009488Anthrax Page 1 of 643 TAB LE OF CONT ENTS Amcrithrax o I. Amerithrax Science Update-04-26/2002-11 /25/2005 2. EC dated 2/S/200S-Technical Review Panel meeting agenda, Ant hrax Review Panel , Amcrithrax Panel Summary 1111412001; 12 page PowcrPoint 3. EC dated 12/21 /200 I-Meeting of Analytical Chemistry Expert 4. 5. EC dated 21l 1/2002- Proposcd Lab Analysis and R&D Strategy Ana lytical flow chart EC dated 11 / 14/2005-Case Agent meet ing (attac hed WFO Forensic & Investi gati on update meeting 11 /05 /200 I) EC dated 11 114/200S-Scientific Review Panel Meeting June 11 -22-2002 6. 7. 8. EC dated 12121 /2001- Exlcrnal Expert Review of Analytical Plan U.S. Anny Medical Researc h Institute of Infectious Di seases. Reference Material Rece ipt Record (I page) 279A -WF-222936-BATrELLE Serial #9 1 (20 pages) 279A -IV F-222936-SC 118 Seri,1 #3 (S pages) 279A-W F-222936-USAMR II D/ BEI Serial # 19 (7 pages) 279A- WF-222936-USAM RIID Serial # 141S (2 pages) 9. 10. II. 12. 13. 14. Dr. Ivi ns USAMRII D Laboratory Notebook #4010 Incoming Shipment Records for the 8 I )ositive FB I Repos itory Samples (origin is FBlR Database) United States Di strict Court Search Warrant Application and Affidav it Case # 07 -524-M-OI 07-52S-M-OI 07-S26-M-OI 07-S27-M-OI 07-52S-M-OI 07-529-M-O I 279A-W F-222936-LAB serial 37 and IA 579 279A-W F-222936-LA B serial 25 and IA 553 279A-WF-222936-LAB serial 65 15. 16. 17. IS. WOIRHEPP Batch 3 Table of Contents.pdf  1  009489Anthrax Page 2 of 643 19. 20. 279i1-WF-222936-Lil B serial 1308 and Iii 6553 279i1-WF-222936-LilB serial 1310 and IA 6554 WOIRHEPP Batch 3 Table of Contents.pdf  2  009490Anthrax Page 3 of 643 Amerithrax Science Update 04/26/2002 Univer s i t y o f Maryland (UMD) Sandia National Laboratory (SNL) Lawrence Livermore Na t i o nal Lahoratory(LLNL) W oods Hole Ocean ogr a phic Institute (WHOI ) FBI Labo ra tory, Chemistry Unit ( FBI Lab ) -betlA'een 04/21-25/2002, USAMRIID shipped or FBI agents hand-carried to the above labs numerous samples of surrogate and anthrax material produced by dif=erent production methods. The material was supplied by Dug'....ay Proving Grounds in Utah. This will assist in addressing the si:icon issue, as '...'ell as . established FBI t s forensic biological/ chem ical analytical scheme. - 13 samples sent to Uf.m -11 samples sent to all the other labs LEAHY MATERIAL under examination by FB I Lab, WHOr, a nd LLNL -anticipate ana l ytical results later today regarding age of Leahy sample from WHOI and LLNL -anticipa t e analytical results from FBI Lab next week USAMRII D: Regarding Contamination -27 environmental isola tes are cur re ntly under examination at the Northern Arizona Univers~ty tor genetic confirmation as a result of the contamination issue at USANRIID -anticipate analytical results today/next week -over 862 environme~tal swabs collected -samp les consisted of severa l Bacillus Anthracis strains (mutants, antibioti c resistance. vaccine strains. etc ) -subpoena issued this ?...'eek to USAMR IID regarding all documentation of contamination, swabb ing, testing. notes and r eports. multiple interviews con d tlcLed wi t h no significant results G[EXWSR B3D1.pdf  1  009491Anthrax Page 4 of 643 Other Testing - 2 sets of thermal stability experime nts conducted o n the Leahy, Daschle, Post evidence have been completed t o date. The prelimina ry resul t indica ted differences in age of spores from are evidence. These results are in the process of being confirmed via additional r ounds of testing. -Colony morphology examinations are in the process o f being conducted t o determine differen c es in the anthrax spores. (The latter two experiments were on ho ld due FBI invest igation during the cu r rent week due contamination issues at USAMRIID) Genetic Analysis -To date 13 /17 genetic samples receiv ed at USAMRIID for r.epository. 10 samples ha ve been sent out to NAU. G[EXWSR B3D1.pdf  2  009492Anthrax Page 5 of 643 Amcrithrax Genetics Update May 15,2002 B. flllthmcis Strain Ames Repository al USAMRJlD S , anthracis (SA) Ames isolates have been identified at sixteen (16) laboratories to date . Those laboratories have a total of 561 Ames isolates. The U.S. Anny Med ical Research Instirute of Infectious Diseases (USAMRJ fD) possesses 449 of those isolates. A thorough review of USA1\'l RITD's isolates is on-going to dctennine their disti nguishing characteristics. Isolates from ten (10) labomtorlcs have been submitted to Northern Arizona Uni versity (NAU) and Los Alamos ational Laboratory (LANL) and genetic analyses are underway. Two (2) laboratories wi ll submit samples by 5117/2002, three (3) labordtories have been requested to resubmit samples. Fina lly, twenty-seven (21) additional BA Ames isolates arc being provided by Dr. Peter Turnbull (formerly with Parton 0 0\\"11). The Instirute of Genomic Research fT iGR) DNA Sequencing of B. anlhracis Ames Isolates Fifteen (is) micrograms of plasmid DNA from the original 1981 SA Ames isolate has been purified at NAU and was shipped to TIGR during the fi rst week of May. TlGR has produced a shotgun clone library of that DNA and peR sequencing is underway. TlGR est imates completion of thaI effort by 513012002. Fifteen ( IS) micrograms of plasmid DNA appears suffici ent to complete the DNA plasmid sequencing, and the level of plasmid DNA purity provided by NAU appears su ffi cient. NAU is working to purify plasmid DNA from selected iso lates of Ames and will submit those DNAs to TIGR for sequence ana lysis. TI GR expects will begin a production sty le process al that point, and should be able to generate DNA sequences at the rate of one per 7-10 days. The 198 1 SA Ames isolate is deemed the gold standard for defin ing the Ames strain and wi ll be used a~ the reference against which all other isolates will be compared. A recent Science puhl ication deta iling data from initial RA Allies sequencing efforts, va lidates our scicll tific approach and prOVides useful infonnat ion that might focus our effort... to locate sign ificant genetic differences between the collected isolatcs of Ames. That paper docs not contain DNA sequenc!;! data from the 198 1 SA Ames isolate. DTM Mutation Rate Study in Collaboration with NA U To gain an hctter understanding of its mutation rate, the 198 1 SA Ames has been passed through 10 transfers at NAU; the eq ui valent of 25 ,000 generations. That cffort resu lted ill the detec tion of two dis tinct mutation events at a homo meric (poly A) tract designated HM - J. /\ second genetic loci, a (AT AT) VNTR showed one mutati on evellt. These data suggesllhat the evidence Ames has mutation "hot spots" . Purther, this lapid nHC afmutation at HM-I provides suppon 10 an observect difference of one (A) mUlalion in a SA Ames isolate from RMtelle. tf G[EXWSR B3D1.pdf  3  009493Anthrax Page 6 of 643 confinncd that Aattelle isolate might be ruled oul as the source of the anthrax in the Amerithrax investigation. NAU is developing the eonfinnalory assay for the HM ~ 1 site. These findings provide encouragement as we muve forward with the gcnetic ana lyses. The following is an outline of the AMERJTHRAX genetic analysis strategy: Phase J : Through Plasmid DNA Sequencing, Oefille Where "Hot Spots" for Mutation Exist. Phase I involves the sequencing of plasmid DNA from the following: Ames 198 1, Ames Ames 1 995~ Knudso n , Grinard isolate, Ames BaC (Banelle), and the Texas Goat 1985~l vi ns, isolate. Phase n. A.) Octennine if all three evidentiary samples are the same Ames sub-strain. 8.) NAU will develop rapid peR assays that can quickly delect the presence or absence of identified mutation spots defined in Phase I. Those assays will be used to more rapidly screen isolates in the Ames repository, samples recovered from the leiters, and clinical samples from the five (5) fatalities. Phase III : Establish the baseline mutation rate and fully validate gcnetic markers . TIlis effon will require genetic analyses of the 198 1 BA Ames isolate at various s iages as it is cultures through 100,000 generations. G[EXWSR B3D1.pdf  4  009494Anthrax Page 7 of 643 Ameri thrax Genetics and Auribution S~ b< b7C IWFO May 15,2002 B. anlhracis Strain Ames Re positorv at USAMRIID The following B. anthracis (SA) .A..mes sub-strains and isolates are identified to date. Sixteen ( 16) Laboratories have a total of 112 Ames sub-strains; 10 labs have submitted and analysis underway at NAU (5 / 1312002); 2 labs to submit or resubmit by SI t 712002; 2 labs to submit or resubmit by 5124/2002; 2 labs to submit or resubmit, date to be set by 512412002 . In addition , USAMRIID possesses 449 potential sub-strains of Ames BA. The Institute of Genomic Research CTIGR) DNA Sequencing of B. anthracis Amel'i Strllins and SulrStrains Fifteen (IS) micrograms of Ames 198 1 plasmid DNA purified at NAU, 512002; 15 micrograms of Ames 1981 pure plasmid DNA shipped to TIGR 5/8- lonOO2; TI GR produced shotgu n clone library of Ames 1981 plasmid DNA 5/ 1312002 ; TIGR undergoing PeR sequencing of Ames 198 1 plasmid DNA to be done 5/3012002. To date, the conclusions are that 15 ug appears sufficient per Ames sub-stra in to complete the DNA plasmid sequenci ng, and the level of plasmid DNA purity provided by NAU appears sufficient. The work for the Ames 198 1 isolate would be completed in approximately two weeks ti me and the above tentat ive conclus ions will be confirmed. At the that point, NAU will begin using the technique developed for the plasm id puri fication to start Shipping various Ames sub-strain DNAs to TJGR for sequence analysis. T IGR expects to ~gi n a production style process at that point , and shou ld be able to generate a suh-strain DNA sequence at the ratc of one per 7-lO days. DTRA Mutation Rate Study in CQllaboration with NAU BA strain Ames 1981 passed for 10 transfers at NAU: the equi valent of25,OOO generati ons. The tiM? I poly (1\) tract shows two distinct mutation events. A second genetic loci, a (AT AT) VNTR showed one mutation event. These data suggest that the evidence Ames recovered in the Amerithrax investigation was not cultured fo r an exte nsive time before sporu lat ion and subsequent wcaponization. Further. this rapid rate of mutation at HM -J supports the minor difference of poss ible one (A) mutation 111 the SaC Ames of Battelle may be real, and that the Battelle BaC su b-strain of Ames is ruled out for invo lvement in the Amerithrax investigation. G[EXWSR B3D1.pdf  5  009495Anthrax Page 8 of 643 Potentia! 4 genomic new mutations identified in clinical isola te of Steve ns NAU undergoing peR assay development for 4 new mutations. TIGR sequencing of Stevens and lab Ames isolate confirm existence of poly(A) mutation of HM?1 marker. NAU undergoing development of confirmatory assay for HM?1 One (Illata!) Battelle Ames isolate (BaC) varies at HM-J from Amerithrax evidence, as well as a new second genetic loci . Note: Battel le has submitted 19 Ames isolales T1GR DNA Sequencing Study Protocol Phase 1 - Where are HOI Spots for Mutation? Ames 198 1 . Ames 1985-lvi05 Ames 1985-Knudson Grinard Ames S aC (Battelle) Texas Goat Phase Ill? Arc all three crime scenes the same Ames sub-strain? (2) Flo,;da (2) New York (2) Washington DC Phase 11.2 o NAU to develop rapid assays for mutation spots defined in Phase I Ames RejX>sitory samples to test with new PCR assays from Phase I 112 16 Labs 449 USAMRlID Crime Seene Samples (xx) Florida (xx) New York (xx) Washington DC Clinical Samples (5) Fatalities Phase III - Establish baseline mutation rate and validate genetic markers - Publicati o n(s) (2) 10 passage (2) 20 passage (2) 30 po-. tssage (Z) 40 passage G[EXWSR B3D1.pdf  6  009496Anthrax Page 9 of 643 EXECUTIVE SU 1MA RY AMERlTKRAX Sa mple Status 3nd Benefit Anal ys is for 5129102 USAMIUID IN ITIATIVE: Under Lab Division contract USAM RlJD provides primary (i nitial) characterizat ion of all suspect samples and serves as the Repository for AMES sample returned by Subpoena from laboratories identified as ha ving AMES. It is the staning point for a ll FB I microbiological analysis. STATUS: USAMRlID's primary support to date has been analyt ical services. Microbiological, immunol ogical and morphological characterization oflhe evidentiary material (Daschl c, Leahy, NY PoSt, Brokaw) is complete. USAMRlI O's CUlTent focus is on the c barac terization of Repository samplcs of Bacillus antbracis Ames collected by Grand Jury Subpoena. There is only I delinquent repository sample (37 Spec imens) (NMRC). Sixteen ( 16) have been received and II have been sent to Paul Keirn, NAU for strain identification. USAMRIfD is also conducting Thennal stability studies of the evidence, analysis of e nvironmental contami nat ion samples within USAl\.1RIID. PCR a nalysis of Anthrax in the human remains from the PA hijackers was inconclusive. AFIP had 17/ 17 negative hijacker specimens (2 x selective media, 2 x PC R). BENEFIT: Characterization ofthe suspect samples and repository samples will provide valuable infomlation on th ennal stabiliry, % viability and morphology. Those assays provide an overall cbamcterization of the DA Ames recovered from the various ma il ings. The repository for AMES samples ensures that representative speci mens from all ident ified labs arc properly archived. Northern Arizona Unh'ersit y INITIATIVE: Dr. Pau l Keirn, a leading authority on the strain identification ufbacteria, provides st rain identification of all FB r anth rax samples by using 15 MLVA genetic markers. STATUS: NAU has identified all evidential)' samples as AMES. The current focus ofNAU is in the strain identification of the repository sa mples. NAU has sent plasmid DNA from the original AMES isolate ( 198 1) 10 TlGR for sequencing. Preliminary (verbal) resu lts o n the repository samples thus far indicates that 2 are not AMES and I is aviru lent. BENEFIT: NAU has a world class reference collection of anthrax samples and developed the 15 MLV A marker system for classifying anthrax strains. NAU provides sample extracts to LANL and TtGR fo r further analysis . The Plasmid DNA ex:tracl<;; may provide a new level of differcntia tion as assigned to USAMRIIJ..). These samples will be shipped directly to CDC for antibody testing. G[EXWSR B3D1.pdf  53  009543Anthrax Page 56 of 643 PHASE II: Du gway Peol-jo g Grounds (DPG) A contract (A21204248) is now in place with DPG ror the production of Bacillus species by a variety or production , harvesting, drying and mill ing processes. The aim oflhis project is to test and refine the techn ical production profile the Amerithrax science investigators have elucidated. A site visit to DPG is planned for 9125/02 to finalize the test plan and to deliver the Leahy dry powd~r evidence which will be used as the seed stock for thc above captioned project. This material wi ll be flownlhand deli vered by WFO and HMRU to VPG. G[EXWSR B3D1.pdf  54  009544Anthrax Page 57 of 643 L _ _ _ _ _---Lo:::co~M~ : l:--~.--~------- ~:.' t . pc.:Rl~""' ~ . . ... : ::~'- .- -, _",' . ~~ge,:1 Co mprehensive Scientific Review: Amerithrax 10/0 1/02 b6 b7C Scientific R (~ vi ew Pan els - I. November 14,2001 - U,S. avaJ Medical Research Institute (NMRI), Southern Research Insti tute Medical Research Institute of In fectious Diseases (USAMRllD), .S. Anny Dugway Proving Ground (DPO). Office of the Secretary of (SRJ)'b Iu.s. Army Defense - CounterprolitTeralion Pol icy, AIjemptur Technology. Ltd. (fonner employee of PortO Il Down, UK) 2. November 15- 16, 200 1: Scientific Representati ves from the Russian Federation 3. Decemb er 3. 2001 : Scientific Representatives from Ponon Down, UK 4. February 6, 200 2: Scientific Representatives from Porton Do wn, UK 5. February 11 , 200 2: .scientific Representatives from Israe li Goverruncnt G. March 20-21 , 2002 : Scientific Rcpresentati ves from the Russian Federation 7. December 7, 200 1 - National Science Foundation, National Academy ofSeiences, Umversi ty Rochester, Lawrence LivemlOre National Laboratory (LLNL), NMRJ, Uni vers ity of Maryland, Sandia Nat ional Laboratory (SNl . .), Los Alamos National Laboratory (LANL), lndiana University, Oak Ridge National Laboratory, Pacific Northwest National Laboratory, Un iversity of Pittsburgh, FBT Labo ratory 8. June 10- 11, 2002 - Northem Arizona University. LLNL. The lnstitu te fo r Genomic Research (TIGR), USAMRIID, SNL, FBI Laboratory. University of Maryiaml, Woods Ho le Oceanogrdphic lnstitutc (WHOJ). NMR[, University of Notre Dame, LANL, Dugway Proving Grounds, Center for Disease Control (CDC), University of Connecticut O rgan ism (evidence) 1. Identified as virulent Bacillus anthraclS (Ames strain) 2. Antibiotic sensitive 3. No known genetic engineering or modification 4. Concentration known (colony fonn ing units per gram) - approx. 1011 - 1011 5. Partic le size known 6. Puri ty of samples known (Scanning and Transmissi on Electro n Mi croscopy) : Daschlc and Leahy - very pu re NY Post - vegetati ve cel ls, debris and matrix 7. Amorpholls sil icon layer present under exosporium 8. Age (e") - modern, approx. 2 years old (confirmed by WHO! and LLNL) 9. Manufacture process known - agar, freeze dried, low energy milling , G[EXWSR B3D1.pdf  55  009545Anthrax Page 58 of 643 ., " o o d O, , ~ . .Pa~ ie'. : 10. stainless steel 316 identi fied in Leahy sample and NY Post sample. potentially present in agar and from stainless tweezers. II. Oxygen and Hydrogen isotope ratio by WHOI . to delenn ine source of water used for produetion( comkted. Water samples are being collected from areas where orkcdltraveled. 12. Dugway Proving Grounds? production methods, harvesting. drying, milling (ongoing) bE l:-7C Assoc iat ed evidence 1. Tape end match between Brokaw (9/18/0 1) and NY Post (9118/0 1) 2. No tape end match to 5 rolls of tape from 3. Enve lope ink match: Brokaw = NY Post, Leahy - Daschle 4. Fibers: Brokaw (black cotton. black wool, black nylo n) NY Post (none) Daschle (brown po lyester) Leahy (b lue wool, yellow and red acrylic, black cotton, red cotton) 5. Fibers compared to 204 pieces of clothing from l k2040 comparisons) wi th no matches 6. Identical trash marks on Leahy and Daschle letters from photocopy process 7. Comparison to hundreds of copi ers throughout areas of in terest? no matc h 8. Collections atl Ifomler apartment - detectio n of Bacillus rhllringensis and Bacillus globigii 9. Analysis for Protective Anti gen and Lethal Factor antibod ies from [ [blood (ongo;ng) 10. AM I re-entry - hot spots located in mail slot (Enquirer Rcscarch) and Dailey work statio n 11. Located mailbox in Princeton NJ I I Repositorv Sampl es ? 1. Co llected 110 isolates from non-USAMRDD labs (15) 2. Collected 451 isolates from USAMRlID 3. Oflhe 561 total isolates: 280 tested as Ames strain? approximately III additional test results are being confirmed. 170 remain to be tested? expoct to be completed within two weeks. 4. Purified DNAs by NAU (0 TIGR for sequenc ing: Nyugen clinical sample Lundgren cl inical sample Brokaw environmental samp le NY Post environmental sample AM r from Stephens' keyboard Trenton NJ post offiee enviromncmal sample Initial sequencing of original 191\ I Anles iso late completed 5. Plasmid sequencing 198 1 Ames isolate compl~t ed ? no difference with AMI 6. Morphological polymorphisms effort - Univ. of Maryland and NMRl G[EXWSR B3D1.pdf  56  009546Anthrax Page 59 of 643 EXECUTIVE SUMMA RY AME RlTHRAX Sample Status for 10/04102 IJSAMRJID Ames Repository: Submission to the Ames Repository is completed. on USAMRIID laboratories subm iued a total of 1 !O iso lates, and USAMRfID personnel submiued 451 isolates for a total of 56 1 isolates. Additionally. two isolates were obtained from the mailbox in Princeton, N.J. All 563 iso lates were shipped to Northern Arizona University for initial typi ng of DNA. Approximately 280 of the 563 samples have been genotyped and shown to be the Ames strain . Approximately 111 test results are being confirmed and 172 isolates remain to be tested. Expected to be completed in two weeks. No r thern A r izoox University (NAU) NAU purified and shipped 5 DNA preparations to The lnstihHC for Genomic Research (rIGR). The DNA preparations are from isolates of various Ames, either Repository samples, or evide.flce from the case. The five samples are: 1) clinical isolate from victim Nyugen, 2) clinical isolate from victim Lundgren, 3) environmental isolate from Brokaw Office, NYC, 4) environmental isolate from NY Post bui lding, NYC, and 5) the original Ames iso late from 1981 . Northern Arizona University is in the process of preparing pure DNAs from the following three samples: I) computer keyboard from AMI Building, Boca Raton, FL, 2) environmental isolate from T renton Post Office ai r filters, 3) environmental isolate from NIOSH cleanup in Trenton, NJ. These ONA s will be shipped to T IGR upon purification and a check for Quality Assurance and Control. T1GR is now us ing the purified DNA from the original Ames isolate to complete (he DNA of its two plasmid DNAs. Los Alamos Natio u:t l Laboratory (LAi'\L) Since there was no detection of genetic engineering in the evidentiary material and in a la rgt! portion of the repository samples, LANL has now redirected their efforts to focus on assays for the detf."Ction of the Israeli plasmid. (n addition, several isolates from the Ames Repository at VSAMRlIO will be tested as pos itive controls due to some of these iso lates a re reported to contain the Israeli plasmid. Northern Arizona Uni versity wil l prepare DNA preparations of al l repository isolates for shipment to LANL. G[EXWSR B3D1.pdf  57  009547Anthrax Page 60 of 643 T h e Institute of Genomi c Resea rch (f IG R) TIle plasmid sequencing from the 1981 Ancestral Ames standard is complete. Results to date show no difference in tht: ancestral Ames plasmid sequenccs ve rses those from tht: Stevens/Florida isolate. The sequencing of the complete genome begun the week of 08126102 [or the 198 1 Ames standan.l. The library has been constructed and an initial set of clones have been chosen fo r seq uencing. This project is expected to take about a month to complcte. Co mm onwealth Bi otechnologies, In c. (CBI) CBt is developing a separate assay for the HM-I homomeric locus on PX02. This locus shows some variation within the Ames strain. C Bfs analysis complements the work o f NAU on the same locus, and may help to localize the source of the evidence from the population of repository samples o f the Ames stra in. This work is expected to take two months . CBl will help deve lop specific markers that arise from the whole genome sequencing effort underway at TlGR. Any variatio n observed w ithin the Ames stra in will be analyzed using a variety o f molecular assays. This effort wi ll parallel the work at TIGR. CBI is also online wi th the 15 MLVA marker system, devc loped at NAU, and will act in the future as an independent center to screen and genotype bacillu~' anrhracis isolates. This new s urge capacity affords Amerithrax science investigators an additional resourcc if needed in the future. Nationa l Science Fo und a tio n (NSF) NSF has agreed to fund research to exploit a potential morphological polymorphism. Thi s polymo rphism is observed in isolates from Amerithrax cv idence and is believed 1 be a unique 0 forensic signature. The fundin g for FY 02 will be $100,000, with the possibi lity o f add itional funding next year. A proposal from the University of Maryland in collaboration with the Nava l Medical Research Center, was vt'l"ittcn and subm itted to NSF fo r review and comment T he proposal is submitted to the NSF by Univers ity o f Maryland with written support by the Naval Medical Research Center. NSF has verba lly agreed to fund for FY02. The work will commence October, 2002 at the Naval Medica l Research Centcr. Univers ity of Maryland (UMD) UMD is also pursuing the differentiation and detection o f tryptic d igests (plants) and pepla nes (acid and heat hydrolysis) of protein. Ini tial results on heme (from bl ood agar or sim ilar p roducts) detect ion in com pl ex biol ogica l matrices (red blood cells. hemoglobin , etc) has been successful. Prior to a nalysis of the NY Post material methods lor the detection of sIXJrcs grown on sheep blood agar (S BA) and calibration curves are being developed . A draft manuscript detailing methods and rt:sults was received on 9/16/02. G[EXWSR B3D1.pdf  58  009548Anthrax Page 61 of 643 F BI Labo ra tory Seventy o nc items of clothing (7 1) were submi tted fo r trace analys is. Add itional itcms were a lso submitted to the Fireanns Unit, Latent Fingerprints (2 books), Photo Processing (disposable camera, slides and ncgati ves) and Documents Unit (tape, pens and indented 'HTiting). FBI Chemistry Unit is continuing with ICPIMS analysis o f surrogate samplcs obtained from vario us sources. Preli m inary results have been provided by the Chemistry Unit and a meeting is schedu le next week to discuss the obscrved resu lts. Other types o f examinations are continuing to date. \\Iood s Hole Occan ogr a pbi c Institut e (WHOI) WHOI has comp leted isotopic analysis of the samples it has received. A final report is in preparation which will describe obtained results in detail. A confidential draft ma nuscript was received from an intelligence agency regarding microbial fo rensics. In cooperatio n with academia this group has demonstrated that oxygen and hyd rogen isotope ratios can be used to indicate the source o f water in which specific cultures were gro\VO. The isotopic distribution in spores has been correlated with thc watcr used to grow the respective sample. Measurements of potential water sources wil l provide a reference point for future comparison of Amerithrax evidence and any sei7 material to potentially eliminate production ..ed in certai n geograpbical regions. Amerithrax is considering examining several possible sources of water (Princeton, Frederick, Baton Rouge, Ocala, Dugway, etc) to detem line the variabi lity in oxygen and hydrogen isotopic ratios in these waters sources. Amerithrax will coordinate with Hazardous Materi al s Response Unit (HMRU) to assist with the co llection of the various water samples. ) Lawrence Livermore Nation a l Labol-ator), (LLNL) LLNL has completed the isotopic analysis of the samples it ha'l received. A final repo rt is be ing prepanxl which will describe obtained results in detail. Ana lys is of W et a nd Dry Co ll ec tion Poin ts at cl_ _--'I Apa rtmcDt Recent d iscuss ions w ith Intelligence CO j Unity ,icntists revealed the presence of potential collection points for biological weapons in former apartment in Frederick, MD. These points included wet drain water traps for the sinks alld bathtub, air vent areas fo r the drains and traps, and several u nique dry aerosol collection points. On 0911 112002, a search pursuant to consent of the apartm ent was conducted and samples taken froll1the coll ection points. Analysis of the samples was conducted for B. alltilracis, B. globigii. and B. thurillgerzesis. Scveral samples werc found to contain e vidence of B. thuringenesis, however, no anthracis or globigii was ident ified . bo b7C Ce nfers for Disease Control (eng G[EXWSR B3D1.pdf  59  009549Anthrax Page 62 of 643 I On 08130/02,1 ~u lunlarily submitted his blood sample at the WFO, Health Services. On 09103/02, hi s blood sample was shipped FedEx to CDC fo r antibody testi ng. On 09/12 /02, Isecond blood sample was taken and shipped FedEx to CDC. The fi nal blood sample w ill be taken on 09126/02 and will be shipped to CDC at which time all samples will be bo b7C analyzed . The lime frame to obtain results is 1 week after tcsting begins. blood serum before and after vaccination as well as his medical records when he was assigned to USAMRHU. These samples will be shipped directly to CDC for ant ibody testing. PHASE II, Dugway Proving Grounds (DPG) 1\ contract (A21204248) is now in place with OPO for the production of Bacillus species by a varicty of production, harvesting. drying and mil ling processes. The a im oftlus project is to test and re fine the technical productio n profile the Amerithrax science investigato rs have elucidated. On 9125102, a site visit to OPG was conduct to finalize tbe test plan and 10 del iver the Leahy dry powder evidence which will be used as the seed stock fo r the above captioned project. nus ma tt!rial was flo\',lftlhand delivered by WFO and HMRU to OPG . On 09/1 7/02, a subpoena Was scrved to GSAMRlID official s to obtainl I G[EXWSR B3D1.pdf  60  009550Anthrax Page 63 of 643 EXECUTIVE SUMMARY AMERJTHRAX Sa mple Status for 10/ 18/02 USAMRJ ID Ames Repository: Submission to !he Ames Repository is completed. Non t JSAM RJID laboratories submitted a total of 110 isolates, and USAMRIIO personnel submitted 451 isolates for a total of 561 iso lates. Additionally, two isolatQ: were obtained from the mai lbox in Princeton, N. J. All 563 iso lates were shipped to Northern Arizona University for initial typing of DNA Approximately 280 of the 563 samples have been genotypt=d and shown to be the Ames strain. Approximately II I test results are being confirmed and 172 iso lates remai n to be tested. Expected to be completed in Iwo weeks. Nort hern Arizona Uni\'crsity (NAU) NAU continues 10 type the repository samples from the Ames slrain. There are 134 samples left to type out of the original submission of 563. Approximately 5% of the samples run to date are showing a distinct ditference ill their genetic sequences compared to that obtained for the evidencc. The estimated time of completion for this project is approximately three weeks. J In collaboration with COl , NAU is confirming the presence of a SNP (s ingle nucleotide po lymorphism) recently found within the large plasmid. Los Alamos Na tional Laboratory (LANL) Si nce there was no detection of genetic e ngineering in the evidentiary materia l and in a !luge port ion of the repository samples, LANL has now redirected their efforts to focus on assays fo r the detection of the Israeli plasmid. In addition, several isolates from the Ames Repository at USAMRlID will be tested as positive controls due to some of these isolates arc reported to contain the Israeli plasmid . Northern Arizona University will prepare DNA preparatio ns ofal! repository isolates for shipment to LANL. The Inst itu te orG ctlomic Resea rch (TIG R) It is estimated that TIGR will complete the sequenc ing of the ancestral Ames strain in the next four to five days. TIGR will then add additional genetic sequencing runs for the Florida evidence iso late obtained from the victim, Stevens. AllY additional polymorphic markers found will be used to elimi nate known so urces of the Ames strain as the contributo r of the ev idence. T IGR is awa iting the delivery of fou r additional samples for full gc:numic sequenci ng. These samplcs will be comprised of two sets of morpho logica lly d istinct cultures obtained from the ev idcntirn)' powders (spores). Assays will be developed for any genetic differences observed in these -)- G[EXWSR B3D1.pdf  61  009551Anthrax Page 64 of 643 !J samples and then applied to the repository samples. The development of these assays will be done by em and NAU. Co mm onweah h BiolechnoloAies, In c. (C BO CBr is developing a separate a.ssay for the HM-I homomeric locus on PX02. This locus shows some variation within the Ames strain. CBJ 's analysis complements the work ofNAU on the same locus, and may help to localize the source of the evidence from the population of repository samples of the Ames strain. This work is expected to take two months. em will help develop specific markers that arise from the whole genome sequencing effort undcnvay at TIGR. Any variation observed within the Ames strain wi ll be analyzed using a variely of molecular assays. This effort will parallel the work at TIGR. CB I is also on line with the 15 MLV A marker system, developed at NAU, and will act in the future as an independent center to screen and genotype bacillw; anthracis isolates. This new surge capacity affords Amerithrax science in vestigators an additional resource if needed in the fu ture . CBI is confirming the presence of a SNP (s ingle nucleotide polymorphism) between the evidence and the ancestral Ames strain. Jf this is confinned then there will be at least o ne marker to J differentiate the ancestral Ames strain from the evidence. Th.is marker can also be used to eliminate known sources of the Ames strai n. CBI is also typing some of the repository samples to confinn the results obtained from the work being conducted at NAU. Unive r si ty or Maryland (UI\1D) A draft final report and manuscript were received on 09/ 16/02. UMD is also pursuing the differentiation and detection of trypt ic digests (plants) and pctones (acid and hent hydro lys is) of protein. Initial testing of an assay to detect heme (from blood agar or similar products) in complex bio logical m:lIrices (red blood ccll s, hemoglo bin, elc.) has been successful. The assay developed by UMD has been able to detect heme at levels as low as 0.5 picomo les (5x I 0. 