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_ _ 2 2 2 93 6 ;USAMRI ID _ go ALL In:?nmun*1:|m r:|:=m*1"i11~1rn| HERE n; 1:5 UI»JELAIFIEII
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g investigation was conducted by Special
The United States Army Medical Research Institute
of Infectious Diseases USAMRIID! Keycard Access records from
1998 through 2002 were queried f r v ' ' ' scientistnd visitinq scien§i§t| E both irgm_Ehe1 who have p eviously been identified as
visiting USAMRIID circa May 1998.
All available records were searched for names
containing| |or| Ior I t all queries met
with negative results.
As previously reported, Bruce Ivins was the USAMRIID
point of contact for| |and| | A query of all available
recor s for "Ivins*" met with positive results. Records indicate
Bruce 'ns showed keycard activity during a date range of
August , 1998 through June 02, 2002.b
Agent SA!E?f:f?%%?%iof the federal Bureau of Investigation b
FBI! on February 24, 2005:rO
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'~_S-'1|:|FD-392 Rev. 1?:-6-95!
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' FEDERAL BUREAU OF INVESTIGATION be
ALL II'IFi:'PI'l@!tTIUI'] EIJI-ITAIIJED by/C
EIEPEEIIJ U1lICL;~s5SIFIEI:~
DATE 13-12-2008 BY ED324 TIC EiiUIIi"DE_.-"HE'S -' _ Date of transcription
Z 2 5 Z 2 O Q 5
On February 18, 2005, an Internet author query on
website: www.pubmed.com for] Iscientists,E::::::::::::] and| |et with positive results.
i lthe following
publication: I ii Z _ |
| |"A Novel Surfactant Nanoemulsion with Broad-Spectfm
Sporicidal Activity against Bacillus Species," The Journal of
Infectious Diseases 1999; l8O:19391949.
WFO Note: Attached hereto and considered part of this
document is a copy of the above-mentioned publication.! ,
Briefly; paqe| Iindicatedl I andl Iare
I + + |
B
Briefly, page three "Material and Methods" section
indicated, "B. anthracis spores, Ames and Vollum 1B strains, were
supplied by Bruce Ivins US Army Medical Research Institute of
Infectious Diseases [USAMRIID], Fort Detrick, Frederick MD ...Four
strains of B. anthracis were provided bl have a patent application
Briefly, page ten "Discussion" section indicated, "BCTP
[the novel surfactant nanoemulsion] and its derivative BCTP 401
appear to have great potential as environmental decontamination
agents of for treatment of exposed persons in either a military
operation or terrorist attack."
Briefly, page ten "Acknowledgments" section indicated
"Bruq%92IvinsJ__H==
. .fo their technical support.]
I |for supplying characterized B. anthracis
strains an space at Louisiana State University..."
mwngmmim O2/25/2005 n Frederick, Maryland
File # 2'79AWF222936USAMRIID 9292Q92 Date dictated N/A
This document contains neither recommendations nor conclusions of the FBI. It is the property of the FBI and is loaned to your agen
it and its contents are not to be distributed outside your agency.
liamouda et al., Sporicidal Actii ofBCTP Nanoemulsion Page 1 of 12 Z
The Journal of Infectious Diseases 1999'180:1939-1949 _ _ _© 1999 by the Infectious Diseases Society oi America. All rights reserved. AL L IHFOHMTI UH - I UHTMHEL0022-1899/1999/18006-00245402 00 HERE I5 i5'3Li** IFIEI1'
' DATE l2-l2-ZUU5 BY 513324 UII E.i5.ll.e"1:'I*i_.*'R'I'5
A Novel Surfactant Nanoemulsion with Broad-Spectrum
Sporicidal Activity against Bacillus Species
Tarek Hamouda,1 Michael M. Hayes,1§ Zhengyi Cao,1 Richard T0nda,1 Kent Johnson,2
D. Craig Wright,3 Joan Brisker,3 and James R. Baker, J r.1
I C enter for Biologic Nanotechnology and Department ofMedicine, and 2Department of Pathology, University 0fMichigan
Medical School, Ann Arbor; 3NOVA VAX, Inc., Rockville, Maryland
Received 10 March 1999; revised 30 June 1999; electronically published 12 November 1999.
Two nontoxic, antimicrobial nanoemulsions, BCTP and BCTP 401, have been
developed. These emulsions are composed of detergents and oils in 80% water.
BCTP diluted up to 1 : 1000 inactivated >90% of Bacillus anthracis spores in 4
h and was also sporicidal against three other Bacillus species. This sporicidal
activity is due to disruption of the spore coat after initiation of germination
_, without complete outgrowth. BCTP 401 diluted 1 : 1000 had greater activity
than BCTP against Bacillus spores and had an onset of action of <30 min.
Mixing BCTP or BCTP 401 with Bacillus cereus prior to subcutaneous
injection in mice reduced the resulting skin lesion by 99%. Wound irrigation
with BCTP 1 h after spore inoculation yielded a 98% reduction in skin lesion
size, and mortality was reduced 3-fold; These nanoemulsion formulas are _
stable, easily dispersed, nonirritant, and nontoxic compared with other
available sporicidal agents. *
==..._. 92>> _,...._,_4..._ ., .._c-i_=.... 1,.,.;.__ s;......__.i ,,,__ K ._.~, ... .»__i. a; _.~.....,_. !:v _;., ,. _,.......~;» ,1 Pv<J1~']' .....w._.~Ae.. .. .,.-.1... ,_.<,»~..
Presented in part: 98th general meeting of the American Society for Microbiology, Atlanta, May 1998 poster A49!; 38th
Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, September 1998 late-breaker slide session
11, LB-9!; 99th general meeting of the American Society for Microbiology, Chicago, May 1999 poster A300!.
The animal experiments were approved by and performed according to the guidelines of the Unit for Laboratory Animal
Medicine, University of Michigan. i A
D.C.W. and J.B. are employees of NOVAVAX, Inc., and have signicant financial interest in the company. NOVAVAX
Inc., is the supplier of the emulsions. J.R.B., T.I-1., M.M.IL, D.C.W., and .T.B. have a patent application entitled: Methods of
inactivating bacteria including bacterial spores.
Financial support: Defense Advanced Research Project Agency contract MDA 972-1 -007 of the Unconventional
Pathogen Countermeasures Program!._ -- Present ailiation: Pulmonary/Critical Care UnitInternal Medicine, University of Michigan, Ann Arbor.
Reprints or correspondence: Dr. James R Baker, Jr., University of Michigan Medical School, 9220 MSRB-HI, 1 150, W.
Medical Center Dr., Ann Arbor, MI 48109-0648 jbakeg'[email protected] !.
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Hamouda et al., Sporicidal of BCTP Nanoemulsion _ Page 2 of 12
92
Bacteria of the Bacillus genus form stable spores that are resistant to harsh conditions and extreme
temperatures. Contamination of farmlands with Bacillus anthracis leads to a fatal disease in domestic,
agricultural, and wild animals [1]. Human infection by B. anthracis usually results from contact with
infected animals or infected animal products [2]. Human clinical symptoms include a pulmonary form
that has a rapid onset and is frequently fatal. The gastrointestinal and cutaneous forms of anthrax,
although less rapid, can also result in -fatalities unless treated aggressively [1, 51]. B. anthracis infection in
humans is no longer common, because of eifective animal control that includes vaccines, antibiotics, and
appropriate disposal of infected livestock. However, animal anthrax still represents a significant problem
because of contamination of farmland. Although a vaccine is available [Q] and can be used for the
prevention of anthrax, genetic mixing of different strains can render it ineffective [Q]. The potential
consequences of the use of B. anthracis spores as a biologic weapon were demonstrated by the
accidental release of B. anthracis from a military microbiology laboratory in the former Soviet Union.
Seventy-seven cases of human anthrax, including 66 deaths, were attributed to the accident. Some
infections occurred as far as 4 km from the laboratory [1]. Genetic analysis of infected persons revealed
the presence of either multiple strains or genetically altered B. anthracis [§].
Other members of the Bacillus genus are also reported to be etiologic agents for many human
diseases. B. cereus is a common pathogen. It is involved in foodborne diseases because its spores can
survive cooking procedures. Local sepsis and wound and systemic infections have also been attributed to
B. cereus [2]. _
»
Disinfectants and biocides e.g., sodium hypochlorite, formaldehyde, and phenols! that are highly
effective against Bacillus spores are not well suited for decontamination of the environment, equipment,
or exposed persons because of toxicity that leads to tissue necrosis and severe pulmonary injury after
inhalation of volatile fumes. The corrosive nature of these compounds also renders them unsuitable for
decontamination of sensitive equipment [L1-Q].
Concerns about these issues have stimulated interest in new types of biocidal agents that can safely
decontaminate Bacillus spores. We have investigated the sporicidal properties of two antimicrobial lipid
emulsions. Nanoemulsions are produced by mixing a lipid-oil "discontinuous" phase with an aqueous
"continuous" phase under high shear forces. The result is an oil droplet of "~*400800 Pm in diameter that
is able to fuse with and subsequently disrupt the membrane of a variety of different pathogens [E].
BCTP is a nanoemulsion made of soybean oil, Triton X-100 detergent, and tri-n-butyl phosphate in 20%
water. BCTP 401 is a mixture of this emulsion and a liposome, P10. P10 is made of water, Tween 60,
soybean oil, glycerol monooleate, rened soya sterols, and the cationic compound cetylpyridiniumchloride. These two compounds have antimicrobial activity against enveloped viruses and bacteria '
through membrane disruption unpublished data!. In the current studies, we examined the ability of these
emulsions to inactivate different Bacillus spores. i
Materials and Methods
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lrlamouda et al., Sporicidal Activity of BCTP Nanoemulsion Page 3 of 12
Surfactant lipid preparations. BCTP is a water-in-oil nanoemulsion, in which the oil phase is made
from soybean oil, tri-n-butyl phosphate, and Triton X-100. Stock solutions contained 80% lipid
components and 20% water. Three different preparations of BCTP, 2, 8, and 16 months old, were tested
for their stability. BCTP 4'01 was prepared by mixing equal volumes of BCTP with P10, the latter being
a liposome-like compound. P10 is made of glycerol monosterate, rened soya sterols, Tween 60,
soybean oil, a cationic ion halogen-containing cetylpyridinium chloride, and peppermint oil. The average
size of these nanoemulsions is in the range of 400-800 mn, as determined by laser light scatter LS23 0;
Coulter, Hialeah, FL!. These surfactant lipid preparations were stable aer boiling for 1 h or exposure to
1 N nitric acid or 1 N sodium hydroxide for 2 h. This treatment resulted in a <20% reduction in the
emulsion mean particle size [1_6_]. These solutions were stored at room temperature and were diluted
before each experiment to the working dilution. All dilutions herein are in reference to the stock solution.
Spore preparation. For induction of spore formation, B. cereus ATCC 14579!, B. circulans
ATCC 4513!, B. megaterium ATCC 14581!, and B. subtilis ATCC 11774! were grown for 1 week at
37°C on nutrient agar with 0.1% yeast extract and 5 mg/L MnSO4. The plates were scraped, and the
bacteria and spores were suspended in sterile 50% ethanol and incubated at 22°C for 2 h with agitation to
lyse the remaining vegetative bacteria. The suspension was centrifuged at 2500 g for 20 min, and the
pellet was washed twice in cold distilled water. The spore pellet was resuspended in trypticase soy broth
TSB! and used immediately for experiments. B. anthracis spores, Ames and Vollum 1B strains, were
supplied by Bruce Ivins US Army Medical Research Institute of Infectious Diseases [USAMRIID], Fort
Detrick, Frederick, MD! and were prepared as described elsewhere [5]. Four other strains of B. anthracis
were provided by Martin Hugh-I ones . Louisiana State University, Baton Rouge!. These strains from
South Africa; Mozambique; Bison, Canada; and Del Rio, TX! represent isolates with high allelic
dissimilarity.
In vilro sporicidal assays. For assessment of sporicidal activity on solid medium, tiypticase soy
agar TSA! was autoclaved and cooled to 55°C. BCTP was added to the TSA at a 1 : 100 nal dilution
and continuously stirred while the plates were poured. The spore preparations were serially diluted 0-
fold!, and 10-FL aliquots were plated in duplicate highest inoculum, 105 spores/plate!. Plates were
incubated for 48 h aerobically at 37°C and evaluated for growth.
For assessment of sporicidal activity in liquid medium, spores were resuspended in TSB. Next, 1 mL
of spore suspension containing 2 >< 106 spores nal concentration, 106 spores/mL! was mixed with 1
mL of BCTP or BCTP 401 at 2>< nal concentration in distilled water! in atest tube. The tubes were
incubated in a tube rotator at 37 °C for 4 h. Treatment of B. anthracis was done at 37°C, which promotes
spore germination, and at 22°C, which does not promote spore germination [5]. After treatment, the
suspensions were diluted 10-fold in distilled water. Duplicate aliquots from each dilution were then
streaked on TSA and incubated ovemight at 37 °C; then colonies were counted. Sporicidal activity
expressed as percentage of killing was calculated as follows: {[cfu initial! - cfu posttreatment!]/[cfu initial!]} >< 100.
The experiments were repeated at least 3 times, and the mean and SE of the percentage of killing
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Hamouda et al., Sporicidal Activity of BCTP Nanoemulsion Page 4 of 12
were calculated by use of StatV1ew software Abacus Concepts, Berkeley, CA!. Analysis of variance
tables and paired t test were used when applicable.
Electron microscopy. B. cereus spores were treated with BCTP at a nal dilution of 1 1 100 in TSB
by means of Erlenmeyer asks in a 37°C shaker incubator. The spore-BCTP mixture was washed with
saline and centrifuged at 2500 gfor 20 min, and the supernatantiwas discarded. The pellet was xed in
4% glutaraldehyde in 0.1 M cacodylate pH 7.3!. Spore pellets were processed for transmission electron
microscopy, and thin sections were examined after staining with uranyl acetate and lead citrate.
