashes and dry mouth after handling mail that was irradiated to kill any possible anthrax spores. So an inquiry was begun into the possible ill-effects from irradiated mail and the clean-up of the Senate's Hart building.” –Washington Post • “At least 87 suburban postal workers who handled irradiated mail have reported health problems including nausea, headaches and breathing problems, union leaders say.” -CNN

Further Problems With Irradiated Mail
• Irradiation can have damaging effects on materials (computer media erased and melted, documents destroyed, precious stones ruined, etc.) • Irradiated mail has been reported to catch fire

Close-up of 1949 Washington quarter dollar showing the bubbling of the plastic encapsulation holder after going through the irradiation process. The coin was “bright white” before irradiation.

Anthrax
• Bacillus anthracis is found in two forms: – vegetative cells (metabolizing rod-shaped bacteria, “awake”) – endospore (non-metabolizing, “dormant” or “hibernating”) • Bacterial spores are able to withstand extreme treatment, can be aerosolized, and used as a biological weapon.

Bacillus Anthracis vegetative cells

Bacillus Anthracis spores

Pics from the Official US Department of Defense anthrax website (Oct 2001)

GOAL
Development of methodology and instrumentation for the detection of bacterial spores in mail

Development of a Method for the Detection of Spores Inside Mail
Intrinsic Fluorescence Detection:
– requires NO reagents (detection relies on optical properties of components found naturally in spores) – requires NO sample handling (no opening of mail or direct contact with potentially contaminated mail) – requires NO sample processing (no time required for biological ‘grow-out’ or chemical steps) – can be adapted to existing mail sorting equipment

How Intrinsic Fluorescence Detection Works
• light of a specific energy is directed towards the sample • components of the spores absorb the excitation light and then spontaneously emit light of a different color • the emitted light is detected

Fluorescence Properties of Spores
Bacterial spores have several intrinsic fluorescent components that can be used for their detection.
B. thuringiensis Spores & Cells Protein and DNA Fluorescence
15000 Cells Spores
350 300

B. thuringiensis Spores & Cells Calcium Dipicolinate Fluorescence
30
Cells Spores

B. thuringiensis Spores & Cells Other Component Fluorescence
Cells Spores 20

Fluorescence

Fluorescence

10000

250 200 150 100 50

Fluorescence

5000

10

0 300 350 400 450 500

0 350 400 450 500 550 600

0 700 750 800 850 900

Wavelength

Wavelength

Wavlength

Protein & DNA

Ca-Dipicolinate

Other Components

Fluorescence Properties of Spores and Vegetative Cells
Spore Germination Spore Core Kinetics
Ca-Dipicolinate Other Component

Spore Germination Cellular Metabolite Kinetics Relative Fluorescence
6000 5000 4000 3000 2000 1000 0 0 10 20 30 40 50 60 NADH DPNH Flavoproteins Hemoproteins

10000 7500 5000 2500 0 0.0

Peak Area

0.1

0.2

0.3

0.4

0.5

0.6

Time, min.

Time, min.

Calcium dipicolinate and other core components are lost quickly when spores germinate. Other fluorophores used for microbial detection are inappropriate for spore detection.

Fluorescence Properties of Paper
Paper also exhibits fluorescence when excited with the light used to excite spore components.

The fluorescence from the paper must also be determined to ensure that spores are actually detected.

Detection of Spores Inside Sealed Envelopes
(5 mg sample size)
100

Fluorescence Signal

75 50 25 0

Envelope Only

Spores in Envelope

The background-corrected signal from spores sealed inside an envelope are shown in the bar graph.

Fluorescence Properties of Paper
The fluorescence properties of 45 kinds of paper and 5 kinds of envelopes were investigated to determine if they would interfere with the detection of spores.
Envelope Fluorescence
150
750

Paper Fluorescence

Fluorescence

Fluorescence

100

Envelope Secruity Manilla Card Stock Tyvek

500

50

250

0 650 700 750 800 850

0 650 700 750 800 850

Wavelength

Wavelength

The wavelength of light emitted from the other components of spores are shown with the red arrow in the spectra above. Wavelengths used to determine the background of the paper are shown in blue. Both the excitation light and emission light are able to penetrate the envelope making detection of spores inside mail possible.

Fluorescence of Various Bacterial Spores
600 500 B. thuringiensis B. megaterium B. subtilis

Fluorescence

400 300 200 100 0 650 700 750

800

850

900

Wavelength

The wavelengths of light used to determine the background of the paper are shown in blue on the spectra of spores.

Fluorescence Properties Between Spores of Different Species
Differences between the fluorescence spectra of bacterial and fungal (e.g., mold) spores can be exploited to differentiate between the species. The method to detect bacterial spores described herein is not “fooled” by mold spores.
Yeast and Bacterial Spore Solutions (Ca-Dipicolinate Fluorescence)
600 500 100 Saccharomyces cerevisiae Spores Bacillus thruingiensis Spores

Yeast and Bacterial Spore Solutions (Other Component Fluorescence)

Fluorescence

Fluorescence

75

400 300 200 100 0 350 400 450 500 550

50

25

0 700 750 800 850 900

Wavlength

Wavelength

Ca-Dipicolinate

Other Components

Hoax Material Interference?
The background-corrected signal from ca. 10 mg of bacterial spores and hoax materials inside a folded piece of paper are shown in the bar graph below.
Detector Signal Output
300 250 200

Signal

150 100 50 0 -50 Spores Jello Baby Powder Corn Starch Powdered Milk Flour (bleached) Powdered Sugar White Cocoa Gypsum

Detector Prototype

Further Work
• determine detection limits of method with various spore species • demonstrate detection with Bacillus anthracis spores • investigate adapting detector to high-speed mail sorters

Master your semester with Scribd & The New York Times

Special offer for students: Only $4.99/month.

Master your semester with Scribd & The New York Times

Cancel anytime.