13). This assay is being used to detect heme in SSA agar grown spores and a calibratio n curve is being prepared for this assay. Samples of spores produced at Dugway Proving Grounds (DPO) will be forwarded to UMD for agar and heme analysis. Sa nd ia National Laborato ry (S NL) A one year con tract extension for ?320,000 has been completed. The contact wi ll allow detailed analysis of the samples produced by Dugway Proving Grounds (DPG). SNL is readdressing the stainless steel issue and searching for additional endospores in the NY Post material. A final report on the evidence is being prepared . - 2- G[EXWSR B3D1.pdf  62  009552Anthrax Page 65 of 643 , I FBI L!tboralory S~vcnly one items of clothing (71) were submitted to the Trace Analysis Unit. Addi tional items were submined for examinat io n by the Fireanns Unit (4 keys), the Latent Fingerprints Un it (2 books), the Photo Processing Unit (disposable camera. slides and negatives) and the Documents Unit (tape, pens and indented writing), Woods Hole Oceanographic Institute (WUO I) WHO I has completed the isotopic analysis of the samples it has received. A fina l report is in preparation. Th e Un iveniity or Utah (UUT) A confidential draft manuscript was rece ived from another agency regarding microbial forens ics. The University of Utah has demonstrated that oxygen and hydrogen isotope ratios can be used to indicate: the source of water in which the cultures were grown. Amerithrax, in cooperat ion wilh HMRU and Field Office ERTs, is in the process of collecting water samples from 20 locations where ~s known to have resided or visited based upon the info rmation availablc al this lime. These samples \'!ill be submitted to the UoiversityofUtah detenninc the variability in oxygen and hydrogen isotopic ratios in these waters sources . I .J Lawre nce Livermore National Laboratory (LLNL) LLNL has completed the isotopic analysis of the samples it has received. A fina l report is being prepared which will describe obtained results in detai l. C enters for Disease C ontro l (CDC) O n 08/30/02,1 Ivoluntarily submitted his blood sample allhe WFO, Health Services. On 09/03/02, his blood sample was shipped Fed Ex to CDC for antibody testing. On 09/ 12102, lsecond blood sample was taken and shipped FedEx to CDC. The final blood sample was takcn on 09n6/02 and was shi pped to CDC for analysis. On 09/ 17/02, a subpoena was served to USAMRITD offi cials to o btainl I blood serum before and after vaccination as well as Ius medical records w hen he was assigned to USAMRlID. Thesc samp les will be shi pped di rectly to CDC for antibody testi ng. hn b7C I CDC has completed the analysis of the above listcd samples for the concentration of the anti-PA IgG (the a nthrax toxin antibody). The results show thai there was no detectable anti-PA IgG in any of the sera laken after 08/26/99, however, it should be noted thot thcre are no kno \l,'11 serulII samples from I Ifor the period of 08/2 6/99 La 81 30102. - 363 G[EXWSR B3D1.pdf   009553Anthrax Page 66 of 643 II Sta tus of Evidence From The American Media In corporated (AM l) Building TIlIce basic types of evidence wert! removed from the American Media Incorporated building in August/September 2002 under the supervision of HMRU. Hr..1RU is currently coordinating the analysis of thl; evidence co llected from AMI . The first evidence type was in the form of quantitative biological samples take n from ddined sites within the bu ilding. The quantity of anthrax isolated from each site will be determined and ploned to produce a three-dimens ional map of contamination. This map will be analyzed to reconstruct the panem of the primary anthrax dissemination within the building (as opposed to secondary dissemination due to foot traffic, etc.). Such information will be matched with wilnes." information and epidemiology data to provide as complete a picture as possible as to the events that led the death of one employee and the illness of another. The completion of the above project requires the fulfi llment of several conditions. First, the extensive data collected at the site must be consolidated into a single searchable database . All of the data were compiled by CDClATSDR. However, it was deemed by FBI personnel that CDC does not have the appropriate level of computer security to perform the work required . C DC, therefore, relinq uished all of the raw data into FB I custody upon completion of on-scene operations. Second, all of the culture plates generated at AMI have been photographed and stored in digital forma t. TIu!se images must be analyzed in detail to provide the final sem iquantitative estimates of contamination levels. The results generated at the laboratory during 011scene operations were produced using a crude grading system meant only to provide an estimate of contamination to be used as guidance for subsequcnt sampling. The majority of cultures obtained at AMI were on contact culture plates, wltich were placed directly on the surface to be tested . The subsequent pattern of bacterial growth observed on the culture plate corresponds to the pattern o f contam ination on that particular surface. Proper analysis of these types of plates requires that the area of bacteriaJ growth be measured precisely. In order to do thi s, specialized software must be purchased. A demonstration of this software is scheduled fo r Tuesday, October J ) 22,2002. The second type of evidence was taken in order to secure spore samples, in particular large, intact spore clumps. Successful reeo....ery of spores and spore dumps wi ll be fo llowed by detailed im aging analysis in order to ascenain whether the spore preparation from Florid a is co ns istent with the spores the fro m New York (Brokaw and Post) maili!1gs and/or the spores from Washington, D.C .(.Daschle and Leahy) mai lings. The items taken fo r spore recovery are known to be contaminated based upon microbiological sampl ing. The itcms taken for testing include metal shelves from mail slots, tops of cardboard boxes, hangiug fi le folders, and pieces carpeting. Each of these items present will require the development of a different recovery technique. TIlese evidence items are currently stored at USAMRJID, wh ich may nOI have the tcclmical capabi lity available to insure optimal spore recovery. Therefore, Ihe items may have to be irradiated to kill thl:! bacteria in order to allow other laboratories to conduct an analysis of these items without the need fo r biol ogical conta inment faci lities, -464 G[EXWSR B3D1.pdf   009554Anthrax Page 67 of 643 The third type of evidence is mail taken from the AMI building which was dated from July 200 1 unt il early Octoher 200 t. It is hoped that mi crobio logical analysis of this mail will provide an indication of the exact day that contaminated mail entered the AMI building, thereby providing a more complete understanding of the events that led to the contraction of anthrax by the two AMI employees. Based upon HMRU's experience in their investigation of the mai l sorting operation in Springfield, Virginia where the Leahy lette r was processed, they detennined that spores stick tenacio usly to paper, and that th~ closer a leuer is to a source of contamination, the more contaminated it will be. Therefore, it is hoped that quantitative microbiologi cal analysis (culturing) will produce a spike of highly contaminated mail on or aro und the datc at which contamination entered the building. This analysis is routine, but will require a significant amount of time and an adequate workspace. HMRU is currently trying to identify a laboratory that can safely JX!rform this task. PHASE n: Du gway Proving G r ou nds (DPG) A contract (A21204248) is now in place with DPG for the production of Baci llus species by a variety of production, harvesting, drying and milling processes. A site visil to DPG was held on 9125/02. A final test plan has been complcted and work commenced week of 10/ 14/02. j - 5- G[EXWSR B3D1.pdf  65  009555Anthrax Page 68 of 643 EXECUTI VE SUM IARY AME RJTHRAX Sample Status for 10/30/02 ~o rlh c l'n A rizona Univers ity (NAU) NAU cOlltin u ~s to type the repository samples from the Ames strain. There arc 67 samples left to type out of the original submission of 563 . Approximately 5% of the samp les run to date arc showi ng a distinct difference in their genetic sequences compared to that obtai ned for the evidem:e. The estimated time o f completion for this project is approx imately three wee ks. NAU was unable to confmn the presence oCa potential SN P on PXOI . CB I con finned that the SN P does not ex isl. NAU is testing the possibility that the mutation occurcd while growing the colonies fo r DNA preparation prior to shipment to TlGR. NAU has isolated and puri fied DNA [rom morphologically distinct isolates obtained fro m the evidence powders by scientists at USA MRlID. The expected date of shipment of the DNAs to T IGR fo r fu ll genome sequenc ing is t 1/04102 . Los Ala mos Nationa l Laboratory (LA NL) Si nee the re was no detection of genetic engineeri ng in the evidentiary material and in a large portion o f the repository samples, L ANL has now red irected the ir e fforts to foc us on assays fo r the detection of the Israel i plasmid. In addition, several isolates fro m the Ames Repos itory at USA MR fT D will bt: t~slt:d as positi ve (;onlro ls uw: to samc of thcse isolates are reported to contain the Israel i plasmid. Northern Arizona Universi ty will prepare DNA preparations of all repository isolates for shipment to LANL. No progress was noted due to the lack of co ntinued funding fro m the FBI. The funding was re-instated on 10128/02 . Work is now in progress. Th e Institut e of Ge nom ic R esearch (fIGR) TIGR provided results of the sequence of the ancestral Ames strain to the FBI on 10128/02 . Analysis of the data is currently taking place at the FBI Academy. Within one week, C HI will ass ist in this errurl . TIGR is now performing add itional geuetic sequencing r UIl S fo r the Florida evidence isolate obtained from the victim. Stevens. T IG R is awaiting the delivery of additional samplcs fo r full genomic sequencing. T wo of these samples arc due to arri ve at T IG R o n 11 /04/02. T hese samples are compri sed of two morphological ly d isti nc t cultures obtained from care fu l examination and puri fication from the evidentiary powders (spores). The estimated time for ge no me sequenci ng of each sample is six weeks. These samples arc the top priority within the TlG R faci lity. Assays will be developed for any genetic d ifferences observed in these samples and then applied [0 the repository samples. The development of these assays wi n be G[EXWSR B3D1.pdf  66  009556Anthrax Page 69 of 643 done by Cill and NAU. Commonwealth Hiotechn ologies, luc. (C 81) e m has developed an assay for the HM-I homomcric locus on PX02 . This locus shows some variation with in the Ames strain. CBI will perform this assay on a port ion of the repository samples ill Lheir possession to assess the level of variation. This work is expected to take four weeks. CD! w ill help develop specific marker assays that arise from the who le genome sequencing effort underway at TIGR. Any vari ation observed within the Ames strain will be analyzed using a variety of molecular assays. This e ffort will parallel the work at TlGR and NAU. CB I is also online with the 15 MLVA marker system, developed at NAU. and will act in the future as an independent center to screen and genotype Bacillus anthraci.f isolates. This new capaci ty affords Amerithrax science investigators an additional resource if needed in the futu rt: . CB I confinned the work ofNAU on a potential SN P at position 175022 within PXOI. This site is nOI polymorphic between Ihe evidence and the ancestral Ames strain. To date. no polymorphic markers have been found between the ancestral Ames strai n and Ihe evidence. Full ge nome sequence infonnation from the ancestral Ames isolate is now under analysis. 2 G[EXWSR B3D1.pdf  67  009557Anthrax Page 70 of 643 .' 07-26 1Jpdate T he Insti tute for Genom ic Research (TIGR) TIGR pr~st:ntcrJ sl.:tjuencing data from the 05/81 isolate to CTFSRU pcrsorUlc! un 7122/02 . The sequences were compiled and compared to the Florida isolate obtained from the victim, Stevens . No differences were found. The cloni ng results were, however, incomplete, as about 20% of the data was mi ssing. This is believed to be caused by the plasmid purification method used by NAU. T1GR is now work ing on fini shing the sequence by focusi ng on areas which did not efficiently clone and sequence . This data should be provided to the CTFSRlJ in about two weeks. Northern Arizona University NAU will shift their emphasis to genotyping the reposi tory samples . The plasmid purification project will be shifted from NAU to CBL Because no diversity has heen found in the plasm ids to date. a reduced set of samples will be s ubjected to plasmid purification and sequencing. {fthese results also show no differences, emphasis in sequencing will be shifted toward idenlifying differences in lhe main chromosome. Any differences identified within the Ames strai n will be quickly converted to specific assays, and tht: ev identiary samples will be lesled. The goal is 10 elim inate specific laboratories known to have hand led the Ames strain as sources of the evidentiary material. eBI CBI will assist NAU in genotyp ing the Ames repository samples. They will also take over the plasmid purification projt:~t fro m NAU. em is developing an assay to test [or the orie ntation o f two large DNA invers ions fo und on the PXOI plasmid. Upon co mpletion of testing and validation oCthe assays. they will be used all evidt:nliary samples. Due: lO d on ing inefficiency, it is no t yet knowll whethe r or nm the 05/81 samples also harbor the inverted segments of DNA on PXOI . G[EXWSR B3D1.pdf  68  009558Anthrax Page 71 of 643 AM ERITlll{,. \X SCI ENCE UPDATE 1lI01 /02 United States /\r"m y Medi cal Research Institu te of Infecrious Disea ses (USAMRIID) Three stable polymorphisms were identified in the BA Ames spo re powder from the Leahy letter. Since these polymorphisms, which are present in approximately 3 to 5% of the total cells are stable, it is believed that they are based in the DNA of the BA Ames and can therefore be subj ected to DNA sequencing. Additionally, the three stable poiymorphisms have also been detected in the Senator Daschle and NY Post powders. These will be independently isolated , propagated , and transported to Northern Arizona University on 11/20/0 2 for DNA extraction Other polymorphism s at very low levels have been detected in addi tion to the three mentioned above. Work is progressing to identifY and isolate these signatures from the three powders . No rthern Arizo na University (NA U) NAU continues to type the repository samples from the Ames strain. Tht:re are 67 samples left to lype out of the original submission of563 . The estimated time of complet ion for this project is approximately three weeks. NAU was unable to confirm the presence of a potential single nucleotide polymorphism (SN P) on the PXO I plasmid. em confirmed that the SNP does not exist. NA t.I is testing the possibility that th e mutation occurred while growing the colonies for DNA preparation prior to shi pmen t to n GR. Wet pellets of the three distinct morphologies of BA Ames from the Leahy letter, which were isolated at USAIv1RJID, were transported to NAU on 10/26/02 . NAU will purify the DNA from these samples. The three DNA preparations represent the W ild type morphology in the spore powder and two morphology types which are also present in the spore powder at low polymorphic level s NAU has conducted quality checks on these samples. The DNA samples were received at TIG R o n 11 /01/02 for sequencing. Los A I:wIOS N:Hiollal La borato ry (LAN L) Si nce there was no detection of genetic engineenng In the evidentiary material and in a large portion of the repository samples, LAl' lL has now redirected their effort s LO focus on assays fo r the detection of the Israeli plasmid. In add ition, several isolates from the Ames Repository at US AMRlID will be tested as positive contro ls due to the fac t that some ofthese isolates are reported to co ntain the Israeli plasmid. Nort hern Arizona Universi ty will prepare DNA preparations of the repository isolates for shipment to LANL. LANL has been given additional funding on 10/2 8/0 2 to continue this project. Amerithrax Squad 2 (AM X 2) is in the process of prio ritizing which samples will be screened for the Isra el i plasmid in an effort to fo cus in on Page I of 3 G[EXWSR B3D1.pdf  69  009559Anthrax Page 72 of 643 spccifi c repository samples. T hc In stitu te of GCIIOI n ic Researc h (TIG R) TIGR provided results of the sequence orthe ancestral Ames strain to the FBI on 10/28/02 . Analysis of the data is currently taking place at the FRI Academy Within one weck, CS I will assist in this effort Assays will be developed for any genetic differences observed in these samples by NAU and COL This project is expected to take two weeks. Analyses of the th ree DNA preparations from the Leahy materia l are the top priority wi thin the TIGR facility. Assays will be developed fo r any genetic differences observed in these samples and then appl ied to the repository samples. The development of these assays will be done by CBI and NAU. T IG R is now performing additional genetic sequencing runs for the Flarida evidence isolate obtained fram the victim. Stevens. C Ollllllonwealt h Biotechn ologies, In c. (C Ol) C S I has developed an assay fa r the 1-lM? 1 hamomeric locus on PX02 . T his locus shows sOllle variatio n within the Ames strain. CS I wlil perform this assay on a portio n of the repository samples in their possession to assess Ihe level of variation. ThIS work IS expected to take fou r weeks. CA l wi ll help develop specific marker assays that arise from the whole genome sequencing effon underway at TIGR. Any variation observed with in the Ames strain witl be analyzed using n variety o f molecular assays. This effon will parallel the work at TIGR and NAU . ca l is alsu online with the 15 rvn.. V A marker system, developed at NAU, and will act in the fu ture as an independent center to screen and genotype Bacillus amhracis isolates. This new capacity affords Amcrit hrax science investigators an additional resource if needed in the futu re . c a l confi rmed the work o f NAU o n a potential SNP at position 175022 within PXU I. T his site is not polymorphic between the evidence and the ancesual Ames strain. To dale, no polym orp hic markers have been found between lhe ancestral .Ames strain and the evidence Full genome sequence information fro m the ancestral Ames isolate is now under analYSIS. Un ive rsity of .\1a ryla nd ( UM D) A QAlQC si te visit was co nducted o n 10/24/02. Ducumentat ion, protocols, evidencc storage and handling procedures were reviewed and documented. The sheeps' blood agar (SSA) plates were received for use in the heme analysis. Su rrogate samples have been produced . It is expected that initial results from the heme analysis will be known within o ne week. Page 2 of 3 G[EXWSR B3D1.pdf  70  009560Anthrax Page 73 of 643 UllivC1 "Sity o r Utah (UUT) In cooperation with HlvrRU and Field ERTs water samples from 20 locations will be collected Special purpose collection kits have been ordered. The samples will be forwarded to the University of Utah for Ol(ygen and IIydrogen isotopic analysis. Sa nd ia Natio n al Labora tory (SNL) Under direction from M1X 2, SNL undertook the task of looking for additional vegetative/mother cells containing spores. A series of growth stages have been observed and the silicon chemical mapping performed . A detailed su mmary report on the efforts to date is expected by 11 / 15/02 S TL wil/ also receive samples from the production project at DPG fo r characterization Dugway Proving Gro u nds (DPG) DPG is und er contract to produce BA Ames by a variety of well defined processes. Small scale culture production, QNQC and pe R analysis of stock cultures began o n 10/30/02 and is expected to take approximately 2 to 3 weeks. DPG is negotiating with the agar company for the production of pre-manufactured agar plates. The pre-manufactured plates will improve quality control , speed up production and reduce the chance of contaminat ion. It is anticipated that the process to produce a sufficient amount of BA Ames fo r further processing will take approximately 1 mon th. Page J of J G[EXWSR B3D1.pdf  71  009561Anthrax Page 74 of 643 AMERITHRAX SC IENCE u p n ATE I ) /U8/U2 Un ited Sla tes A rrny Mcdict l Resea rch l iistitute or Inrections n i se"se.~ (USAM tUI D) isolation of morphologlcaHy and biochemically dlstmct colomes from the evidenuary material of the Leahy, Daschlc, dnd I\'ew York Post spore powders continues. There are several o f these variants within the powders; however, th ree (3) distinct isolates of interest were collected from the Leahy material based o n specifi c morpbological and biochemical characteristics. One of these colony types represents the predominant "wi ld-type" population. The other two have been selected for study based , in part, on the ability to differentiate them from othel' variants in the sample. The phenotypic differences o f these two variants on several growth media and u nd er various growth conditions have been assessed to maximize the ability to identify the same variants in oth!;!r samples. T he ability 10 produce spores under various laboratory conditions has also been examined ; spore production/morphology on certain sporulation media are essential to the process of di fferentiating these isolates. The characteristics of these particular strains are cu rrently being useU to screen the Daschle and New York Post materia ls for the same variants. Cu lture material prepared from the three Leahy material isolates was sent to NOl1hern Arizona University for nucleic acid extraction. Nucleic acid extracts will be sent to The Institute fo r Genomic Research (TIGR) fo r genetic sequencing. Genomic and plasmid sequencing will be used to explore genetic variation between the original AMES isolate and the isolates o f interest. Specific assays can then be developed toward any genetic variation, in order to more effectively screen tht! hundreds o f samples in the repository; this will continu e as other samples are fully characteri zed . Nor'lh er'lI A rizo na Un ivers ity (NA U) N AU co ntinues to genotype the repository samples from the Ames strain There are XX sa mples left to analyze out o f the o riginal submission o f 56 1 The estimated time of completion for thi s project is approximately two weeks. Wet pellets of three distmct morphologies obtained from the Leahy leHer powder, isolated at USAlvlRHD, were Iransponed to NAU on 10126/02 NAU conducted quality checks on these samples, pUrified the DNA, and shipped the Dl\"A to TIGR on 10/30/02 T IGR received these samples on 11 /01102 . The three DNA preparations are from the dominant morphology and two mino r mo rphology types which are consistently found at low levels in the spo re powder from the Leahy letter. Los Ala mos Natio ll a l La bor atory (LA NL) Because no evidence of genetic engineering has been fou nd in the evidentiary ma terial, LANL has now redirected thei r efforts to focus on assays fo r the detectIOn of the Israeli plasmid Page I of J G[EXWSR B3D1.pdf  72  009562Anthrax Page 75 of 643 Several isolates from the Ames Repository at USAMlUlD WIll be tested as positive contro ls since they are reported 10 contain the Israeli plasmid. NAU will prepare D A from these isolates and ship the DNA to LANL. In an effort to focus in on specific repository samples, Amerithrax Squad 2 (AMX 2) IS in the process of priorit izing the samples to be screened for the Israeli plasmid Th e lnslil ul e o f Genomic Resea rc.h (TIGR) T IG R has begun the process of whole genome sequencing the first mo rphological variant obtained from NAU on 11101102 This process is expected [ 0 take six weeks T IGR will then begin analysis o f the second morphological variant. Upo n completion of each project, the data will be mdependently analyzed by Commonwealth Biotechnologies, Inc. (CS t) and TIGR. TIGR provided raw D A sequencing results of the ancestral Ames strain to the FBI on 10128/02 . Analysis ofl he data is currently taking place at both the FBI Academy and Commo nwealth Biotech nologies, Inc. (COl) If any genetic differences nre observed, assays wiil be developed to test for the presence of Ihese va l; ants within the Ames Repository. This work w ill be conducted joint ly by NAU and CBI In order to fill in small segments of missing sequence data from the Florida Isolate, TtGR is now perfo rming additional sequencing runs from this samp le The completion of the Florida project will ensure that no potentia! genetic differe nces within Ihe Ames strain are missed. This effort is expected to take four weeks. Commonwealth Biotechn ologies, Inc. (Cnr) On I IJ04J02, cm received the whole genome sequence chromnlograms from the ancestral Ames strai n sequenced by TTOR. In conjunction with the FBI Labo ratory, CBI is currcntly III the process o f assembling an d analyzi ng thi ... data. This elTon is expected to take approximately three weeks. ell! will develop specific marker assays arising from any variants observed between the whole genome sequence of the ancestral Ames strain and the Florida isolate. These difierences will be assayed lI si ng a variety of molecular techniques NAU W II! develop separate molecular assays to co nfi rm the presence o f any differences fou nd. Once confirmed, the reposito ry samples will be screened usi ng these techn iques. CB I is developing and valid ating mo lecular assays designed 10 detect the presence o fa minor DNA sequence variant in a mixture of two or more sequence variants. If needed, this technique w;1I he appl ied to the Ames R epo~ ito ry samples In an effo rt to identi fy the prese nce of DNA sequences found in the mo rphological variants currently being seq uenced at Tl G R. Page 2 of ) G[EXWSR B3D1.pdf  73  009563Anthrax Page 76 of 643 University of M:tryla nd (U MD) The heme analysis is currently undenvay A small number of diagnostic peptides have been identified from tryptose-based cu lture media. Woods Role Oce;tn og rap hic In stitut e (WHOn A QNQC site visit was conducted 11 /04/02 Docu mentation, protocols, evidence storage and handling procedures were reviewed and documented. Sandia Na ti onal La boratory (S NL) A detailed summary report on the effons to date is expected by 11 / 15102. Additional vegetative cell s in various stages of growth have been identified SNL will also characterize samples received from the production project at DPG. Du~w ay Provin g C r ounds (OPG) DPG is under contract to produce BA Ames by a variety of well defined processes. Production of the seed stock has been mitiated and pre-manufactured agar plates have been ordered It is an ticipa ted that the process to produce a sufficient amOUR! of BA Ames for further pro<.::essing will take approximately I mOllth. PageJ of J G[EXWSR B3D1.pdf  74  009564Anthrax Page 77 of 643 AME RTT HI~,( SCI ENCE UPDATE 11 /15/02 United Sta les Ann y Medica l Research Institute of Infeclious Diseases (USAi\ IRffiJ) Dr Worsham at USAMR1TD continues to isolate morphologically and biochemically disti nct co lomes from the evidemiary material cfthe Leahy, Daschle and New Yurk POSt powders USAMRJID has isolated three phenotypIc vanants from the Leahy and New York Post spore powders. USAMRl lD has isolated two of these three variants from the Daschle material and is currently working on the final variant from the Daschlc spore powder The three variants i~olll t ed from the Leahy spore powders were delivered to Northern AJizona University on October 25, 2002 The corresponding \'ariants from the New York Post and Oasch le materials are schedul ed for delivery to NAU on November 20,2002 USAtvlRJl D is preparing 10?1 2 mg of Le:ahy and NY Post evidence for irradiation and shIpment Universlty of Maryland for heme and peptide analysis . Shipment should be done hy late next week (Q Etlons for screening the repository samples for the distinct colonv momholo glcs are cgQIj o"i oo I IWIIl be meeting with Dr. Worsham,Li-::----cc-:-.,..--..,..--.,..-:-:;:--:---' on Friday (November 15, 2002), to develo p th e strategy for accomp lishi ng this project . Topics such as time fr ame for completion, additional resourct!S , laboratory space, and quality assurance/quality control issues will hI! di scussed . T he analytical test plan for th IS project I~ being revlcwed . No r-the.?n Arizo na Ulli"cr~ il y (NAU) All o i Ihe Ames repository samples have been run through the ML VA 15 assay They are being scored and double read The results should be ready for dissemination by next week A final report should bc aVai lable withi n two weeks Dr Keirn is cammuing to work o n developing a..