Germination inhibitors or enhancers. B. cereus spores fmal concentration, 106 spores/n1L! were
suspended in TSB with either the germination inhibitor D-alanine nal concentration, 10 mM! or the
germination enhancer L-alanine nal concentration, 5 mM! [H712]. This suspension was then
immediately mixed with BCTP nal dilution, 1 : 100! and incubated for variable intervals. Then the
mixtures were serially diluted, plated, and incubated overnight. The next day, growth on the plates was
counted, and the percentage of sporicidal activity was calculated.
In vivo toxicity testing. Mice were exposed to various concentrations of the different emulsions by
means of different routes of administration. The highest concentrations that produced no gross or
histopathologic lesions in mice were reported. Exposures included subcutaneous or intramuscular
injection of 100 FL, open wound irrigation with 2 mL of the emulsions, and intranasal instillation of 25
FL/naris. The emulsions are relatively viscous when not diluted, so toxicity testing in the nares was
conducted at the highest concentration that would not suffocate the animals. Three to four mice were
tested for each concentration of each compound, and the experiments were repeated on at least three
occasions.
In vivo sporicidal activity. Two animal models were developed to conrm the sporicidal activity of
the emulsions in vivo. In the rst model, B. cereus spores suspended in sterile saline! were mixed with
an equal volume of BCTP to a nal emulsion dilution of 1 : 10. As a control, the same B. cereus spore
suspension was mixed with an equal volume of sterile saline. Next, 100 FL of each of the suspensions,
containing 4 >< 107 spores, was then immediately injected subcutaneously into CD-1 mice. Nine mice
were inoculated in each group, and the experiment was repeated on three different occasions. .
In the second model, a simulated wound was created by making an incision in the skin on the back of
the mice. The slcin was separated from the underlying muscle byblunt dissection. The pocket was
inoculated with 200 I-L of saline containing 2.5 >< 107 spores and closed by use of wound clips. One hour
later, the clips were removed, and the wound was inigated either with 2 mL of sterile saline or with 2
mL of BCTP : 10 in sterile saline!. The wounds were then closed with wound clips. The animals were
observed for clinical signs. Gross and histopathologic examination were done when the animals were
euthanized 5 days later. The wound size was calculated by the following formula: é a >< % b >< rr, where
a and bare two perpendicular diameters of the wound. Five mice were used in each group, and the
experiment was repeated on three different occasions. Both sets of animal studies were also conducted
with BCTP 401 at identical dilutions.
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Results
In vitro sporicidal activity. To assess the sporicidal activity of BCTP, spores from four species of
Bacillus genus B. cereus, B. circulans, B. megaterium, and B. subtilis! were tested. BCTP at a 1 : 100
dilution showed 97% sporicidal activity against B. cereus and B. megaterium in 4 h gure 1!. B. 1
circulans was less sensitive to BCTP, showing only an 83% reduction in spore count, whereas B. subtilis
appeared resistant to BCTP in 4 h. The other nanoemulsion, BCTP 401, was more efficient in killing the
Bacillus spores. At a 1 : 1000 dilution, it showed 99% killing of B. cereus spores in 4 h compared with
50% with a 1 : 1000 dilution ofiBCTP!. BCTP 401 at a 1 1 1000 dilution resulted in 96% killing of B.
subtilis spores in 4 h, in contrast to its resistance to BCTP. Bleach diluted 1 : 100 i.e., 0.0525% sodium
hypochlorite! showed 98% sporicidal activity against B. cereus in 4 h. There was no signicant
difference in sporicidal activity against B. cereus between BCTP diluted 1 : 100, BCTP 401 diluted
1 : 1000, and bleach diluted 1 : 100 P= .23!. '
Figure 1. Spoiicidal activity of BCTP against 4 different Bacillus species compared with that of
- - . . BCTP 401 against 2 Bacillus species. BCTP showed signicant sporicidal activity aer 4 h of
"* ~"""-ls * ' treatment against Bacillus cereus, B. circulans, and B. megaterium spores but not against B. ~
~. subtilis spores. BCTP 401 showed more eective killing against B. cereus in 4 h and also had
Lyn H sporicidal activity against B. subtilis that was resistant to BCTP. Bleach diluted 1 : 100 was used as
.. positive control and was comparable to BCTP or BCTP 401 at same dilutions.
r-'1:I:<* 1:7* , ~
Testing the stability of BCTP. Three different preparations of BCTP, stored for 2, 8, and 16 months
at room temperature, were evaluated simultaneously for sporicidal activity against B. cereus spores to
detennine the stability of the emulsions. BCTP was diluted 1 : 10 and 1 : 100 for the experiments :t_igu_re_
2!, and there was no signicant difference in the sporicidal activity of the preparations P = .94 and .77!.
- vi Figure 2. Comparison of sporicidal activity of 3 different preparations of BCTP aged 2, 8,
; T _, '_'_; ff_ ;_; and 16 months. Preparations have equivalent sporicidal activity, showing that BCTP is stable for
i ~.. 8 Hp 110 16 IIlOI1t11S. iy
e __" " "1
. . -.I.»A> ..,.-»»--».-.- .._..
B. cereus sporicidal time course. An 8-h experiment was done to analyze the time course of the
sporicidal activity of BCTP diluted 1 : 100! and BCTP 401 diluted 1 : 1000! against B. cereus.
Incubation of a 1 1 100 dilution of BCTP with B. cereus spores resulted in a 77% reduction in the number
of viable spores at 1 h and a 95% reduction after 4 h. Again, BCTP 401 diluted 1 : 1000 was more
etfective than BCTP diluted 1 : 100 and resulted in an "95% reduction in count in 30 min gure 3!. The
improvement in killing between BCTP 401 diluted 1 : 1000 and BCTP diluted 1 : 100 was statistically
signicant up to the 4-h time point P < .05!.
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_Hamouda et al., Sporicidal Activity of BCTP Nanoemulsion Page 6 of 12
-
Figure 3. Time course of nanoemulsion sporicidal activity against Bacillus cereus. Incubation
with BCTP diluted 1 : 100 resulted in 95% killing in 4 h. Incubation with BCTP 401 diluted l : 1000
resulted in 95% killing in only 30 min. Difference inkilling between BCTP diluted l : 100 and
BCTP 401 diluted 1 : 1000 up to 4-h point was signicant P < .05!.r
Sporicidal activity of BC]? against B. anthracis. After initial in vitro experiments, the sporicidal
activity of BCTP was tested against two virulent strains of B. anthracis Ames and Vollum 1B!. We
found that BCTP at a 1 : 100 nal dilution incorporated into growth medium completely inhibited the
growth of 1 >< 105 B. anthracis spores. Sporicidal assays in uid media, after 4 h of incubation with
BCTP at dilutions up to 1 : 1000 with either the Ames or the Vollum 1B spores, resulted in 91%
sporicidal activity when the mixtures were incubated at 22°C and 96% sporicidal activity when the
mixtures were incubated at 37°C L.
M 5 Table 1. Sporicidal activity of BCTP against 2 different strains of Bacillus anthracis spores
V '1 as determined by colony reduction assay % killing!.
A
B XL ,.
Sporicidal activity ofBCIP 401 against B. anthracis. Because BCTP 401 was effective at higher
dilutions and against more species of Bacillus spores than BCTP, it was tested against 4 different strains
of B. anthracis at dilutions of up to 1 : 10,000 at 22°C to prevent germination. BCTP 401 showed peak
sporicidal activity between ~1' : 1000 and ~1 : 5000 dilutions t_ae_2!. It was less efcient at
concentrations >1 : 100.
M he i * C Table 2. _ Sporicidal activity of BCTP 401 against 4 different strains of Bacillus anthracis
* . representing different clinical isolates. .
G = _.~..<
» !-
Electron microscopic examination of the spores. We used B. cereus because it is the most closely
related to B. anthracis. Transmission electron microscopic examination of B. cereus spores treated with
BCTP diluted 1 : 100 in TSB for 4 h revealed physical damage to the B. cereus spores, including
extensive disruption of the spore coat and cortex with distortion and loss of density in the core gure 4!.
Figure 4. Electron micrographs of Bacillus cereus spores before top! and after bottom! treatment
with BCTP. Note uniform density in cortex and well-defmed spore coat before treatment withBCTP.
Spores after 4h of BCTP treatment show disruption in both spore coat and cortex, with loss of core
components.
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Hamouda et a1., Sporicidal Activity of BCTP Nanoemulsion Page 7 of 12
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Germination stimulation and inhibition. To investigate the effect of initiation of germination on the
sporicidal effect of BCTP on Bacillus spores, the germination inhibitor, D-alanine [11, 1§_], and
germination enhancer, L-alanine [1_9, 20], were incubated with the spores and BCTP for up to 1 h.
Percentage of killing was calculated at different time points. The sporicidal effect of BCTP was delayed
in the presence of 10 mMD-alanine and accelerated i11 the presence of 5 mML-alanine gure 5!. All of
the individual time points showed a signicant difference in killing between the three treatments P
< .002!. -
' Figure 5. Effect of germination inhibition and stimulation on sporicidal activity of BCTP diluted
1 : 100 against Bacillus cereus spores. Sporicidal activity of BCTP was delayed in presence of 10
l t mM D-alanine germination inhibitor! and accelerated in presence of 5 mM L-alanine germination
E enhancer!. All time points show signicant difference between 3 treatments P < .002!.
i In vivo toxicity testing. CD-1 mice injected with BCTP diluted 1 1 l0 in saline did not exhibit signs
of distress or inammatory reaction, either grossly or histologically gge 6A, $52!. Identical results
were obtained when the toxicity of BCTP 401 was tested in mice subcutaneously. Intramuscular
injection of the BCTP or BCTP 401 diluted 1 : 10 did not have any toxic effects in the form of
inammatory reaction, edema, or necrosis in mice. Open wound irrigation with 2 n1L of the emulsions
did not result in any pathologic damage. Intranasal instillation of the emulsion was less tolerable because
of its viscosity; however, there was no injury from BCTP diluted 1 : 50 and BCTP 401 diluted 1 : 25.
Oral administration of 10% BCTP mL/kg of body weight daily! in rats for 1 week did not result in any
gross or pathologic changes, and the rats maintained normal weight gain during this period data not
shown!. In these tests, pathologicexamination of local tissues and internal organs was done, and no
abnormalities were detected.
Figure 6. Gross and histologic photographs of animals injected subcutaneously with different
combinations of BCTP and Bacillus cereus spores. A, B, animals injected with BCTP alone at
~~ <"»-n. dilution of 1 : 10. There was no gross tissue damage, and histology showed no inammation. C,
1 *0 0 D, animals injected with 4 >< 107 B. cereus spores alone subcutaneously. Large necrotic area ,
5* 1 resulted, with average area of 1.68 :!:O.35 cmz. Histologic examination of this area showed
la essentially complete tissue necrosis of epidermis and dermis, including subcutaneous fat and
r» Q 7:7-ii muscle. E, E mice injected with 4 X 107 Bacillus spores that had been immediately premixed
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Hamouda et al., Sporicidal Activity of BCTP Nanoemulsion ~ Page 8 of 12
withBCTP nanoemulsion at fmal dilution of 1 : 10. These animals showed minimal skin lesions,
with average area of 0-02 :1: 0.01 cmz "98% reduction from lesions rwulting from untreated
infection with spores; P < .002!. Histology of F indicated some inammation; however, most
cellular structures in epidermis and dermis were intact. All histopathology is shown at ><4
magnication.
In vivo sporicidal acvily. B. cereus infection in experimental animals had been previously used as
a model system for the study of anthrax and causes an illness similar to experimental anthrax [2, 2, 2_1_
-24]. Two animal models of cutaneous B. cereus disease were developed to assess the in vivo sporicidal
activity of BCTP. A suspension of 4 >< 107 B. cereus spores was mixed with saline or with BCTP at a
nal dilution of 1 : 10 and then immediately injected subcutaneously into the backs of CD-1 mice. Mice
that were infected subcutaneously with B. cereus spores without BCTP developed severe edema in 6-8
h. This was followed by a gray, necrotic area surrounding the injection site at 1824 h, with severe
sloughing of the skin present by 48 h, leaving a dry, red-colored lesion gure 6C, Q!. CD-1 mice
injected with B. cereus spores premixed with BCTP never developed such a necrotic lesion, and edema
and inammation were minimal gure 6E, Q!. The size of the necrotic lesion in BCTP-treated mice
was "98% smaller than the necrotic lesion size in untreated mice from 1.62:1: 0.35 cm2 to 0.02 :1: 0.01
cmz; P < .002!. Similar results were observed with BCTP 401 diluted 1 : 10.
In additional studies, a 1-cm skin wound was infected with 2.5 >< 107 B. cereus spores and then closed
gme 7A, E!. For some of the animals 1 h later, the wounds were irrigated with either BCTP diluted
1 : 10 or saline to simulate postexposure decontamination. Irrigation of experimentally infected wounds
with saline did not result in any apparent benet gure 7C, 112!. BCTP irrigation of wounds infected
withB. cereus spores showed substantial benet, resulting in a consistent 98% reduction in the lesion
size from 4.84 :1: 0.48 cmz to 0.06 :1: 0.03 cmz; P < .001; gure 7E, IL!. This reduction in lesion size
was accompanied by a 3-fold reduction in mortality 'om 60% to 20%! compared with that in
experimental animals receiving either no treatment or saline irrigation. Similar results were observed
with BCTP 401 diluted 1 : 10.
