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2002 powerpoint on work in same lab.
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Detection of Anthrax Detection of Anthrax Spores in MailSpores in Mail
Florine C. ClearyFlorine C. Cleary
20022002
AnthraxAnthrax• 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 Detection of anthrax spores has recently become a concern for us all.concern for us all.
These are the three letters These are the three letters sent containing sent containing AnthraxAnthrax
““This is nextThis is nextTake penacilin nowTake penacilin nowDeath to AmericaDeath to AmericaDeath to IsraelDeath to IsraelAllah is Great”Allah is Great”
““You can not stop usYou can not stop usWe have anthraxWe have anthraxYou die now.You die now.Are you afraid?Are you afraid?Death to AmericaDeath to AmericaDeath to IsraelDeath to IsraelAllah is greatAllah is great””
““This is nextThis is nextTake penacilin nowTake penacilin nowDeath to AmericaDeath to AmericaDeath to IsraelDeath to IsraelAllah is GreatAllah is Great””
Background
• Five peopleFive people dieddied in America last fall as a result in America last fall as a result of handling anthrax-tainted mail.of handling anthrax-tainted mail.
• Two of the people killed were Two of the people killed were postal employeespostal employees who worked at the Brentwood facility, which who worked at the Brentwood facility, which processes mail for the federal government processes mail for the federal government
• ThisThis led to a massive campaign by the U.S. Postal led to a massive campaign by the U.S. Postal Service Service to sanitize mailto sanitize mail, especially that destined , especially that destined for politicians on Capitol Hill.for politicians on Capitol Hill.
Potential Health RisksPotential Health RisksInvolved With IrradiationInvolved 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 theirradiation process. The coin was “bright white” before irradiation.
AnthraxAnthrax• 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.
Pics from the Official US Department of Defense anthrax website (Oct 2001)
Bacillus Anthracis sporesBacillus Anthracis vegetative cells
GOALGOAL Development of methodology and Development of methodology and
instrumentation for the detection of bacterial instrumentation for the detection of bacterial spores in mailspores in mail
Development of a Method for the Detection of Spores Inside Mail
Intrinsic Fluorescence Detection:
– requires NONO reagents (detection relies on optical properties of components found naturally in spores)
– requires NONO sample handling (no opening of mail or direct contact with potentially contaminated mail)
– requires NONO sample processing (no time required for biological ‘grow-out’ or chemical steps)
– can be adapted to existing mail sorting equipment
How Intrinsic FluorescenceDetection Works
envelo p esp o res
exc itatio n light emitted light
• 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 SporesFluorescence Properties of SporesBacterial spores have several intrinsic fluorescent Bacterial spores have several intrinsic fluorescent components that can be used for their detectioncomponents that can be used for their detection..
B. thuringiensis Spores & CellsProtein and DNA Fluorescence
300 350 400 450 5000
5000
10000
15000Cells
Spores
Wavelength
Flu
ore
scen
ce
B. thuringiensis Spores & CellsCalcium Dipicolinate Fluorescence
350 400 450 500 550 6000
50
100
150
200
250
300
350Cells
Spores
Wavelength
Flu
ore
scen
ce
B. thuringiensis Spores & CellsOther Component Fluorescence
700 750 800 850 9000
10
20
30Cells
Spores
Wavlength
Flu
ore
scen
ce
Protein & DNAProtein & DNA Ca-DipicolinateCa-Dipicolinate Other ComponentsOther Components
Fluorescence Properties of Spores Fluorescence Properties of Spores and Vegetative Cellsand Vegetative Cells
Spore GerminationSpore Core Kinetics
0.0 0.1 0.2 0.3 0.4 0.5 0.60
2500
5000
7500
10000Ca-Dipicolinate
Other Component
Time, min.
Pea
k A
rea
Spore GerminationCellular Metabolite Kinetics
0 10 20 30 40 50 600
1000
2000
3000
4000
5000
6000NADH
DPNH
Flavoproteins
Hemoproteins
Time, min.R
elat
ive
Flu
ore
scen
ce
Calcium dipicolinate and other core components are lostquickly when spores germinate. Other fluorophores usedfor 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.
envelo p esp o res
exc itatio n light
sp o re emis s io n
p ap er emis s io n
Detection of SporesInside Sealed Envelopes
(5 mg sample size)
0
25
50
75
100
EnvelopeOnly
Spores inEnvelope
Flu
ore
scen
ce S
ign
al
The background-corrected signal from spores sealed inside an envelope are shown in the bar graph.
Fluorescence Properties of PaperFluorescence Properties of PaperThe 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
650 700 750 800 8500
50
100
150Envelope
Secruity
Manilla
Card Stock
Tyvek
Wavelength
Flu
ore
scen
ce
Paper Fluorescence
650 700 750 800 8500
250
500
750
Wavelength
Flu
ore
scen
ce
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.
The wavelengths of light used to determine the background of the paper are shown in blue on the spectra of spores.
Fluorescence of VariousBacterial Spores
650 700 750 800 850 9000
100
200
300
400
500
600B. thuringiensis
B. megaterium
B. subtilis
Wavelength
Fluo
resc
ence
Fluorescence Properties Between Fluorescence Properties Between Spores of Different SpeciesSpores 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.
Ca-Dipicolinate Other Components
Yeast and Bacterial Spore Solutions(Ca-Dipicolinate Fluorescence)
350 400 450 500 5500
100
200
300
400
500
600Saccharomyces cerevisiae Spores
Bacillus thruingiensis Spores
Wavlength
Flu
ore
scen
ce
Yeast and Bacterial Spore Solutions(Other Component Fluorescence)
700 750 800 850 9000
25
50
75
100
Wavelength
Flu
ore
scen
ce
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
Sp
ore
s
Ba
by
Po
wd
er
Co
rn S
tarc
h
Je
llo
Po
wd
ere
d M
ilk
Flo
ur
(ble
ac
he
d)
Po
wd
ere
d S
ug
ar
Wh
ite
Co
co
a
Gy
ps
um
-50
0
50
100
150
200
250
300
Sig
nal
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