~ s ays far the Hlvl-I homomeric locus o n PX02 as well as other quick screening assays Los Alamos Na ti o n:'!1 La boratory (LANl) The assay ror the Israeli Plasmid has been developed and is in the process of lJt:ing validated . Dr .Iilcksol1 has requested positive and negatIve com rols to va lidate the Israeli Plasmid assay AMX 2 has identified the appropriate cont i ols from the repository samples to be sem to Dr Jacksnn from NAU Dr KeuTI will be advised th!s week which samples shou ld be shipped 10 LAI\rt... fo r Page I of J G[EXWSR B3D1.pdf  75  009565Anthrax Page 78 of 643 Dr Jackson's assav validatIOn. Once thIs assay has been validated, all of the evidence and climcal s.1mples (a total of 25 samples) wIll be screened [f the results are negative, then only those repository samples that are known to contain the Israeli Plasmid will be screened . If the results are positive, then all oflhe repository samples (a total of approximately 563 ~amples) will be screened The Ins titut e orCenom ic Hcscarch (T IGR) TI GR begun the process of whole genome sequencing the first morphological vanant obtaIned from NAU on 1110 1/02 This process is continuing and IS expected to take five weeks T1 GR will then begin analysls of the second morp hological variant Upon complet ion of each project, the data will be independently analyzed by Commonwealth Biotechnologies, Inc (CB [) and lIGR. TIGR provided raw DNA sequencing results of the ancestral Ames strain to the FBI on 10/28/02 . Analysis of the data is cu rrently taking place at bo th the FB I Academy and Commonwealth Biotechnolog!cs, Inc (CS I). if any genetic difference" are observed, assays wi ll be developed to test for the presence: of these variants wi thi n the Ames RepoSItory. This work will be conducted joi ntly by NAU and eBl [n order 1 fill in small segments of missing sequence data from the Florida Isolate, TIGR is 0 conttnUlng to perform additiona l sequencing runs from this sample The com pletion of lhe Florida proj ~L:t will ensu re that no potential genetic differences within the Ames stmin are missed . This effort is expected to take three weeks Co mm on wenlth Bi utec hnologies, In c. (e DT) On 11/04/02, CBt received the whole geno me sequence chromatogra ms from the ancesllal A1no..::s strain sequenced by TIGR . In conjunction wilh the FAJ Laboratory , CEt is co ntinuing the process of assembling and analyzing this data This effon is expected to take approximately two weeks. eSI wIll develop specific marker assays arising from any vanants observed between the whole geno me sequence of :he ancestral Ames strain and the Flonda Isolate. These dIfferences will be assayed using a variety or molecula r techniques NAU will develop separate molt!Cular assays to confirm the presence of any differences found . Once confirmed. the repository samples will be screened uSIng these techmques . presence of a minor DNA sequence variant in a mixture of two or more sequence vanants . tf nceded. this technique will be applied to the Ames Repository samples in an effort to identify the presence of DNA sequences fnunrl in the murphological variants cu rrently being sequenced al T IGR. eBI is developi ng and validating rr.olecular assays designed to detect the Page:1 of 3 G[EXWSR B3D1.pdf  76  009566Anthrax Page 79 of 643 I1 niversity of M:u1'I:lll d (UM D) The heme an ...lysis assay IS cu rrently undergoing validation Only 4 mg of spores are required for the analy:;;is A new negative control sample of SA Sterne o n sporulat ion medIa IS bemg prepared after the prevIous negative COl1lrol was found to contain a co ntanunant After validatio n of the assa)" UMD will be begi n analyses on the Leahy and Post matclial o nce it is received from USAMRlID . AMX 2 is prepanng the nccc~5a ry paperwork for transfer of the evidentiary material to UMD from US AMRIID A small number of diagnostic pept ldes have been identified fro m tryptose based culture mcd ia and harvested (spent) sporulation CIlllure media previously supplied by the FOT A teclmical and cost proposal is e~ected to be provided to AMX 2 next week This proposal will allow the lJ}...ID to continue the work on the peptide analysis into 2003 . Sandia Natio na l L:l boratory (SN L) A detailed draft summary rcpo rt originally expected by 11/15/02 has becn delayed The repo rt is currently going through a thorough editorial review and Will not be finalized until next week. SNL will also he characterizing the BA Ames samples receivcd from the production project at DPG o nce DPG has produced sufficient amounts fo r shipment to SNL Du gway Provi ng Grounds (OPG) DPG is under contract to produce BA Ames by a variety of well defined procedures. Produ clia n of the seed stock has been initiated and is anticipated ro be completed in 3 weeks Pri ~lr to the BA Ames production, a biosafety and environmental plan must be approved and in place. These plans are expected to be approved In approximately I wet=k . This stock culture will be analyzed by pe R to verify that IllS consistent with the BA Ames . It will also be assayed for concentlatio n and an aliqu ot will be [r07en for archival purposes . Pre? manufactured agar plates have been ordered and Will Improve quality control. specd up production and reduce the chance of contamination. A new glove box neceSSilry for handling the dry powders has been ord ered It IS anticipated that the process to produce a sufficient amount of BA Ames fo r further processing will take approx lmalc1y I month Un iversilY o r Uta h (UUT) In cooperatio n with H1v(RU and F;eld ERTs. wi'.ler samplt=s fro m 20 locations will be collected Special purpose collection kits have been o rdered and are expected to bc Shipped out 10 tht! Field ERTs alo ng with the sampling protocol within IWO weeks. The Field ERTs are being directed to send the samples to H1v1RU instead of University of Utah. HMRU will maintain the water samples received from the Field ERTs and o nce all samples are recclved. HMRU wi ll ship them to the Umvcrsity of Utah for analYSIS . Page) of 3 G[EXWSR B3D1.pdf  77  009567Anthrax Page 80 of 643 AMERITHRAX SC IENCE UPDATE 11/22 /U2 United S la tes A rm)' M ed ica l Rest:lrch Ins ti t ut e or Infec tious Oise:lses (USA MRllD) USAMRlID continues to isolate morphologically and biochemically disti nct eolontes from the evidentiary material of the Leahy. Daschle, and New York Post spore powders. Three phenotYPIc variants each from the Leahy and New York Post powders have been isolated. Two of the three variants have been isolated from the Daschle material and isolation of the third varian t from this material is currently underway. The three variants isolated from the Leahy material were del iveroo to NAU on October 25,2002 The corresponding variants from the New York Post and Daschle materials are scheduled for delivery to NAU on November 25, 2002. The phenotypic screening of the Ames repository was discussed at a meeting on November lS , 2002. A recommendation was made to confirm genetically that the respective variants iso lated from each cvidenliary sample are the same, prior to initiating the repository screening process. In the absence of genetic information, the fatty-acid composition of each variant isolated to date will be analyzed using gas C hromatography. Experiments will be conducted to determine if the gas chromatograph can be used to detect differences between the variants and the wild-type organism; in add itio n, to prove its potential to demonstrate an association between respec;tive variants If this work proves fru itful, the instrument may be utilized as an in-house validation toward selection of future variants. Also under investigation are ways to minimize the effects Ihat BA vegetative cell g rowth will have on performing seri"l dilutions. Nort h e rn Arizo na Un ive rs it y (NAU) NAU has finis hed the pri mary analysis of the repository samples. Of the 561 total sa mples, 117 samples remai n 10 be reviewed by a second reviewer. 43 samples from the total must be reanalyzed due to amplification failure during the first round of analy~is The results indicate that the majority of the reposi tory samples are Genotype 62, the Ames strain genotype. Tht! three DNA samples, containing the wild type (major type) and two minor morphological types, have been analyz.ed . The three DNA preparations are from the dominant morphology and two minor morphology colonies consistently found at low levels in the spore powder from the Leahy leller. All tbree are Genotype 62, the Ames strain genotype. These DNA samples were shipped to T IGR fo r full genome sequencing o n 10130102 Los Alamos Na t io na l Labo ra tory (LA NL) Because no evidence of genetic engineering has been found in the evidentiary material, LANL ha..'> now [edirected their efforts to focus on assays to detect the presence of the Israeli plasmid . Thirteen to tal samples from the Ames Repository at USAMRIID will be tested as positive Page I of 3 G[EXWSR B3D1.pdf  78  009568Anthrax Page 81 of 643 controls and negative controls. NAU has prepared DNA (j?Cln these isolates [ 0 ship to LANL. LAJ\f"L is in the process of finalizing the CDC requirements fo r shipment of Select Agent DNA from NAU to LANL The Institute of Gt' li omic Resea rch (TlC R) TIGR has begun the process of whole genome sequencing of the first morphological variant obtained from NAV on 11/01/02. Upon completion, TlGR will then begin analysIs of the second rnorphological variant. T IGR provided the DNA sequencing results of the ancestral Ames st rain to the FBI on 10/28/02 Analysis or the data is currently taking place at bot h the FB I Academy and Commonweal th Biotechnologies, Inc. (CBl). To date, two possible variant positions have been identified . These positions will be confirmed by both CBI and NAU. Assays will be developed on any confirmed variant posit ions. These assays will then be performed on the repository sampl es in order 10 eliminate potential sources of the evidentiary material. C om monwealth Biotechn ologies, In c. (CBI) CB l continues to process and analyze the Ancestral Ames whole genome sequencing data. Currently, two potential differences between the Florida isolate and the Ancestral Ames sample have been observed. These potential differences will be funher evaluated by separate assays at buth CB I and NAU. C Br continues to develop rn ol~cula r assays designed to detect the presence of a minor DNA sequence variant in a mixture of two or more sequence variants. If needed, this techn iC]ue will be applied to the Ames Repository samples in an effort to identify the presence of specific DNA sequences found in the morphological variants currently bei ng sequenced at TIGR. UniverSi ty of !Vlaryland (UM O) Validation of the heme analysis assay continues A new negative control sample of SA Srerne on sponJ latioll media was prepared and new positIve controls of BA Slerne and R cerell.{ were harvested rrom sheep's blood agar. AMX 2 has reC]lIcsted that UMD submit the required EA Ia I for transfer of the Leahy and Post materials to UMD from USAMRlID. UMD will begin analyses on the evidentiary material once received from USAMRl ID. A small number of diagnmtic peptides have been identified from approximately 15mg of culture media and harvested (spent) sporulation culture media previously supp lied by the FB I. UMD is continuing to optimize the Chromatographic procedures fur desalting the samples. In addition, a com prehensive literature search is being conducted for specific enzymes, hyd rol ysis processes, and plant and animal sources of peplones. tryptos e~ba se d Page 2 of ) G[EXWSR B3D1.pdf  79  009569Anthrax Page 82 of 643 Sa ndia N:lt ioll:ll L:tboralory (SNL) SNL's thorough editorial review of the detai led draft summary report is o ngoing. ANtX2 has not been notified ofa targel finalization date. The report was originally expected by 11 / 151 . 02 Dugway Provin g Grounds (O PG) The first "batch" of seed stock will be harvested this week and heat shocked for percent viability determinatIon. Essentially, this will be the first QNQC assay of the DPG seed stock . Production of tile remaining seed stock is anticipated to be completed in 2-3 weeks Approval and initiation ofbi osafety and environmental pLans are required prior to the full production of BA Ames. Approval of these plans is expected at the next blosafety committee meeting, scheduled fo r Dec 2 Once production of flA Ames is underway. it will take approximately one month to generate sufficient material for the drying and milling steps. University of Uta h (!JUT) In cooperation with f-l}...{RU and Field ERTs, water samples from 20 locations will be collected. Purchase Order A3LD002 has been processed. Field ERTs have advised WFO and HMRU that some loca tions derive their water from several sources According to Dr. Ehlringer, who has studied the Sail Lake Ci ty Water system extensively, the differences are expected to be within the measurement precision. Special purpose collection kits have been prepared and were sent to the 16 ERTs along with the sampling protocol by FcdEx on 1\/19/02. The Field ERTs are being directed to send the samples to f-Th.fRU wilhin2 weeks of receipt of the sampling kit. Page J of 3 G[EXWSR B3D1.pdf  80  009570Anthrax Page 83 of 643 AI\1EIUTHRAX SCIENCE UPDAT E 11126/02 United States Army Medical Research Insti tute of Infecti ous Discases (USAM RlID) USAMRIID has completed isolating morphologically and biochemical ly distinct colonies from the evidentiary material of the Leahy, Daschle, and New York Post spore powders. Three phenotypic variants each from the Leahy, Daschle and New York Post powders have been isolated. The three variants isolated from the Leahy material were delivered to NAU on October 25,2002 and is currently being seq uenced at TIGR. The corresponding variants from the New York Post and DascWe materials arc scheduled for delivery to NAU on November 25,2002. The phenotypic screening of the Ames repository was discussed at a meeting on November 15, 2002. A recommendation was made to confinn genetically that the respective variants iso lated from eaeh evidentiary sample are the same, prior to init iating the repository screening process. In the absence of genetic informat ion, the fany?acid composition of each variant isolated to date will be analyzed using gas chromatography. Experiments will be conducted to determine if the gas chromatograph can be used to detect differences between the variants and the wild?type organism; in addition, 10 prove its potential to demonsrrate an association between respective variants. If this work proves fruitful, the instrument may be util ized as an in?house validation toward selection of future variants. Also under investigation are ways to min imize the effects that BA vegetative cell growth will have on perform ing serial dilutions. USAMRIJD has been instructed to proceed with preparations fo r repository screening while confirming the fatty acid analysis. As a result of the presence of these variants in the evidentiary material, USAMRlID scienti sts conducted preliminary phenotypic screening of the Ames repository during the viabili ty checks. Amerithrax 2 (AM X2) has received the data from this screening. OCthe 563 s~ml p l es, 106 samples have shown to contain an additional colony type other than the wild?type colony morphology. USAMRIID is the contributor of the majority of the 106 samples. T his efforts has helped to prioritize the screening o f the repository. A meeting with USA MRIID scientists is scheduled for initiation of screening the repository samples. ovembcr 27, 2002 to discuss the No rth ern Arizona Unive rsity (NAU) NAU has fi nished the primary analysis of the repository samples. Of the 563 total samples, 117 sam ples remain to be reviewed by a second reviewer. Forty-three samples from the total must be re?analyzed due to amplification fail ure during the first round of analysis. The results indicate that the majority of the repository samples are Genotype 62, the Ames strain genotype. The three DNA samples from the Lea hy materi al, contai ning fhe wild type (major type) and two mino r morphological types, have been analyzed. The three DNA preparat ions are from the Page I of 3 G[EXWSR B3D1.pdf  81  009571Anthrax Page 84 of 643 dom inant morphology and two minor morphology colonies consistently found al low levels in the spore powder from the Leahy leuer. All three are Genotype 62, the Ames strain genotype. These r1NA samples were shipped to T IGR fo r full genome sequencing on 10/30/02. Purified cultures of the wi ld type and two morphological varianLS from the Daschle letter and the N. Y. Post lelter were delivered to NAU on 11 1251 02. DNA will be purified from these cultures and shipped to T IG R for whole genome sequencing . Purification and quality assurance testing s hould take approximatc ly one week. Los Ala mos Natio na l La bor a tory (LANL) Because no evidence of ge netic engineering has been found in the evidentiary material, LANL has now redirected their efforts to focus on assays to detect the presence of the Israeli plasmid. Thirteen total samples fro m the Ames Reposi tory at USAMRlID will be tested as positive controls and negative controls in the validation of the assay. NAU has prepared DNA from these iso lates to ship to LANL. NAU shipped thc!;c DNA samples to LANLon 11(25/02 . T he Instit ute of Geoomic R esearch (TIGR) T IGR has begun the process of whole genome !;equencing of the fi rst morphological variant obtained from NAU o n 11 /01102. Upon completio n, TIGR will then begin analysis of the second morphological variant. TlGR provided the DNA sequencing results of tile ancestral Ames strain to the FBI on 10/28 /02 . Analysis of the data is currently taking place at both the FBI Academy and Commonwealth Biotechnologies, Inc. (CBI). Any potcntial positions of variation will be confinned by both CBI a nd NAU. Assays will be develo ped on any eonfinned variant positions. These assays will then be perfonned o n the repos itory samples in order to eliminate potcntial sources of the ev identiat)' material. Scnior representatives from tht! NSF and NIH, and FBI scienti sts, met with TIGR ma nagement on 11 121102 to discuss the time line of DNA sequenc ing of FBI evidentiary material. TIG R senior management indicated that new DNA sequencing equipment is due to arri\e at TIOR on or about 12131102 . Once installed and operational, this equi pment will be devoted to the FBI sl:q ucncing project. This added throughput capac ity should reduce the time requi red tor whole genome sequencing fro m si x weeks to about three weeks. Co mmonwealt h Bi otechn ologies, [nco (CBI ) CB I continues to process a nd analyze the Ancestral Ames whole genome sequencing data obtained from TIOR. Currentl y, a number of potential differences between the Florida isolate and the Ancestral Ames sample have been observed. These potential differences are prelim inary, and requi re furt her eva luat io n. This fmt her evaluat ion is currently in progress at the FBI Laboratory. as we ll as CBI and NAU. Page 2 of 3 . , G[EXWSR B3D1.pdf  82  009572Anthrax Page 85 of 643 CB I continues to t1evdop molecular assays designed to detect the presence of a minor DNA sequence variant in a mixture of two or mo re sequence variants. If needed, this technique will be applied to the Ames Repository samples in an effort to identify the presence o f spccific DNA sequences found in the morphological variants currently being sequenced at TlOR. Unive r sity of Maryland (UMO) Val idation of the heme analysis assay continues. A new negativc control sam plc of 8A Sterile on sporulation media v..?as prepared and new positive co ntrols of 8A Sterne and B cereus were harvested from sheep's blood agar. AMX 2 has requested that UMD subm it the required EAIOI for transfer of the Leahy and Post materials to UMD from USAMR.lID. UMO will begin analyses on the evidentiary material once received from USAMRllD. A small number of diagnostic peptides have been identified from approximately 15mg of tryptose?based culture media and harvested (spent) sporulation culture media previously supplied by the FB?. UMD is continuing to optimize the chromatographic procedures fo r desalting the samples. In addition, a comprehensive literature search is being conducted for speci fic enzymes, hydrolysis processes, and plant and animal sources of pcptones. Sandia Nat ionall..aboratory (SNL) SNL's thorough editorial review o f the detailed draft sununary report is ongoing. AMX2 has not been notified of a target finalization date. The report was origi nally expected by 11115/02. Dugway Proving G r ounds (OPC) The fi rst "batch" of seed stock will be harvested this week and heat shocked for percent viability determination. Esse ntially, this will be the first Q!VQC assay of the DPG seed stock. Production of the remaini ng seed stock is anticipated 10 be completed lu2? 3 weeks . Approval and in itiatio n ofbiosafety and environmental plans are required prior to the full production o f SA Ames. Approval of these plans is expected at the next biosafety committee meeting, scheduled fo r Dec 2. Once production of BA Ames is underway, it will take approximately nne month to generate sufficient material for the drying and milling steps. Univer sity of U,,,h (UUT) In cooperation with HMRU and Field ERTs, water samples from 20 locations will be collected. Purchase Ord cr A3LD002 has been processed. Field ERTs have advised WFO and HMRU that !lome locations d erive their waler from several sources. According to Dr. Ehlringer, who has studied the Salt Lake City Water system ex tensively, the differences are expected to be within the measurement prec isio n. Special purpose collection kits have been prepared and were sent to the 16 ERTs along \yith the sampli ng protocol by FedEx on 11119/02 . The Field ERT.s are being directed to send the samples to HMRU within 2 weeks of recei pt o f the samp ling kit. Page 3 of 3 G[EXWSR B3D1.pdf  83  009573Anthrax Page 86 of 643 AMERITHRAX SClE"ICE UPDATE 12/03/02 United States Army Medic:a l Research ?n sti tul e of In fectious Disea se... (USAMRUU) No rthern Arizona Un iversi ty (NAU) NAU has finished the primary analysis orthe Repository samples. Repons are currently being prepared fo r the last balch of 11 7 samples F0I1y-three samples from the total must be reanalyzed due to amplifi cation failure during the first round of analysis. The resuhs indicate that the majority oflhe Repository samples are Genotype 62, the Ames strain genotype. NAU is developing a molecular assay 10 identify the nUnor component of two or more DNA sequences present in a mixture. This is being done in anticipation or ONA sequence differences found between the major type and the minor type within the morphological variants. The DNA sequencing work is being conducted at T IGR Once developed and validated, this assay will be applied to the Repository samples to identify which, if any, contain the same mixture as the evidentiary material. Purified cultures o f the wild type and two morphological variants from the Daschle letter eessing of the anthrax contaminated mailbox found in the Princeton, New Jerscy area by members o f the HMRU, CBSU and FBI Laboratory examiners is set to begin on 4123n003 . AMX?2. HM RU and C OSU personnel will meet on 0412212003 with laboratory personnel at the United States Army Chem ical Transfer Facility. Aberdeen, MD (Edgewood) to conduct a trial run and to discuss safety protocols for this operation. Spores will be collected using contact plates to detcnnine the distribUlion pattern. The morphological characteristics of the spores co llected from the mai lbox will be examined using SEMffEM to dctennine whether or not these spores resemble the spores in the New York Post or Leahy/Daschle lettcrs. IIMRU wi ll det:ontaminate the mailbox prior to any forensic examination. FBI Laboratory examiners from the Trace Evidence Unit (fEU), SPU and the LFPU will examine the mailbox fo r potential fo rens ic evidence of value after HMRU personnel certi fy that the mai lbox has been decontaminated. Northt!rn Arizona U nive~ity (NAU) NAU's ini tiative to develop two PCR ba.<;ed assays to detect the SNP found in the Leahy morph B is sti ll on going. It should be noted that the assays developed by CBI will be different from those being developed at C BI. Once developed, these assays will be used. to screen the Ames B. anthracis Repository samples located at USAM RJID . This initiative is still on?going. Th e Institute fo r Genomic R~sea rch (TIGR) TlGR's initiative to completely ( 100%) sequence the 1981 Ames B. anthracis standard and the Leahy colony morphologies is on going. This is TIGR's top priority and this initiative is still on? going. Com lll onwea lth Biotechn ologies, Inc. (e m) CBl's initiative to develop two PCR based assays to detect the SNP found in the Leahy morph B is still on going. It should be noted thallhe assays developed by em wi !! be di fferen t from those being developed at NAU . Once developed, these assays will be used to screen the Ames B. anthracis Repository samples located at USAMRIID. Th is ini tiative is still on?going. Pagel of 3 G[EXWSR B3D1.pdf  98  009588Anthrax Page 101 of 643 DU b/way Pro\'ing GrOllll(.Js (OPG) On 0411612003, AMX-2 and CBSU personnel met to funher rcfine the DPG SA production plan. This comprenensivl:: analysis plan will include BA production processes which are covered in I Inotes as well as the protocols used by Ms. Abshire in the production of BA-Ames at US AMRllD. This plan includes producing SA using different types of growth media, including those with and without sheep's blood agar, as well as utilizing different washing, drying and milling procedures. Aliquots will be taken during each step of production. Once the samp lc aliquots have becn irradiated and cenified as safe to work with. they will be transported to USAMRIID for SEMffEM analysis as well as to other designated laboratories for further analyses. OPG scientists will submit add iti onal detailed protocols fo r the key steps in this production process which will include the type of equipment they wi ll use during crit ical steps of thi s process. These protocols will be thoroughly reviewed by AMX 2 and CBSU personnel before they are implemented by OPO scienti sts. Un ivt rsity of Utab (UUT) UUT has completed the isotopic analyses of20 drinking water samples collected from areas throughout the United States. The geographic distribution of the water isotope values were generally consistent \.\ilh the known trends for water in the United States. Five out of eighteen c ities had similar isotopic ratios to include Columbus, OH; New York, NY; Princeton, Nl; Frederick, MO and Fon Dawson., WV. However, these water samples were distinguishable from other cities such as Dugway, UT; Livermore, CA; Ok lahoma City, OK and Miami, FL. In preparation for the water sample collection from foreign locations by the Legat Offices, AMX2 wi ll discuss with UUT sc ientists which countri es will be significant to ensure an adequate sampling. Several Legat Offices cover numerous countries and to ensure a sufficient sampling is conducted, specific countries need to be identified and samples collected from those areas. Page 3 of 3 G[EXWSR B3D1.pdf  99  009589Anthrax Page 102 of 643 AMERITIlRAX SCIENCE UPDATE 5/01103 Un ited Stat~ Army Medical Resea rch Institute of Infuriolls Diseases (USAMRlID) N::ntal Medical Research Cent er (NI\.'fRC) On 03/13/2003, FBI-LAB Hazardous Matenals Response Unit (HMRU) retrieved 19 wet sam ple preparatiuns o f B.a. Ames from USAMRHD These samples were produced and maintained by Ms. Teresa Abshire. Research Scientist. Aliquots were taken from each vial after the 19 samples were inventoried, photographed and processed for latent fingerprints The fo llowing analyses of the samples will be conducted by the respective laboratories. Strain Identification Single Nucleotide Polymo rphism (SNP) Colony Morphology SEMITEM Elemental Analysis Carbon 14 Dating AgarlHeme Detection Gas Chromatography Northern Arizona University (NAU) Commonwealth Biotechnologies, Inc (CBl) NMRC USAMRIID Sandia National Laboralof)' (SNL) Woods Hole Oceanographic lnstitute (WHOI) University of Maryland (lIMD) FBI-LAB Chemistry Unit UMD Due to the limiterl sample amount, approximately I milligram per pellet, lIMO will condu ct o nly the heme analysis of the samples . This w ill serve as a validation study before the evidencc is submitted for analysis. WHUi Several of the 19 samples contain phenol, in additio n to residual materials from density grad ient purification. Sample clean-up prior 10 Carbon-14 analysis is necessary, as these substances may hinder accurate mea.<;tlremenIS. WHOI has provided sample clean-up suggestions Nor" them Arizona University ( AU) NAU is d eveloping a SNP-specific assay also targeting the morph 0 sequence Thei r approach uses molecular beacons rather thar. 11NAs Sensitivity anti .specificity are similar to those found at CBI. Development is continu ing. An Ames-specific SNP assay using primer extension has been developed . This assay wI ll allow rapid specific testing fur the Ames strain o rBA. SOP preparation is in prog ress. The Institute for Ge llomic Researc h (TIGR) Page 1 of 2 G[EXWSR B3D1.pdf  100  009590Anthrax Page 103 of 643 T IGR is co ntinuing closure of whole genome sequcnc~!I for the morph variants obtained from the evid ence Common we:llt h lliot echnolog ies, Inc. (C BI) COl has d eveloped a SNP-speci fi c assay targeting the morph B sequence at position 95078 . The assay involves the usc o f a r NA to suppress the amplification of the wild type while allowi ng amplifi catio n of the minor component. Additional validation tests are in progress. Sensitivity is at 0.5% Specificity is in the range o f 2-3 CT values. Cell lines will be transported to CBI on Thursday, June 5 fo r further validat ion wo rk. Upon completion o f vahdation work, eS I wi ll be sem a test including mixtu res o f both types and cells prepared from the evidentiary powders. Following successfu l completion of the lcst, the reposito ry will be screened fo r the presence of the minor SNP. DNA preparation work at NAU will be required prior to repository screening . Dugway Proving Gro und.? (DPG) OPO has experienced inst rumental problems with their T ransmission Electron Microscope (TEM). TEM analysis of B.a. Ames materials produced by DPG will co minue once instrumental issues are resolved. A request has been made ofDPG to provide protocols for drying and grinding of the spore ma lerials. Univers it y of Utah (UUT) Page 2 of 2 G[EXWSR B3D1.pdf  101  009591Anthrax Page 104 of 643 AMERlTHRAX What's Hot This Week 0712412003 l. On 07121103 CBSU hosted th e weekJy "sc ience group" m eeling al QT. Additional di scussion regarding the tentative identification of Bacillus licheniformis from the first sel o f mailings was discussed. A trip is plannoo o n 7/30/U3 10 NOVOZY.' 1ES BlOT EC H~OLOGlES. Davis, CuJiforni ~ for co nfirm:JliOIl of th e identification of th e isolate. SSA I ~onducted a site visit with \VFO personnel at NOVOZ)111es, Inc., Davis CA on 07110/03. Novozymes will conduct tests to verify tb e tentative finding of Bacillus liche"iformi5 on 7130/03 - 8/03/03. Funding for these initial tests has been obl.lIiucd. If the tentative identification is confirmed, Novozymes will pcrfonn genetic characterization of the isolate. Additionally. consideration is being given to sequencing the isolate at TIGR . b6 b7C II . C BSU h os t~d a meeting on 6126/03 with Dugway Proving Ground (DPG). WFO, HMRU. Ft. Detrick and CRSU personnel to finalize details of the anthrax production cffort at DPG. OPG develo~d a slightly revised plan and submittcd it for approval. That plan has been reviewed and it was dctennincd that the plan does no t contain the level of detail requested at a meeting fommunicntcd this information to nPG on 6/27/03. conducted on 6126103. 