- , ~1,_,...a~-92»,j , _ _ , gure 7. Gross and histologic photographs of animals with experimental wounds infected
__ 3 -._- with Bacillus cereus spores. A, B, mice with experimental wounds infected with 2.5 >< 107 B.
""~'"~"~1 P _, ,_ ,, cereus spores but not treated. Histologic examination indicated extensive necrosis and marked ~ _ ~ - ~ inammatory response. C, D, mice with wounds that were infected with 2.5 >< 107B. cereus .
" spores and irrigated 1 h later with saline. By 48 h, large necrotic areas surrounded wounds, with
1 ii average area of 4.86 :1: 0.48 cmz. In addition, 60% of animals in this group died as result of1. .. ~ _r-~. _., . . . . . infection. Histologic examination of these lesions indicated total necrosis of dermis and
subdermis and large numbers of vegetative Bacillus organisms. E, F, mice with wounds infected
with 2.5 >< 107 B. cereus spores and irrigated 1 h later with 1 : l0 dilution of BCTP. There were
small areas of necrosis adjacent to wounds .06 =1: 0.03 cmz!, 98% reduction compared with
animalsreceiving spores and saline irrigation P < .001!. In addition, only 20% of animals died
from these wounds. Histologic examination of these lesions showed no evidence of vegetative
Bacillus organisms and minimal disruption of epidermis. All histopathology is shown at X4
magnication.
http://www.joumals.uchicago.edu/JID/joumal/issues/v180n6/990281/990281.text.html 2/ 1 8/2005
Hamouda et al., Sporicidal Activig of BCTP Nanoemulsion Page 9 of 12
Discussion
In these studies, we demonstrated that BCTP and its derivative BCTP 401 have effective sporicidal
activity against a variety of Bacillus spores, including B. anthracis. BC'IP diluted 1 : 100 has a
sporicidal activity against B. cereus, B. circulans, and B. megaterium, whereas 1 : 1000 is effective
against B. anthracis in 4 h. BCTP 401, aBCTP-P10 mixture, appears to have a more rapid and broader
sporicidal activity than BCTP. BCTP 401 diluted 1 : 1000 killed 95% of B. cereus spores in 30 min at
37°C, compared with a 70% reduction achieved by BCTP diluted 1 : 100. BCTP 401 diluted 1 : 1000
was also effective in 4 h against B. sublis spores that were resistant to BCTP for up to 24 h. BCTP 401
did not show effective sporicidal activity against B. anthracis at dilutions of <1 : 100, contrary to the
original BCTP, which showed killing at dilutions between 1 : 10 and 1 : 1000. The fact that BCTP 401
requires dilution to be etfective againstB. anthracis spores suggests that BCTP 401 needs dispersion by
water to minimize its aggregation and to facilitate direct contact with spores.
Comparison of the sporicidal activity of BCTP against B. anthracis at 22°C, a temperature that does
not promote spore germination, and at 37°C, at which germination occurs as conrmed by microscopic
examination!, indicates that complete spore germination i.e., outgrowth! is not necessary for the
bactericidal activity of the emulsion. The small difference observed between the sporicidal activity at 37°
C and 22°C may represent the killing of additional organisms from a few germinating spores. Sporicidal
activity was also conrmed in water, a condition unsuitable for B. anthracis spore germination data not
shown!. The sporicidal effect seems to start almost immediately and occurs within 30 min of incubation
with the emulsion. Factors facilitating germination resulted in acceleration of the sporicidal activity of
BCTP. Inhibition of the initiation of germination with D-alanine delayed BCTP's sporicidal activity. On
the basis of these observations, we hypothesize that the sporicidal action of these emulsions occurs
through initiation of germination before complete reversion to the vegetative form, leaving the spore
susceptible to disruption by the emulsion. The initiation of germination could bemediated by the action
of the emulsion or its components, but the emulsion appears necessary, as spores do not initiate
germination in its absence. The results of the electron microscopy studies show disruption of the spore
coat and cortex with disintegration of the core contents after BCTP treatment. However, the exact
mechanism of killing is unclear and requires future investigation. Sporicidal activity appears to be
mediated by both the Triton X-100 and tri-n-butyl phosphate components, because nanoemulsions
lacking either component are inactive in vitro data not shown!. This unique sporicidal action of the
emulsions, which is similar in efficiency to that of 1% bleach, is interesting because Bacillus spores are
generally resistant to most disinfectants, including many commonly used detergents [Q].
Animal studies demonstrated the protective and therapeutic eect of BCTP in vivo. B. cereus
infection in experimental animals has been used previously as a model system for the study of anthrax
[21, Q, l5_]. The disease induced in animals experimentally infected with B. cereus is in many respects
similar to anthrax [2, 23]. In this study, we demonstrated that mixing BCTP with B. cereus spores before
injecting the spores into mice prevented the pathologic eect of B. cereus. We also demonstrated that
BCTP treatment of simulated wounds contaminated with B. cereus spores markedly reduced the risk of
infection and mortality in mice. Because the emulsion appeared to lose sporicidal activity when diluted
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Hamouda et al., Spoiicidal Activity of BCTP Nanoemulsion if Page 10 of 12
0
92
past 1 : 100, higher concentrations of the emulsions : 10! were used for the in vivo studies to make
sure they remained efective after dilution with body uids. Other experiments show that testing BCTP
401 in mice under similar conditions demonstrated similar effects. These results suggest that
decontamination of spores prior to or after exposure can effectively reduce the morbidity and mortality
from B. cereus infection. This appeared to be a valuable application, because unlike other sporicidal
agents, BCTP or BCTP 401 did not demonstrate any toxic effects, grossly or by histopathologic
examination of the mice [2_§]. Other tests in mice showed that these emulsions are nontoxic if
administered intrarnuscularly, intranasally, or orally, providing other potential sites for treatment.
BCTP and its derivative BCTP 401 appear to have great potential as enviromnental decontamination
agents or for treatment of exposed persons in either a military operation or a terrorist attack. The
inactivation of a broad range of pathogens, including vegetative bacteria, enveloped viruses [21]
unpublished data!, and bacterial spores, combined with low toxicity in experimental animals, seems to
make it suitable for use as a general decontamination agent that can be deployed even before a specic
pathogen is identied. The nanoemulsions can be rapidly produced in large quantities and are stable for
many months unless frozen, which causes separation of the oil and lipid phases. Undiluted, they have the
texture of a semisolid cream and can be applied topically by hand or mixed with water. Diluted, they
have a consistency and appearance similar to skim milk and can be sprayed to decontaminate surfaces or
potentially interact with aerosolized spores before inhalation. These properties provide a flexibility that
will be useful for a broad range of decontamination applications. Further studies are warranted to
determine the exact mechanism of the sporicidal effect of BCTP and its derivatives, and this may lead to
further improvement in formulations.
Acknowledgments
We thank Shaun B_. Jones, Jane Alexander, and Lawrence DuBoise Defense Science Office, Defense
Advanced Research Project Agency! for their support; Bruce Ivins, Patricia Fellows, Mara Linscott,
ArthurFriedlander, and the staff of USAMRHD for their technical support and helpful suggestions in the
performance of the initial anthrax studies; Martin Hugh-J ones, Kimothy Smith, and Pamala Coker for
supplying the characterized B. anthracis strains and the space at Louisiana State University Baton
Rouge!; Robin Kunkel Department of Pathology, University of Michigan! for her help with electron
microscopy preparations; and G. Morris and A Shih for their technical assistance with manuscript
preparation.
References ,'
1. Dragon DC, Rennie RP." The ecology of anthrax spores: tough but not invincible. Can Vet I 1995;
36:295-301. First citation in article I PubMed
2. Welkos SL, Keener Tl, Gibbs PH. Differences in susceptibility of inbred mice to Bacillus anthracis.
Infect Immun 1986; 511795-800. First citation in article I PubMed .
3. Franz DR, Jahrling PB, Friedlander AM, et al. Clinical recognition and management of patientsexposed to biological warfare agents. IAMA 1997; 278399-411. First citation in article I PubMed
4. Pile JC, Malone JD, Eitzen EM, Friedlander AM. Anthrax as a potential biological weapon. Arch
Intern Med 1998; l58:42934. First citation in article I PubMed
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Ivins B, Fellows P, Pitt L, et al. Experimental anthrax vaccines: efficacy of adjuvants combined with
protective antigen against an aerosol Bacillus anthracis spore challenge in guinea pigs. Vaccine
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Mobley IA. Biological Warfare in the twentieth century: lessons from the past, challenges for the
future. Mil Med 1995; l60:54753. First citation in article I PubMed
Meselson M, Guillemin J, Hugh-Jones M, et al. The Sverdlovsk anthrax outbreak of 1979. Science
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Jackson PI, Hugh-J ones ME, Adair DM, et al. PCR analysis of tissue samples from the 197 9
Sverdlovsk anthrax victims: the presence of multiple Bacillus anthracis strains in different victims.
Proc Natl Acad Sci USA 1998; 9511224-9. First citation in article I PubMed
Drobniewski FA. Bacillus cereus and related species. Clin Microbiol Rev 1993; 6:324-38. First
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Alasri A, Valverde M, Roques C, Michel G. Sporicidal properties of peracetic acid and hydrogenperoxide, alone and in combination, in comparison with chlorine and formaldehyde for ultraltration
membrane disinfection. Can I Microbiol 1993; 39:52-60. First citation in article, I PubMed
Beauchamp R0 J r, St Clair MB, Fennell TR, Clarke DO, Morgan KT. A critical review of thetoxicology of glutaraldehyde. Crit Rev Toxicol 1992; 22: 143-74., First citation in article I PubMed
Hess JA, Molinari IA, Gleason MI, Radecki C. Epidermal toxicity of disinfectants. Am J Dent 1991;
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Lineaweaver W, Howard R, Soucy D, et al. Topical antimicrobial toxicity. Arch Surg 1985;
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Morgan KT. A brief review of formaldehyde carcinogenesis in relation to rat nasal pathology and _
human health risk assessment. Toxicol Pathol 1997; 25:291-307. First citation in article I PubMed
Russell AD. Bacterial spores and chemical sporicidal agents. Clin Microbiol Rev 1990; 3 :991 19.,
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Wright DC. Antimicrobial oil-in-water emulsions. US patent no. 5,547,677; 1996. First citation in
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Titball RW, Manchee RI. Factors affecting the germination of spores of Bacillus anthracis. I Appl
Bactetiol 1987; 62:269-73. First citation in article I PubMed
.,Foster SJ, Johnstone K. Pulling the trigger: the mechanism of bacterial spore germination. MolMicrobiol 1990; 4:137-41. First citation in article I PubMed
Shibata H, Takamatsu H, Tani I. Germination of inactivated spores of Bacillus cereus T. Effect of
preincubation with L-alanine or inosine on the subsequent germination. Jpn I Microbiol 1976;
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Preston RA. Douthit HA. Functional relationships between L- and D-alanine and NH4Cl during
germination of spores of Bacillus cereus T. J Gen Microbiol 1988; 134:3001-10. First citation in _
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Burdon KL, Wende RD. On the differentiation of anthrax bacilli from Bacillus cereus. I Infect Dis
1960; 1071224-34. First citation in article
Burdon KL, Davis J S, Wende RD. Experimental infection of mice with Bacillus cereus: studies ofpathogenesis and pathologic changes. J Infect Dis 1967; 1171307-16. First citation in article I
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Fritz DL, Jaax NK, Lawrence WB, et al. Pathology of experimental anthrax in the rhesus monkey.
Lab Invest 1995; 73:691-702. First citation in article, I PubMed
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> a
24. Welkos SL, Friedlander AM. Pathogenesis and genetic control of resistance to the Sterne strain of
Bacillus anthracis. Microb Pathog 1988; 4:53-69. First citation in article I PubMed
25. Lamanna C, Jones L. Lethality for mice of vegetative and spore forms of Bacillus cereus and
Bacillus cereuslike insect pathogens injected intraperitoneally and subcutaneously. I Bacteriol
1963; 85:5325. First citation in article -
26. Cao Z, TondaR, Morris G, Hamouda T, Johnson K, Baker JR Jr. Sporicidal effect of novel
surfactant lipid on B. cereus [abstract C-300]. In: Proceedings of the 99th general meeting of the
American Society for Microbiology Chicago!. Washington, DC: ASM, 1999. First citation in article
27. Chatlyyne LG, Brisker J, Wright DC. A lipid nanoemulsion with eective viricidal activity against
HIV -1 and other common viruses [abstract 351]. In: 3rd Conference on Retroviruses and
Opportunistic Infections: program and abstracts Washington, DC!. Alexandria, VA: Infectious
Diseases Society of America, 1996:118. First citation in article
I
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F N
| .;.in p
FD-302 Rev. 10-6-95!
-1-
~ FEDERAL BUREAU OF INVESTIGATION
ALL IIJFUFLI-LELTICIN I:|:lI92TT§kI]*IED _
HEIREII-I I3 TIJIILAESIFIED
DATE 1.2-l2~2ElElS EFF IE~I35EE-l UC BMI.fI*E¬_#PFf3
GRAND JURY MATERIAL DISSEMITATE PURSUANT TO RULE 6!
_ On March 19, 2o03,| I bornl IDate of transcription Q 3 Z 2 2 Z 2 Q 0 3
I Iplace of employment, the Unite States Army Medical Research with Social Security Account Number! lwas interviewed at
Institute of Infectious Diseases U AMRIID!, Fort Detrick,
and Special Agents SAs!| Iandl
all of the Federal Bureau of Investigation FBI!.
provided the following information:Mar land. The interview was conducted by Inspector in Charge IIC!
/ / / //61? 1/_ ;=
Ek# 279AWF-222936USAMRIID 279A/WF222936-LawdmwSA ' In I SE7by SA I
This document contains neither recommendations nor conclusions of the FBI. It is the property of the FBI and is loaned to your agency;b2b3
1:6
1376
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107E
hwmemwon O3/19/2003 at Fort Detrick, Marvland W//95¢7c
I 1V-A , .K: 1 U r-
FD 302 Rev. 10-6-95!
_ I -
FEDERAL BUREAU OF INVESTIGATION
ALL II~IFU13J{i1,TItIlI~I EIJLITAIIJED
HEREII-I IS TJIJCLASEIFIED
DATE l2~lE2ClCl3 Ff E6324 UC BAUI.»*DH":I OnI bornI I I
with social security account nube home address._-w- __ _ 4F! home telephoneI35
Dmedummmm 0242442005 b7C
According to the polygrapher t gm
. . I e<f*/I The interview was conducted by US Posta
Inspector PI! and Supervisory Special Agent I
SSA! of the Federal Bureau of InvestigationTnmtm_ I cellular phdQeI Iwas L_Jinterviewed-after completing a voluntary ' 'at the Frederick, Maryland offsite of the #$%F%
FBI!. Present for a ortion of the interview was FBI Special "
I I ' ' d f h ' Agent SA! After being advise o _t e
identities of the interviewing agents and the purpose of theinterviewI::::Iprovided the following information: '
I Iis currently employed asI I
in theI Iat the United States
Army Medical Research Institute of Infectious Diseases' | USAMRIIDL, BiidinqI I Fort Detrick, Maryland, phoTe number
was
formerly known as theI I
supervisor is
I phone number
currently shares an office with
92[:::] beganI I employment at USAMR ID inIshortly after earning degree inI Iat theUniversity ofI Ibackground is limited to
has never streaked an a ar late with bacteria at9 p
USAMRIID. _
d inI Isuites during themonths of employment at USAMRIID. [;;::]primaril worked on pro ects with and recalls wor ing insuite[::E]of Building gained access to thecontainment suites in Iafter[::] immunizations
were complete. .
has worked in rooms I I andI A ofBuilding has been in a walkin cooler located on he
Investigation on 02/15-17/2005M FIECIGIICK, Maryland
Fh# 279AWF222936USAMRIID-92m§g Daemmm N/A
I I Po tal Inspector ~bY- SSAI
This document contains neither recommendations nor conclusions of the FBI. It is the property of the FBI and is loaned to your agency;
it and its contents are not to be distributed outside your agency.06
own
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in this wa in coo er in 2001 or 2002.
recalls the walkin cooler was lined with shelves full o racks
of samples from the aerosol challenge laboratories.
[:::]has also worked inEff%QQQgfjite of Building[:::::][::]first entered this suite with to ain experienceworking in a containment suit. assisted[i¬:]withHgggg::::]related toI aerosol challenges. hasalso worked inI Ioutside of the containment suit at the
hoodline in the "gray area" of the ' recalled
thad once ot temporary access from to an airlock
in Building o ick u fro
has use
I One of
hel ed w' ' 'I p I I ith this in aI I abora oryin BuildingI I. The materia resulting from e purification
attempt was returned to does not have access toSuitesE:] in Buildin I Ib ma have been in the I <»Q- I-g w=|:|_y _-non~containmen "cold"! areas of those suites to pick up
material. '
The only p recalled seeing Ba in an oddlocation was in Room of BuildingE::::] This is a lab where
I I and gwere working on a Johns Hopkins
roiect<invol§inq the use of aerosol samples of Ba in a study of»[i I The labeled tubes ofa samples were stored in a refrigerator w ' ' '
glass door where[:::Ihad intended to place
I Iworked on a number of projects at USAMRIID. One
project involvedI Iat
USAMRIID. I Icontrolled aerosol system. I I
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I Iwas also involved in a studv to determine the
this research usingIThis research was funded b |InI IaskedI Ito continue
1| H
I Iworked on another project associated with [:;:::::]I The qoa was
toI I
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Continuation ofFD-302 of _ , on 02 / 1 5 / 2 0 O 5 , Page 4
' recalled thad Ionce attended a classified meeting
withI a Iand
also attended the meeting.
I Iand received Ba from DUGWAY
PROVING GROUND DUGWAY!. DUGWAY p pared the Ba from stock
that was provided to them by BRUCE IVINS. IVINS is a[::::::::::][::::] and is an "odd character". '
t on 9/ll/200l,[:£2%¬§]in t
unit in Building when
observed th ' t plane strike the World Trade Center ontelevision.E:fiffwas atI I
thought initially that a light plane had struck the World Trade
Center left unit and be an work in theof Bui assistingin the room when thev were interfupted by[;:]I em to warn t
to leave the suite immediately because the World Trade Center
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was attacked and a plane was headed for Fort Detrick. I I5 b6
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I I I I Iwas initially alarmed_by tis violation of
safetv nrotocoll |J.a_te.r learnsrl thai
[::::] S8M!E::::::;;:::::] dressed in black in the hallwaynear the cage washing mac ine. ther occasion[::]saw anindivtdnaj whom| Ithonght was in a containment suit in
suite conducting research or may
also hav in the sui e. ometime after eUSAMRIIDj:?fffobserved[:::]loadin old hoodlines into a truckfrom an area outside Build1ng[:::i that was near the
smokestacks.[:::]could not recall the color of the truck nor any
other'details.
[::::]related thatE::::lhas put forth theories to[;;:::]- regarding the mailing of t e an hraxlaced letters. E:::] c aims
USAMRIID has nothing to do with it and that a foreign government
or an intelli ence communit misha is res onsible for themailings. E::%] referred toI I as I I
Referring to the investigationl Itold IthatI I is a nice guy and thatI I
together.
I IwasI IraterI I
WLI Iwho periodicallcontacts fo advice and assistance inI Istudies. [::::::ijb6
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hasI Iexerience and is interested in ¬oining[::::]visited atI I but has not
visited Approximatel I years agod
USAMRIID eople have voiced concerns to
about hasI Iexperience.
" [:::;:kas shown a prestamped Federal Eagle envelope
similar to t ose used in the mailing of the anthraxlaced
letters in 2001.[::] has never used this type of envelope and
has never seen one like it at USAMRIID.
I Iresides inI
~ 92
InI Irode on a train through New Jersey to
InI I attended a conference at[:::::::1_____1
The conference was sponsored by the Center of Biomaterials.
[:::] was less than impressed with the security at
USAMRIID when[:] arrived there. was very concerned about
safet recalled that once trans orted Y-|:| , P
plague bacteria between buildings by the "VIP" method, which
means "vial in pocket", instead of the proper packaging and
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7 Continuation of FD-302 of _ , On O 2 / 1 5 / 2 O O 5 , Page
transport procedures. never observed piggybacking in the
hot side. Howeve has observed piggybacking in other areas
and recalled that recently piggybacked behind[:::]through
the front door.
The investiga ' f the anthrax-laced letter mailin sis a sensitive issue toI IbutF_1 is not sure whv.
panicked feeling about the perception
associated with the facility or person responsible for themailings.E::]was very concerned and] ' I
I referred to the investi ative and QLand the ' ' s had on| |
believes and does not want the
same thing to happen to was not a
During the polygraphj Ibecame aware ofI I
| Iabsolutely had no
involvement in Ehe mailings.
At this point in the interview, SSAI I toldI I
I Ithereafter
provided the following information:
/
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| knows
that there are lyophilizers in cold areas of Building atUSAMRIID, but[::]has never used a lyophilizer at USAM D.b6
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read in the media that the mailer of the anthrax
I After was initiall
interviewed by aqentsI IcontactedI in
I I and advisedI Ithat aoents mav call Ito veriff information aboutI I
Sometime before 200lJ IcontactedI Ireqardin aproject involvinqj 1
r
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During the interv' was told[:::Las free to leave
the interview at any time. did so to use the room.[::::]was offered and accepted bottled water to drink.[fffTwas told _that[:] could take eak f ch or bring in a lunch[::]hadbrought witd Iid Icar.I Ielected to continue the
interview I
Ill
SSAI Iexplained the procedure of attempting to
determine whereabouts and activities during the windows of
opportunit for the mailings of the anthraxlaced letters in2001. E:::fdescribed numerous types of documentation that[::¥;
might want to search for to help the investigators.E:::]agree
to provide archived emails from[:::]home and office computers,
calendars, credit card records and phone bills, travel and leave
records, laboratory notebooks, and any other documents that
could help determine[:::]whereabouts.
OnI Iwas recontacted by PIwho requested thatI rIcoTe back into the office for a "
On
brought with| Iseveral of the requested items to help with| I
timeline of activities during the windows of opportunity for
the mailings. I ISSH Iand PII Italked with| I to determine if any new ideas had
occurred t other than the three issues disclosed onEl c Iprovided the following comments regarding the previously-
discussed issues:
[::::] concerns about the dried Bg work were not
necessarily ethical concerns
I The International
Convention says that as long as the work is on pathogenesis or
vaccine efficacy, the work is not considered offensive in '
nature.
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[::::]corrected comment thad::] had onlbeen to New York City on one occasion. ¬
I I has theI Ifor this travel
and will provide it to the investigators. '
Another issue that may have been worrying is that
IH Iwas told Eha Iwas free to leave, II
I _ _ _ __ _ provided thefollowing in response to questions asked by SSAI I
has not trained any other individgals tp usee ui men or proceduresE::Ihas used toI I I eThe onlv tra1n1nqr__1gave was Qn the] tj
! .
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There was a big push to do studies in thel I
after the mailings occurred. | |was doing
work ror | and[:] is not sure if the work was
classified or not.
I |visited| |in| to give a
talk. I was there.| éwasl saidL__jhad called theFBI numerous imes about_Ehe use o i |[:::::] in the mailed anthrax, but the FBI would not listen.
O
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Continuation of FD-302 of
l:::;] ' ost any f r i n visiting scientists at
USAMRII . n t t t t' f th wen o o a mee ing o e
with[::::] There was aI group o |Scientists from] at the meeting. The
After thinkin about it more,E:::]cannot clearly recall k,w| IQ h k h h dl' '£ ° ifl | reallv sa t t e truc wit t e oo ines or 1 STAq remembers seeintwo times in the hallwa in suit E:::] and[:::isocialized with[:::::::1 but[::E]did not. went to the
| USAMRIID librarv to check out a book called|
_ I It is a "really neat" book.|
name was on the library checkout sheet, and] |had checked out
the bo
aerobi
notebo
The mi
ii
the nook prior to the mailings. The book is specific to
ology.
got really upset when| |found that a laboratoryok was ost.k< l b6ssing n is Ehe one which had ¬he| I b7C
n it. was the technician who ma e en ries in .
tebook,|does not know what happened to the notebook.
SSA askedl Iabout the requisiti?n_£grm[::%]_provided for I |had worked with. t e
In regard to the original purpose of the [:::::::::::::]Iparticipated in, the anthrax spores in liquid form which
were being used in aerosol challenges wereI I|had wanted to
92
92 - be
27 9AWF222936USAMRIIDi ' O Q
FD-3'02a Rev.10-6-95!
Continuation ofFD-302 of _ ,0 02 /15/2005 ,Page 14
move toward using dried anthrax in the challenges. I I
g:::::;]could not think of any other issues- otentially
relate to t e word "anthrax" which might bother| hnd might
have contributed to| Iwants to help
the investigators| land will do
whatever is needed.| |agreed to provide additional
_ whereabouts at the time of the mailings. agreed to come backinformation and documentation regarding[EEEg?ctivities and
to the offsite for formal handwriting exemp ars.
:< ~, -t' b 6Foébiemiosss
-1-
FEDERAL BUREAU OF INVESTIGATION 106
ALL Inrnnnarrnn conrarnat < blc
HREIH I3 UHELA53IFIED '
vita ta-iz-zuqa at auaza um aimsuayars
Date of transcription Q 3 Z O 1 Z 2 O ! 5
i p MM __q¢myH _ A home address|| 7 elephone number]social securtv number date of birth] L1
s| work tele hone number] Iwas interviewed - P .
at the Federal Bureau of Investigation's FBI! offsite location in
Fr d ' k M r l d. Aft b ' d' d f Ch 'd t't f th e eric , a y an er eing a vise o e 1 en 1 o e
interviewing agents and the purpose of the interview, L
provided the following information: I
" has conducted work for BRUC¥;ELQ§ on| I
at USAMRIID. | B | |
Investigation on O2 /2 5/2 O O 5 at FI¬d¬IiCk , Maryland
File # 2 '79IAWF22293 6USAl/[RI ID 92?QQ Date dictated N/A
SA
by Postal Inspectorl I k
This document contains neither recommendations nor conclusions of the FBI. It is the property of the FBI and is loaned to your agency;
it _nnd its contents are not to be distributed outside your agency. '
x ~ -
W » O O
FD-302:1 Rev. 10-6-95!
279AWF222936b6
b7C
[:::::::]did not recall workinq with any specific Ames
or
| IVINS or i
would be able to provide more information about Ames work
and storage locations at USAMRIID.samples at USAMRIID inContinuation of FD-302 of ' I , On O 2 / 2 5 / 2 O O 5 , Page 2
I Ihad no recollection of any plate count comparison
study with IVINS or Ifor BioPort or B
[::::::::] had never applied for funding from Princeton
University, nor did[:] know anyone who attended or was employed
there.
>-- vP » ~ Q O
FD-302a zev. 1046-95!
279AWF-222936
Continuation of FD-302 of I , On O 2 / 2 5 / 2 0 O 5 , Page1:5-
137C
_°>_
There was no one at USAMRIID nvhom|:|suspected of
being involved in the 2001 anthraxla.ced letter mailings.
FD-302 Rev. 10-6-95!