1 III. Atomic Force MicroscollY (AFM): During the science group meeting held on 061, 6103 a r esent3tion was given on AFM. During SSA WFO, it was agreed that CBSU sho ul d subscquent discussions between prepare a written assessment of the value of AFM to the AMERlTHRAX investigation. CBSU is working with Sant.lia National Labor-nary to draft an assessment oflne potenlial value of AF~ in the examination of spores. ue l POd No response from Sandia National Laboratory to date. IV. Mill: The Stt!rilitc was returned to WFO. ,,' G[EXWSR B3D1.pdf  102  009592Anthrax Page 105 of 643 Submission to the FBI Lab for Latent Fingerprint examination pending. bo v. Isotopic Analyses: h7C Samples from Terry Abshire's (USAMRJID) collection have bet:n analyzed by the Univ. of Utah . h as conducted an initial re view of the data with Dr. Ehleringcr. CBSU anticipates a fina l analysis urthe data wi thin days. I The Woods Hole Oceanographic Institute estimates completion of requested carbon?14 analyses in September, 2003 . CBSU has contacted WHOI 10 request more expedi tious analysis. Addit ionally, casu has requested that LLNL provide eXJXditious analytical services if necessary. VI. Bethesda Apartment : The Bethesda apartment was searched on 07/0 )/0]. NMRC received approximately 63 swabbings. whieh are being tested for anthrax, plague and Bacillus hchenijormis. All samples were negative for BA and PCR negative for plague (culturing continues). No Bacillus lichenijormis has been identified to date, although screenmg continues at NMRC. VII . AM I Eviden ce: C llSU has ordered the reljuired glove boxes, which are scheduled to arrive at USAMRIID on or about 7/30/03. AMI mail folders will be moved to the ChemicaJ T rans fer Facili ty (CTF), Edgewood. MD. fot processing. CDSU and HMRU will coordinate the construction of a new containment structure in (he CTF and will use the new glove boxes for additional containment. VIII . NAU I em, CBI has refined its SN P assay to detect the morph?B SN P at a level of 0 .5% The assay wi ll not be used to screen repository samples unti l TIOR exhausts the possibili ty of other SN Ps. COl wil l perform vali dation tests of thi s assay on materials from the letters. These material s must be del ivered to CBI from US AMRJID. This delivery will commence on or about 08iOll03 CAl IS cu rre nt ly developing screening assays tor the tandem duplil.:liuns n-ga rding muta tions on tbis scale. \Ve mu sr th erefore assess relalh'e a",'anlages o f peer review !lnd scie ntific consensus aga inst the damage of d isclosure - l\1W X. FBr Laboratory ICPMS Analysis: WFO has expressed concern about the fact that the Laboratory's ICP MS will not be install ed and on line for some time. CBSU has requested that the Chemistry Unit (CU) considcr using the ICPMS located in Building 12 to analyze samples prepared in the CU. X I. Culturing BA on various growth media: Dr. James Buraru; has di rected the c ulturing of BA on various types of growth media The purpose of this initiative is to dctennine whether or not the nutrient media contributes to the finding oft in, sil ica, and iron in the spore coat of the evidentiary materials. Samples have been grown in various commercial media are now be ing dried and prepared for irradiation. Following irradiatio n, the SOStl B h )K.aW mailings are Bacllllu. SUhiilt'i lud not 11a... llllfe IJch.m~f{)rmJ-", ~s " was indicated by initia l t ~<; t.;: . Thio; strain ofsuhtihs !ormF mllCOld t.:oIO!lie;: which are rypicatly no ! .;:een In Fubtliis. A 16S R:s'A sequencing of the ba~t eri..i ill." confirmed it 3S \"uhrrli ... Novozymes Biotechnologies Incorporated (NBI), Davis, California is currently performi ng a genetic characterizatio n of the iso late. Additionally. consideratio n is being given to full genome sequ encing the isolate at TIGR. n. CBSU hosted a meeting on 6126/03 with Dugway Proving Ground (DPG), WFO, I [MRU, Ft Detrick and casu personnel to finalize details of the antm-ax proollction effort a l DPG A revised SOW was received and accepted_ SAs ITom WFO visited DPG on 7/27?29/03 to relay the exact na ture of the studies that arc requi red I Imlvekd to DPG on 8/ 18;0.3 and prepared 4 qq 150mm NC'A' Sporul::iliof"! Medium (NS\1) plart!s. The plates were inoculated on 81::0./03 .:md will be harvested on Sat S:'23':01 I wi !1 a!so ~lrovide on?sitc oversight of the OP(; drying and grinJ ing processes. New b!ender cups. mortar and pestl::s ;;nd <'I .spr.edvac were o rdered I bC"? n l. A lom ic Force .\1icroscopy (AFM) : A It!chn ical assessment entit led "Celll\'lorphology and Membrane Characterizatio n using Scanning Probe M icroscopy: Capabilities. Current Understanding and Issues was received from Sandia National Labo ratories o n 7/29/03. M b7C Ld i~~ r-n t~ia t~e-b e t-,,?e e-nJmaterial from the first mailings (post and Brokaw, 91 18/0 1) and the second 7 ffe- e- - ~- -mailings (Oaschle and Leahy, 10/910 1) . This information could assisl in determining whether the spores recovered fro m AMl are more like the first or !;econd preparatio ns. A preliminary test on non.evi dentiary material was suggested by ( Hn order to validate the met hod. Sandia National Laboratory and the laboratory o~ I Loyola University in, Chicago are being co nsidered for the initial studies. The Lab Division considers the tec hnique 10 be patenti3l value. Ih lwe,?er Ihr method ha~ not bCCr. lt":{tcd fo r thi;:. purpo<;e Pre!imill.u y s.mdies ",:iiJ be requi ri:d "nO!" I HMRU, revie>ved the assessment and feel s that the technique may be able 10 or G[EXWSR B3D1.pdf  117  009607Anthrax Page 120 of 643 R/2 1/2003 It') ?\FM appli:alion to {hI! c'v,dencc material which ",:!li he;in to \'alidulc: the ,echtliqu~ for li:->e Ih:L1; >;lIgges!ed a ~el I)f initial experiments 1)0 bo b7C thl' evidence: 2 G[EXWSR B3D1.pdf  118  009608Anthrax Page 121 of 643 8/21 /200) J Iso top ic A n a l ys~s: V. University of Uwh: A group of 19 (Abshire) samples was submiucd to the UniVClslty of Utah as validation samples fo r hydrogen and oxygen isotopic analysis Th~ samples were all grown and processed at USAMRlfD. Fo ur (4) samples contai ned iru.1Jfficient material for analysIs . Seven (7) sam ples clearly showed contamination due ( 0 the presence of other organic material. The remaining 8 samples provided result s that generally agree with the expected values. Additional ,,::unp!e mJ.terial w a ~ re.:e-i\\!d on S/:! 1 by UtJ.h fo r purification and anaJysi.; LJt",h has requC';; [cd a ::..ampil! of rence.al to prepare some surrogate.; to t;\o'aiuO:l.!e their c'(tractit)n l1focedurc: USA'v1R Iro d011hle di s~il!cd deioniud laboralOry water. us:!d to grow the"e l:~\ores. b\!f'dn a 48 hour jtlcub:ltl~)n and equi!ibr.l(ion on 8il I') and i~ expected to he anl'llyzed on Sn.2" The previ()Us water samplet: fm m Frederick. ;..I sample \,,"heel and will he ;".!.nalyzed ll V. Bet hesda A pa,-Imenl: T he Octhesda apartmt!nt was searched o n 07/0 1103" NMR C received appro) from \1PH and Jelivered Ihem II) Nr" IHC NMR C wi ll be tasked with growing these samples as assessing whether o r not they contaill the morphological variants observed in the Leahy. Post, and Daschle Idlers. This testing will replace the planned swabbing of lne AMI mail folders, which was designed 10 address the same question V il . NAU I C Ol: CSI has refined its SNP assay to detect the morph-B SNP at a level o rO.5%. T he assay will nOl be used 10 screen repository samples until TIGR exhausts the possibi lity of o ther SNPs. e BI wi ll perfonn validation tests o f this assay 0 0 materials from the letters. These materials must be delivered to CDt from USAMRHD. Thi s delivery will commence on o r about 08/0510 3 CB I is currently developing screening assays fo r the tandem duplicat ion discovered by T1GR in the morph A samples. Their approach is independent from the approach taken by TIGR. NAU is independently develo ping another assay for the mo rph-B SNP using mo lecu lar beacons . Preliminary information suggests that the sensitivity and specificity of this test is comparable to CB I's assay. A SOP is currently being written. NAU \,riil be provided the same set of validat io n samples that were provided to CBI to assess the level o f sensitivity of the assay, and determine whether the test is sufficient ly sensltive to detect the presence of the morph-O SNP directly fro m the evidentiary material fO VafiJ,HIOI1 of::he CAr Morp h-B SN1' .lS::ay i;: ex-peeled by the end of AugU);f Plannin:: i" lJllden\.ay move appropriate reposit(\ry samples [,1 eEl follo;.....;!~S the vdljda;:JOn o f the \'lorph-B S \:P [!~~.ly VllI. T1G R: " meeting Wit h TI GR (Ook place on 07/28/03 . TIGR has completed the entire DNA sequence of the Ancestral Ames iso late. as well as the mo rph 0 isolate from the Post. The Ancestral Ames strai n will now serve as the reference strain for futu re comparisons . The only difference found between the two isolates is Ihe pre?!iously identifi ed SNP (morph B SNP at positio n 95078) The nex t sample to sequence is a third morphological variant, called LL6 . T his vari ant is distinguished from the wild type, as well as the first two morphological variants, so-c alled morph A and mo rph B. This variant was discovered in and isolated from the Lea hy letter by G[EXWSR B3D1.pdf  120  009610Anthrax Page 123 of 643 8/2 1/200) microbiologists II I USA..fvlRJlD . TIGR has initialed development of an assay to screen for the prest!J\cc oflhe tandem repeat found in morph A in all the identJficd locations obscl ved in the letters Once completed, they wi ll screen all the available strains of BA?Ames, as well as other, non-Ames stf:l.i ns, for the pre sence of this pattern in the genome. Similarly. they will screen all available strains for the presence o f a SN P discovered in the Post morph A sam ple (but not the ot her morph A samples) . These analyses wi ll provide information regarding the rarity oflhese markers. TIGR has a set of 10 samples corresponding to the ancestral Ames strain aner passage through ten generations. This study was conducted at Dugway Proving Ground and included fundi ng from the FBI. These samples will be tested for the prl!Sence of the morph A duplication, as well itS the morph A (Post) and mo rph B SNP. This information will hel p establish the rari ty o f these mutations . TIGR will contact Paul Keim's laboratory at NAU and provide the information necessary for u sing SNAPSHOT tech nology to confirm t he presence of the Morph Band M o rph A SNP in all the strains mentio ned above. lX . FBI Laboratory I e PMS Analysis: T he Chemistry Unit ICPMS laboratory has been completed and installation of the instrument is nea rly (.;ompiete. It is anticipated that the t race metal analysis on the 19 Abshire samples will ue initiated during the week of !oIi:'l7-22, barring any installation a nd valida tion issues with lhe new rCPMS . A set ('>fwaler ::ampl e~ ffl)m USAJ\.? RIl D is also :;ch ~dlllt- to be analyzed. 1 X. C ulturing UA on various g rowth media: Dr. James Burans has dIrected the culturing of BA on lwe!ve types o f gro\\1h med ia Tht! purpose aflhis inttiative is to determine whether or not the nutnent media contribu tes to the findin g o f tin, silica, and iron in the spore coat of the evidentiary matl;!l iars. The plate .;ml nts :lfe .;ompleted M!t.! the ..:;p ,m~~ hav? been harvcsted The plate: C(lunts ;lV{'rageJ .o I 75)( I 01~ Cfu:'1'1! Oil 8114, Dr Bu ran~ h ega ~) the 'illiHdfllpling. He expe.::LS to ;,end rhe male.rial [0 SandiJ NatiOl131 Lab~ I SNL) tn 7- 10 day~ tor $EM.TEM anal),si:; ar;J to the FB r Lahoratory for tr[!c far, the opposite is {me. On ly a few percent of the spores in each sample show a 5i layer and it is often not as well defined or complete . The results thus far suggest that the evidence was not grown on NSM but definitive conclusio ns should wait until all the sampl es are anal)'7..ed and a complete report is received A briefupdatc with images was receivt:d on 7/3 1/03 (It is of interest 10 nOie that these Sandia mapping techniques were recently awarded an IR&D Magazine Top 100 Award, a very pre.<;tigious scientific and indus tria l award.) ~andil'l e'(pect.; ,;ompleul)n or [J1e : estimates a..Ild spe.:ilic mil~s[ nne.; . .F:nan ce Di\.'ision has sent all RFP to the labs for the requi red ioforma.tion. allowin.!? only live Jays h1r re<;ponse. X IV. lli fTerc nti a ll)ltenot),pic Colo ny E:tpression 6 '" G[EXWSR B3D1.pdf  122  009612Anthrax Page 125 of 643 S(~Si:!I)03 AMERlTllRAX Fo rensic Science Upd :l.t e 08i! i1!U03 Du gW:l)' Pr'oving Gro und SAs from \?FO visited DPG on 11'17?29103 to relay the exact nature of the studies that are required I \traveled to DPG on 8/18/03 and prepared 499 I)Ornm New Sporulalion MedIUm (NS\'I) plates. The plates were inoculated on Sl20/03 and harvested on Sat 8/23/0; Three hundred and thirteen (313) plates produced 45 grams o f SA paste. The i!.riditional 153 NSM plates were inoculated on 8/13/03 The yield from these plates. harvested on 8116/03. was 24 8 grams The total to date is 15.8 grams of paste from NS M. Two hundred and seventy four (274) plates ofKSM were poured on 8125103 and were Inoculated on 8/27 /03 These plates of are to be harvested on 8130103 The drYing phase of the project that was tentatively scheduled to begm on 8/26/03. will not begIn o n the c'iC H1vCRU has scheduled the G5 transport plane to LiSA.ivlRJ ID for irradiation rO t Q118/0J , in order tn transfer the malenal Ato mi c Force i\'li..:rosco p y (AFM): . .\ techmcal assessment entitled "Cell r-.. l u rp holo~y and Membrane Characterizatio n U';;ing Scnnnlrl\; Probe Microscopy Capabilities. Current Understanding and Issues" was receIved from SilllJi<.1 N,l liolldl Laboratories on 7i29/03 I I Hrw ?RU. reviewed Iht! asscssmc:J.t and feels thal the techniaue In :,C,Ible to ditfercnUJte between matcnal from the tirst G[EXWSR B3D1.pdf  123  009613Anthrax Page 126 of 643 noted however, the met hod has nO l been tested for t!'li s purpose Preliminary studies ..... 111 be requ iled prior to AFM application to me eVidence matenaL Ihas suggested a set o f :nitial experiments wilich will begin to v"lidate the techr~que for use o n the evidence I bo b7c Iso top ic An:llyses : UIf/\'l!/",mvof (jtah: J\ group of 19 (Abshire) samples was submitted to the University of Utah as validatio n samples for hydrogen aod oxygen isotopic analysis rhe samp!es were all gro\\' n and processed at USAl\;OOID , Four (4) samples contained insufficient matenal fo r ; malysis Seven (7) samples clearly showed comammation due to the prese:1ce or other organic material , The remaining 8 samples provided results that generally agree with the expected values, Addit ional sample material was received on 8/21 by Utah fo r purificatio n and analysis USANIRIID double distilled dei onized laboratory water, used to grow these spores, began a 48 hour incubation alld equilibration on 8/ 19 and is expected to be analyzed or: 8/22_ The previous wau~ r samples from Frederick, MD were city ar.d well water. Utah has requested a sample of renocal to prepare some surrogates to evaluate their extraction procedurc_The extractio n procedure must be tested prior to using it on tile remaming Abshire sample!> CBSU opines that the sa mples o f SA Ames grown on 12 types of media at USAMR1ID by Dr Burans would represent better \'aJidat ion samples Since :he evidence does not contam rcnocal developing a rcnocal cleanup procedure would nOI prove 10 be useful A .~et of the Rur1[(:, to provide: A TSDR personnel ....... jth the computer ha rd drive containing the mappmg dat:!. and :0 disOlSS 3dc!itio na! details fer the project NA U I CBr, CR I has retined its SNP assay to detect tbt: morph-B SWat a level of 0 5% The assay will not be used to screen repository samples until TIGR exhausts the pOSSibility of other SNPs C81 wil[ perform va lidation tests of this assay on materials from the letlecs. These materials must be del ivered to eB I from U5ANOOtD . Th!s delivery will corrunence o n or ilbout 08/05/03 em is currently developing screening assays for [he tandem duplicatlo;} discovered by T1GR in the mo rph A samples. Their app roach is independent from the approach laken by T1GR. NAU is independently d eveloping another assay to r the morph-B SNP usi ng molecular beacons Prelimmary info rmation suggests that the sensitivity and speci ficity of this test IS comparable to CB I's assay. A SOP is currently being written. NAU will be provided Ihe same ::.et o f valid ation samples that were proVlded to em to assess the level of sensitiVity of the assay and determi ne whether the test is suffi ciently senSitive to deteCllhe presence o f the morph-B SNP directly fro m the evid~ n tia ry ma terial Plnn ning IS underway to move appropriate repository sa mples to C D! followi ng the validati on o r the !'vlorph-B SNP assay (131 has contiJln!!d the presence ort he morph [3 SNP in cells derived from the Leahy Th~refcre, they hl ... e centirmed that the mcq:h 9 S}(P c:m be detected d irec: l)" from .hl! e'lidentiary material withoLit the need tor Isolation and punfication of the morph B van ant. They .Ire line-tuning Ihe dSs..1.y to incfe::J.se the senSitivity. :mt.l t::'(pect:o:'e rcady for a validat IOn t!\.,,'TC ISI! by the end o f September, 2003 t''.),... dc!- rS I has .llso .:::o ntirmed th~ prescnce of the ma rph A sequence{s) m onc of tile ..amp It:::. Ii-LIm wh ich It IS dcm ed {Leahyl They are currently deslgnmg DNA assays lu test tor the presence I.l l ,my o t lhc patterns so far discovered. (\1r1CUrrclltly, they are co nl irming the presence oCthe morph .-\ pauern m the other eVidentiary materials ~s disco ....ercd at T IGR Additional matem.ls co matning mOl ~ h A variants Will be fo rwarded [0 CRI from USAMR1 ID du ring the \ ~eek llf 91S/03 G[EXWSR B3D1.pdf  125  009615Anthrax Page 128 of 643 8/}3n003 T IGR: .-\ meeting ?),.'\th TIGR took place on 07/28/0 3 TIGR has comoleted the entire DNA .ie:qu;::n.;e uf the AJ:ct:::.trdi A. . nes isointe, as weB as thl:: morph B Isolate from the POSl. The ?\nces!rai Ames slr:u!'. !I! no w serve as :he reference strain for future compagc through ten generations ThIS study was conducted at Dugway Proving Ground and included funding from the FB I. These samples will be tested for the presence o f the morph!\ duplicati o n. as well as the morph A (Post) and morph 13 SNP Thi s info rmation WIll help establish the rarit y o f these mutal10ns TIGR will conta.ct Pau l Keirn's laboratory at NA U and provide the tnformation fo r U:illlg S:-J .-\.PSHOT rechiic[ogy to . ::ontir:t1 the preseCKC: oJfth~ \Iorph B and M u rph .1. SNP til all the .iIr:1ins menti oned J.bove nec~ s sal!' The ~\) ll t ~llllll1dnt round tn [he eVld~nua.ry material Will be :iubmitted 10 T1GR tor genetic sequenclIIg rl3r LahOl':ltol'Y ICP(\ IS .-\.1\;\I)'s is: rile ChC:lll islrY utlit ICP~ I S labocJLOry has been compieted ami m~t.lll:llion of the 1I1stnlllleill is Ilt!:!ctv complet e Borh ofr he FB I's lCPMS instruments Me ~xpected to be \J p~rJ tiona l by ~);: : 2003 Tht: CSAlvlRllD water samples. and a set of I:: samples grown by Dr Hurans. tlnder :1 v:'(oely of ':l.lItun ng .::ondttlons. Will be ill1alyzed by- the FB I .Anothe r sc t of 500 ~ rowth media sJmples wt ll be a nal ~' zed by a cont ract lab. J G[EXWSR B3D1.pdf  126  009616Anthrax Page 129 of 643 S!2SJ:20:)j OTHER FBl LAB .\ NALYS IS The Pond ":\' .:ontamcr <:nd tts relaid Ite ms have b~ tn e:~amm ed by the Tra..:t: Evidence Chemm rl as well as latcnr fingerprin t Units with negattve r:sults_ C ulturin g ijA 0 11 various g ro wth med ia : Dr James Burans has directed the culturing o f BA on twelve typt:s of growth media. The purpose of thiS iruu3! ive is to determine whether or not the nutrient media contnbutes to the fi nding o f tin. silica. and iron 10 the spore coat of the evident iary materials The plate counts are completed and the spores have been harvested. The plate counts averaged - 1. 7S x l Olll cfu/mi. On 3/14, Dr Burans began the sub-sampling The samples are being irradiated at USAMRlID They wllIlhen be sent to Sandia National Labs (SN"'L) fo r SEytrfEM analys is, Univers ity of Utah :or Isotopic analysis and to the FBI Laboratory for trace metals analysis. The results Will be com pareJ to those to und in the eVidentiary powders. Elemen t,, 1 i bpp iflg " A total of ten ( 10) of the 19 Abshire samples. a ll grown on New Sporu lation Media (NSMi. have been analyzed uSing TEM elemental marri ng techniques. In genera l, these 6 s-ost letter is unique and not found in the ongi~al A,ncs stram of OA isolated and scm to USAMRll D in 198 1. The samples used were the 1981 Ames aga r slant and Ihe Ames derived from the Leahy Len er spore powder Screening the 198 1 Ames sample will determine whether or not the phenotypic colony expressions exists. This project i~ expected to be completed in 2 weeks, however, prelimmary results indicate that the original 198 1 isolate of Ames is fr ee of the multiple morphology variams, while the mUltiple variants arc rcproducibly found m the Leahy Letter Ames. Thus, the morphology variation ident ified in the Leahy, Daschle and NY Pust Ames indicates multiple genetic variation nOI foun d in the original 198 1 Ames, and multiple potenti al forensic "signatures" . Battell e i\1 emor ial In stit ute (R M I): r---,S M] wi ll ident iry the species of the material in the flask which was co ll ected as evidence in = ., L,--,-~I.apanment . The flask is bel ieved to contain Bacillus thurmgiensis (BT). BMI will confirm the initial test results and providt: details about the specific strain of 8 r The results wi ll allow Investigators to compare thel ~ask: material to several commercial 8T products. The FB I Lab, Chemistry Unit \...?ill also evaluate any inert ingredients in the fla sk which would be expected to be mixed wi th the spore material, if it was a commercial horticultural product. AM[ Buildin g, Boca Ra ton, FL: BA Spore Sample.v: Repl esentatives from the Laboratory Division and WFO visited the Miami Publ ic Health Laboratory (MPHL) on 7111 /03 . It was decided that samples from the AM] building in the possession or the MPHL are adequate to test for the presence ofthe colony morphologies fo und in the materials from the letters. HMRU has obtai ned the samples from MPH and delivered them to NMRC. Select samples with high co lony count s will be transported to USAMR1I D and assessed whether or not they contai n the mo rphological variants observed in the Leahy, Post, and Daschle letters 3-D Mapping: Preparation of a secure area for the three-dimensional mapping project is currently underway al the Agency for Toxic Sub stances and Di sease Registry (ATSDR) in Atlant a. A site visit by AMX-2 and HM.RU personnel is anticipated fo r mid to late October to ensure the security of the site, (0 provide ATSDR personnel with the computer hard drive containing the mapping data and to di scuss ad ditional details for the project The goal of this project is to detenn ine the pri mary source and rclease of anthracis spores in the NvO Bu ilding. These mappmg results will also assi st III corroborati on of witness accounts. 9/24/01 G[EXWSR B3D1.pdf  140  009630Anthrax Page 143 of 643 AIVIEruTf] ~,iX __ Forensic Sd ence VpLl : tt l 10/03/ 200J Is olOllic Allalyses: Umllf!r.nl\l of {flah' The overall goal of the isotopic conchment studies 15 to aHcmpt to link the hydrogen and oxygen isotopic ral ios in the evidence spures 10 a likel\' source of water and 'or gro'''1h medIa which was used to grow the evidence spores. Since only 28% of ,he hydrogen In spores comes from the waler, tht:: tulturc media may make a sigmflcam difference in the isotopic results. casu opines, and Utah agrees, that the sam ples of BA Ames grown on 12 types o f media at USA.V[RlfO by Dr. Burans would represent good validation samples. A second issue to be resolved is thaI the predictive models currently used are based upon B. """hl/lis rather than B. allfhraci .. It is expected that there would not be a sign ificant diITercnee in the curves bul it has yet 10 be tcsted. Wllh answers to these 2 questions iOlerprelation orthe evidentiary results will be more meaningful. TIH: UniYel'sily ofI.Jrah ,,,,ill begin loe analyst:> [lrOtilld the wed.. . of 11)/6 Results are e'(pect~d approximalely two weeks aner thlll nllS :,;un:=. laboratory has conducted a si!;nilkalll lim: o r irwc<;ti!!ation whi.::h :.:orrclarex a "pnrc'<; nitrogen siable isotope .;:ompn<;ilion "..v1 lh the nitrogen isotope ratins in it<; f;WWlh media \ validillinll st udy i" being Jlublished in the neYI fev," '.vecks It may be Vnssible l\} elCc\lllle a l'{Ju r.;;: ofgnw"lh medi" ~s a 1ll1lrient SOUTce for a s pecilic b"t...:h u(spon!:>!.",sed on Ihe nilrogen r:llio..; No nh e rn A r;Zo na University \ NAU) / Co mm o nwea lth Uio\og iC:l 1 Ius til lrl e (C nll: CHI has designed two probes to detect the presence of twQ of the morph A patterns wit hin the evidence . These assays are designed to detect morp h A variant -speciflc sequences without Ihe need for supp ression of the major type. T hey have tested these probes on {ile evidentiary material provided to them from thl! Leahy letter. They have confi rmed lhe presence ofbolh of the patterns in the evidcm.:e T his fi nding strongly suggests {hat the evidence contains mo re than a single lI10rph A va riant patt ern, and is in ract a mixture of many morph A varia nt s These assay have the advantage afnor requi ring suppression of the major type Additional material s containing fIIo rph A vanants \"111 be forwarded to CBI from USA..'vlRIlD NAil IS mdependently developmg assays for thl! morph-B SNP lIsing molecular beacon and prllTl!!r extens10n techmques Preliminary information suggests thaI the sen ",rivlty and specifiCity of this testIS com parahle to CBI's assay NAU W ill be provided the same set of valida tion samples that were provided to CBI to assess Ihe level orsensitivity of the assay, and determme whether the le~t IS sufficiently sensitive 10 det ecl lhe presence of the morph-B S rp directly rro m the evidenttary material. NAU wrll be provided with the identity ora newly discovered SNP in Ihe morph C sample 1' fo r conlirmaLioll. T his 51\1 was discovered by , 'lGR in the morph C genome. It IS currently Ihe o nly high quali ty difference tound in this morpholo~ical va riant (see TIG R U prl thai h;lV:': heen .;:niie;;rcd fix Lile Sl!ppiier:; u:;i ng L rCP-Erni;sinl1 inSHllmeJ1l . Tho;! ile ini!i:.lt study ,.".it! uernnma raLe the v~lriabiJilY from bal..:h (0 batch in the metii,l;uppi ied by a nWIHlfact urer. Two elements of interest in the evidentiary maten al, silicon (Si) and tin (Sn), present mt eresting analytical challenges at trace levels The Chemistry Un il is preparing an ana lytical plan 10 discuss samplt: preparation issues, instrument selection and expected analysis times . It IS expected that it \Yill take about I month to develop and validate the samp ling preparation and to analyze s of heme I,) the lilt.!!' Thn:e ddtlitivnill /iller m Pl"Op(,scd " change in solvo!lll strength to 20% :.Itet()n;lrilc. \-Vh ole Genome Amplifi c:lIi on Propo5<'1is ha,,~ ~en reviewed fro m three laboratories, Law rence: Livermore NatIOnal Lahonllory, the Un iversity of Cincinnati, and ISIS PhannaccUl icals. Statements of Work (SO W) have been drafted. and funding reque:steti Contracts wit h the 3 laboratories are being i s:>ucci A six4 month time fram e is specified in the SO W, LaboratOries will he t(lsked speci li ca!ly \vith development of a protocol to obtain typeable human DNA from minute amounts expected from handl ing paper similar to the evidentiary material. CBSU and WFO stafl' will be meet ing Ihe laborato nes In a series o f "kick off' '' meetings to imtiate the validation sludic:s. If the val idat ion studies a l t! successful, then one ofthc laboratories will ue selected to conduct the WGA tcchnique on the actual eVidence 4 OifTel'c n tb l Ph enotypic Colony Express ion On 9/4/03, W FO removed 2 BA Ames stocks thai have investigative and hlstnric..l1 significance to the ongolll?; invesugation that wili be plilted out from the repository These samples are the 198 I Ames and the Lea hr materral Screening the 198 1 Ames sample w1 11 determme whether or nOl the Ill phenotypic colony expressioll s exists The Leahy material W be used as it contrul for this project Th is project i~ expected to be completed in 2 weeks B:lIt cllc Memtwia / In Slitute 8M! will identify the SpeCies of the materia l in the flask which was collected as eVidence in r--'=~~part mcnt The flask is believed to contain Bacillus f}lIIr mgiellsls (BT) Rlv[l wi ll co nfirm the imtlal (e5t resu lts Clnd provide detaIls about the specific stram of BT The results will allow Ilivesugarors to compare the flask matt!lIal to several commercial BT products The FA! D b6 bi e G[EXWSR B3D1.pdf  143  009633Anthrax Page 146 of 643 I_ "['OfC!tOry. Chemistry VI :ill also evaluate any inen IIlgfl!ulem th\! l1ask wh ich wo u ld be c.-.:pecten In b.~ . 1111'1(~9 Wlln th~ spore m;ttcrl;t l. if il was n COlUlllt:Jcial hortlcullUlal prOdUCl" "' ."1 1 ? " idcnce: 8A Spore Samples ? Representatives from the L2boratory Division and WFO visited the Miami Publ ic Health Laborat ory (MPHL) on 7111 /03 It was decIded that samples from the /\.,.