..]_.. 1
FEDERAL BUREAU OF INVESTIGATION
ALL INFDRIILETIIJN CDIJTAII-i'ED
I-IEIREIN IS TIIJIILELSSIFIEIJ
DATE l.Z.I.22EiEiB BY 60324 TIC BMJ.-*"DE{fP.f5,so
137C
x Date of transcription O3 [ 1 5 Z 2 O O 5
ch 14, 2005,I I date of birth
'a Y IJLQEinterviewed at residence, I_ce_]I1ular e noneI Ijpwefg IbY g_ I an Also resent dur ' n? theb1Ete r'v':?ewt7~r 29.s=IiIof theI-p IResident Agency
RA! I .provided assistance with theI Iof I Icomputer hard drive. After being a vise o the identities of
the interviewing Agents and the purpose of the interview;
provided the following information:
At the outset of the interview signed and
dated a FD94l, Consent To Search Computer s! form. A copy of
the consent form is attached and the ori inal is maintained inthe 1A section of the case file. informed the Agents
thatI:I used "password" to access the Windows 98 operating
system onI:I computer. .
Investigation on 0 3 / 1 4 / 2 O O 5
File # 2 7 9AWF2 2 2 93 6 USAMRIID _ . Date dictated
SAby SA I
This document contains neither recommendations nor conclusions ot the FBI. it is the property ot the FBI and is loaned to your agency;
it and its contents are not to be distributed outside your agency.
FD-W -'26-51! t itLL ILIFUFLI-UiTIUIl EEII-lTJiIl~1ED
EEEFEIIII IS TJIJCLASSIFIED $
III-t'I'E 1.2-12-BEIGE BY 50324 UC B.t1&.IiT_rDIZ;P."f5
CONSENT TO SEARCH COMPUTER S! 5%
1,_l , have been asked by Special Agents of the
Federal Bureau of Investigation FBI! to permit a complete search by the FBI or its designees of any and all computers;
any electronic and/or optical data storage and/or retrieval system or medium, and any related computer peripherals,
described below:
Storage o{ Retrieval Media, Computer Peripherals92 LPU lVlaEe Model SE Senal'Number Tl ava1la5le!
and located atl , whichl own, possess,
control, and/or have access to, for any evidence of a crime or other violation of the law. The required passwords, logins,
and/or specic directions for computer entry are as follows: /1///4
AI have been advised of miiigo ret'f1se F61'c5h§¬nt't'othisisearch, and I give perniissioiifor tE;searcli,ireely
and voluntarily, and not as the result of threats or promises of any kind.
I authorize those Agents to take any evidence discovered during this search, together with the medium in/on which
it is stored, and any associated data, hardware, software and computer peripherals.
D te
DaZ?
ESignature
;
Signature of Witness /
| I
Printed Full Name of Witness
r
Location
,5 -
-I 0 E
FD-302 Rev. 10-6-95!
-1-
bf
FEDERAL BUREAU OF INVESTIGATION bvc
ALI. IIIFEIII-711-LLTICIK CUNTAIIITED
PIEPEIII IE1 TIIJIILETLSSIFIEID'0
D|!i.TE _2l22E92C92i: BI SO32-11 UII BJ1J.~I,.~"I1K,"P.fS Date of tmnscpon O3 :02 C2005
On February 2 nd March 1 2005 [::::::] I II I date of birthI I, Social Security AccountNumberl I If I was interviewed atI Iresidence,
I I cellulartelephone I I b Special Agents SAs!Ia IandI Y1. After beingadvise of the identities 0 the interviewing gents and the
purpose of the interview,[:::::::] provided the following
information:
in obtaining a position with the United States
Army Medica ' e of Infectious Diseases
.
92USAMRIID! I
characterized the working
environment as friendly and professional.I I
I IVINS exhibited no inappropriate
behavior at Ehe time. He was very concerned about crossin a
boundary between professional and personal life.
investigation on O 2 / 2 8 / 2 O O 5 at I I
File # 27 9A-WF-2 22 9 3 6 -USADIRIID - 92 Date dictated
SA
W SA
This document contains neither recommendations nor conclusions of the FBI. it is the propeny of the FBI and is loaned to your agency;
it and its contents are not to be distributed outside your agency.
I
M 6 E
_FD-302a Rev. 10-6-95!
279AWF222936USAMRIID
Continuation of FD-302 of ___| I , On 0 2 / 2 8 / 2 O O 5 , Page
The overall work environment in the[:::::::::::]
Division was friendly. U rsonnel were both friends andco-workers. According toEf%E?%ifja group of scientists, by
definition, was an unusual grou of people, thus personalityconflicts were not unexpected. F H
IVINS was friendly, but also conducted himself in a manner that
made it clear thaq | IVINS was
very moody, but coufd not be faulted tor his behavior, as he
"always had a hear Qf gold," Accord to[:::::::] IVINSalways meant well. icould not determine the
cause of IVINS mood swings.
I Ihe did not have many friends.| |felt bad
that IVINS lacked friendsL]
| Irecalled discussions with IVINS
regarding] |during| lat
USAMRIID.
' I Iemployment at USAMRIID ended inl I
of
c .
~
' FD-302a Rev. 10-6-95!
2'79AWF2Z293 6USAMRIID
Continuation of FD-302 of __| I , On Q2 / 2 8 / 2 O O 5 , Pageb7 tb 6
3
I IVINS was always a little unusual,
IVINS discussed his own personal issues[:::::::::::][::::::::] He discussed the difficult relationship between
himself and his wife, and difficulties with his teenage
children. He expressed insecurities about being without friends
and being perceived as strange or weird. IVINS indicated that
he started to see a counselor. Issues of depression, rampant
insecurities, history of s chiatric illness in his family werealso discussed by IVINS[:¬:%:::::::::::] According to[::::::::]
IVINS was insecure personally, not professionally.
IVINS informed[:::::::] that he was labeled with
bipolar and schizophrenic disorders: however, the disorder
labels did not fit withl |so| Idisregarded them. | Iwas not clear whether the labelswere assi ned by IVINS or is doctors[:£EEEEEE%:]did not recallif was aware of these l . labe s thought that
IVINS had a personality disorder which did n oss into "frankpsychosis." Nothing in IVINS behavior madeEi:ffworry about the
technical aspects of his job performance. His condition
affected his social interactions; however, his disorder did not
prevent his functioning at work, nor his community involvement
.and work with children. IVINS was too critical of himself. He
analyzed every word said to him and all of the possible meanings
implied by statements directed toward him.
IVINS discussed his family background with[::::::::]on
He grew up in Lebanon, Ohio, where his father was a
. id not believe that IVINS mother workedthe home.i |
took trips out West to visit National Parks. Soccasion.
pharmacist
outside of
The family
visited anu
aunt] |
did not recall whether the visits were family outings. IVINS
never referenced any traumatic occurrences in his life. He
~ ¬ &
FD-302a Rev. 10-6-95!
b6
b7C
279A-WF2Z293 6USAMRIID
Continuation of FD-302 of J I , On O 2 / 2 8 / 2 O O 5 , Page 4
indicated that he had few friends in high school and college.
He mentioned that he was very awkward around women and was never
"super popular." IVINS had been married only one time. IVINS
attributed his mental issues to the way in which he was raised.
I would sometimes worry about IVINS mood,
> was seeing a counselor to discuss his issues.
indicated that IVINS continued to have both go nd baddays, but that the counseling did quite a bit of good.T%:%::::::]
did not know the duration or extent of IVINS counseling.
| IVINSbehavior was hard to describe to someone who did not know him, |
IIVINS did not like to
talk on the telephone.|
~
FD-3029. Rev. 10-6-95!
b6
b7C
279AWF222936USAMRIID
Continuation ofFD-302 of _l I , On O 2 / 2 8 / 2 O 0 5 , Page 5
Idid not recall specificroblems or pressures associated with thei Elprogram.indicated that tension was usual y grea er| I
around the time of an aerosol challenge, as there was
' a signi icant amount of work to accomplish within a set time
frame.
regardin contamination issues outside of the USAMRIID hotsuites. F |"this is what is
going on at work" type of information.| Irecalled concern
over contamination in office areas, but did not recollectspecific details. [::::::;;]xmas under the impression that the
whole department was invo ved in the swabbing of all of the
office spaces. id not remember what prompted the extensive
. ' nor did recall the time frame of the event.
|believed that occasional swabbing outside of the hot
suites was conducted as a quality control; however,[::] neverobserved such swabbing during[:::]tenure at USAMRIID.
5/
FD-302a Rev. 10-6-95!
2'79AWF22293 6USAMRIID
Continuation of FD-302 of __| I , On O 2 / 2 8 / 2 0 O 5 , Page1?
b7!
_8_
verbally consented to a review of any items by the interviewing
Agents during a return visit.
During the interview,[::::::;Jread, signed and dated
an Non-Disclosure Agreement regarding t e Amerithrax .
investigation. The form is maintained in a 1A envelope.
92
92
rx1i -4 !.
at
FD-302 Rev.me@$ I
-1- b6
b7C
FEDERAL BUREAU OF INVESTIGATION
ALL II1IFEl1l'Q|f'LkTIE92I1Z CEIIIITAII-]'EIIHEREIN IS UNCLASSIFIED I D f _ _DATE 12-12-zone BY 50324 UE BAUEDKERYS °° "m m"° O2 22 2 5
92
_ _ "Date Qir ' 'Social Secur' nt Nu er_4SSA§:j£b_ work telephone
: , was inte viewed, as pre~arranged, at| Elace
o employment, room :_ orter Street, the UnitedistatesArmy Medical Research ' ' ' es USAMRIID!,
Fort Detrick, Maryland on the a ternoon of February 17, 2005.
b ' advised of the identlt of the interviewing PostalKfter eingInspector and Special Agent SA!,[E::::]provided the following
information: ~
provided the interviewing Postal Inspector and SA a
' e had received on| I at agproximatelv
from] Ireqardinql |
WFO NOTE: Enclosed in the FD34O attached to this communication is
the above captioned facsimile.!
.Through investigation[::::::]is described as: 6Ei%?
:
FIRST NAME;
ALIAS :
rt |
DATE OF VISIT:"§
R :
SUPERVISOR :_____....
Investigation on O 2 / 1 7 / 2 0 O 5 at FIECIEII Ck , Maryland
w# 279AWF-222936U - Dmdkmmd N/A
Posta Ins ector
t contains neither recommendations nor conclusions of the FBI. It is the property of the FBI and is loaned to your agency; This documen
it and its contents are not to be distributed outside your agency.
-?92.9 /-5,
Q G
FD-302a Rev. 10-6-95!
2'79AWF222936USAMRIID b6
1976
__2._ Continuation 0fFD-302 of _ , On O 2 / 1 '7 /2 O O 5 , Page
MISCELLANEQIIS: Accessg Ugh/IRIID Suite
with an worked
_with Amesw train of Ba.
0 0
v FD-302 Rev. 10-6-95!
-1-V
FEDERAL BUREAU OF INVESTIGATION
b aALL IEFURI-IELTIUBI CI:lI~TTAII*EI'J bgw
HERE IIII In TJIJ| L§a5IFIED v
DATE 1.2-12-zone Ff @0224 TJC B.MI_.*[3H;'P. Date of mnscption O2 [1 8 [2 0 0 5
|:| Date of Birth <1>oB>= |:|
Social Securit Account Number SSAN!:[:::::::::::] work telephone# I | ' t ' a - d 1;|:l 1 = was in erviewe , as pre arrange , a p ace
of employment, room| |Porter Street, the United States
Army Medical Research Institute of Infectious Diseases USAMRIID!,
Fort Detrick, Maryland on the afternoon of February 17, 2005.
After being advised of the identit of the interviewing PostalInspector and Special Agent SA![:f::::]provided the followinginformation: '
[:::::]advised has beeni |Division
[:::::::::]for approximately years.
[:::::]adyised.[::] was previously interviewed regarding
the anthrax laced ailings of September and October, 2001circa August 2003.iifEif:Tadvised[:::]could not recall any
additional details from the August 2003 interview that would be
helpful to the investigation.
Y
44
l~ '
advised only vaguely recalls seein at
USAMRIID. advise has heard rumors that wou
"come over to our laboratories" to observe research procedures
unrelated to discipline.[::::::]further advised[:::]did.not know if had approval from either[::]supervisor or the p
Division Investigator conducting the resear hobserve. advisT%E§§§2fpuld not recall what[::::::¬::E¬::]
Division laboratories was known to frequent in particular.
WFO NOTE:[:::::]was provided a printout of USAMRIIDIelectronic foreign scientist recordsL_____1 I
Investigation on O2/18/2005 at Frederick, Maryland
, 1=i1¢#
79AWF222936USAMRIID- '59 _ l Date dictated N/A
Po tor
This document contains neither recommendations nonconclusions of the FBI. It is the property of the FBI and is loaned to your agenc
it and its contents are not to be distributed outside your agency.
'< ~ 0 0
FD-302a Rev. 10-6-95!
* 279A~WF222936USAMRIID
I: °2/18/2° 2
Continuation of FD-302 of , On , Pageb6
b"/C
Q lab" for
further adv" a list of afl foreign scientists hatwho per USAMRIID foreign scientist records, was to have accesséto
Ivisited the Division from January 1999 to present. |
WFO NOTE: Enclosed in the FD340 attached to thiscommunication is the list of foreign visiting scientists[::::]had
provided to the interviewing Postal Inspector and SA.!
| |adrised
Ilist of foreign scientists visiting the[::::::::::ji:]
Division from January 1999 to present.
WFO NOTE:[::::] contacted[;;;;;;;;;;;]telephonically.!
| | BRUCE IVINS was possibl theUSAMRIID POC for| ladvised,t%:::][::::::::]the visit was cleared through the "front o fice" and the
request for the visit came from "command."
WFO NOTE:[::::] contacted[::::::::::::::]
telephonically.!
WFO NOTE:[:::::]contacted BRUCE IYINS telephonically.!