\{I bUlldmg In the possession ofl he MPHl.. are adeq uat e to test for the presence of tne colon) morphologies found in the materials from the letters. HMRU has obtai ned the samples fro m MP II and delivered them to \I?l.1RC. NMRC is be tasked with growing these samples and assessmg whether or not they contain the mOJphological variams observed 10 the Leahy, Post, ilOd Dasch!e letters 3-D t'vtappmg: Preparation ofa secure area fo r the three-dimensio nal mapping project IS currently under..vay at ATS DR in Atlanta. A Site visit by A1\1IX-2 and I [MRU personnel is anticipated for late October to ensure the security of the site. to provide ATSDR personnel with the compUl l!1 hard dlive contai ning the mapping data and to discuss additional detaIls fur tbe project G[EXWSR B3D1.pdf  144  009634Anthrax Page 147 of 643 A M EIHTIlRAX Fo rensic Sci ence Upd ate 10/1 012003 Iso top ic Analyses: Umversl/yof (Jirik The overall goal of the isotopic enrichment studies is to attempt to link the hydrogen and oxygen isotopic ratios In the evidence spores to a likely source of water andl or growth media which was used to g row the evidence spores. Since only 28% of the hydrogen in spores comes from the water, the cultu re med ia may make a signifi cant difference in the isotopic resu lts. o pines, and Utah agrees that the samp les of SA Ames grown on 12 types of media at USAMRJID by Dr. Burans wou ld represent good validation samples A second issue to be resolved is that the predictive mod els currcnlly nthracis. It is expected that there would nut be a used a re based u pon B. sub/ilis rather than 8 . G significant difference in the curves but it has yet 10 be tested. With answers to these 2 ques tions interpretation of the evidentiary results will be more meaningful. The University of Utah has begu n 1ht: analysis. Results arc expected approximately three to four weeks. casu This same lab has conducted a significant line o f invesl igatlOn which correlates a spore's Nitrogt:n stable isotope I;ompositio n and with the Nitrogen isotope ratios in its growth media A valid ati o n study is being published in the next few weeks. II may be possible to exclude a ?source of growth media as a nutrient source for a specific batch of spores based on the Nitrogen ratios T he Ins titute fOI"" G enomi c Research (TI C R): Random shotgun sequencing has been completed on LL6. a third type of morphological variant fo und in the evidentiary material (rnorph C). A si ngle high quality difference has been lound between this isolate and the maj or type. The change is fro m a G to A at positio n 2 13922 . This change interrupts a rending-fra me. creating a Sl Op codon within a known gene coding for a sensor histidine kinase. Preliminary screening resu lts from the another morphologil;a l variant, LL7, suggests that thi s same g ene is als.o effected in this sample. The LL7 (morph D) variant has a deletion o f 264 base pai rs in this :.ame gene relative 10 the major typc. If confi rmed , an assay to detect this variant will be developed by C BI . T IGR has agreed to perfo rm w hole genome sequencing o f the non-13A co ntaminant ( presumably Bacilllls sub/ilis). Thi s sample will be delivered to TI G R on or about 10/ 15/03 1\'OI"Ih enl A rizo ll a Ulli versil}, (NA U) I CO nllll o nwe:t lth Bi ologica linsfitlli e (C Ol) : C B I has designed two probes to detect the presence OflWO separate mo rph A patterns found in the evidence. T hese assays nrc desig ned to detect morph A varianl-spe(:ifi c sequences without the need for suppression of the major type. CB I has tested these probes o n the evidentiary mat erial provided to them and have confirmed the presence of both o f the patterns in the evidence This finding strongly suggests that Ihe evidt::lh..:t:: cont ains more than a si ng le Illorpn A variant patt ern , and is in faci a mixture o f many morph " variants. These assay have the G[EXWSR B3D1.pdf  145  009635Anthrax Page 148 of 643 advantage of not leljUi rir pression of the major Iype Addition A variants will be forwan.H"u [0 r.BI from I j ~AMR lrn erials contJ ining morph CD! is performing internal validation of the mOl ph A . b7C products. The FBI Lab, Chemistry Unit wIll abo evaluate any inert mgredients m the tlask which would be expected to be mixed with the spore material, ifit was a commercial horticultural product. AM I Ev idell ce: Represcntatives from the Laboratory Division and WFO visited the Miami Public Ilcalth I.aboral ory (MP HL) on 7/1 1/0 3. It was decided that samples from the Mfl bui lding in the possession of the YfPHL are adequate to test for the presence of the colony morphologies found In the materials from the letters HMRU has obtained the samples frolll tvtP H and delivered them to NMRC r-..J"tvlRC is be tasked with growing these samples and assessing whether o r not they cont ain the morphological variants observed in the Leahy, Post, and Daschle letters. 3-D Mapping: Preparation of a sccure area for the three-dimensional mappi ng project is currently underway at ATS DR in Allama. A site visit by AMX-2 and HMRU personnel is amicipated fo r mid to late September to ensure the security of the site, to provide ATS DR personnel with the computer hard dnve containing the mapping data and to discuss additional details fo r the projec!. o G[EXWSR B3D1.pdf  148  009638Anthrax Page 151 of 643 t\MF:RITHHAX Furensic SciellCC U(>d :He 10/3 1/ 2003 [SO lO pi c Ana lyses: [/tlll'ersltv or Ulah The overall goal of the isotopIC ennchment studies is to attempt to link the hydrogen and oxygen IsOtOPIC ratios In rhe eVldenc~ spo res to a likely source of water and/or gro\vth media which was lIsed to grow the eVldcnce spores. The University is completing the analysIs ufthe 12 samples grown by Capt Burans on different media This will assist in determining what effect the media has on the isotopic compositioll ill the spores since the same wat er was used. These samp les will be followe d up by a g roup of samples produced al Dugway Results from the Buran)' samples are expected nc.'(1 early week has begun background literature searches on other ~tab l e isotopes that may be of assistance. These include Magnesium (Mg), Silicon (Si), Calcium Ca). Iron (Fe) . Zn (Zinc). in identifying additional and Tin (Sn). In addition, casu is aSSisti ng SA sporulatio n media fo rmulations for consideration T he Institute ror Genomic Research (TIGR): Preliminary screening resu lts from the another morphological variant, LL7, suggests that this same gene is also effected in this sample The LL7 (morph D) variant has a deletion of264 bas~ pairs in this same gene relative to the major type If confirmed, an assay to detect this vanant Wilt be devcloped by CO L TiGR has agreed to perform whole geno me sequencing or the llun- BA contaminant (presumably Baed/lis .wlb/dis) Upon receipt of sufficient genomic DNA, T IGR wi!! begin JLbrary constructi on casu b. bie fo r sequenci ng. No rth cnl t\1"i"Zona U niver~ it y (NAU) / CO llllll on wea l1h Bio logic:l1 In stitut e (CB I): C13I has designed two probes to detect the presence of t\\'o separate morp h A patterns found in the evidence . These assays are designed to detect morph A valiant-specific sequences wit hout the need for suppressio n orthe major Iype eBI has tested thes.e probes o n the eyident inry material provided 10 them and have confirmed the presence of bo th ort he patterns in the evidence This finding strongly suggests that the evidence cumains more than a single morph A variant pall ern, and is in fact a mi:\."ture of many morph A variants These assays have the > CBl presented time course testing to demonstrate the optimum time for cul ture g rowth of evi dentiary samples to eliminate potential loss of morph A signal. 06 iJ te 55AI Imet wit h CB Ton 12103IOJ to review the SOP drafts for the torph A assays. CB I anticipates readiness to proceed with assay erificatio n on 1_/0 /OJ Pend ing satisfactory co mpletion of the veri fication test, CBI anticipates beginnin::> the blind valida ion test on 12/1 0/03 A ~I-:-:-_ ......j holds the key to the blind validation sal pies that have been delivered to CB L AnalYSIS of the evidence reposilolY ~alllples \ ill begin following FB[ confirmation of blind validatiu n le~IS at BI and pending FB [ approval o f the evidence analysis plan ......:--__ G[EXWSR B3D1.pdf  157  009647Anthrax Page 160 of 643 _._---_._ _._ .. Isotopic Ana lysis The University of Utah, Stable Isotope Laboratory is conducting a set of experiments with the overall goal to link the hydrogcn and oxygen isotopic ratios in the evidencc spores to a putative source of water andlor growth media. Thc results from 12 spore samp lcs grown on dificrcnt mcdia by Capt. Burans, Dcpt. ofHomcland Securi ty, and at Dugway by FBI persoIU1cl will assist in detennining what effect the growth media and water source has o n the isotopic composition in the spores. Upon complction of this line o f cxperiments, the examination of the evidence could be expected by mid? January, 2004. The Utah Isotope laboratory will be continuing with other val idation studies to further clarify the interpretation of this exami nation. -Ibc laboratory h conc luded the instrumental examination of the Burans and Dugway samples, and is now conducting final calculations prior to submitting a reIX)f1 to the fBI. lbe Utah Isotope Labo ratory is a lso finishing a set of experiments designed to demonstrate any differences in water metabolism between Bsubtilis and B. an fhracis. Whole Genome Amplification (WGA) Lawrence Livermore Nat ional Laboratory, the Un ive rsity of Cincinnati , and ISIS Pharmaceu ticals have begun indcpendent approaches to altempt to use WGA to amplify human DNA which may be present o n the ev idence letters. Eac h laboratory is tasked with devdoping a prol experience, knowledge, and expertise in developing molecular markers wi ll be used in this and ot her Amerithrax efforts_ Oth er C ultu re Prt p:n ,ttio ns CBSU and WFO arc continuing researching to find other growth media preparations which may have been used by the IIIdIViduaJ(s) in the Amerithrax attack . CBSU is arranging for the u!>t! of a BL?) suite (0 conduct additional growth experiments using a new series of potentiai growth media. A series of experi mnets using 5 different media, with and with out ant i-foam has been proposed . The growt h experiments should begin in the next few weeks at Edgewood Chemical and Biological Center. B3D1.pdf G[EXWSR  166 .'  009656Anthrax Page 169 of 643 ISO lopi c A na lnili Goa l: To determi ne the geographic region of production o f the evidence through the use of Hydrogen and Oxygen stable isotope ratios. and to iciclltify the class of media through Carbon ll and Nitroge n analysis. I'rug ress: Isotopic analysis ofco ntrol!ed samples grown on different media has been co mpleted. The resu lts are consistent wi th expectations. (See fo otnote I) Isotopic analYSIs o f2 0 domestic water samples has been completed T hirty (30) international water samples arc pending analysi,> Timeli n e: IsotopIC anal ysis or the Leahy evidence is scheduled fo r next week. 1121-2:;/04 H cme Ana lvs is Goa l: To determine whether or not heme was present in the g rowth media. Prog ress: Assays for heme detectio n have been dcvelo lJt:d . Final validat ion o f the extractio n and analytical method IS underway Tililc lin c: If the final heme validatiun study is successtl.JI. the New York Post eVidence will be nnalyzcd during the week of 1/ 19-23 /04 rB I Labnral On' Ic r AII :l I\'sis Goa l: T n delelllline the growth conditions used by the suspecr(s) by ,ma lyzmg the elemental pro file o f the cvidencc. typical growth media. and spores g rowli under controlled lest condi tions. Prog ress : The FBI Lab htls tes ted several sets of B .(llllllracl.'? (SA) samples whic h were glown o n agar, using a va riety of co nditions (2 ). r hese experimental sampl es do nO{ have a n elemental profile co nsistent with the eVidence. The experl mellts have prOVided info rmation to suggest o ther types o f g rowth merl ia may have bee n used [0 grow the evidence These resu lts. alo ng with the S EM ti ndi ngs. shou ld determine whether o r nOt [he pelpctrmor used one o rthe w hure ,111 {hying me:hods that were repiicat~d d con tmue to co nduct trace metal analysis on selected growth IIk'llLa. A to pnOlltl4t the remamlO3 sampics ilnd to rCVlew rc~ult5 alld cnmpansiollS among the media types Anether groWTh e: of selected samples of each growth expcnment. It does nm appear that all o r l he submitted samples require the full 8-hour analYSIS (3) T he ~amples arc bemg prioritized to allow for the qUIck lurn-arou nd of spo re preparations from the next growt h experiments. Wh ole Genome Amplification (WGA) Goa l: To tl clermine whether trace amounts of human DNA may be present on the leiters. I'rog r?ess: T hree contract laooratories arc performing studies on amplification o f trace amounts of DNA from human skin cells. A dilution series o f human DNA was prepared and senl 10 all th ree contract laboralories Ti lli e line : Studies .~ho ll i d be compleled by May, 2004 . At that time II wi ll be decided whieh WGA procedure should be applied to Ihe envelopes used in the mailings . ll iffHI' lIlial Pheno ty pic Co lonv [ .t pression Co al : To observe the evidenlU"lt)' morphological variants in cullures of l ht! repositury samples . IJrogn?ss: Selelll lSIS from USA1VlRl lD and NMRC have collaborated on a procedure and are contlnulOg to retine a standardized procedure lor morphological screemng oflhe reposllory Three scientists: frolll 10 assist In this effo rl An addilionallCcilnicmn from NrvLRC has been tht: proJect. Fou r vanant s identified at NMRC have been Iransponed (0 USAMR IID tor observation and characteriz."lIIOn New variants will be S to T IGR lor whole genome sequencIng ClII USAM RIID have been identified add~d to Time line: Screemng should commence in February. 2004 and last approximarely thrt:e months Gene tic C hal?acIUi1:3 Iioli of morphologic:11 \':lI?ianlS Goa l: To idenufy the source of Ames stmin BA usetl in the leners S I: lllI S: Imerna! validation of three ('-13 and N-IS prelimmary resulTs <11< ClHlslste1l1 with the use of a C-3 plant/anllnal tvpe medin.. the most likely or C0l1111101'1 type being so\" based The C -I \ \ ;J!ue IS idenllcalto the value measured dt WHOt as rart ()rtlH.: ..:arbon tlall'lg TimcJin c: I"'Llt:t:~" UClta fo r the O-IR and H- 2 isotopes are expectcd by 1/ 15/().1 The isOtopic analysis of the thiny b6 b7C mternallunal water s.1Inples is also currently in prog:rcss, lI e n~~ A n!l lvsis Ilo t Goa l: To determine \'< hether or heme was present in the growth mcdill . l' nI2"ess: Assays lor heme detection IlaV~ b~n de, ~'isi f cd LLN L and IBIS ill CalitorTII:l du ring wee" of ! Cg - ~91O-l P,dimil1<1 ry result s IIldiCs nllh' ,11l1phlied at LL \J I .o1IIU I B[$ . IBIS is try to do D NA repair 0 11 Irradiated cells ~um::llt l \ \.-lsiting U or Cillcinnati to gel a techmc<.1 Jpdate I b-5 b7c I Is Time linc: Studi!!s shuuld he completed by the ~nd offl~COO4 At that lime 11 \\?ill he dtx:lded wllll..:h \VG:\ prcceLi urt! should be L - I_ - - - - ' ~.!.l-'1A .!..",:-. 1.").1.. -, I U. \'I\.~' :':') rogress: Com monwealth Biotechnologics. Inc. (CBI), Richmond, VA has concluded cxteillal validatio n o f three molecular assays targeting morphological variant s A I, A2 , and A3 . The A I and A3 assays exhibit robust hehflVior when tested against cells from the New York Post, Leahy, and Daschle letters. The ex ternal bli nd validation test results were sat isfactory for assays Al and A3 . Emergency Co nlr(l CI Review Board approval granted on 03/04/04. A plan for Iranspol1ing the Repository to CB I on 03110/04 is in place Ti me lin e: Co ntract]s expected to be fin alized by 3/10/04 Screening o Cthe repository fo r the A 1 and 1\3 va riants is expecten ]0 comm ence Ihereafu.!1" and end in 06/2004. Genetic Charar.tcriultion oft h ~ Aarilltl'i Contaminant Fo u nd in New York ~ I ni li llgs Goa l: To full y characterize the BacjfJlIs .whl ilis co ntaminant found in the evidencc. Progress: DN A scquencmg of the evidentialY matenalls in progress at T IGR Preliminary sequence IIIfo rmatio n has identifi ed numerous genetic differen ces netween an eVidentiary contaminant and th::: H ,"/lbMIS 16K reference stra in. Targeting DNA assays to these genetic difference ~ may result in a fingerprint assay for the isolate identified III the evidence. (fsuccessful, this approach may provide informatio n Ihal will identify the o rigin oftne contallllnaling st rain recovered from the New York mailings. CBSU is developing addi tional gcnotyping assays for strain identification and differentiation of B. .Y llh/dIS isolates Ti me line: Assembly of the genome seq uence and compariso n to the publ ished sequence IS due o n Assay devdopmcm at the FBI Laboratory is III progress (02/2004) and will continu e until sllmcient genetic resolution is obtained 03/05104 .. G[EXWSR B3D1.pdf  193  009683Anthrax Page 196 of 643 JlMXWeelct ufmurphologrcal variants discovered in Ih~lr The-sc \il,nlgllt:d " . G[EXWSR B3D1.pdf  195  009685Anthrax Page 198 of 643 J AMX Weekly Science Update March 12, 2004 targelm3 thc.;c variable regions An SOP and budget for Repository <;cfcen ing ha~ been 1 t!Lei\'cd li'oll1 USA" IRI 1D Time line: Repos!tory screening for morphological variants oh~ervcd ..:ommen\:l.: m 03/:0(;4 and la5t appro",imately ili!i.:c mG!1th~ i tl :hc e\ tdenliary matena ls shou ld Genetic C h:lra crcl'iz:u ion of Morph ological Varia nts GO:II : T o Identity the source or Ames strain S A used in the letters Prog r ess: Commonwealth Biotechnologies, [nc (e81). Richmond, VA has concluded external validation of three molecular assays largcling mo rpholog.ical variants A I, A2. ane! A3 Two ass..-tys pJ.ssed t!'l:ternal bhnd validation Ames main smnpJes housed in the I{cPOSltory ill CSAMRI TD were tla1l!)1x)[1ed III CA l on 03/ 10/04 Tim e lin e; Contr2cI wi:h CS t was finalized on 310810?1. Screening 11f t ":': Repositury fo r the A I '-lnt! A} variants is expected (0 comm~ncc o n 03115/04 G('llctic Ch :lr:tc t el"iz:lIioll of th e Bacill us Co ut:lIl1 ill:lIIl fo und. in New " o d.: Mailings Goa l: To fully charact~nze the Bocil/If.\ .m!JIlIi.\? contaminant(s;) found In the New York maihngs Prog r ess: DNA sequencmg o rthe /j{u..:rfllf.\ suh,"is isol 'lte recoverccl flam Ihe 1'.' c\'..? Y(lrk Post Ictler \vas completed Oil 03109/04 T1GR. wi ll present lindings 10 FB I scientists on 03il l /04. Assays ....,[11 be dc\eJoped at TIGR and the FBI L2.boratory targeti ng areas of vanabiJ ity . Ifsuccesstul. this ;;!red from tht: :-.1L'\\. Y rk \l mailings. is developing additiona l gennt:ping assays for strain ident ificat ion and ditlcrcnti atioll of H. .II/hllh.\" isolates. casu Ti lli e liu t': As~ay development at tite FBI laboratory is III plogr~~) t021:00-') InlOrmatlo n tfuillthl! TIG R D NA sequence trOtn the evidentiary isolate \\.0111 be mle~rated mID t!1e ongoing project iO Id.:ntify variable target seC]l1enc~s G[EXWSR B3D1.pdf  196  009686Anthrax Page 199 of 643 , " Isotopic Analvsis Goal: To detcnninc the geographic region of production of the evidence through the usc of Hydrogen and Oxygen stable isOtope ratios, as well as identify the class of media through Carbon I} (C ?1 3) and Nitrogen IS (N15) analysis. Isotopic examination of the Leahy evidence has provided data which requires additional work in order to draw meaningful conclusions. Prugress : The analysis of 46 Legal wate r samples ha'i been complt:tt:d. As ex pected, the Oxygen and Hydrogen isotopic profile fit the mcteroic water line. The first phase or a series o[cxpcriments to dctennine ir D18 is isotopically enriched as a function of growth time has been completed at the BFAC. Determination of cfulgram and irradiat ion of sufficient sample for distribution has taken place and samples arc being prepared for shi pment. Protocols and interview questions for waler s.ampling (i ncoming, d istilled, laboratory and purified) arc being prepared for the 16 U.S. laboratories known to have the Ames strain of BA. Isotopic analysis on these samples will then be perfomlcd . Time lin e: Samples ofthc extended growth experiments conducted al the I3FAC wil l be prepared and distributed to Sandia NL and the Universi ty of Utah as blind validation samples for J Ierne analysis during the week of 03122/04. Heme Analysis Goa l: To dctemline "hether or not heme \" present in the growth media. ,'as Prog ress: nle heme detection assay developed at the University of Maryland was transfl:m:d to the U.S. Anny Chemical and Biological Forensic Analysis Cen ter, Edgewood. MD o n 0211712004. The performance of the a,>say wa,> val idated at the ECBFAC on 2/25104. Supplies have been received 10 condw':l a blind vali dm ion with samples produced at the 13FAC. Time line: A set of blind unknown samples, gcner.:Hed fro m the extended growth experiment at the BFAC, wi ll be analyzed prior to the analys is of the New York Post evidence. The est imated ti mc of complction fo r the New York Post evidence is the end of 0312004. t<: lementa l A nalvsis Goa l: To determine the growth cond itions used by the suspcct(s) hy analpiug the d l.:mcntal profi le of the evidence, typical growth media, and spores grown under controlled test conditions. , ' G[EXWSR B3D1.pdf  197  009687Anthrax Page 200 of 643 2 AMX Weekly Science Update Ma rch 25 , 2004 Progress: Sample analysis by ICP/OES of media components used to grow tC'it samplt:s at ora are complete. The analysis of24 prioritized samples from a collection of more than 500 media components are compl ete. It has been determined by the Chemistry Un it Ihallhe hydrofluoric acid used fo r sample d igestion is de leterio us to the quartz plasma torch creating a higb blank reading of sil icon. Therefore. the data collected measurin g silicon i suspect Alternative methods are currently being explored by the Chem istry Unit. T ime li ne: Alternati ve analytical methods for Si stich as graphLte furnace a nd name atomic absorption arc bei ng explored. Elemental Mapping Goa l: To provide high resolution SEM and TEM elemental mapping of spores. Progress: TEM grids conlaining four sterilized Ames samples grown at UPU in liquid culture \\li th a n anti ? foam are complete. i\nalysis of the 4 samples d id not show the Silicon around the spore coat as obse rved in the evidence. However. lht:: ami-foam product used at DPG to prepare these samples has been ddl!nnined not be a bEUR S ilicon-based product. hi(: Samples of a Hacillus species which first showed Si on the spore coat in the 1980 and 198 1 University of V A. A puhl icalions have been donated to the FBI Laboratory by co-author co llection o f 35 different spore prcparations from those early experi ments were acquired on 3/ 10/04. Additional University of Cambridge, histori ca l data on the actual spore production wil l be sought from l I I I U.K. Time lin e: Grids [or the appropriate samp les wi ll be prepared at USAM RI ID next week and shipped to Sand ia NL for analysis. Whole Ge n ome Am nlifica lion (WGAl Goa l: To determine whether trace amounts o f human DNA may be ext racted and typed from the evidentiary leners. 11rogr ess : Three contracting lahoratories art:: developing singlc-cell detection and amp lificatio n capabi lities. Prel im inary results indicate that an amount or unirradiated DNA equivalenllo that found in a single cell can be successfully amplified and typed . One contracting laboratory. IBIS. is attempting to perform DNA repair on irradiawd cell s. It appears that irradiation has a negati ve impact on the abil ity to amplify trace amounts of DNA rrom affected cell s. DNA repair studies conducted to date have had limited success. Time line: Stud ies sho uld be completed by the end of 05/2004. At that time it will be decided whi ch WG A proced ure should be applied to the evidentiary envelopes. Differe ntial Ph enot\' pic Co lony Expressio n Goa l: To observe the evidentiary morpho logical variants in cultures o f the repoSitory samples and to Corn;l(1lt: these p:J.nems with those observt=d in the evidentiary materials. G[EXWSR B3D1.pdf  198  009688Anthrax Page 201 of 643 3 AMX Weekly Science Update March 25 , 2004 Prog ress: NMRC has prepared a new set of morphologic.al variants discovered in their laboratory. These variants have been transported to USAMRJID for observation by technically competent scienti sts with ex perience identi fying variants from the evidentiary powders. SOP has been prepared for USAMRII D s(;icn tists to begin processing the Ames repository samples. Appropriately defined and characterized variants wi ll be se nt to TlGR fo r whole genome sequenci ng. Assays will be designed targeting these variable regions. Tim e line: Repository sc reening for morphological variants ubserved in the evidentiary materials should com mence in 03nOO4 and last approximately three months. Genet ic Charaete.-anti" " or Morphologica l Variants Coal: To identify the source of Ames str.lin SA used in the lellers. Progress: Commonwea lth Biotechnologies. Inc. (CSl). Richmond, VA has concluded extemal ....alidation of three molecular assays targeting morphological variants AI . A2, and A3. Two assays passed external bli nd validation. Ames strain samples housed in the Repository al USAM RII D were transported to CR I on 03110/04 . DNA has been extracted from approximately 200 Repository samples. Additional equipment required for screening has been installed. Time linc; Screening for morph A variants A I and A3 is expected to conclude hy 07n004. Genetic C haracteriza tion of th e Bacillus Co nta minant Fo und in New York Mailings Goa l: To fully characterize the Bacillus slIblilis contam i n an t (~) found in the Nt=w York mailings. Progress: DNA sequencing of the Bacillus subri/is iso laL recovered from the New York Post leiter was e completed on 03 /09/04. TIOR will present findin gs to FBI scientists o n 03/ 1 1/04. Mo re detailed report s listing DNA sequence differences will be provided to FD1 5(;icntists on 03n 6/04 . As~ays will be developed at TlGR and the FB I I..aooratol)? targeting areas of variabili ty. CBSU is developing additional genotyping assays for strain identi fication and differentiation of B. .rublilis isolates. Time line: Assay development at the FBI Laboratory i~ in progress tOm004). Infonnation from the T1GR DNA sequence fro m the evidentiary isolate will be integrated into the ongoing projcct to identity variah lc target sequences. G[EXWSR B3D1.pdf  199  009689Anthrax Page 202 of 643 - " AMX Weekly Science Update --~----~ ----.---~--------------------- -- April 2, 2004 i -- '" >--- Isotopic Analvsis Goa l: To dctennine lhe geographic region of production oflhe evidence through the use of Hydrogen and Oxygen stable isotope ratios. as well as identifY the class of media through Carbon u (C- 13) and Nitrogcn l5 (N\5) analysis. Isotopic exami nation of the Leahy evidence has provided data wh ich requires addit ional work in order to draw meaningful conclusions. Progr ess: A validat ion experiment was conducted at the BF AC to investi gate the relationship between longterm grO\V!h on agar p lates. the enrichment of the staning water and the isotopIC enrichment noticed in the spo re evidence. T ime lin c: Sam pl es of the above experim ent have been received at the Univer.:;i ty of Utah. Analysis is scheduled for next week. Heme An al )'s i ~ Goal: To determine whether or nOI heme was present in the growth media. Progress: The heme detection assay developed at the University of Mary land was transferred 10 the U.S. Army Chemical and Dialogical Forensic Ana lysis Center (EC B/FAC), Edgewood, MD on 02/ 17/2 004. The perfo rmance of the assay was val idated at the EeB/FAe on 2125/04. Slight improvements to the method were made by ECBfF AC T ime line: A set of bl ind unknown samples, generated from the extended grov.'lh experiment at the BF AC \\I?jll be used as validation study farthe ECB/FAC melhod. The extraction at BFAe and analysis at ECB/ FAC is planned for next week .. If the validation work appears ta be successful , then the Leahy evidence will be schedu led fo r analysis. Elementa l Ana lysis Goal : Ta determine the grawth canditions used by the suspect(s) by ana lY7ing the ekmental profi le of the evidence, typical gro\v1h media, and spores grown under cOnlrolicd test conditions. Progress: CBSU and Chemistry Unit met to discuss a series of validation studies to clarify the previously co llected data. The elemental 4uanrification will incl ude the use o f IC P/DES and Gr/AAS to provi de two G[EXWSR B3D1.pdf  200  009690Anthrax Page 203 of 643 2 AMX Weekly Science Update Apri l 2, 2004 approaches for the same measurement_ Uni t chiefs or CTFSRU, Chem istry and CI3SU, \vill be briefed on the proposed plans. Ti me line: Once initiated, the validation work is expected to be completed in 4 months. it is then cxpcc.:ted that the Leahy Evidence will be fe-examined. J<:lemencal Ma npin::: Goa l: To provide high resolut ion SEM and TEM elemental mapping of spores. Progress: TEM grids containing three samples of a Bacillus species which first showed Si within the spore coat in the 1980 and 1981 publications, have been forwarded to Sandia. Sandia is also running a sam ple be obtained froml Iof USAMRIID. b?r.; T ime line: Resu lts arc expected by the end of April. Whole Genom e Amplification (wGAl Coal: To determine whether trace amounts of human DNA may be extracted and typed from the evidentiary leners. Progress: Three contracting laboratorics arc developing single-cell detection and amplification capabilities. Preliminary results indicate that an amount of un irradiated DNA equivalent 10 that found in a single ce ll can be successfully amplified and ryped. One contracting laboratory. lBl f.!. is allcmpt ing to perform DNA repa ir on irrad iated cells. II a ppears that irradiation has a negative impact o n the ability to amplify trace amounts of IJi\A from affected cells. DNA repair st udies conducted to date have had limited Sliccess. Time line: Studies should be comp leted by the cnd of 05/2004. At that time it will be decided. which WGA procedure should be applied to the evidentiary envelopes. Differential Pb en otypic Co lonv Expression Go al : To observe the evidentiary morphological variants in cultures of me repository samples and to correlate these patterns with those observed in the evidentiary materials. Progress: An SO P and budget fo r Repository sl,;n::tning has been receivcd from USAMRl ID . Two additional tec hn icians have been assigned to this project. Equipment needs have becn included in the submitted budget. Time line: Repository screeni ng for morphological variants observed in Ihe evidentiary materials should commcnce within two weeks and last approximately three months . Ge netic C h aracterizatio n of Moqlholoeical VarianlS G[EXWSR B3D1.pdf  201  009691Anthrax Page 204 of 643 3 AMX Weekly Science Update April 2, 2004 Goal: To identify the source of Ames strain BA used in the letters. I}rogress : Ames stroin samples housed in the Repository at USAMRlID were transported to CBI on 03/10/04. Approximately 400 samples have been gro .....n and the 0 A has been extracted, quanti tated, nonna lizcd and frozen. Screening the DNA for the presence of the varianrs is due to commence on or about 04/0612004. Time lin c: Screeni ng of the morph A I and A3 \'arianlS is scheduled to proceed through 0612004 . C hara cterizatio n Qf th e Bacillus Co nta minant FOUlld in New York Mailin!!.s Coa l: To fu lly characterize the Bacillus subrilis contam inant(s) found in the New York mailings . Gl~ netic Prog ress : DNA sequencing of the Bacillus sub/i1is isolate recovered from the New York Post letter was completed on 03109/04. TIGR has presented these fi ndings to FBI scienlislS. DNA assays have been designed targeting variation observed between a reference strain and the evidentiary stram. CJ3SI J is developing additional genotyping assays for strain identification and differenl iation of B. slib/dis isolates. Dozens of strains of B. SUblili will be provided hy the U.S . Depan menl of Agriculture. These strains will be genetically characterized and compared 10 the evidentiary isolate. Time line: Assay development at the r Bl Laboratory is in progress. When sufficient resolution is obtained, large collections of B. sub/ilis will be screened using assays designed to target vari ation fou nd wi thin the evidence. G[EXWSR B3D1.pdf  202  009692Anthrax Page 205 of 643 Isotopic A nalvsis G oal : To determine the geographic region of production of the evidence through the usc of Hydrogen a nd Oxygen stable isotope rdtios. as well as identify the class of media through CarbonI} (C -13) and Nitrogen lS (N15) analysis, Isotopic examination of the Leahy evidence has provided data which requires additional work in order to draw meaningful conclusions, Ilrogn!ss: A validation experiment was conducted at the BFAC to investigate the relationship between longterm growth on agar plates. the enrichment o f the starting water and lhe isotopic I'llIichmcnt noticed in the spore evidence, Time line : Anal y~i~ of the samples of the above experiment are underway at the University of Utah, H eme Ana lvs is Goa l: To determ ine whether or not heme was present in the growth media , P rogr ess: The heme detection assay developed at the University of Mary land was transferred to Ihe U.S. Army Chemical and Bio logical r:orensic Analysis Center (EC B/FAC). Edgewood. MD on 02117/20 04 , The performance of the assay was val idated at the ECB/FAC on 2nS/04 , Slight improve ments to the met hod were made by ECB/FAC. Extraction and analysis o f these samplc=s has been coord inated with the BFAC and ECBlFAC. T ime line: 1\ set of blind unknown samples. generated from the extended growth experi ment at the BFAC were analyzed o n 4/ 12/04 . A report is expected in a few days., If the validation work appears to be successful. then the Leahy evidence will be schedu led for analysis, Elemental Mapping Goa l: To provide high resolution SEM and ,,'I':M elemental mapping of spores. b6 b"iC I Progress: Sandia has observed Si on the spore coat of 16 of 136 ( 12% ) spores exam ined in detail from the IsampJe I; 1030 This is a significant finding in that this is the first lime that the Si has ix"'Cn observed in samples other than the evidence. Sample 1030 is a mix of 13 separate productions of the Ames strain over a G[EXWSR B3D1.pdf  203  009693Anthrax Page 206 of 643 2 AMX Weekly Science Update April 22 , 2004 1 year penod fro m 12195?11196.. Sandia is do ing a statistical analysis of the c ... identiary samples fo r compar.:l.ti \Ie purposes T ime lill c: Statistical analysis of the evidentiary samples will be completed by May 1, 2004 . Add itional samples related lO # 1030 arc expected to be irradiated and shipped to SNL on or about 4/26104 . Elemental Ana h'sis Goa l: To determine the growth conditions used by the suspect(s} by analYLing the elemental profile of the evidence, typical growth media, and spores grown under contro ll ed test conditIOns. Progr ess: CBSU and Chemistry Unit met 1 discuss a series of validation studies to clarify the previously 0 collected data. Chemistry Unit will order the necessary Si hollow cathode for the GF AAS and the pol ymeric com ponents fo r the ICP/OES. These two techniques will be compared for Si measurements T im e line : Once initiated, the validation work is expected to be completed by 07/0 1/2004. W hole G enome Am plifica tio n (W GA) Goa l: To determine whether lrace amounts of human DNA may be extracted and typed from the evide n tiary lettcrs. Prog ress : Three contracting laboratories are developing singlc-cc ll detect ion and amplification capabi lities. Preliminary results ind icate that an amou nt of unirradi ated DNA equivalent to that found in a single cell can be success fully amplified and typed. One contracting laboratory. IBIS. is attempting to pcrform DNA repair on irradiated cell s. It appears that irradiation has a negative impact on the ability to amplify trace amounts of D1\'A from a ffec ted cells. DNA repair studies conducted to date have had limi tcd success. Tilli e line: Stud ies should be completed hy the end 01' 05 /2004 . At that time il wi ll be decided which WGA procedure should be applied to the evidentiary envelopes, Difreren tial P henoty pi c Colon y ExpressiQ n Goal : To observe the evidentiary morphological variants in cultures o f the reposilOry samples and to correlate these pattems with those observed in the evidentiary materia ls. Prog ress: An SOP and budgct for Repository scree ning has bt:cn recei ved from lJSAMRlID. Two additional technicians have be~n assigned to this project. Equipment needs have been included in the submitted budget. Tim e line : Re pository screening fo r morphological variants observed in the evidentiary materials should commence w ithin two weeks and last until the end of 06/2004. G[EXWSR B3D1.pdf  204  009694Anthrax Page 207 of 643 3 AMX Weekly Science Update April 22 , 2004 Ge neti c C haract~ri.za t ion of Mornholoeica l Va ri ants Goal: To identify the source of Ames strai n S A used in the leiters. Progress : On April 7. 2004. SA l Ireported that 28 additional Ames strain slants were entered into the FBI repository. Duplicates of these slants wefe sent to ~AU's Keirn Laboratory fo r genotypic characterization. Additional sampl es from Sweden will be shipped on 4/27/04 10 USAM RIID. The UK defense lab will provide samplcs to FB IR. Legal is recontacting the defense lab to set up shipment detai ls. Ames strain samples housed in the Repository at USAMRI ID wefe transported to CB I on 03110/04. To date, 638 samples or 58% of the repository has been extracted for analysis. In addition, Taqman analysis was started au the extracted. To date, 380 samples, or 17% of the repository has been screened using the Morph A assays. SA l land visited CBI on 4122104 for a briefing on the progress. boS b7 : sli I Time line: Screening of the morph A 1 and AJ variants is scheduled to proceed through the end of 06/2004. Genetic C haracter iza tio n of the Bacillu s Conta minant Fou nd in New Yo rk Ma ilines Goa l: To fully characterize the Bacillu.f subtilis contam inant(s) found in the l'\'ew York mai lings. Progr ess : DNA sequcncing of tile Bacif/us subrilis isolate recoverl!d from the New York Post letter was compl eted on 03/09/04. TIGR presented these findings to FBI sc ientists o n 3/26104. UNA assays have been designed by TlGR which should target variation observed txtwl!en a re ference strai n and the evidentiaty strain. On 4/7/04 PCR testing of 23 peR assays for specific ampl ificat ion of genomic regions of thc NY Post evidence 8. Sublilis isolate was startcd. As of 4/22/04, TlGR and CBS U have completed screening the putative Post contami nant specific assays against the refcrence 168 B. sublilis strain. All but six of the assays apJk'ared to be speci fi c forthe cvidence strain. CBSU has begun processing an add itional 65 B. sublihs samples for screening usi ng the Post speci fi c assays. This spcc ifici ty panel of isolates was obtain~d un 4122104 from the US DAINCAU R. Peoria, lL. On 4/23/04 TlOR sent data regarding comparative analysis of the Post and Brokaw B. silbrilis isolates. Rased upon their analysis, the two B .w btilis isolates apJk'ar idemical. Time line: We originally reported that the specificity panel studies would be completed by 5/31/04. Completion of all the strains from the US DA eolletion may take an additional 2-4 weeks. G[EXWSR B3D1.pdf  205  009695Anthrax Page 208 of 643 Isoto pic A na lys is Goa l: To detemlinc the geograph ic region of production of the evidence through the use of Hydrogen and Oxygen stablt= isotope ratios, as well as identify the class of media through Carhon l3 (C- 13) and Nitrogen ' ; (N15) analysis. Isotopic examination of the Leahy t!vidence has provided data which requires addit ional work in order 10 draw meani ngful conclusions. I)rogress : A validation experiment was conducted at the BFAC to investigate the relationship b~ l wecn longtenn growth on agar plates. the enriciuncill of the starting water and the isotopic enrichment noticed III the spore cvidl.:nce. Timc lio e: Analysis of the samples of the above experiment are underway at the University of Utah . Hemc Ana lvsis Go al : To determine whether or not he me was present in the growth media . Progr ess: The heme detection assay developed at the Un iversi ty of Maryland was transferred to the U. S. Army Chem ical and Biologica l Forensic Analysis Center (ECBlFAC). Edgewood. MO on 02117/2004 . Thlo! performance o f the assay was validated at the ECD/F AC on 2125/04 Slight improvements to the m ethod were made by EeD/ FAe. Ext raction and analysis of these samples has becn coordinated with the SF AC and ECB/ FAC. CBSU expects the results from this latest validation experiment b} 51712004. Tim e lin e: A sct of blind unknown samples. generated from the extended growth experiment at the OF AC were ana lyzed on 411 2104 . A n:pon is expected in a few days. If the validation work appears to be successful. then the Leahy evidence will be scheduled for analysis. Elemental Mal)lliu~ Goa l: To provide high resolution SEVI and TEM elemental mappi ng of spores. Prog r'css: Sandia has observed Si on the spo re coat o f7 3 of 111 (66%) spores examined in detai l from the Daschle evidence. Sandia is perform ing a statistical analysis of the other evidentiary samples fo r comparative purposes. G[EXWSR B3D1.pdf  206  009696Anthrax Page 209 of 643 2 Tim~ AMX Weekly Science Update April 29 , 2004 line: Statistical analysis of the evidentiary samples will be completed by May 1,2004. Additional samples related to # 1030 are expected to be irradiated and sh ipped to SNL for elemental mapping. Elementa l Analvsis Goa l: To determine the growth conditions used by the suspcct(s) by analyz.inl; the dt!l1lcmal profi le o f the evidence, typical growth media, and spores growll under controlled test condit ions. Prog r"ess: Chem istry Unit has made the necessary instrument enhancements to eliminate the Quartz components. This has significantly reduced the background Si level. Chemistry Unit is planning to use (wo different instrumc mal approaches to quantify the 5i level in the evidence spores. Time lin e: Once initiated , the val idation work is expected to be completed by 07/0112004. Wh ole Genome Amplification (WGAl Goal: To dctermine whether trace amounts of human D:-.JA may be extracted and typed from the evidentiary leiters. Progr ess: Three contracting laboratories are developing low copy number amplification capabilities. Preliminary results indicate that an amount of unirradiated DNA equivalent to that fo und in a single cell can be successfull y amplifi ed and typed. It appears that irradiation has a negative impact on the ability to ampl ify trace amounts of DNA from affected cells. One contracting labomtory, IBIS, is attempt ing to perform DNA repair on irradiutcl.l cell s. More success has been ac hieved using mitochondria l DNA (mtDNA) as the target for WGA amplification rather than nuclear DNA loci. Time line : Studies shou ld be completed by the end of 0512004. At that ti llle it wil l be decided wh ich \\IGA proced ure should be applied to the c\'identiary envelopes. Differep tial Phenotvpic C:olollv ExprcSllion Coa l: To o bserve the evidentiary morphological variants in cultures of the Repository samples and to correlate these paucms with lhose found in the evidentiary materials. I)rogress: An SO P and budge t fo r Repository screening has been prepared. Two uddi tio naltcchnician .. have been ass igned to this project. Equipment needs have been included in Ihe subm ith;:d budget. Time line: Repository screening for morphological variants observed in lhe evidentiary materials should conunencc with in 0412004 and be completed by the end 0(0612004 . Gcnctic C har a ctcri~;ltion of Morphological Variants Coa l: To identify the source o f Ames strain Dt\ used in the leu r:s present in minor variants. wing g\,::m:lil; Yilrialion found in mutations G[EXWSR B3D1.pdf  207  009697Anthrax Page 210 of 643 AMX Weekly Science Update April 29 , 2004 Progress: Ames strain samples housed in the Repository at USAMRIID were transported to em on 0311 0/04. To date. approximately 65% of the Repository samples have been extracted fo r analysis, and approximately 20% or tne Repository has been genetically screened using the two Morph A assays. Additional RepositOry samples will be shipped to CD I on 04128/2004 fo r inclusion and screening. Kcsults from validation studies for implementing new instrumentation into the morph A screening will be submil by eBi on 051 0212004. T ime lin e: Screening of the morph A I and A3 va riants is scheduled 10 prm:ced through the end 0(06/2004. GenCl ic Cha r acterization of the Bacillus Co ntamin a nt Fou nd in New York Ma ilines Coa l: To fully characterize the Bacillus .~uhliJis cOUlaminant(s) found in the New York mailings and use this characteri zatio n to screen other samples of 8 . sllbrtlis to determine the sourcc of the evidence. Progress : DNA sequencing of the Bacilllls Sublilis isolate recovered from the New York Post letter was completed on 03 /09/04 . DNA assays have be!;!n d!;!signed by TlGR and the FBI Laboratory (CBSU) targeting variation found within the evidentiary strain. Testing was started on 04/0712004 . TIGR subjected the isolate recovered from the Brokaw leiter to this same suite of assays. The results indicated !hat the B. subrilis found in the Brokaw lener is the same as that found in the N. Y. l'ost letter. CBSU has begun scree ning an additiona165 8. :mbrilis samples using a set of evidence-specific assays. This spec ificity pancl of isolates was obtained o n 4/22/04 from the USDAINCAUR. Peoria, lL. Although some o f the t!vidence-specifi e markers are positi ve in other strains, most of the types are very di fferent in the other strains suggesting that the markers will be very informative for identi fying strain(s) that arc closely related to the evidence. Additio nal samples of B. sublilis arc being identificd for collecti on a nd testing within tht! FBl Laboratory . T im e linc: The specificity pand studies will be comp leted b}' 5/31 /04. Comp letion of all the strains from the USD.I\ collect ion may take an additional 244 weeks. G[EXWSR B3D1.pdf  208  009698Anthrax Page 211 of 643 Isotopi? A nal vs is G oa l: To determine the geographic region of production ofthe evidence through the use of I1ydrogen and Oxygen stable isotope rati os. as well as identify the class o f media through Carbon i3 (C-13) and Nitrogen l5 (N15) analysis. Isotopic examination of the Leahy evidence has provided data which requires additional work in order to d raw meaningful conclus ions. l'trog ress: llle Oxygen-IS isotopic profile orthe evidence suggests that it was grown under conditions in \vh ich evaporation oftnc water in the media occurred during grov.1h. An experiment was designed to attempt to replicate this behavior. The resuhs indicate that the experiment failed to replicate the isotopic profile noted in th evidence. Ti me line: A meeti ng is sched uled for next week wi th Captain Bura ns of the BFAC to discuss the results of the recent experiment and conside r a fo llow up design that may rep licate the evaporative conditions which are bel ieved to account for the isotopic profi le of the evidence. H cmeA nal~ Goa l: To determine whether o r 1101 heme was present in the growth media. Progress : The heme detcction assay developed at the Uni ... ersity of Maryland was transferred [0 the U.S. Army Chemical and Biological Forensic Analysis Center (ECB/FAC). Edgewood. MD on 02/ 17/200 . L The performance of the assay was validated at the Eell!FAe 011 2125/04. Slight improvements to the method were made by ECBIF AC. Extraction and analysis of these samples has been coordinated with the BF AC and ECBIf AC . The MAL DI-TOF-MS analysis of the recent val idation study has generated some technical questions. CBSU and ECS/FAC will review the mass spectroscopy dala on 5!12104, and cOllsidcr additional validation work o n the assay. Ti me lin e: A set o f bl ind unknown samples, generated from lhc exte nded growth experiment al the BFAC were analyzed on 4112/04 . If the recent validation experiment proves to be sutliciently rel iable, tht:n C BSU will proceed with plans to extract and cxam ine the I,eahy Ev idence. Elem enta l Mapping Goal : To provide high resolution SEM and TEM elemental mapping of spores. G[EXWSR B3D1.pdf  209  009699Anthrax Page 212 of 643 2 AMX Weekly Science Update May 6, 2004 Progress : Sandia has observed Si on the spore coal of 73 of 111 (66% ) sporl!s ~xamincd in detai l fro m the Dasc hle evidence. Sand ia is performing a statistical analysis of the OIher evidentiary sam ples fo r comparal ivt: purposes. Time lin e: Sta tis ti cal analysis o f the evidentiary samples wil l be compielc:d by 05 / 14nOO4. Ongoing investigati ve efforts are expected 10 produce morc samples for comparison in the nenr future. Element'a l Ana lys is Goa l: To determine the gro\\,1h conditions used by the SUSpeCI{S) by analyzing the elemental profi le of the evidence, typical gro"-1h media, and spores grown under controlled test conditions Progress: The Chem istry Unit (CV) of the FBI Laboratory has made the necessary instrument enhance ment !) 10 el im inate the quartz components. This has significantly red uced the background SI Icvel. The CU is pla nni ng to use the one instrumental approach to thorough ly validate the Si method and then quanti fy the Si level in the evi den tiary material. Thc growth of a reference spore samp le by CTf SRU is expected 10 be completed during the week o f 5/ 10/04 . This spore preparat ion wi ll serve as a d igestion blank and internal standard . Time lin e: The validation work is cxpected to be completed by 07/0112004 . Whole Ge nome A m plifi cation (WGAl Goa l: To determ ine whether trace amounts of human letters. nr-:A may be extracted and typed from the evident ial)' I'rogress: Three contracting laboratories are developing low copy number ampli /icalio n capabi lities. Prel iminary results indicate that an amount of un irrad iated DNA eq ui valent to that found in a single cell can be sllccessfully amplified and typed .. Limited success has been achieved usi ng mitochondrial DNA (mt DNA) as the target for WGA ampJilicatio n ralher than nuclear DNA loci. Time linc: VaJ idJ tion samples are being prepared by the FBI Laboratory for shipment to Ihe contracting labs . Theses studies should be completed by the end of OS/2004. At that time it will be decided which WOA procedure should be applied 10 the evidentiary envelopes Differential l'henon'pic Colony Express ion Goal: To observe the evidentiary morpho logical variants in cu ltures o f the Reposi tory samples o.nd to corre l;Jte these pat1em s with those found in the evidem iary materials. Prog ress : Targeted growth of Repository samples wil! initiall y focus on those samples that the molecular assays idemifyas containing the variants. Time lin e: Screening fo r morpho logical variant s observed in the evidcmiary maleria ls sho uld be completed hy the end 0(06/2004 . G[EXWSR B3D1.pdf  210  009700Anthrax Page 213 of 643 3 AMX Weekly Science Update May 6,2004 Genetic C haracterization of Morphologica l Variants Goa l: To identify the source of Ames strdin BA used in the letters using genetic variation found in mutations present in minor variants. Pr o~ rc ss : Ames strain samples housed in the Repo~ i tory 31 USJ\MR IID wcr..: transported 10 CBl on 03110104 . To date, approximately 80% of the Repository samples have been extracted for analysis, and approxi mately 30% of the Reposi tory has been genetically screened using the I wO Morph A assays. Additional Reposi tory samples wcre shipped to C BI on 04/2812004 for incl usion and screening. Resu lts from validation studies for implementing new instnllnelllation into the morph A screening process wcrc subm itted by e m on 05/0212004 and approved by FBI personnel fo r implelll~1l1alion inlo the screening protocol. A limill!d number of l{epository samples have tested positive for onc or both o f the morph A patterns. Investigat ive efforts are ongoing to identify thl! complete history ofthesc isolates. Time line : Screening of the morph A i and A3 variants is scheduled to procl"Cd through the end of 061200?1. Genetic C haracterizatio n oftbe Bacillu s Contaminant Found in New York Mailin::.s Go~l: To fu lly characteri ze the Bacilfus sub/iUs contaminant(s) found in the N..::w York ma ili ngs alld use this characterization to screen other samples of B. sublilis to determ ine the source o f the c vidence. Progress: DNA typing of markers found in the evidentiary material has revealed thai these patterns are generally not observed in other isolates of 8. sub/ilis. Analysis of the <;train o f 8. .l"IIblilis fou nd in the Brokaw letter revea led that this isolate has the same molecular characteristics JS the isolate recovered in the N. Y. Post letter. C BSU has begun screening an additional 65 B sub/illS samples using a set of evidenct:.specific assays. Fewof the evidence-specific markers are found in other strai ns, s uggesti ng that at least some of these markers will be very infomlativc for identifying strain(s) that arc closely related to the ev idence Additional samples of 8. suhlilis are being identified for collection and testi ng withi n the FB I Laboratory. Time lin e: The s pecificity panel studies will be compit:ted by 5131 /04. Completion o f all the strains frOI11 the: US DA collection may take an additional 2-4 weeks. G[EXWSR B3D1.pdf  211  009701Anthrax Page 214 of 643 Isotopic An a lysis Goa l: To determine the geographic region of product ion of the evidence through the use of Hyd rogen and Oxygen stable isotope raL as we ll as identify the c1ac:;s o f media through Carbon 13 (C?13) and Nitrogcn ,j (N? ios, 15) analysis. Isotopic examination of the Leahy evidence has provided dara wh ich requires additional work in order to draw mean ingfu l conclus ions. Progr ess: The Oxygen-18 isotopic profile of the evidence suggests that it was grown unde r condi tions ill which cvaponuion of the water in thc media occurred during growth. An experiment wa.c:; desigm:d (0 attempt to replicate this behavio r. The resuhs suggest that the experiment fai led to replicate the isotopic profi lc noted in the e vidence. A new sct o f extended growth ( 12 days) samples grown under "evaporative" I:onditio ns has been startcd at the N RFACC. T im e line: The new ex tended growth samples should he harwsted, sterilized and sent to the Un iversity of Utah by 06/ 11/2004. H eme Analvsis Coa l: To determ ine whether or not heme was present in the growth media . I'rogress: Extraction and MALDf-TOF-MS analysis of the recent validation study hDd generated some 2004. An insllfficient Dmount of the technical questions that were discussed in a meeting at EC B/ FAC on 051181 last validation samples was available for replicate analysis. eBSe and ECB/ FAC agreed that additional validation samples would be grown. harvested and extracted at ECOfF A(' thus reducing thc turnaround time . Sam ples of Bacillus amhracis Ames and Stem strain.. were sent from USAM RIIO to ECB/ FAC o n 05126/2004. T im e line: A new batch of spores will be harvested on 06/01 /2004, steri lizt::d, and be ready fo r MALDl?TOf analysis by 06/04/20 04. E lemental Mappinc; Go:li : To provide high resol ution SEM and T E,\1 elemental mappi ng o f spores. Progress : Sn ndia Nati onnl Lab (SNL) has observed Si o n the 66% of the ~ porc coat from tht!' Daschlc evidence, 76% from Leahy, and 12% fiom sample 1030. SNL is perfonning a slallstical analysis of the other sam ples fur comparative purposes, G[EXWSR B3D1.pdf  212  009702Anthrax Page 215 of 643 2 AMX Weekly Science Update May 27. 2004 Time line : Investigative effons continue to generate additional samples for analysis. At least new 19 samples are to be steri lized during the week of 05/3112004. The samples will be prioritil.:t'd before sending to SNL Elemental Ana lysis Goa l: To detennine the growth conditions used by the suspect(s ) by analY-Ling the elemental pro ri le of the evidence, typical groWTh media, and spores grown under controlled ieSI conditions. Prog r ess : 'r hc reference spores cultured by CTFSR U , and delivered to Chemistry. included 1.7 grams or Bacillus cercus T (non pathogcnic) strain spores and 4.8 grams of spores grown with and without Silicon antifoam , respectively. Preliminary sam pl es fesuils shows deleclablt! levels of AI. Ca. Cu. Fe. Mg. Mo. Zn and Si. Reproducibility experiments are underway to detenninc the minimum sample size rcquin:d . 4 Tim e line: The val idation work is expcclt.:J to be completed by 0710 1 12004. Whole Genome Amplification (WGAl Goa l: To determine whether trace amounts of human DNA may be extracted and typed from the evidentiary lelters. I'rog ress! Three contracti ng laboratories are developing low copy number amplifi cat ion capabilities. Preliminary resu lts indicate that an amount of un irradiated DNA equivalent to that found in a si ngle cell can be successfully ampl ified and typed. Lim ited succcs..<; has been achieved usi ng m itochondrial DNA (mtDNA) as the target for WGA amplification rather than nuclear DNA loci. Time lin e: Validation samples have been shi pped to the contracting lahs . These sample:> consist of DNA in variou."i forms and prcsc/H at different conce ntrattons. 11\1;: validation studies should be completed by Ihe e lld of 05/2004. At that time it will be decided which WG/\ procedure should be appl ied to the e\'idence, ifany. Oiffcl-e ntial Phenotypic Colonv Exp r ess ion Goa l: To observe the evidentiary mo rpho logical variants in euhures of the Repository samples and these patlt!rns with those found in the evidentiary matt!rials. (0 correlate Progress: Targeted growth of Repository samples will ini tially focus on those samples thai the molecular assays identi fy as conta ini ng the variants . A sub-set of the remaining Repos itory samples will be grown and tested . Time line : Screening for morphological variants observed in the evidl.!nt iary materia ls should be completed by the end of 0612004. Genetic C haa-actcrlzat ion or Morph ological Va riants G[EXWSR B3D1.pdf  213  009703Anthrax Page 216 of 643 3 AMX Weekly Science Update May 27, 2004 Goa l: To identify the source of Ames strain AA used in the letterS usi ng genetic variatlon fo und in m utat ions present in minor variants. Progr ess: Ames strain samples housed in the Repository at CSAM RIID were transported to CB I on 03110/04 . To date. all of the Repository samp les have been extracted for analysis, and the ONA has be~n no rmalized for inclusion into the test stream. To date. approximately 40% of the Repository has been genetically scree ned using the twO Morph A assays. A limited number of Repository ~mpl es have tested positive for one o r bot h of the morph A patterns. Investigative efforts are ongoing to identify the comph:te history o f these isolates. Additional variants have been identified that appear to be closely related to the known morph A patterns. Time lin c: Screening ofthl.! morph A I and A3 variants is scheduled to procet'd thro ugh the end of 06/2004. Genetic C h a racteriz ation of th e Bacillus Co ntamin a nt Found in New York i\1ailinl! ~ Gua l: To fully charac terize the Bacillus subtilis contaminant(s) fou nd in the New York mailings and use this characterization to screen other samples of B. suhrili.f to detenn ine the source of the evidence. Progress : DNA typing markers found in deri vative evidentiary materia l has revealed patterns not generally observed in isolates of B. suhriJis. Analysis of derivative isolates of the strains of B. .whlilis found in the Brokaw and NY Postleners revea ls that the isolates have common molecular characteristics. These observations are based upon D A typing markers recently developed in collaboration with T IGR . CDSU has begun screening an additional collection o f 70 8. sublili:)? samples usi ng a set of 48 PC R assays designed to test for the presence ofs~cific rcg ions present in the New York strains and not present in the B. s ublilis 168 type strain. As o f 05/2612004, 43 B. Sllhlilis strains have been typed usi ng fhe evidence spccitic peR marke rs. and none have been an exact match to the NY strains. At the conclusion of sc reening the 70 isolates. a sub-set o f pe R based assays will be chosen to spec ifica lly identify B..Whlilis strains thai m:llch the NY evidentiary stra ins. T ime line: Genetic characterization ofapproximatcly 40 B. J/lblilis strai ns, used in the specific ity panel studies. will be completed by 5/3 1/04 . Characterization of all 70 strai ns from the US DA eollt:ction may take an additional 2 -4 weeks. or , . G[EXWSR B3D1.pdf  214  009704Anthrax Page 217 of 643 Isotopic Analvsis Goal: To determine the geographic region of production of the cvidence through the use of Hydrogen and Oxygen stable isotope ratios, as well as identify the class of media through Carbonll (C?1 3) and >Jitrogen lS (N. 15) analysis. isotopic exami nation of the Leahy evidence is completed_ The isotope ratio data is be ing used to attempt to replicate the gro\\,1h conditions noticed in the c::vidence. Progress: lnc Oxygen-I 8 isotopic profi le of the evidence: suggests that it was grown under condit ions in which c::vaporation of t he watcr in the media occurred during gro\\,1h. An experi ment designed to replicate this fi nding failcd to produce the isotopic profile noted in the evidence. A follow-u p experi ment will usc a more rigo ro us sct of "evaporative" conditions to attempl 10 simulate the water metabolism observed in the evidence. The experi m ent w ill be co nducted through coordi narion with the NBFAC in a BSL-3 su ite that is sui table for open containers o f dry spores for severa l days. T im e line: In the last update, a growlh expcriment was predicted to begin on 06/01 /2004 for shipment on 0611112004 to University of Utah. The lac k of a suitable lab has caused the experiment to be re?schedu led to begin al the NBF ACC by 06/ 1512004. followed by shipme nt of sampl es to University of Utah. Hfm e A na l\'sis Goa l: T o detenn ine whether or not heme was present in the growth media. Progress: ECB/FAC is cOlllinuing the efforts 10 \ alidate the MALDI? TOF?MS examination protocol originally developed by University of Maryland. The anal}1 ical method has s how n to be reliable for the measurement o f heme extracts from spores. Work during the week uf 0617-1 0/2004 has focused on the production of a sct of standards, heme?conrai ning and heme-free spores, that can be compared against the evidence. A new batch o f spores was harves ted o n 06/0212004 and washed and extracted on 06/0712004 . These were fou nd to have no hemc. Further consultation with the origi nal scient is t who developed the assay at University of Maryland indicated that the spares must be al lowed to dry on the blood agar plates for 7 to I 0 days prior to extraction and ana lys is. Time lin e: The new re ference mmcrials will begin to be cultured on 0611412004. wi th harvest between 06/21? 