I ladvised IVINS recalled the individual referred as| |and the circumstances of[::] visit to USAMRIID.
agreed to contact the interviewing Postal Inspector
or SA should recall any additional pertinent information.
lI J-.1 D
FD-302a Rev. 10-6-95!
' 279AWF222936USAMRIID bi
Continuation of FD-302 of , on , PageO2/18/2005
Through observation
gAsmsNAME= ,
E-I-RS.1I:=NAME =
MLQQLEELNIZ-.[£.AL =
O,CCUE__.§_'_J._=L%92T :
..El."B.1.-QQLER =
WORK ADDRE S S : -za=sr.i.=..;T:*": ;<".i._.-~- ~ ~
WORK TELEPHQ§§M5;»-»»* and interview[::::] is described as92
QSAMRLID
Qiics
[;;;:lPorter trget
FortqDe§rig§;m_aryiandlhf
Through investigation[::::::::]is described as:
§ QQ.-UNIIRY :
LAST NAME:7§IRsI¢NAME=
QCCUEAETON:
AFFILIAIQQN:
11-ATE OlF,.é4l§=L."-I =
PUREQSE oE_v1sIT= _......~.,.._. . -... ._ ML -,
K -
USAMR¥£B=RQQ:
§QE§ZEX=LASIMNAME:
g;§§Tm5ME:
g§£IEi§TggN=
D§.11.E.@QF~_-VI §,IT =
PURPQSENOF VISIT:1-"'_.:'_"__v_. >2 =-T..f.T~>~§92l
se§¬%EEEi§§§i§5jii§§EF
Through investigation is described as:
I' ""* , _V ,___._April 200092- June"200i
~ 0 a
FD-302a Rev. 10-6-95! _
b6
~ 279A-WF222936-USAMRIID b7@
I l O2/18/2005 4
Continuation of FD-302 of Q , on ' , Page
USAMRI ID POC :
I
Through investigation|:| is described as:
QQUNI§¥=
E5§?rEME
FIRST'NAME:
SEX:
AFFILIATION:
15..-i_S.§s@e111ber 30 er...
PURPOSE OF VISIT:
USAMRI ID POC :
Through investigation s described as:
J I,
FD-302a Rev. 10-6-95!
Continuation ofFD-302 of 279AWF222936USAMRIID
I: <12/18/2005
,On ,Pageb6
b7~
AFFILIATION:
*ipv_é:mr@;_z;>,.,i1.999PURPosE oF VISIT: 7' w
USAMRIID POC
Through investigation |:| is described as:
COUNTRY:w=m==$=~%;
LAST§g¥E:
E.LRS.T,_192I'A1v1E =
AFFILIATION:
aQ§IE QEmMLSQI: Z §Qgns£;l6$2p¢m19&2 *'>1
PURPOSE WF VISIT:_$,é==e1.._ ears -~~O
USAMRIID POC:
Through investigation is described as:
QQHEIRX;
LAST,N§ME:
E;RST~NAME:
AEEILIATJQN :
-DAEE-9F*WESlT: August £§;ZUL I599
PURPQsE_oE_Ml§;T=
USAMRIID POC:
,,_~_»*~__<92
Through investigation|:|is described as:
QQUN$RY:
,1-"'* *1LASTWNAME: I |
J I w
FD-302a Rev. 10-6-95!
279AWF222936USAMRIID
Continuation of FD-302 of| 02/18/2005
, On , Page
FIRST_NAME:_
OCCUPATION:
A"FFI_;IATIoN=
Oygs N%%embé; 23Lw;99gm*b6
107C
7
DATE OF >a?_:
PURPOSE OF VISIT:
1:-r'"'-"" *'"" """~ "T-
USAMRIID POC:
Through investigation|:|is described as:
COUNTRY:
LAST NAME:
FIRST NAME:
OCCUPATION:
AFFILIATION:
",___§=_ -"9
_____ 1_. 'J_DATE OF VISIT: 0 segtember_g§T30;;1999
'P :URPOSE OF VISIT
r'""""""'__-'-*-_:-F
UsAMR;;D POC:
Through investigation|:|is described as:
._QQUNfI'.R-Y-.2
I;_A_S.T.¢.NAME Z
F I
MIDDLEL INI TIAL/NAME:
A WTION:vv""'s"' "92
FD-302a Rev. 10-6-95!
Continuation of FD-302 of
USAMRIID POC:
;.~ '* Y-~ 1, ' , 0 v
02/18/2005
, On , Page
c._,,, , :..- ' **
Through investigation is described as:
COUNTRY:
-LAS$N%ME:
FIR$T ggyE=
KFFILIATION:-E-.-_ ~.~-_~--~ ,-._
DATE OF VISIT: Febfary O§$*2UUI: "*"*~ 'PURPosE"6F"Es1T= *'_ m *
¬ """ "1
UsgMRIIDigoc=
J-
Through investigation[::::::]is described as:
COUNTRY:
F§§ST NAME:' _;A$I_QmAFFIL
QAIEsOF@NlS$E;
PURPOSE QELYISIT:_§_,, ' r. w*92b6
279A~WF222936USAMRIID b7C
2 weeks Scertins in duiy 2331
FD-302a Rev. 10-6-95!
. 279A-WF222936USAMRIID
Continuation of FD-302 of ,On ,Pagebo
7
9
USAMRIID POCL
Through investigation: is described as:
-QQUNIRX=
L£éQl}§¥ E=
FI§§ZIN3ME=
AFFILIATIBN:
iffy '.ZT';~i
DATE OF VISIT:9-=--==rrr_"~1* » 71"-'1 ~:= Uu1I3=2~¢;2-9 '
PURPQSEMQF VISIT:'-i-:72, »- _._I..>T_r.1:;;._;_,,
_=USP~iY1RI;ID:.2Q.c;.
DAZEIQE » VI s,I.T,, I4; I ! =
PURP9§¬lI._I>;
POC 11 -
Through investigation |: is described_ as:
ignmw;L§§I_NAME=
§iRsm_NAM;=
AEEL§¥£EQN= i,,oDATE_QE_ML§lT: ~@;¥§;k§g;;g;¢1a in JuQe_300l
PqggggEioEay;§IT=
1! E
FD-302a Rev. 10-6-95!
0
136
279AWF222936USAMRIID b7
Continuation of FD-302 of , On , Page
US1¢92y1.1=s.1;.1; 129;
. Through investigation|:| is described as:
COUNTRY:
li¥$1;NAME:
FIRST NAME:
RFFILIATION:
DATE OF VISIT:
PURPOSE OF VISIT!__ qMay 1s9219 2000
USAMRI{D*POC¢
Through investigation|:|is described as:
cggNTRg=
~ LAST NAME:rr<""'_ ~ "
FIRs1_gAME=AF§fLIATIoN=
Q,-F-~~"" *"r~@
DATE QB v;s;$= MEV 189219 2000 "
PURPOSE OF VISIT:I='-'""" '_' ' * a-
0
USAMRIID POC:
Through investigation|:| is described as: ..
FD-302a Rev. 10-6-95!
279AWF222936USAMRIID
Continuation of FD-302 ofI O2/18/2005
, On , Page
<_lQU1.1T'IR¥,5_
;5§IlN¬¥E=
FIRST NAME:¢£':""" _ I74
Sxgn
AFFILIAIIQQ:av-"' " MB-
JEJ$PGF~V§SET: Mav 1s~19 200006
073
ll
PgB£9§EiQEiysIT=
USAMRIID POC:¢r-"'" '* ' "***'";:-:1.
Through investigatiodis described as:
COUNTRY:
j££¥l§§¥E=
FI3s1_g§ME=
AFFILIATION: .
"Y*~*T*-zw
DAFEK Maw o
$§bosE_oE;y1sIT= > ' =' 5;!
USAMRIID POC:
Through investigation[:::::]is described as:
ERA! =
E;;§§ME:.
§E_X,-=.}
AEEl._Ll.Z92TI ON =E" ' *7
DATE__ T w@y;is»19$2oo0,JOF v1s; =
PUggosEeoEiyg§;T=
USAMRIID POC:tr.-.-yr ' 1 '-
Through investigation |is described as:
CQUNTRY: Y" R-irq
LAs$eNAME=
§£B$EsEAME=
92., > a>
l
I
I
rFD-302a Rev. 10-6-95!
b6
b7f.i
279AWF222936USAMRIID
Continuation of FD-302 of
k
92-»->I | O2/18/2005 12
AFFILIATION:
DATE OFWXISIT:
PURPOSE OF VISIT:
',_,__..._._--=-------7Q,On .Page i |I §2yj;B-I9 ZUUU
.UsAMR,I.I-ms:-vgc =
QMM Thro1:rgh investigation is described as:w-"" -:---I
,_C,QUI92ITRY:
LAST NAME:
éMiE=
AFF1L;g__;LoN.=_ H A_ M ODATE OF VISIT: ""4 "¬¢'é§1?E starting in June 2001g-r~PU'RP~OSE__QE__ VISIT:j-~<'r";"*'I" " ' ""' "*;=
USAMRI ID poc=
Through investigation Z is described as:
_C;QIJ192ITRY:, ,
LAST NAME:
FIRST _,1§IZ¥"1E =
i1l"FI;.;AT1oN=
__. ..,7.¢1;_2_gr_1-g OF visit; May
PURPOSE OF VISIT:
W I
USAMRIID POC:
'-.--<:*~ ~-'4' M »
Through investigation |:| is described as:
" ' O Q
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Case ID #: 279AWF222936USAMRIIDv// Pending!-VGA
Title: AMERITHRAX;
MAJOR CASE 184 .
Synopsis: To provide a periodic update for the ongoing project
to review USAMRIID laboratory notebooks. This update summarizes
information obtained from additional laboratory notebooks and
folders belonging to Dr. Bruce Ivins and Ivins' research group,
located in Ivins' office at the United States Army Medical
Research Institute of Infectious Diseases USAMRIID!, Fort
Detrick, Maryland. Notebook numbers 16 or 3464!, 1599, 4240,
4237, 3745, 3233, 3919, 1748, 2064, 3465, 3269, 1670, 3080, 3114,
2013, 1511, 3563, 3234, 1844, 1599, 1670, and folders were
reviewed. Reference EC dated July 14, 2004, serial 882.I36
b7C
Enclosure s!: Enclosed is a Microsoft Excel spreadsheet listing
numerous laboratory notebooks and folders located in Ivins'
office.
Details: Between July and October 2004, a second set of USAMRIIDlaboratory notebooks and folders were reviewed. Brud§¬%y%§_and
researchers in Ivins' group used this set of notebooks. Numbers
were assigned by the USAMRIID library to all laboratory notebooks
issued to Principal Investigators, and the folders had a
handwritten title summarizing their contents. These notebooks
and folders were reviewed to identify any individuals who had
access to Ba Ames and were not already under investigation,
previouslyunknown places where Ba Ames was stored, people within
USAMRIID or people and places outside USAMRIID to whom Ba Ames
was distributed by this research group, and any other details of
interest.
Various Standard Operating Procedures SOPs! were
copied, including an SOP for the "Production, Harvest, and
34 giving
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I Re: 279AWF222936, 03/O8/2005
.
' Purification of Bacillus anthracis Ames S ores for AerosolChallengei'_; a March 1.997 SOP Qrenarsd had I I
' I I a May 1995 sop re ared by
the USAMRIID Bacterioloqy Division entitled I I
an April I997 SOP prepared by Ivins entitled
"Preparation of Bacillus anthracis Spores for Aerosolization"; an
April 1997 SOP prepared by Ivins entitled "Quantitation of
Bacillus anthracis Colony Forming Units"; an April 1997 SOP
prepared by Ivins entitled "Preparation of Bacillus anthracis
Spores for Testing Aerosolization Efficiency"; and a March 1997
SOP prepared by Ivins entitled "Preparation of Bacillus anthracis
Spores for Aerosol LDSO Determination". '
In theI Ifolder, a Ba Ames Primary
Subculture was shipped to Battelle in April 2004. Ba Ames
"read -to-s r " h d y p ay spores were s ip e to Battelle between Apriland Se tember 2004 Furthermore EE:::::::::::::::]Iv' ' p . , ins visited
Battelle for observation and advice between May and September
2004.
Notebook #16, also labeled #3464, contained an entry by' July 27, 1994. Ivins reported that[:::::;;;]<of[ifiEf:ff]was performing work with Ames spores in ra its. Ivins
also attached an August 17, 1994 graph showing a gamma-
irradiation kill curve for Ba Ames strain spores. The kill curve
was produced by]
I _ IOn or about November 22, 1994, an ekperiment
was to be conducted to
and Ivins were to do the skin testing. Ba Ames was one
o t e strains used in this experiment.I _
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Notebook 4240 revealed that on March 5, 2002, Ivins
gave 'lliliters of Ames spores for a
challenge with ra ' On Q; about May &, Amesspores were also provi e toI I
Notebook 4237 included pages, dated in 2000,
referencing RMR 1029, and provided the latest count of unheat-
shocked spores as 4.3 x 10 to the 10/milliliter. Additionally;
page 23 displayed an email, dated May 10, 2000, to Ivins from
I W I This e-mail
designated Ba strain identifications for isolates that
to be Ameswanted to Fhip to Ivins. One of those strainsI I is known
I Ahandwritten note by Ivins stated the types of Ba strains sent| I[:::::::::::]but it did not appear that Ames was among them.
A folder entitled I Irevealed that
This plan involved studies of DNAbased
immunization against anthrax. It was unclear whether the anthrax
studied was the Ames strain.
Other individuals with possible access to Ba Ames not
reviouslv identified were included in a folder entitled
Iindividuals assisted Ivins with a
protocol involving the testing in guinea pigs of Vaccinia virus
and Baculovirus strains, making Ba protective antigen as
prototype vaccines against human anthrax. The starting date of
this experiment was to be June 19, "8" [writer believes this may
mean 1989], and the ending date was to be December 31, 1990.' ' ' previouslv identified isI
A folder entit1edI Ib6
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3
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- Re: 2'79A-WF222936, O3/O8/2005
b6
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"key personnel" working on an
enhancement of anthrax vaccine efficacy with immunostimulatory
oligonucleotides. It was unclear which strain of Ba was used.
' I I showed that on October 13 2000 B6 IIvins attempted to send Ba Ames for research purposes toI I E29FThis D
request was denied on February 7, 2001 by the United States '
Department of Commerce on the grounds that it would be
detrimental to United States foreign policy.
Notebook 3745 contained information about RMR 1029 _
experiments conducted between May and December 1999. In D mber bg1999, Ivins gave some postaerosol challenge sera to [::::fff::::] b7C
for PA determination.
Notebook 3233 contained a request made in November 1990
from IandI I of I IThey were attempting to design aI Itest for Ba and
as e Ivins to provide some Ames spores, germinated spores, and
vegetative cells. '
Notebook 3919 c ntained electronic mail sent onFebruary 24, 2000 fron1E:ii:::EEEEEE5:] of USAMRIID to various
other researchers at USAMRIID. was scheduling a multiagent
vaccine study; Ames spores were used in the study.
Notebook 1748 divulged that in March 1983, concentrated
Ames supernatants were given toI I
Notebook 2064 contained information about a June 1987
experiment conducted by Ivins,| I
They were attempting to obtain some "hot" spore preparations and
4
To-: Washington Fig From: Washington Field _Re: 279AWF222936, O3/O8/2005 iéIv
determine the virulence of some of their other spore
preparations. Subsequently, Ivins attem ted to 'virulence of some "Mav 87" Ames sporFs;
Experimental Protocol written on December 8,
1993, had an objective to
IExperimentalProtocol written on December 8, 1993,
entailed a] I
Ex erimental Protocol written on July 29, 1994,comprised an? I
Notebook 3465, issued September 16, 1992, included
several protocols utilizing Ba Ames with as-yet unidentified
individuals listed as ri l t t 1 ' ' ' ' p 'ncipa inves iga ors: Protoco [:::::]
written September 1 1992 and Protocol written on May 11,
1993, included a Protocol written on December
9, 1992, include a and Protocol [::::] written on
April 1, 1993, included a This notebook also
contained a Standard Operating Procedure for Lyophilization of
Reagents, written February 24, 1992.
Notebook 3269, issued 2 1990, listed an
experiment Ivins performed for to determine whether
anthrax spores germinate in R-medium or R-medium and 10% unheated
horse serum. Ivins also gave some Ames spores to[:::::::::::]on
June 9, 1992 and on September 23, 1992.
Notebook 1670, issued June 29, 1982, contained an entry
written on March 3 1983. Ivins gave Ba Ames concentratedsupernatants uo[:i::::::::]for LF and PA determination.
Notebook 3080, issued November 20, 1987, contained
information regarding an experiment conducted on or about January21, 1988 by and in which the two
tested the Ba Aro strain as a vaccine against Ames s orechallenges. On or about April 20, 1988, Ivins and[fi:::::::::] .
5
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vTo-: Washington Fig From: Washington Fieldl
Re: 279A-WF222936, 03/08/2005
injected some "British" Ames spores into Hartle uinea pigs.The Ames spores were obtained fron92[:::::::::::f:j
Notebook 3114, issued Ma 26 1988, contained an entryon June 29, 1988, in whichl suggested that Ivinsl
92 | which involved Ba Ames. This idea was suggested
ecause Ivins deemed his lasmid isolation procedure inadequate.Subsequently ran Ivins' | |
Seven folders Dugway Spore Harvests #1, Dugway - 2nd.
spore shipment, 3rd spore shipment, 4th spore shipment, -5th
spore shipment, 6th spore shipment, and 7th Dugway Shi ment contained information about spore shipments in 1997 fromE::::
E::::::]of Dugway to Ivins. Some of the information included
shipment dates, what was done with the spores after receipt, and
how the spores were processed at Dugway.
' The "Dugway Spore Information" folder contained
information entitled "Scope of Work - Bacillus anthracis Ames
Spores", detailing the Dugway spore shipments, the last of which
was to be shipped no later than June 30, 1997. Inside the folder
were faxed copies of quality control assa results between April1997 and September 1997, initialed by[:::E::::::]~ Also included
was a copy of the Reference Material Receipt RMR! 1029
inventory, dated October 22, 1997.
There were multiple folders containing numerous pages
of information pertaining to | Fjexperiment.
This was an eightpart, longterm rabbit study using RMR 1029.
The study began in April 2000 and concluded January 2002. Copies
1 of spore preparation forms indicating plate counts and
concentrations were obtained. Similar forms were also obtained
in the folder labeled "Long-term Efficacy Study; 12-month rabbit
study".
I Folder contained the previously-unknownname of referencing a challenge [it is
unclear whether this is an intramuscular or aerosol challenge]
and testing two types of anthrax vaccines. Notebook 2013 issued|_De-cemmr 6.. 1.285.. mentioned] and|:|Iwere mentioned in studies with
B. subtilis and B. thuringiensis. | |was consulted by
Ivins in Notebook 3167, issued May 19, 1989, on using polymerase
chain reaction PCR!| I|:| v
The folder entitled[::::::::::::]had different
spreadsheets indicating various information, including: agent
6
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Re: 279AWF-222936, 03/O8/2005
name, ID number, room location, principal investigator, date
acquired, facility from which the Agent was acquired, etc.
Another spreadsheet in the folder was labeled "Bacillus anthracis
strain collection", and had a handwritten line on top stating
"Perry's Collection" [writer believes this to be Perry Mikesell].
This spreadsheet contained information such as Ba number, name,
notes, and references. However, there were no dates on this
collection. A spreadsheet dated July 22, 1991 had a list of all
anthrax strains in Perry Mikesell's collection. The final pages
in the folder described the type and location of particular Ba
strains, dated between March 2002 and April 2004.
Notebook 1511, with entries beginning November 2, 1988|containedI k1 I It also mentione alon
with Ivins, as having harvested
on January 12, 1989.
In Notebook 3553, issued April 8, 1993, Ivins referred
to "old" and "new" batches of Ames in December 1993. Other pages
also mentioned these two batches of Ames, as well as the
experimental protocols.
Notebook 3234, issued March 16, 1990, was en '
Iand featuredI I"Making B. anthracis spores for
transductants". Two pag ' '
is occurrence was dated July 25, 1990.
A folder entit1edI Icontained a copy of a memorandum dated January 4, 1993 from
I J I IandI Iwere namesI mentione in Notebook I I issued October 20, 1983.I
mentioned in relation toI
Notebook issued October 29 1981, mentioned [::::][::::::::]and Ivins gave[::::::::::]su ernat t f p an s o
different Ba strains, including Ames, on June 2, 1982, in order
to perform anI I The notebook
7b
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also contained information on the efficacy of certain media on
growth, concluding that agar was a better support matrix than
agarose.
Notebook 1670, issued June 29, 1982, stated that on
September 1 year unknown!] | it is not known
h l d! h B. w erqE::::::]is emp oye gave Ivins B. t uringiensis,
subti 1S, and S. fecalis.
The folder entitled
contained a lo of the amount of eceived the amoun 9 I t
used, and the dates of use. The log was begun October 27, 1997.
The folder entitled "RMR 1029- Highly Purified Ames
Spores - 3x10 " contained a set of e-mails between Ivins,
IIdated between
October 12, 2001 and November 9, 2001. The emails documented
numerous individuals to whom the Ames strain was sent and the
dates in which it was sent. '
A folder entitled "Harvesting Spores - + GLP Spore From
Dugway" contained "Acceptance Criteria Test Forms", which tested
and plated RMR 1029 on March 18, 1998. The last page featured an
email sent from Ivins on Januarv 17. 1997 to the following
rprincipal investigators: |In the email, Ivins calculated
the amount of cultures needed and the time it would take to
produce enough spores for aerosol challenges of 1000 rabbits and
200 monkeys. Ivins concluded that the concentration of spores
for each animal was based on what he and others in his group,
administered or tried to administer! to the monkeys and rabbits
in F96-16 and F96-17, or 3.0 E9. Ivins prepared 8.5 milliliters
of aerosol per animal, or about 8 milliliters per tube.
Ultimately, Ivins calculated that it took 13 runs to generate
about 3.0 E12 Ames spores for the "current batch". Since they.
needed ten times that amount,-it would take them 130 runs with
the flasks if performed with 2 liters per run, as they currently
did. Therefore, it would take 130 weeks at one run per week or
65 weeks at 2 runs per week. The total amount of culture needed
to produce the spores would be 260 liters. Below the email was
Ivins handwritten note describing what was needed, when, and in
what solution the spores would be delivered. -
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Notebooks:wan».-
fi Principallnvest.LocationCopied
1 12 iiiIvins' officeSomeComments i
NamesnotalreadyidentiedasPOI/Access if
9216043464 - Bruce Ivins ~ .vins'officeSomeInstances inwhich Anrcs was given to someone _
9210043080 Bruce Ivins lvins o feeSomeShipping fonns ip
rromrc! Bruce iuins 1vins' ofticeSomeSuspicious entries abruptly stopping around 9/01-10/01!
nor-1r #4241!
tvinsofficeNoAnythingwit1rBa1739,A0462,BA1004,74,1029-1030
17143465!
triesoffice SomeStorage locations H
rs anon!
1vins'office Some
4 Iivinsvim! office Some
Bruce Ivins inst otceSome1
roro
zonpn!
onBruce Ivins ms!attics19
2064 Brncelvins92otceSome
no _t .V111ruins
3 8'officeSome1/Ivins
14n #o2so! Bruo 1' Wiip e vmsvins'92officeSome
1 3745 i W Brucelvins ,ms!officeSome
3919Bruce Ivins inst ofC0Some
1599p i |'_[nnu.vins'officeSome
4240 Bruce Ivins .vins'omC0Some
42sr_Brueelvins .Ivins' otceSome
3685 Bruce Ivins .vins'uo1:ceNo
31670113!Bruce Ivins ins ofceSome
3234 Bruce Ivins ..vins'92otceSome
1519 Bruce Ivins ins/'ofuceSome
"Anthrax Toxin", 2 Nov. 881 e
Biuceivins 1.vins' otce No
3563 Bmce Ivins Ivins'ofC6
3160 Bruce Iivins.1.vins' orceSena
No0 1511 Brnceivins inst ofceSome
3920 Bnrcelllvins .vins' otiiceNo
4420 W Bmceivins vms
IoiceNo
4562 Bruce Ivins vins92
otce No
4103 ZBruceIvins 1vins'office Some§ppre*ee1uiuo1encycai1cu1ations of BaZinbabwe and Amos
4306 Brucellvins Ivins'-r
officeSomeMedia comparisons, effects of temp. on spore count if
4281 Ivins 1vins'onC0MostEffect of storage conditions on spore coimts in suspension
§pore cormts on differentsolidmedia
Percent encapsulation of sporesinpreps W
Pour plate versus spreadplate comparison W 113ioPoitpersom1e1
r 1Y
i
I2p
Comments Folders: P mwnotclready identied as P0l1Acccss
1ldllldjuvantdomparisonExperiment#2Bmce lvins lvins ofliceNo lnstancesi11whichAmeswas giventosomeone
115'- Live Strain vaccination i if W Bruce lvinslvins otbceNo ppilgfonns
Protocol 112 - MPL Titration/Adjutant and_rlmigenPreperetion Bmcelvins lvins otlice No Suspiciouscntiies abiupdgtoppipgaround9/01-10/01!
i92/i0I1i ¬!l__Ci10ii¬1lg/tl_-_i.5-2 year MDPH-C!