24/2004. Extracli on and analysi s will fo llow twO days after harvest. The analys is of the evidence can be accomplished in three days, after it is determined that the above experiment is successful. G[EXWSR B3D1.pdf  215  009705Anthrax Page 218 of 643 2 AMX Weekly Science Update Elemental Mappine Goal: To provide high resolution SEM and TEM elemental mapping of spores. Progress: Sand ia National Lab (SN L) has observed Si in the spore coats of 66% of the spores in the Daschle ~ viden ce, 76% of spores in the uahy evidence. and 12% of the spo res in samp le 1030. SNL is continuing a statistical analysis of the other samples for comparative purposes. Time li ne: Recent investigative developments continue to generate additi onal samples for analysis. A t least 19 addit ional samples are bei ng prepared a l NM RC. These samples will be sent to the NBfAC C fo r ultimate shipment to SN L SNL will then conduct elemental mapping to detect any simi lari ties with the evidence. E.lemental Analvsis Goal: To ueterminc the growth conditions used by the suspcct(s) by analyzing the elemental profile o f tne evidence. typical growth media. and spores gro wn undc:r controlled test conditions. Progress: The referc:nce spores cultured by CTFSRU and del ivered to the Chemistry Uu it included 1.7 grams of Bacillus cereus T (non-pathogenic) strain spores grown with Silicon-containing anti- foam and 4.8 grams of spores grown witho ut Silicon anti-foam. Prelim inary results revealed detectable level s of AI. Ca. Cu, Fe. Mg. Mn . Zn and Si. Reproducibility experiments arc undern'ay to detem1illc the m inimum sample size req ui red fo r anal ysis. Tillie line: The val idation work is ongoing. technical questions are being reso lved. Signifi cant improvements to thc analytical met hod were made on 06/0912004. This study is expected to be completed by (J7/0 112004. Who le Gcno lll l! Amplificat ion f W GA) Cooal: To detennine whether trace amounts of human DNA may be extracted and typed from the evidentiary Icuers. P rogress: Thn:c contracting laboratories are developing low copy number amplifi cal iUII capabi lities . Prel iminary results ind icate that an amount ofun irradiatcd DN A cqui voem in 06/2004. Time lin c: Screcning of the morph A I and A3 variams should proceed through the end of 0612004. Gen etic C haracleri1..3tion of the Baci llus Conta minant Found in New York Mailinl!.s Goa l: To fuUy characteri7..e the Bacillus slibrilis contaminam(s) found in the New York mmlings a nd use this characteritation to screen other samples of 8 . slibri/is to determine the source o f the l!vidl!ncc. Progr ess: DNA typing of markers found in derivative evidentiary material has rcvc=aled pa llern.~ not generally observed 10 isolates o f B. suhri/is. Analysis o f derivative isolates of the slrains of 8 . Juhtilis found in the I3rokaw and N Y Pust letters reveals that the Isolates have common mo lecular characteristics. T hc=~ observations are based upon DNA typing markers recent ly deve loped by CBSU in collaboration with T IG I< . CBSU has begun screening an add itio nal collect ion o f 708. subrilis samples using a SCI of 4 R PCR assays designed to test for the presence ofspccific regions presenf in the Nc=w Yo rk mains and no t prescnt in the 8 . .mblilis 16g type strain . As of 06/1712004,49 8. SIIhrilis strains have been typed using the evidence- spcciflc PCR markers; and none arc an ex act match to the evidentiary stra ins. A sub-~t:t 01 PCR-based assays will be chosen thut WI ll most eftkiently sn~en B. ~lIbli1iS strains In a high throughput mode. A co mple mentary strain- G[EXWSR B3D1.pdf  221  009711Anthrax Page 224 of 643 4 AMX Weekly Science Update June 17,2004 typing teclmiqut! produced by DiversiLab has also revea led genetic patterns in the evidence that are different from other strains of B. subtilis. Time line: Genetic charaCleri7..3t ion of approximately 60 B. SlIbli/is slrains. used in the specific it y panel studies. wil l be completed by 07/01 /2004. G[EXWSR B3D1.pdf  222  009712Anthrax Page 225 of 643 Isotopic Ana lvsis Coa l: To detennine the geographic region of production of the evidence through the use ofl-Iydrogen and Oxygen stable isotope ratios, as well as identify the class of media through Carbon I) (C-13) and Nitrogen l5 (N15) analysis. Isotopic examination of the Leahy evidt!nct: is completed . The isotope ratio data is being used to attempt 10 replicate the growth conditions noticed in Ihe evidence. Prog ress: TIle Oxygen- 18 isotopic profile of the evidence suggests Ihat It was grown under condi tions in which evaporation of the water in the media occurred during growth. An experiment designed 10 replicate thi s find ing failed to pn.x.l uce the isotopic profile noted in the evidence. A follow-up experiment, was started 011 6/ 14/04 to use a more rigorous set of "evaporative" conditions to altempt to simulate the water metabolism obser\'ed in the evidenct!. The experiment was conducted through coordination with the N Dr AC in a SS L-3 suite that is sui table for open containers of d ry spores for scvcnll days. A proposal. for additional experimental work , to help refine our interpretatio n of the data is being reviewed. T illi e lin e: All samples currently at Utah have bt."Cn analyzed. A report o n the isotopic ana lysis of the water samples submi tted by For Legats is complete. The existing contract is being updated. The new "evaporative" growth sam ples wi ll be selll to Utah by July 30. 2004. E lemental Mapping Coa l: -10 provide h igh resolution SEM and TEM elemental mapping of sport!s. P rogress: Sandia National Lab (SNL) has observed Si in the spore coals of 66% of the spores in the Da<>ch le ev idence, 76% of spores in the Leahy evidence, 67% of spores in the NY Post evidence, and 12% of the spores in sample USAMRJID sa mple 1030. The Y Post sample a lso contai ns cellu lar dcbns. Timc li n e: Recent investi gati ve developments continue 10 generate additio nal samples for analysis. A group of 19 samples is undergoing analysi s at SN L. Four samples of significant investigative lead value are expected to be comp leted by 8/30/04. Elem enta l Ana lysis Goa l: To determi ne the growth conditions used by the sllspect(s) by anal yzill g the elemental profile of the evidence, typical growth media, and spores grown under controlled test conditions. G[EXWSR B3D1.pdf  223  009713Anthrax Page 226 of 643 2 AMX Weekly Science Update Progress: The reference spores cultured by CTFS RU and delivered to the Chemistry Unit incl uded 1.7 grams of Bacillus cereus T (non-pathogenic) strai n spores grown with Silicon-containing anti-foam and 4.8 gram s of spores grown without Si licon anti-foam. Preliminary results revealed detectable levels of AI. Ca. Cu, Fe, Mg. Mo. Zn and Si. )0 mg of irradiated Leahy material was analyzed by Chemistry with rcsu hs duplicating those from 2 years ago. Time lin e: All samples submined to Chemistry hay!;: been \:ompleted . Whole Genome Amplifica ti on (WGAl Goa l: T o dctennine whether Irace amounts of human DNA may be extracted und typed fro m the evidenti ary teners. Prog ress: Validation studies at lhree contract laboratories have been completed and reviewed. One laboratory's performance was favorable. A detailed analytical SOP has been submitted by the selected laboratory for review. A site visit has been conducted and il was determined that minor internal validations requi re completion. Planning to submit evidentiary samples is on?going. Time line: Analysis of evidentiary matcrial is schedu led 1 begin on or about 09/0 1/2004. 0 Differential Phen otypic Colon y [ xpression Goa l: To observe the evident iary morpho logical variants in cultures of the Repository samp les and to correlate these patterns with those fo und in the evidentiary materials. Progress: Targeted growth of Repository samples will ini tially focus on those samples that the mo lecular assays identify as containi ng the variants. A sub?set of the remaining Repository samples will be grown and tested. Tim e line : Screen ing for morphologica l variants observed in the eviden liary materials should he com pl eted coincidentally wi th the completion of the genetic characterizations. Genetic C har.J cteril.::ltion or 1\1orpholo2ical Variants Goa l: To identify the source of Ames strain BA used in the lellers usi ng gCIll'tic variation found in mutations present in m inor variants. Progress : Thc entire Repository has been genetically screened using the two Morph A assays. Re?sequeneing and confiml at io n o f seqllcnce r~sult s should be completed by the end 07/30/200 4. In add ition , 28 samples in a second set o f sampl es have all been c ultured and DNA has been extracted. Analyses arc currently underway and should be completed in two weeks. /\ limi ted number of Repository samples have tested positive for one or bOl h of the morph A variants. InYcsligalivc cffon'i are ongoins to identify the complete history of these isolates. Additional variants huy..; b,,;clI identified t hat a ppear to be closely related to the known morph A patterns. G[EXWSR B3D1.pdf  224  009714Anthrax Page 227 of 643 3 AMX Weekly Science Update T ime line: Screening of the morph 1\ I and 1\3 variants within the first 600 sample set should be completed by the end 0[07/2004. Additional pending analyses should be completed within twu w~eks. Genetic C haracterizat io n orl he Bac illus Co nta mina nt Found in New York Mailin!!s Goal : To fully characterize the BQcillu~ sub(ilis contaminant(s) found in the New York mailings and use this characteri zation to screen other samples of B. subtilis to determine the source of the evidence. Progress: DNA typing of markers found in deri vative evidentiary material has revealed patterns not ge nerally observed in isolates of B. sub/ilis. Analysis of derivative isolates o f the strains of B . .5Itbtilis found in the;;: Brokaw and NY Post letters reveals that the iso lates have common molecular characteristics. These observat ions arc based upon DNA typing markers recently developed in collaboration with TIGR. CBSU has completed screeni ng a diversity panel of 70 B. sublilis ~amples using a set o f 48 PCR assays designed to test for the presence of spectfic regions present in the New York strains and nor prescnt in the B. ~'/lbli1is 168 tyIX strain. All of the B. slIbtilis strains have been typed using the evidence? specific PC R markers. and none are an exact match to the evidentiary strains. A complementary strailHyping technique produced by Bacterial Barcodes, Spc=ctral Genomics. Houston, TX, has a lso revealed genetic patterns in the evidence that are different from other strains of B. subfilis. A sub?set of 4 PCR?based assays were chosen that will most efficiently ide ntify B. sub/ilis stmins that match the t!vidcntiary strai ns. CBSU hlis deSigned 4 T liljMan real?time peR assays designed to detect the 4 specific genetic sites in the B. s ubtilis evidence strams. As of 07/28/04. the Tag Man assays have been optim iLeJ fo r reagent concen trations. Assessment of their specificity fo r the evidentiary genetic markers will begin mV02/04 by performing the assays on the 70 B. sub/ilis strains mentioned above. In addition, the assays will be tested to determine the limit of detection of the evidentiary B. SlIblilis strain wi thin a high background of 8. an/hracis UNA. Time lin e: Specificity testing of the evidentiary B. sublilis TaqMan assays against the 70 H. panel should ~ completed by 08/06/04. .\"uhtifi.~ diversity Analysis ofsampJcs collect ed OeON S, for the presence of Bacillus antllracis Goal: During May 2004, thc FBI conducted an OCONUS site exploitation in furthcmncc of the Ame rithrax investigation. Over 440 samples were collected during this mission at three locations. These samples were prioritized for processing at the National Bioforcnsics Analysis Center (NBFAC). All samples have been processed for analyses at the N BF AC. The NB FAC focused on culture analysis while sam ples prepared for PCR analysis were transferred to the NMRC for analysis. Three samples from site three were positive for the presence o f DNA from three unique markers fo r Bacillus anthracis. Two of the samples contained only trace levels. All Culture analyses wil l be completed by 08102/04. Tn date NM RC has completed approxirlla tdy 225 peR analyses. G[EXWSR B3D1.pdf  225  009715Anthrax Page 228 of 643 4 AMX Weekly Science Update An analytical plan is bei ng developed at Northern Arizona Uni versity fo r further testing (strain dClcnnination) of the samples that yielded positive peR results. A sile visi t bas oc"\!n conducted . Tim e line: All microbial culture results will he completed by 08/02104. Specimens to be surveyed by pe R ana lysis for the prcSt!nce o f threat agents, have been processed and ex tracted. The Nn FAC and NMRC expect to complete the peR analysis within the ne",' two week::.. Strain typing analyses wIll reqUIre approxima tely 6-8 weeks. , G[EXWSR B3D1.pdf  226  009716Anthrax Page 229 of 643 - " AMX Weekly Science Update -------- - - -------- ------- - -- . August 5, 20nA I - -- ~ Isotonic Analysis Goal : To determ ine the geographic region o f productio n of the evidence through the use o f Hyd rogen and Oxygen stable isotope ralios, as well as identi fy the class of media rJuough Carbon l3 (C-13) and N itrogen ' S (N. 15) analysis. Isotopic examination oflhe Leahy evidence is completed. The isotope ratio data is being used to anempt to replicute t he growth co nditions noticed in the evidence. P rogress: The Oxygen- IS isotopic profi le orthe evidence suggests thai it was grO\YJ1 under cond itions in which evaporatio n of lhe water in the media occurred during growth. An experime nt designed 10 replicate this fi ndi ng failed to produce the isoto pic profile noted in the evidence. A follow? up experi mcnt, was started on 6/ 14/04 to uSt' a more ri gorous set of "evaporative" conditions to attempt to simulate the water metabo lism o bse rved in the evidence. T he experiment was conducted through coo rdinatio n with the N BFAC in a BSlr3 suite that is suitable for o pen conta iners o f d ry spo res for several days. t\ proposal, for add itiona l experi mental work, to he lp refi ne our interpretation o f the data is bei ng reviewed. Thc spores produced in the st udy were mailed to the University o f Utah on 8/04/04 Tim e line : Al l samples c urrently at Utah have been analyzed . A report on the isotopic analysis of the water samples s ubmitted by FBI LegalS is complete. The existing contract is being updated . Th e anlaysis of the spores in the above experi ment will be completed by 9130/04 . Elementa l Mappi n e. Goa l: To provide high resolution SE\1 and TEM e lemental mapping or spores. Progress: Sandia Nationa l Lab (S L) has obst!rved Si in the spore coats of 66% orthe spores in the Dasch!:: evidence, 76% o f spores in the Leahy evidence. 67<'.r. of spores in the NY Post evidence, and 12% of the spores in sam ple USAM RJ lD samp le 1030. The NY Post sample also contains ce llu lar debris. Results from the SEM work on the research sam ples obtained from the 1980 autho rs, Som lyo el aI , indicates that the samples do in fact have silica in the spo r~ coat as earlie r reported. CBS U will conduct fo llo w? up investigation to attempt to d etennine the biological factors responsible fo r this sil ica incorpor final rt::port o n their effons 10 validate the MALDI -TOF-MS examination . ) .) .) ;:1 ~ ( , . \),: I protocol originally developed by l"nivcrsity o f Maryland. In ! :~.,.;. i () I.:.... \. AMES stra in. heing non-helro lytic. did not lyse heme in the n Despite the lack of heme adsorption by [he AMES stram the r bo Ol her debris. may still ha ve helllt! present. Further validation ~ L_ _ _ _.J rl-----, -- J..,"7C T ime line : Pending review o f ECBIFAC's fmal repon CBSl define the detection conditions and limits. Elemenl'll Ma pping Goa l: To provide high reso lution S EM and TEM elemen tal . Progress: Sandia National Lab (SNL) has observed Si in the evid ence, 76% of spores to the Leahy evidence, and 12% of t G[EXWSR B3D1.pdf  231  009721Anthrax Page 234 of 643 2 AM X Weekly Science Update the 67% of spo res in the ~y Post matt:rial comained 5i. Re-analysis of sa mple 1030 fro m a different s ub sample and grid sho .... ed 6% orthe spores have Si in their sJXlre coats. Time line: Detailed analys is o f the "Barnard" cmd 1029 samples are underway and afC (,!xpcclcd to be co mp lete by R/27 . T welve other samples will be fe-prioritized based upon Ihc..'iC resu lt s. Elemental .'\ nalvsis Goa l: T o determine the growth conditions used by the suspect{s) by analyzing the elemental pro fi le o f the evidence, typical growt h med ia, and spores grown under t:ontro lled test conditio ns. Progress: 30 mg of irradiated Le ahy material was analyzed by Chemistry with results duplicat ing those from 2 years ago. All samples currently in CU are complete. Ti me Iinc: II is anticipated that Al\l1X will submit 4-5 additional AlIA3 positive morph samples lor c1enX:llIal analysis by 8/30. Whole Genome Amplification {WGAl Goal: To determine whether trace amount s of human DI'A may be ex.tracted and typed from the eVide ntiary letters. bo Progr ess: IBIS Pharmaceu tica ls, Carlsbad. CA, has been se lected to proceed with validation of their methodology. Drs.1 and SAl visilcd IB IS on 07/27/2004 for a prog ram review. Draft SOP's have been reviewed and should be finalized at another meeting at Ii3 IS 0 11 08/3112004. b7c I l Ti me line : Pending approval o f SO P's at thc. 08/3 1 12004 meeting. IB[ S w ill co mpletc va lidatio n testing ofthcir me lhooology. and the cvidence will be Iransponed to IBIS to r WGA analysis. OifTert'n tial Phenoh'pic Colom' Expression Goal : T o o bserve the evidentIary morpho logical variants in cultures of the Repository :-.a mp le.~ and to (;orrclate these pallerns wit h t hose found in the evide nt iary materials. Prog ress : Targeted growth of Reposito ry samples is focused on those samp le!. that the mo lecu lar assays identify as co ntaining the A I and A3 variants. Thirteen samples are current ly under exuminalio n. A s ub-set or five addilio nal Reposilory samples will be submitt ed for examinat ion rollowing co mpletion o f pend ing examinations. Time line: Screening fo r morpho logical variants ohserved in the ev identiary materia l" should be (;omplctcd by November I, 2004 . Genetic C ha raclcri7.at ion of M orph ologica l VA riants G[EXWSR B3D1.pdf  232  009722Anthrax Page 235 of 643 3 AMX Weekl y Science Update Goal: To identify t he source of Ames !>Imin SA used in the leiters using gen.!l ic vuriatio n found in mutation, present in minor variants. Progress: All of the Repository samples have been ext racted for analysis. and the DNA has been normalized fo r analysis. T o dale. lhe ent ire Repository has been genetica lly screened uSing the two Mo rph A assay~ . Final rcpom arc being assembled fo r each Repository sample. A limited number of Repository samples have tested positive for one or both of the morph A variants. Investigative effons are ongoing (0 identify the complet e history of I he~ isot,nes. Addit ional variams have been identified that appear to he c losely rcialetllo the known morph A patterns. Time line : Screening of the morph AI and A3 variants within the frrsl600 sample is complete. Several hunored additional isolates seized by WFO at USAMRlID and Du gway Provillg Grounds (DPG) arc being processed into t he repository. These samples will be analYLcd at CB I in a manner co nsistent wit h prevIO us analyses. Genetic Characterization of the Bacillus Contaminant Found in few York Mai lines Goal : To full y c haracterize the Bacillus .mblilis contammant (s) found in t he New York mailing.. and use this charactcrizal ion to screen other samples of B. sublilis to determine the source of the evidence. Progress: O.'lA typing o f markers found in tlerivative evident iary materia l has revealcd patterns not generally ohservcd in isolates o f B. :>ubrj/js. Analysis of derivat ive iso lates o f (he strains o f B. :,ubrj/i.~ fo und in the Brokaw and NY Post lett ers re veals that the iso lates have common mo lecular characte ristics. These obscrvm io n~ are based upon DNA typing markcrs recently develo ped in co llahoration with T IGR. Time line: Testing of assays against a panel of 72 8 . s uhti li.~ slmins describe.d aho ve will be completed by 09/3/04 . Sensit ivity of the assays will be determined by 0911512004. The assays shou ld be rcady fo r validat Ion sl ulhe~ by 09/30/04 . Ana lysis of samples collected OCONUS. fo r the presen ce of /1acillu!; fl lllhracis An OCON US mission was conducted by FB I personnel from the WFO, HMR U, and partners from ot her intelligence agencies. Numerous samples were taken from several site..:; IIlcluding builtlings, wells , and buried latrine structures. The samples were tran:.poned CO~1US and were accessioncd by the NHFAC. Time line : All mic robial cuhure results were completed o n 08/02104. No viable B. Wlthracis was recovered . Specimens are currently undergoing PCR analysis for the presence of threat agents. Final results are expected by 08127 /2004 . As of this report, three samples have tested weakJy positive using pe R analysis. Pend ing complet ion of validatio n stud ies, positive "ample" wi ll be fo rwarded to Northern Arizona University (NAU) for strain geno typing. G[EXWSR B3D1.pdf  233  009723Anthrax Page 236 of 643 Iso topic Ana lvsis Goa l: To determine the geographic region of production of the evidence through thc use of Ilydrogen and Oxygen stable isotope ratios, as well as identify the class of media through CarbonI) (C-1 3) and itrogen H (N15) analysis. Isotopic examination of the Leahy evidence is completed. The isotope ratio data is being used to attempt to replicate the growth conditions noticed in the evidence. I'rogress: The Oxygen-18 isotopic profile of the eVidence !;uggests that it was grown under conditions in wh ich evaporation of the water in the media occurred during growth. An experiment designed to replicate this finding failed to produce the isotopic profile noted in the evidence. A follow-up experiment, was started on 0611412004 to use a more rigorous set of "evaporative ~ conditions to attempt to simu late the water metabolism observed in the evidence. The cXpt!ri mem was conducted through coordination wilh the NBFAC in a BSL-3 suite that is sui table fo r open containers of dry spores for several days. A proposal, for additional experimental work, to help refine our interpretation of the data is being reviewed. Time line: All samples currently at the Un iversity of Utah (UU) have been ana lyzed. A report on the isotopic analysis of the legal water samples is complete. The exist ing contract is bei ng updated. The new "evaporative" growth samples will be sent to UU by 07/3012004. Elementa l M a ppin~ Goal: To provide high resolution SE~1 and TEM elemental mapping of spores. Progress: Sandia l\"ational Lab (SNL) has observed 5i in the spore coal~ of 66% of the spores in the Daschlc evidence, 76% of spores in the Leahy evidence, and approximately 12% of the spores III samp le \ 030. SNL has reported that the 67% of spores in the NY Post material contained Si. The NY I)ost sample also contains significant amounts of ce llular debris. The Sandia results from the validation/contro l samples obtained from the principal investigators in literature references from \980- 1981 were consistent with the original publications. Time line: Recent investigative dt:vc lopments continue to generate additional sam ples for analysis. A group of 19 sam ples is undergoing analysis at SNL. The samples of significant investigati ve lead value arc expected to be completed by 08130/04. Elcmental Analysis Goal: To determ ine the growth condilions used by the suspect(s) by anal)'zing the elemental profile ofthl! cvidence, typical growth media, and spores grown under controlled lest conditions. G[EXWSR B3D1.pdf  234  009724Anthrax Page 237 of 643 2 AMX Weekly Science Update Prog ress: TIle ref~rence spores cultured by CTFSRU and delivered to the Chemistry Unit (CU) included 1.7 grams of Bacillus cercus r (non-pathogenic) strain spores grown with Si licon-comaining ami-foam and 4.8 gram of spores grown without Silic;:on anti-foam. Prel iminary results revealed detectable levels of AI, Ca, Cu, Fe, Mg, Mn. Zn and Si. 30 mg of irradiated Leahy material was analyzed by CU with results duplicating those from 2 years ago. Time line: All samples submitted to CU have been completed. Whole Genome Amplificatio n (WeAl Goa l: To dctcnnine whether trace amounts ofhwnan DNA may be extracted and typed from Ihe evidentiary letters. Prog'rcss : IBIS Pharmaceuticals, Carlsbad. CA. has been selected to proceed with validation o f their ;;,(, 0?0 methodol ogy. Drs.1 08131 12004. land SA! Ivisited IBIS on 0712712004 ro, a ano th~r program review. Draft SOP's havc been reviewcd and should be final ized at meeting at IBIS on T im e lin e: Pend ing approval o f SOP's at the 08/31 /2004 meeting, IBIS wi ll comp lcte validation testing of their methodology. and the evidence will be transported [0 IBIS for WGA analysis. Differ entia l Phenotypic Colo n), Express ion Goal: To observe the ev identiary morpho logical variants in cultures of the Repository samples and to correlate these patterns with those found in toe evidenliary materi als. Progress: Targeted growth of Repository samples wi ll initi ally focus on those samples that the molecular assays identify as conta ining the variants. A sub-set of the remaining Repository samples will then be grown and tested . Time liue: Screening fo r morphological varian ts observed in the cvidentiary materials should be complcted coincidentally with the completio n of the genet ic characteri/.ations. Genetic C haracterizat io n of Mo rpholo gica l Va riants Goa l: To identify the source of Ames slr(lin BA used in the leners usi ng genetic variation fo und in mutations present in minor variants. Progress: All of the Repos itory samples have been extracted for analysis. and the DNA has been nonnalized for analysis. To date, the entire Repository has been genetically screened using the two Morph A assays. Final reports are bei ng assembled for each Repository sample. A limi ted number or Repository samples have tested positive for one or both of the morph A variants . Investigative efforts are ongo ing to identify tho complete history oflllese isolale:i. Addilional variaml have been identified that appear to be closely related to the known mo rph A pauems. ,. G[EXWSR B3D1.pdf  235  009725Anthrax Page 238 of 643 3 AMX Weekly Science Update Ti me line: Screening of t he murph A I and A3 variants within the fi rst 600 sample set s hou ld be completed by the end of 0712004. Genetic C haracte riza tiun of rhe Bacillus Co ntaminant Found in New York .'\1 a ilin~~ Goa l: To fully characteri ze the Bacillus J.'u.blilis contam inant(s) found in the New York mailings and use this characterization to St.:reen other samples of 8. subtilis to dt:termine the source of the evidence. Progress: DNA typing of markers found in deri vative evidentiary material has revealed patterns not generally observed in isole Network (LR :"J) protocol fo r identiriCation o f BlIdlllls llIUhTilcfS po int s OU I that hemolysic; call bt! u~erved in con fl uent g rowth of agmg cu ltures. The eviden tiary iso tope result s !oIuggcst exte nded (evapora tive) growth. Furthermore. the N Y Pust material. known 10 eomai n 6% agar and mher dc hris, may s lill ha ve traces of heme present. if a blood media was used in iI's p! oduction. T he validalion siudics al ECB/ FAC have demonstrated lhal Ihe MALD I? TOF?MS is able (0 quantify heme In Slc rnc spore extraCb . Time lin e: s tudies. The ECB/ FACs lina l repJrI is e xpected by 09/30/2004. II w ill aitJ III pl:.!nning add ilio nal validalion G[EXWSR B3D1.pdf  245 ..  009735Anthrax Page 248 of 643 2 AMX Weekly Science Update Elem enta l Mappin!,! Goal: To provide high resolution SEM and TE\ll elemental mapping of ~pore~. b6 Progress: Two samples forme rly attributed to l Iwere negati"c for Si incorporation as was sample bi: 1029 . Sample 1029. a mj,:lurc of DPG and USAMRI ID produced mmerial. W,I." density graLiient purifi ed with rCl1ographm. Renograph in is a large mo lecule which is 50% by weight Iodine (I). No iodine was detected in sample 1029 or any other s3mpll!' sent to SNL using the clerno::ntal mapping technique. Sandia National Lab (SN L) has observed 5i in the spore coal of 66% of the ~pores in the Daschle ev idence. 76% of spo res in the Leahy evideoce. SNL has reported thallhe 67% orIhe spores in the I Y Post matc:nal cOnlain 5i. The results indicate thaI about 10% of the ..pores present in r.;ample 1030 contain Si in the ~ pore CO trains descr ibt!d above is completed. Se nsiti vity of the assays wtll be determined by 09/1512004. The assays should be ready for validat ion studies by 09130/04. The validat ion lest plan is currently being drafted by CBSU. Analvsi s of sa mples eoUce.cd OCONUS. for (h e I)resellce of Bacillus Dlllhracis An OCONUS mission was condUt.:ted by FBI personnel from the WFO. IIMRU , and partnl;'rs from ot her intelligence agcocies. :"lumerous samples were laken fro m several siles including buikhngs. wells. and h uried latrines. The sa mple~ were transport ed CONUS and were accessioncd and proccs:'.Cd by lht: I\BFACC. T ime lin e: A ll ffiKTobial cu llure resulls were completed on 08102104. No viable B. llIlfhrm:is was recovered . Three samples have tested positive using PCR a llal~is. Two sample!; are weak , and one !>amrle is inte rmed iatl.! in signal imensity. All three samples were t ransported to Northern Arizona University (NAU ) on 08127/2004 . Pe nding complet ion of \'alidation !;t udics, 'JA U will perform SNP detection assays deSigned to both con firm that the samples cont ain the !.ignawre for B. all/Macis. and lest whether or 001 the ~amples are the Ames strain. The fu ll complement o f genetic assays will be completed by early October. pendmg validation o f one additional Ames specific genelic ~1 award orientation was completed on 1/27/05 . Eviden<.'e tr:msport to IBIS is :-;cht:d u led for 21 15/05. The conlrac[ timclable requires completion of all analyses and reports nn ' he ~t!vr:n sa mples by 4/30/05. DiI1"e rentia l Phenotypic C o lony Expression Goa l: To o b~crve morphological variznlS in cultures o f FB I 811ciflu~? {//llhraci!i 10 corre late these patt erns wit h t hose fo und in the evidentiary mat erjal~. Rep()~i ' ory (FB IR) sa mples and Progress: Analysis of FBIR sampks i~ focused on those s am plc~ thai have been identified as containing the 1\ I and/o r the 1\3 mutat ions. Twelve sampks have been transferred Dr. Wors ham (\ JSAMR II D) for analys is. Pheno typic is at ~~1R C. 10 the BFI\C for G[EXWSR B3D1.pdf  259  009749Anthrax Page 262 of 643 4 AMX Weekly Science Update In addition. this efron. casu has completed contracting aClions to obligate funding 10 NAU required to support of T ime lin e: Approximately 800 survey samples have been through analysis at the BFAC and NM RC. No posit ive cultu re isolations or molecular detection:. for 8 . lIJuhracis have been repo rt ed 10 dalt!. G[EXWSR B3D1.pdf  260  009750Anthrax Page 263 of 643 AMX Weeki Science U dale March 25, 2005 Iso lopic Analvsis Goa l: To determine the geographic region of production of the evidence through the usc of Hydrogen and Oxygt::n stable isotope ratios, as well as identify the class of media through Carhano (C? I J) and N il rogcn'~ (N?1 5) analysis. Isotopic examination of the Leahy evidence is completed. Tht:: isotope ratio d:J.la is bei ng llsed to Dltcmpt to repl icate the growth condilions noticed in the evidence. Il rogress : CBSU is arranging 10 have a video teleconference with Dr. Ehlcringer on April 1, 2005 to brief AMX and the LD on all of lhc eITons 10 dale. The Salt Lake City Division is assisting with the technical details. A series of gro\vth experiments wi th isotopically altered med ia is underway althe :'>JBFAC to e lucidate the specific fractionation Illodel of isotopic incorporatio n into SA Ames. The analysis of 10 OCONUS water samples is complete. Sample 1029 and its sllpemalant wcre also sent [0 Utah. Analysis of the isotopic profile of thc cvidentiary envelopes is pending rece ipt of samples from the USSS. Tim e li n e: Sample 1029 is scheduled fo r the next solids auto sampler run. The Uni versity of Utah will bcglll the analysis o f the evidcllIiary envelopes upon receipt. Elem ental M~lp pin2 COll i: To provide high reso lution SEM and TEM elemental mapping of spores. Progress: . NSF AC prepared a complete set of grids from Sampl es 1029, 1030 r og r('ss : t\ collection o f SEM grids for the Dugway contribution to S by peR. Fu nher strai n typing resu lts rrom the!:.c samples revealed that these samples contain DNA from the Ames strain of Bacillus lJmhracis, consistent with the evidence. An OCONUS mission to collect addi tional items of potential evidentiary value was conducted in November. 