MDPH-C Manusc_r_iptWBruce lvins Z
Bruce lvinslvins 0
vinso No
NoAnything with Ba 7739, AO4p6p2,pl3el1004, 74, 1029-1030
Storage locations __'p'0l"*l1dDPH-1-Anima1Protocol-1991 7Z Bruce lvins
inso
92 APPF°t@4LFPI°01L1rZ111l4 Z___Bntce lvins
11150do _
No
Microencapsulation Work - Protocol 114 Bmce lvins 11180
Some W Z
Protocollld - Multikine 1i.Bruce lvinsv 1
v' T
' ' ft
vindofce
lied
117 - Adjuvant Comparisoiilixperirnent #1 Bmce lvins vine olliceNo P
lylatrix - lll and CPG Studies Bruce lvins
No
lriformationonantliraxsteeringcomniittendprogressandtmnsof
investigators including a. Anthrax steering committee proposal reviews-Btuce lvins
Bmce lvinsvins ollice
vins ofceNo
No
2003 and b. Anthrax progress reports! _ __ Biucelvins vins' otiiceN_ef§W"*
F95-09 mtlnan udjuvant study in monkeys Bruce lvinsv; officeNewStrainirifonnation Z __Z__ Z1Bmcelvins_ 10? ___
vins' olliceSome
rPAsteemigconirnitteeinformationta. rPA research plan, user name Bnicclnms vies otcedo
bivins, password docsnivis4, b. Plans for FY2003-rPA, and c. rPA Biucellvinsins olliceNo P M
progress reponspFa11 2003! Bruce .|.v1nsv
Ivins ofceNew P
QPGinguineapigs itBruce lvinsromeoNo
Anthrax spore production proposal for 2004 Bruce lvins lvins ofceNo K 1
Animalprotocols P 1
Biuoelvins lvins otceSome
Protocol 113 - Detox and BaculoPA detox and delta-Steme PA Bruce lvins lvins otkiceSome P
Biucelvinslvins oimN1 ,2Protocol 121 Z mmWW W
0 Q ompztrisonoflvlDPH PAwithAlhydrogel PA Bmcelvins
Bmce lvinslvins oiiig
lvins otliceNew
no
Bmce lvins lvins olceMost W§. j7 . .vrsrtnesocieppyy 17 _Spore studies wi
B97-04 Bruce lvins lvins ofcePage1 K
2003 CPG Research Plan Bmcelvins lvins ofce All
Strainsto| 1Ivins olceAll
In-housesttainlransfersofanlliraxspores 'Bruce lvins
Bmcel L Ivins ofceNewer 77 , W, ,
"Old" fonneldeliyde study in rabbits; animal protocol B01-11 Biucelvins lvins otliccNo
F09-02, Long-term monkey study not yet in notebook [Ivins note]! Prucelvinsone ofce No
Longtenn efcaoy study- 12 month rabbit study Bruce lvinslvins oll@Some Z W ____p___
I |
t
l
I3
094-09, 0.5, 5, and 50 microgram PA vaccine in monkeys 7
lvins' ofceSomeComments _ Z
studies
Ivins ofceNollarnes not aheady identied as POI/Access
tudy F96-17; Rabbits active irrrrnupization studies! Z
hips officeNoInstances in which Ames was given to someone
study F96-l6 - active immunization studies - monkeys
Ivins officeNoshipphgihmsff *0
MDPH Potency Data
NoSuspicious entries abnrptly stopping around9/01-10/0l_!
nor-os g W g
Ivins offlce
vins' ofceup _rl_nythingwithBa7739,A0462,BA1004,74,1029-1030
~ 2003rPA ResearchPlan H Bruce Ivins NoStoragelocations W if p Long-termefcacyd-monthi W
Bruce Ivins ms ofce All
Bruce Ivinsvins' ofce
V l
vins ofceNo
Brucelvius SomeD99-02 sic!fsporestudies0 Reseatc ans- a. esearchPlans-Jan.200l,b. Researclf
Plan Review-2001! i ppBruce Ivinsvine ofce
vins oice No
Anthrax SOPs and SSPs ~ Bruce Ivins Ivins ofceAll
SEC-9llrelief P Bruce Ivins Ivins ofceNo
Fprpemnental Protocols involving Ames strain 1987-1995 PBruce Ivins Ivins oiceNoGLP studies spores, etc.! if
GLPsporesWBnacc Ivins
Bnrce IvinsIvins ofce _
Ivins ofceIlsa,
No
Making spores for MBPI - BioPort Bruce Ivins Ivins ofcelfo
Dugway spore infonnation Bruce Ivins Ivins officeSome
Harvesting spores- and GLP spore information Dugway! _ __Bruce IvinstamarindNo
7th Dugway shipment Bmce Ivins Ivins ofceSome
Dugway- 5th spore shipment Bruce Ivins Ivins ofceSome
D_ugway-4thsporeshipment _Z W Bruce Ivins Some
Dugway- 3rd spore shipment Brucelvirrsvins' ofce
vins' ofceSomeflu I
4
i
8
133 - Dietlianoloniine PA versus Ammonium Acetate PA Bruce Ivins Ivins ofceNoComments
132 - Efcacyof different emulsions and PA in guinea pigsribBruce Ivins1viiis' officeNames not already identied as P01/Access H Z
128-Encapsulated live vaccine trial widl Istrains Bruce Ivins' ' s'officeInstances iii which Ames was given to someone
PASSI1/ll92/l3LlT Brucelvins1-1"L_n
lviiis' ofce
StiiinsntrvAC2lMlvl2-Eiipeiiirientwitlil J H if Bruce lviiis ivins' olceSuspicious entries abmptly stopping around 9/01-10/01!
OLl_ /1PL.126-Efficacy lcstofMPL+PAinEmulsionfromJuue 1992tintedlvinslviiis ofceAiiytliing with Bit 7739, norm, BAl004, 74, iota-ioaom
Freezirig/Storing/Lyoopliziiig sic! buffer for PA Storage 125 g _____Bnicelviiis lviiis officeStorage locationsProtocol/124-PAandEmu1sionIMPLBruce Ivins lviiis' office
123 llvcsttalnsBruceIvinslVlI1S'"QlllC6122 Yicroencapsidited PA WBruce lvins Ilviiis' office
. PASS11i/1M2Eiiptc Bruce lvins lvins office No
Protocol 119 - Mu1tilriiie2 Z g__ Bruce Ivins lviris' oflice No
Compatluoliiill/lDPl'l-PAWitl1All1ytl10gel ttI1<lPA ButrilviulllP$i9l@°§tNoaridPA-Protection/Iiifo. toAit _ K lllll WBnice lviiis lIvins' office No
Vacciiiest Vaccine Candidates Reports gBYl°Pl1lltnIvins ofceNoPresentation to vaccmewor1riiig_group;Qct. '92 PBiiicelvinslvins officeNo
Passing out CR4 and Processing Bruce llvinslvins officeNo
PAl,PA2,PA7 WBnice Ivins Ivins oceNo
Aro-1aiidAro-2Bruce IvinslIviiis' ofme i92lo_g__ W _
Monkey protocol bacteiemias g__Bmce lviiis ivins' officeNo
RAB-GPRabbitsti1dyandG.pigstudyBruceIvinsIvviinoffitinNo
Summary statistical data formassive computation Bruce lvins Ilvins office No
BAWG Talk- 1999; ASM 1988 Z _BmceIvinslviiis' officeNo
Survivors0l2P1§S,g9/10Alliydrogel-PA,9/91/IDPHA1/A,ZD94-01Bruce Ivins iviiis' ofce
B inslviiis'ofcle*A9,, in
No
Clinical Trialsruce Iv
Biucelvius Ilviiis ofce NoAdjuvants USCtll11A11ll1It1X Vaccine Preclinical Studies and Vaccine _ Z
. Kll.-l,G.plglIl1I11tI11lZll0l10AIO-Sll6lll1S WBrucelviiislviiis' officeNo
Protocol137-Imniiinizatmncliveandiiradiateddelta-Steme pPA102!sporesBruce lviiislvins' officeNo
136-LT stimulationliy different anthraiivaccines ZEvieIvinsIvins officeNo
2002Research Plans WBruce Ivins lIvins' officeNo
lI111n11I1l2tlll0nPJlil1PAltttgltlclllllltlolllcf¬XpBIl1'6I1§ti llD-324Bmcelvins Ivins officeNo H
Vaccine Efficacy Studies-Protocols 98-102; Protocol1ll3Z- PA titration Biucelvins lvins' officeNo
Aio Eiipeiiriients cl__lBruce IvinsIvins officeNoB90-03,199t1Aiiirna1Protoco1s-G.pigninimiizaiion/LD505Bruce Ivinslvins office-No
Mouse safety of Aio KlLl2 i Bruce Ivinslvins' ofce No
B00-03 |§iiperiments Parts1-7!Bruce lviiis Ivins ofce Some
9
92 informed of the identit of the interviewing Agents and the purpose-4v , .,~ _.
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HERE IN LTICLAb LI--IFIED Date of transcription O 1 [2 9 1 2 0 Q 5
DATE 12-12-znna BY 50224 UC EAHKDKKRYE
I I DOBI |
I l Llhome telephone numberL___________J wasinterviewed at the Federal Bureau of Investigations Office,
After being
of the interview1::::::E:::]provided the following information:
is currently employed with
Iarea. L_________liesides at the above address with
| Iwas assigned to the United States Army
Medical Research Institute of Infectious Diseases USAMRIID!.
Within USAMRIID rked as ain .r within t »
Ill
[::::::::] believes[::]may have been involved in
challenges using the Ames strain of Bacillus anthracis Ba!I L but could not be sure since most test sampIes
were not marked with particular strain information, nor was it a
common practi the Principle Investigators PI!, Doctors Bruce
IVINS and to inform the testin individuals of sam le
I ~ strain types. f [used Ames strain Ba elieves[:ft]
more than likely worked with it in rooms and[::::::::j Icould not remember ever working for, or with,
|could not remember ever working with Ames
strain Ba ma e at or by Dugway Proving Grounds DPG!, Utah.
|believ worked with Ba conducting challengestudies in "hotside" rooms and[:::]I06
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ch ' the hotside, but after October 2001, most of the£::]I were stripped away to conduct testinq on unknown mail
Istated that on many
occasions] Iand IVINS wouId_be present during the
challenges, but not during all of these experiments
For challenge experiments,[:;::;::::land other[:::]
would begin preparation for es ing he week prior to the
actua start of testing by decontaminating the work areas. Normal
decontamination procedures involved the use of paraformaldah de todecontaminate all work areas. At the start of testing,[::::E::::::]
received all samples and animals into the hot area and transported
them to the test area. For challenge experiments, agent samples
were placed into all- lass im ingers AGI! and aerosolized into thetest animal cages. [:?:::::::would regularly collect air samples
for later plating, to determine the uantity of agent the animalswere exposed to during the testing.Ig
I Iremembers using anti-foam in aerosol
' challenges, but could not remember its brandname or whether it
contained silicone. Anti-foam was added to the AGI to keep
materials inside the nebulizer from bubbling over.
[:::::::::]could not provide any information concerning
added materials since solutions for challenges were pre-mixed
before being provided to the testers.~
k Ilknowledge of any substitutes for anti-I oam used in Ehese aeroso challenges. I
I Iconducted tests forI IBRUCE IVINS, d_a_|during| Iemployment at USAMRIID, buL had no knotffff:2f:thim:jw ing antifoam or olive
oil to their . as already gone from
USAMRIID when started working there.
[:::;::g::] believed there was spore materials left overafter many o t ese challenges,I I92
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At the completion of the challenge, the remaining materials would
be poured back into the original test tube and placed into the
autoclave for destruction.
To the best of E:;]knowledge, no one ever askad[;;;;;::::]
for, or took, any of the e t over material or plates out o e
hot area.
A _ _ |Trash was the only item from the hot
which was taken to the basement for additional autoclaving.
[:::::::::]believed the only access points for the
basement included one stairwell and one elevatorn [:::::::::]never
observed anyone carr ing autoclave bags out of the b As
stated previously, did not work at USAMRIID while was
there. Therefore, could not comment on whether was ever
around Building _
[:::::::::]observed what[::]belie d t b 1 ve o eyp ates
containing Ba during late 2002, but could not be sure whether they
were Ames strain or not.
[:::::::::]had no knowledge of olive oil ' ' anytestin or mixtures during his tenure at USAMRIID.?fiff:Eff§:iF[:::::::%::::]may have observed a container of olive ' dside, but could not provide any time frame on when|iUiUiiUiiUiib
remembered ' uartsized, greentinted bottle with some t eof label. [fffi%¬:i:?believed this bottle was located in the[:::f¬:]
suite, possibly suite but could not provide any further
details about the bot e. believed the bottle to contain
olive oil only because it looked visually like a bottle of oliveoil, but[:::::::::]never verified the bottle's contents.area '
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[::::::::::] could not provide any information about the useof Bertolli brand olive oil in testing while[::]worked at USAMRIID.
Phosphate Buffered Saline PBS! was purchased by
from a company called Gibco and came in 10X concentration
solutions. n diluted down with distilled waterexperiment. Eff:%ii:fEfhad no knowled e of the addition of 9
the PBS or the use of antifoam in relation to the silicon
Of the PBS.the case
for each
oil to
content
Any documentation about whether antifoam was used or not
in challenges would be located in laboratory SOPs at USAMRIID.
in November 1997, and did not observetE:iff:ffin Building[:::::::::]was not involved ' the Ames challenfes
during that same time period. '
[:;:;;::::]had no knowledge of anyone at USAMRIID_keeping
left over c a enge spore preparations and could not speculate on
who might do such a dangerous
t cou no
might be related to the theft
other aspects of the mailings.thing.
Ba was delivered to[:::::::::]for challenge testing
double bagged, plastic 10 milliliter ml! Falcon tubes.
V [::::::::::]believed the liquid Ba samples were mixedprovide any further information
of Ames strain Ba from USAMRIID which
or to
in
bythe laboratory assistants for the testing primary researcher.
[::::::::] observed Dr. IVINS mix samples on several occasions. All
leftover samples were destroyed by double autoclaving. Testing
samples were stored outside of the lab before testing, inside of
the challenge hoods in test tube racks, and placed inside of -
autoclave bags at the completion of the challenges. Samples were
always labeled with concentration information. ~b F3
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DATE l2l2E2EJEiB BY 603211 UII B.'11.TJ.r"DK;P.YS
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Pursuant to the AMERITHRAX investigation, a trash cover
search regarding items discarded by BRUCE IVINS was conducted on04/O1/2005 and O4/05/2005, at his residence,[;:;]Military Road,
Frederick, Maryland. The items were discarde y IVINS at curbside for
Frederick Department of Public Works DPW! pickup on regularly
scheduled trash pickup. The trash route for his residence was
scheduled for Tuesday and Friday pickup.
I There was no trash A
placed at the curb at| |Military Road. Two trash pails were observed
in the driveway, adjacent to the residence, but it was unknown whether
or not they contained garbage. 1
I II Five plastic baqs werecollected from the residence of IVINSIb2
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Item 1: Papers consisting of advertisements and information
for an orchestra and events relating to the orchestra.
Item 2: Information packet titled, "Friends Welcome" and '
8.5" by ll" map of a resort community named Bethany Bay, with
writing on the map.
Item 3: Several rinted e-mails sent to various recipientsfrom[::::::::::::f:]regarding an orchestral quartet.
Item 4: Manila envelopes with names of orchestral quartet
members printed on white labels.
Item 5: One ziploc bag with brown residue, one empty box of
gelatin mix, and one receipt from Lee Nails, 1700 King
Fisher, Frederick, Maryland.
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