2004. To date, approximately gOO samples obtained from some of thcsc itt!ms have been sent to the NBFAC for microbial culturi ng and peR anal ysis. No posi tive results have been obtained to date . Scporale ly obtained samples from the S:llne OCONUS IDem ion by another US Government agency were stra i/H yped at Nonhem Arizona Universi ty. These samples wcrc obta ined prior to {he May, 2004 sampling mi ssion. These rcsuJ ls are consistent with the earlier strai n typing resu lts described ::Jbovc. Time line: Sampling of the remaining items obtained from the November. 2004 search will be completed In Apnl. 2005. Addi tional proces!:.ing of the Hems oblained from the mitial search o f the location is in the planning stages. G[EXWSR B3D1.pdf  263  009753Anthrax Page 266 of 643 Isotopic Analysi.'" Coal: To determine the geographic rcglOn of production of the evidence through the use of Hyd rogen and Oxygen stable isotope ratios, as wdl as identify the class of media through Carbon 1.1 (C-13) and ;-.Iilrogen l S (NIS) analysii>. Isotop1c exami nation of the Leahy cvidence is completed . The isot ope milo data is being u ~d hI attempl to replicate the: growth conditions nOliced in the evidence. Progress: CBS U hdd a video teleconference with Dr. Ehleringer on April I. 2005 to brief At\1X and the LD on all of the efforts 10 dute. The Salt Lake City Divis io n assisted wilh the technK:al del aiJs_ Dr Ehleringer con tinues fo revise the mathematical models thm may give more information ::sbout the water thaI was used to gro w the spo res. Analysis of stable isotopes incorporated in spore fait), acids has begun. A series of growth ex periment s wit h isotopically altered media is underway at the NBFAC to e luc idat e the specific fractio nation model of isoto pic incorpo rat ion into 13A Ames. The ana lysis of 10 OCONUS water samples is compktc. Sample 1029 and i t ~ supernatant were also scm to Utah. Analysis of the iso topic profile of lhe ev identiary envelopes is pending receipt of samples from the USSS. Time line: Sample 1029 is sc heduled for the next solids auto sa mpler run. The Uuiversity of Utah will begin the analysis o f the eVIdent iary envelopes upon receipt. Elementa l Mapping Goa l: To provide high resolutio n SE~1 and TEM elemental mapping nf .\pores Progress: . NBFAC prepared a complete sct of grids from Samples 1029, IOJU and a reference .\amp1e from DPG to evaluate any density effects through the entire sample pellet as it rclatc~ to Si containing spores \'5. nonSi containing spores. The recently produced DPC! reference mat eri allaYl: r ~ 2,5 and 8 showed 26%,11% and 29% orthe spores containing Si respectively. Layered samples of 1029 at l ayer~ 2,5 and 8 all showed ill&. of the spores co ntaining Si. Sample 1030 showed result.", of 6%,0% and 6% fo r layers 2, 5 and 8 respectively. Ttl recall. the amou nt of Si contain ing spores fo r the evidence are 66% (Daschlc) 67% (NY Post). and 76% (Leahy). Time li ne: SNL has been tasked wilh co mpleting a forma l report on the lindings. "lemelltal Analysis: Coa l: To determine the growth conditions uo;ed by the suspect(s) by ana lYling the elementa l profile oflhc ev idence. Iypical growth ,ned ia. and spores grown uncler controlled le.\t cOlldit io"". G[EXWSR B3D1.pdf  264  009754Anthrax Page 267 of 643 2 AMX Weekly Science Update Progress: A co lleclion of SEM grids for (he Dugway contribution 10 Sample 1029 are currently undergoing SEMlEDX analysis in the Chemistry Unit for the presence of Silicon. Sample 1029 was submitted to CU this week fo r ICP/OES elemental analysis. Time line : Sample 1029 is expected to ~ completed by 412912005. Who le Gl:nume Ampli fi cation jWGAl Goal: T o determine whether Irace amounts ofhuITh1n 0 the evidt!nliary leners. A may be ~x tracled, harmacculicals. Carlsbad. CA, was selected 10 process the evid t!m::e. SOP's have been reviewed and approved. Cont racting action was completed on 0 1/ 1812005, and funds have been obligated to pro\'Klc for the analysis of seven samples along with control... Ti m e line: A post award onentation was completed on 0112712005 . Evidence transpon 10 IB IS was comple ted on 0211512005. IR IS requt:StecJ a mplt!s IS focuse d on those samples that have been identified as containing the A I and/or the AJ mul:u ions. T welve sarr.ples have been transfe rred to Dr. Wo rsham (USA1\1tR lID) for .In;] lysis. Phenotyp ic analysis of all twelve ~amp l es has been completed. From the twelve samp les. scventy-seven (77) morpholog ical variants have been selected for molecular analysis. Cell suspensio ns were made from tnese vNn Time line: Additional morphologIcal variants present in RMR 1029 and ii, dcrivalives are being isolated and characterized . a31 ISSV13Nn :l G[EXWSR B3D1.pdf  269  009759Anthrax Page 272 of 643 3 AMX Science Update Genetic Characterization of Morphologica l Variants Goa l: To identify thc source of Ames strain BA used in the lellers using genetic variation found in mutations present as minor variants. Progress: Screening of the morph Al and A3 variants within the first 600 samples is complete. B?SN P and Ddeletion assays have been evaluated at three laboratories, MR1, CSI and IITRL Contract action has been completed at MRI and IITRI for the D-deletion assay. Funher B-SNP development work is ongoing at eBI. Shipment of Repository samples to MRI and IITRI for f)?del etion assay screening is Sl:hcduled to be co mpleted by 12/02/2005 . Time lin c: Additional OCONUS samples obtained from the UK have expanded the current Repository. These addi li onal ~ample!'; will be processed for the A I and A3 variants at em upon completion of a blinded proficiency test. The expanded Repository will be presented to !VIRI and IITRI for D-dclction testing. Genetic C llara cterizati on of th e Bacillu s Co ntaminant Found in New York Mailin e;s Goal: To fully characterize the Bacillus subliJis contaminant(s) found in the New York mai lings and lise this characterization to screen other samples of B. sub/ilis to determine the source of the evidence. Progr ess: DNA typ ing of markers found in derivative evidentiary material has revealed pallerns not generally ohserved in isolates of B. sublilis. Testing of real-time PC R assays has successfully identified a set of markers that are present in the evidentiary strains but are not found in a pane l of 72 H. sllbtiiis strains. Three of these markers were chosen fo r validation. These validation studies are complete. CBSU has delivered the procedure to NMRC, along with reagents to conduct the assays. Time line: NMRC successfully completed a blind validation test of these transJi.:rred assays provided by CDSU. NMRC will begin testing evidentiary material currently siored and prepared at the NBFAC during the week of 12/12120005. Ana lysis or s!lmp les coll ected OCONUS, for the presence of Bacillus (IIltltrllcis An OCONUS mission was conducted by FBI personnel from the WFO.IIMRiJ. and partners from other intelligence agencies in May, 2004. Numerous samples were takc=:n from ::;everal sites including buildings, wells, and buried latrines. The samples were transported CONUS and were accessioned and processed by the NBFAC. "nlfC C samples tested posi tive by peR for the presence of fJacillus {m/bmeis. Further strai nlyping results from these samples revealed that these sam ples contain DNA from the Ames strain of Bacillus anthracis, consiste nt with the evidence. As a result of these findings. an OCONUS mission to collect addit ional items of potential evidentiary value was conducted in November. 2004. A Hazardous Evidence Stomge Fm;ility at ECDC, Aberdeen Proving Ground. Maryland was lIsed li MRU ilnd CBSU pf0cessed the evidence at the ECBC. DNA extrm;tion and peR conducted on a subset of these samples at MR.l revealed results consistent with the May. 2004 miSSion. to receive and process the collected malt:liti s from {he second OCONUS mi55ion. 270 G[EXWSR B3D1.pdf   009760Anthrax Page 273 of 643 4 AMX Science Update Time line: Research and development on optimized D}JA extraction methods targeting the collection devices and evidence recovered in these missions is ongoing. G[EXWSR B3D1.pdf  271  , 009761Anthrax Page 274 of 643 ---- Work~ng Copy ---- Page 1 Precede n ce: To : R OUT I~~ Date, Attn : 0 2 /0 8/ 2 00 5 Washingto n Field SSA/ rIC '----------' From : Laboratory Chem Bio Co n t a c t: lc;ences Un i t Approved By: Drafted By: Wilson David L Case 10 #: 279A-WF-222936-LAB Title: ( Pe nd i ng ) AMERITHRAX; Me 184; TECHN I CAL REVIEW PANEL MEETING Synopsis: On 14 December 20 0 1 , the Was h ingto n Fi el d Of fice convened a panel of leading anthrax authorities t o review laboratory analyses performed to that date. The expert panelis t s pro v ided comments on the d ata , inferenc es bas e d on their experience , and proposed additiona l analyses. Enclosure (s ) : Technical Review Panel Meeting Agenda, Technical Revie ..... Panel Me e ting Summary, Non Disclosure Forms Details : On 14 December 2001, the Washington Field Office convened a panel of leading anthrax authorities to review laboratory analyses performed to that date. A list of participants is provided at the end of this document. The Technical Review Panel Meeting Agenda (enclosed) included discussion of possible sources of material, the expert i se and equipment required for various production techn ~ ~Jes, and possible chemical signatures. The expert panel i sts pro vided comments on the data , inferenc es based o n the i r exper i enc e, and pro p o sed additi o nal analyses. Proceedings o f the meeting are summar i zed in the enclosed RTechnical Review Panel Meeting Summary. " Based on the discussion, the Laborat o ry Di vis ion i s usi ng n o n-patho gen i c bacterial spores to develop additional analytical methods which may be used to provide investigative and / or forensic leads. bE b7C I Attendees: Non-FBI sRI. f ormer head USAMRIID I Case ID : 279A - WF - 222936 - LAB Serial : 115 3 G[EXWSR B3D2.pdf  1  009762Anthrax Page 275 of 643 -- - - Working Copy --- Gr ounds retired, us Offensive weapons Program t WAMR TTD Page 2 and J ormer head of US Ant~rax program Peru b6 I CO Lent:r ~ US &:m L M dical Research Detachment, Lima, l b7C ; r A pplied Microbiology R&D, Porton Down, UK sonnel Forensic Science Research Unit Trace Evide nce Unit L".,..,.=c=---,==:.-..-J i Forensic Science Resea r ch Unit ~ 80n , Dav ~ L .; Washington Field Office LEAD (s) , Set Lead 1; (Adm) Washington Field (AMX - 2) For information only. G[EXWSR B3D2.pdf  2  009763Anthrax Page 276 of 643 Age nd a Technical Review P;:I U K , ;1/'< ... .... 0./1-< ~ ~ v:. "" 1 -t1 ..... l::j 1. r o'] I b6 UJ'1fM !-II J ~ - .PBI tuFO I I C-luYI- . I'Mu ~ 7C C!_ -:.!..::;?: "". 'J4M~8cJ01V5 . 17. I I II 05";dI /(c. Ij OS l:) ) i'bu..c.., ~I "5Rt !tAM I ':{', J: D G[EXWSR B3D2.pdf  4  009765Anthrax Page 278 of 643 AMERITHRAX Technical Review Panel Meeting Summary 14 Nov 2001 The Washington Field Office convened a panel of leading anthrax authorities to review laboratory analyses performed 10 dale. The agenda included d iscussion of possible sources of material, the expertise and equipment required for various production techniques, and possible chemical signanlres. The expert panelists provided comments on the data, inferences based on their experience, and proposed additional analyses. Based on the discussion, the Laboratory Division is using non-pathogenic bacterial spores to develop additional analytical methods which may be used to provide investigative andlor forens ic lcads. be b7C CPT lames Burans. Ph .D. Peter Turnbull, Ph.D. FBI Panici pa nts SSA David L. W ilson co US Navy Medical Research Detachment. Lima, Peru Centcr for Applied Microbiology R&D, Porton Down, UK WFO FBI Lab, FSRU FBI Lab, FSRU FBI Lab, SAS G[EXWSR B3D2.pdf  5  009766Anthrax Page 279 of 643 G[EXWSR   Envelopes and Letters o No Envelope found associated with human anthrax infections in Florida . Onset of infcction consistent with timing of New York letters o Envelopes from NY Post and Tom Brokaw letters were postmarked 1g Sept 01 from Trenton . NY Post letter was a photocopy of Brokaw letter o Envelope from Senator Daschlc letter was postmarked 9 Oct 0 I o Sides of all enve lopes were taped Contents of Letters o Brokaw letter - Originally processed at CDC - On ly 2 particl es sent to USAM RlIO 6 o NY Post Leiter o Granular ? Tan o Dasch!e [cncr 0c..:rftl ..,j /n 5 o Ju.7)ol") o Li ght Inn powder C-!1 ~ 10"rI rt ...... r,v - Mi ldly electrostatic ? Peaks at 1.5 and 2S microns <<5% 1.5 microns) o B3D2.pdf 009767Anthrax Page 280 of 643 G[EXWSR Scanning and Transmission EM Results o Insuffi cient sample rrom Brokaw letter o Contents or Post letter - Scanning EM: spores lind matril< debri~ , - Transmission EM : spore~, vegetati ve cells, debris and elongated crystalline mntcrial o Contents or Daschle letter - Scanning EM demonstrPtcJ clumps ohpores agglomerated together - Transmission EM : pure spores no vegetative cells sccn o Scanning EM EDX analysis ro r Si, CA, CI, P, Sand 0 are ongoing   Bacteria Isolated from Letters o B anlhrocis ~va s isolated from allleLters: - Gram + baci llus - Non-hemolytic - Penicillin sensitive - Non-moti lc - Gamma phage sensitive - Inducible capsule - PA , LF and Cap gene sequences amplified by PCR 7 2 B3D2.pdf 009768Anthrax Page 281 of 643 G[EXWSR Additional DNA Analysis of B a1lthracis Isolated from Letters o No ev idence o f genetic engineerin g o No DNA evidence of antibiotic resistance to penicillin , doxy cycl ine or ciprofloxacill in Concentration of Spores in Slim pies o Brokaw leuer had insufficient sa mple   o NY Post letter contained : 8 - 1.33 x 10" c fu/gram o Dasehle letter contained: - USAMRIl D: 2.1 x 10" cfulgram - Battelle: 4.6 x 10 10 cfulgrnm o Homo logous to B an lhraci.~ Ames based upon 17 loci in the protective antigen and edema factor genes " ) B3D2.pdf 009769Anthrax Page 282 of 643 G[EXWSR Possible Sources of B. anthracis o State sponsored terrorist group. terrorist group related or unrelated to II Sept, 200 I attack (OCONUS) General Expertise Required (At. Minimum) o BII5;C knowledge nbout B, Anlhracis o Ability to IIcquire ;so]tlte of B. Alflhracis or isolnte from Ihe environment o Basic to IIdvrmced bilcteriology knowled ge:   9 o CONUS based group, mil itias, survivalists with or without OC ONUS ass istance o Unabomber type of indiv idual, " lone wolf' o Diopcst icide product ion experience o Sterile technique o Cu lt ure techniques (Medill selection , prepnrat ion) o Ferme nta ti on techniq ues o Spo re prod uctio n o Harvesti ng techn iques - Drying tec hniques 4 B3D2.pdf 009770Anthrax Page 283 of 643 G[EXWSR   Possible Sources of Expertise o Tnined bllcteri ologbt. US - PhD level, leu lor maltrainin g requ ired irind ivid ual hIts lnduatrlal or medieAI up er lence Juch u blo_peu ld de production or vllcd ne dcvlopmel1t o CAn utili 7.C InrormaliOn fro m nreD lite rA ture A internet, nd FAD minuAI f or blo ll~ l k ld e produ ction o Ope n lite ra ture from fo rm er US OW prog rll m, books, newspl!.per Nu mber of IndividulIls NcccsslIry o Small scal e production (less than 20 li ters of liquid med ium or solid med ium, yield +/- 2 grams) - Possible with one person o Large scnle production (more than 20 liters of liquid or solid medium, yield greater than 2 grams) - Additional people necessary (2 - 3) 10 articl es and Inlernct - Clin ica l mlcrobl ololY manuA ls - COAc hin g/trAin in g from tec hnics l expert s with 8W A gent productio n experience 5 B3D2.pdf 009771Anthrax Page 284 of 643 G[EXWSR Possible Cultivation Methods of B. Anthracis in n Powdered Form o Small scale - Petri dishes with agar media - Roux bottlelTompson bottle with agar media - Flask culture with liquid media - Small scale (mini) fermenter o Large scale - RouxITompson bottle (large) - Flask culture (large number) - Fennenter (greaccr than 10 liters) Possible SOUl'ces of B. ""thracis Powder in Current Scenario OCONUS Liquid   C,_._. Pu" .. td Po ...... . S.. ,II S"I. "', : :" .. M . . . ... CONUS CONUS c"."' .._ ~. 11 Sm.1I Sulo ; \::~~:::, ~, j L Liquid Powder ( Dry rorm) 6 B3D2.pdf 009772Anthrax Page 285 of 643 G[EXWSR Technical Requirements and Signatures for Each Prod uction Method o OCONUS Powder/li quid o Technical requirements ror transport - Plastic, glass or cardboard sealed containers - Containers can be small - Nothing specia l Technical Requirements and Signllture. for Each Production Method o CONUS s mn llllnrg. stil le AgRr meth od o Techn icAl requirements - Incubator or hcn ltd room - Co nlain ~n to hold medium   12 - Glau or plastic: Roul type bolliu or nasks - GlaS! or plu tie petri dishes - Inn oc ubtln g loop! phu lic, mehd, swab, o AK Hl ed ho nr o Sca le - A uloclave or low tech method to s lel'ilize medium o Signatures - Contain ers with liq uid or powder - No labels, fi cticious labels 7 B3D2.pdf 009773Anthrax Page 286 of 643 G[EXWSR Technical Requirements and Signatures for Each Production Method o CONU S small/large scale "gnr met hod o Signatures and Waste - Dirty glass ware - Waste pl astic wnrc - Waste agar - Innoculating loops, swabs, pipettes - Asso rted Jab supplie.'1 Technical Requirem ents and Signatures for Each l' roduction Method _ _ o CONUS s mall scalcllarge scale liquid media method . o Technical requirements - Incubated shake rs - Incubator or low tech heating method - Small or large rermenter - Fl asks up to 1 liter in size. glass or plastic - Pipettes - Broth medi um or low tcch substitute with salts   13 8 B3D2.pdf 009774Anthrax Page 287 of 643 G[EXWSR Technical Requirements and Signatures for Each Production Method o CONUS small scale/large scale liquid media method o Signatures and waste All P roduction Methods Produ ce [I Liquid Produ ct ( of vnrying co nce nt rution s 10 8 - 1011 spores/till) o Must concentrate spore suspens ion ' o CaCl1 o Centrifugation - Filtration through membranes o Methods of conversion from concentrated liquid form to powder:   14 - Waste liquid med ia - Dirty plasticware and glassware - Used assorted lab supplies, pipettes - Air dryin g - Spray dry ing - Lyophilization 9 B3D2.pdf 009775Anthrax Page 288 of 643 G[EXWSR Technicnl Requirements and Signatures ror Liquid Concentration Methods o Centrifugation or filtrati on o Technical requirements - Centrifu ge - Filter concentralOrs and assoc iated apparatus o Signatures and waste - Centrifuge tubes - Nitrocellulose membranes Technical Requirements and Signatures ror Dry Powder Production Method o Ai r drying. ~pray drying or freeze drying o Technieal requirements - Hel\lll\ mps - Filter paper - Solven ts such us aectone - Lyoph il izer - Spray drying equipment - Heat lamps - Filter paper - Glass vials, bottles or ampu les   15 10 B3D2.pdf 009776Anthrax Page 289 of 643 G[EXWSR Technica' Requirements and Signatures for Dry Powder Production Method o Air dry ing, spray drying or freeze drying   o Signatures and wasle ? Bentonite 16 ? Silica ? Sugars - Glutamate - Ski m milk - Acetone or other so lvents 11 B3D2.pdf 009777Anthra>< Page 290 ais?. 3: 5, 2 ,3 23; sgig .2 17 B3D2.pdf 009778Anthrax Page 291 of 643 (Rev, 10-01-(999) FEDERAL BUREAU OF INVESTI GATION Pre c edence: To: From: ROUTINE A t tn: Date, 12 /21/200 1 washington Field Washington Field [ A= A= =l == k I~ S~ C:=B =e C=Ismith A-2 Conta c t : Approved By : Drafted By , ~--------------~ Wilson David I I Case ID #: 279A-NF-222936-LAB "'- (Pen ing ) Title: Ar1ERITHRAX; Me 184; MEETING OF ANALYTICAL CHSr-1:STRY EXPERTS ::3' 1, bo On 7 December 2001, a panel of experts from the natio~al laboratories, the Nationa: Academy of Science, and the National Science Foundatio~ met with FBI inves=iga tors and scientific perso~nel at the Washington Field Office to develop an analytical scheme for the chemical characterization of a~thrax evidence. Details: On 7 December 2001, a panel of 20 experts from the national laboratories, the National Academy of Science, and the National Scie nce ?oundation met with 13 FBI investigators and scientific personnel at the Washington Field Offic e. The purpose of the meeting wa s to develop an analytical scheme for the chemical characterization of anthrax evidence. Selection of attendees from the Department of Energy was coordinated by l I I 1 Chief science and Technology Advisor, Department of Just1ce. Se~e ction of attendees f ~ om academia Nas coordinated by l l an~ _ l of the National Sc ience Foundation and National Academy of SC1ence, respectively. A list of participants is provided at the end of this document. The flow chart below summarizes the consensus forensic approach frcm the meeting. The draft approach will be reviewed by a subset of those attending and by other experts. The approach covers all four aspects of physical characterization of a sampl e: microscopic morphology, bulk analysis, elemental analysis, and molecular analysis. Minimization of sample consumption was a prime consideration. The minimum amount of sample that would be required to conduct all analyses is 55 mg (45 mg consumed) . Synop s is: b7c IIlA Y_N.I ATcL WOIRHEPP B3D3.pdf  1  009779Anthrax Page 292 of 643 To: Re: vlashington Field From: Washington Fiela 279A - WF-222936-LAB, 12 /21/2001 I Forensic Characterization of a Bacillus Sample I IM01j)0GY I I -, Elemental Ash "!yo., I [!J TEM LM \ I o D' SF.MIEDX ~ /l t? Aqueous phase Molecular " " Water Extr. '1 7 "'" ~ CH2Cb Extr GC'~S ' u 7 I Bulk Analysis ICPIMS' ~: ablation,'MS ..!.J.;> -, H~dspace SIMS:...? , , D "" Centrifuge / MALDI " Optical spcctro~copy SEMlEDX' TIM/ED' Laser a.blatlon/MS 2..' TXRF' JJ JSSc,lld rha.~c lonC~mphy" ~"nc:rsprav MS,?10 ! I XPS" MAW'" Ai;ar&!cUi. _..It, to p"p.uk '<>':01 pX02 +/4 specific 10 H. (1IIIlt l'fll'iS 9 spore 511:111(', q ua lity 10 11l1I?lil'l l' si Z(' II slrlii n id e ntification I! ' Jl l? ~? ilh- 1.\ :.!~? I h? l k ,' '':!ill l' ~' ri ll !! 1-1, an' im' f'('s is lal1 (,{, l~ :.!,'Ul' iill' lIlilil'a li ulI ;!.-IU" '-.'- I (I "ump ll'll' s{'(lu ence B3D4.pdf 009789Anthrax Page 302 of 643 WOIRHEPP   5 Federal Grand Jury Subpoena of AMES Samples Amerithrax Reposi tory USAMRIID $225,500 1 I. Anthrax Repos itory 2. Salllpk ('haraCIl' ri /ali ll ll o ,i'IIJiiiiIi" slant .., _. I. ... $8 7, 60 7 ~ DNA .I . -1 ,1. , Strain Identification Paul Keim, N.A .U . $ 142,546 2 15 cx i ~ li l l l! \ ' ~ T R s \ 100 2 Iso la le /) N \ I(' r I ./\ ~ I. :?' Isql atc l'i a'llI id..; Ii )!' T ICi R? I'C'R 'S:\ I' . \ ."; ' ,; 1\"; R& D' ~ \' l'\\ \ " \ I J{, 1{,'\:/)" "1'.:$. ~~ur, Genetic Protil e Paul Jackson, LAN L I. ') o ,...,,~:.<..,.. J4SiI<~ .''-. ... plasmids 1 t.iSIii oo ~ tI Results support R&D I'CRlSN P Assay .> . ., Anti bioti c R~'~i s l ,l nl'" . ~ X. c lollil lg Vl',' lor, I " I (, b l '- ( I Li 0 I H. 11 .\ .......<-,.-' _ 1 J1 a /I i '!: It"; oJ :..... , ...... -t. ......... '-e., Cl'rco lvs il1 ;-\ II . o:s."'" ;.i:~ .... ,,~ ' .v.., _ ~ !C l'l1 l' ........", ,,-"'d""""........ l{,\ I. 1 1. l ~ l\:.r ) I(Hid..;'_~ '.,.'lht 11..'.1 ,' ..; " ,,1. "I ""'.'" D '" ' llOK Sequencing TI G R $ 1. 125M I () \ I'l as l1 lll! \ ,Ii 1 ion ,1:11 '7 ~ 1 \() I p.\ii.' ;..'ql!~' nccs ' ---- .o "j ~ l. ~ ""';"" ,,...,. ........v o \ )p ....?;,ro tl ; ~ :d hwd ;. 'L';'-l!'''~ \ '\, ~' .o. C\ II 'i:' 5f1 (..;: .:.1 .....? . .;;> 1 "P-ItI: .I:\~'I,; ~"11 o i ' I . !:{I:,\. [ I , .I 'I K B3D4.pdf 009790Anthrax Page 303 of 643 (Rev. ! O-O! -1 999) FEDERAL BUREAU OF INVESTIGATION Precedence: To: ROUTINE Date : 11/14/2005 (AMX Washington Field Attn : SSA ~~er i thrax lIC e~~~~~~?~1 C I 2) From : Laboratory Chern B:.o Contact : L r~"," e " r s....U".o".i.l: c ;".,,,::.e"5 ; r _ _ _ _ _ _ _ _ __ ---, Approved By : Adams O;d ht E pen- D/V '" Drafted By : Case ID # : 2 7 9A-WF- 222936 -LAB Tit1e : AMERITHRAX; ( Pending ) /30'8" bE b7C MC 184; CASE AGENT M EETING Nov . 5 , 2001 Synopsis: On November 5, 2001 the vlashing ton Field Office convened a meeting of viro and NK field inves tigat o rs , anthrax and ep idemiological expe~ts to discuss anthrax production and vic tim epidem iology _ Enclosure(s) : M eeting Ag enda , List of Partici pant s , sli des , and notes (lA) Details : On Noverrber 5, 2001 the Was~ington Field Office convened a pa nel of field investigators, anthrax and epi demiological exper~s to anthrax product ion and vict im ep id emi olo gy_ The Meeting Agenda (encl o sed ) included presentations on the technical requ irements f or the production of anthrax by Capt _ James Burans (U S Navy) and John Ezzell ( USMJRI I D) _ A discussion o f resu l ts obtained by the partner l ab oratories t o d ate, possible sources of material, the expert i se a nd equipment required for vario u s production techniques, and possible chemical signa t ures. I I presented and overv iew of epidemio log y . A summary of t h e mee ti ng is enclosed . WOIRHEPP B3D5.pdf  1  009791Anthrax Page 304 of 643 To : W ashington F1~ l d Fr om : Laboratory Re : 279A- Wr- 222936- LAB. 11 / 14 / 2005 The participants ( list enc:osed) recommended a series o f additional analytes to con si der for detailed c haracterization of the evidence. 2 WOIRHEPP B3D5.pdf  2  009792Anthrax Page 305 of 643 To : Re , Washington Field From : Laboratory 279A-WF - 222936-LAB , 11/14/2005 LEAD (s) , Set Lead 1: (Adm) ( ~~X - 2 ) Washington Field For information only. cc, . 1 - Dr. Adams, R!T\ . 1200, ,BI Lab , QT Rm . 1210, FBI Lab, QT 1 Rm. 1214, FBI Lab, QT 1 Rm 3112, FBI Lab, QTtwo 1 Rm 3110, FBI Lab , QT 1 - ~~~ Rm 3110, FBI Lab, QT Dr . 1 be b7C - 3 WOIRHEPP B3D5.pdf  3  009793Anthrax Page 306 of 643 WFO Forensic a nd Investigative Update M eeting 1115/01 ,----,ICDC Q?NKj was attended by l OIJTTF; J. Ezzell, USAMRIID I I WFO,l INK;I IWFO; L IHQISIOC; IWFO; I Burans, US avy, D. Wilson, WFO; c:::J I WFo;:J;j::;::;;;:;::::::;I~WFolsIOCI'----' J. Burans, US Navy presented a through overview of the technical requirements for the production of Anthrax by liquid and Agar methods in both small <<10 L) and large batches. 1.-,,_ _--', CDC presented an overview of the epidemiology. The data suggests that the Florida (never recovered) and NY Post letters were mailed contemporaneously. Based upon the evidence recovered from the letters it was the group's consensus that the material was likely produced in small batch or batches by someone skilled in the an of microbiology. Due to the propensity of contamination in liquid preparations, even by those very skilled in the art. it may suggest an agar-based production. Pre-sterilized agar plates, inoculation loops, etc are commercially available thus further reducing the need for autoclaves. The concentration of spores is consistent with a liquid and agar production but would require a deliberate concentration step from the liquid. The electrostatic behavior of the Daschle material during laboratory handl ing may be caused. by the apparent lack of electrostatic dissipative additives which distinguishes it from large scale state sponsored production. be b7C The single color Daschle material is suggestive of the careful removal of the top layer observed in the centrifugation of anthrax preparations (by USAMRI.ID) while the tri?colored material in the l\ty Post letter is suggestive of the use of all three observed layers or an air?dried preparation. A review oftbe analytical results to date raised the following questions from NK investigators and t.be group. Action to be 2ddressed by: 1 Wbat ~ .o es the presence of Si and 0 in the samples suggest about the preparation? ~o contact lab for cJarifiC2tion. L-_-' ?. 8 WOIRHEPP B3D5.pdf 2. What are the crystals observed in the sample? Is the crystal sbape indicative of air drying or a lyophilized preparation? Could the crystals be artefacts of the sample preparation procedure for SE..M? Are there SEM photo micrographs that can be used by rs during interviews? (digital images OK)? Photos on the wa,' by FED EX. now up wit~ kLD) on crystal growth. may be a good source as well. L I_ _ _ _ _ _- - - ' 3. Is tbere a validated surface sample collection protocol that can be used in Newark to resample tbemail collection boxes. Does USAMRIID have a validated procedure for the culturing and peR of swabs? J. Burans, USN is aware of a validated procedure developed at Battelle, Columbus OB for another US government client. It is ,uggested that a siogle cotton swab b< ultd (0 >ample a IlIfJ:t art. (dol!, air venu,  4  009794Anthrax Page 307 of 643 fans , etc). A control swab and a" known blank" should be su bmitted with each samples. to 4. Can we obtain the repons from Dr. Keirn? Has he compared the isolates with those in his library? How many loci have been detennined? Can blinded samples be accurately identified ie Ames from severaJ sources compared to vollum? Keirn's current results are known to th e LD and contact (procurement) action is undenny to obtain funher use ofKeim 's expertise. 5. What is the identity and plate count (relative concentration) of the heta?hemolytic gram positive rods in the SPS 02.44.01 sample? Does CDC have the expenise to speciate non B. anthracis Baccilli? Sample is bein g shipped to C DC for identification. 6. Have the Keirn and Jackson laboratories been subjected to blind samples? LD to discuss 7. Can we obtain a list of attendees at the last International Anthrax. Conference? contacted IUMRU a nd material will be provided to NEWARK I LI_ __ -' 81 IWilson and Burans wiU review draft and coordinate with Newark for comments, corrections, additions. Meeting held 9am Tues 1116 at WFO b6 b7C 9. What is the current status of the specialized testing? Can we get an update and time line fo r expected (future) results? I ko check status with I I HMRU who has been administering the specialized testing. Contract la b can only release results to LD AD. 10. NK, WFO have suggested a Lab to Field EC summarizing results of testing. EC dated 11/06, 279A-WF-222936-LAB 1 I. Can the group propose additional analytical testing to characterize (develop a "signature/fingerprint" of ) the growth process and post culture processing. Suggested Analytical T esting (attached) forwarded to LD for tasking. It should be noted tbat contact lab was only provided 24 mg of sample. WOIRHEPP B3D5.pdf  5  009795Anthrax Page 308 of 643 Detailed Sample C haracterization A. Post C ulture Processing (Following a phase separation of spores from other matrix components) conduct appropriate analytical exams to determine the presence of. 1. 2. 3. 4. 5. Sugars (sucrose, dextrose) Skim milk powder Sodium Glutamate Antioxidants (thiourea) Hydrophobic, hydrophilic silicas (philadelphia Quartz Co) 6. P~ethyleQe~EG) r vc r~ " ~~ 7. Specific Gravity R. G rowth Media Components (conduct appropriate analytical exams to determine the presence of: Peptones Protealytic digests (tryptacase. casein) Meat protein digests (brain heart infusion, meat infusion) Agar residue Sugars (lactose, dextrose) 6. Salts (Nael, phospbate buffers) 1. 2. 3. 4. 5. C. Additional Suggested Analytical Measurements 1. pH 2. Anion & Cation Ion Chromatography/CE 3. Fatty Acid Methyl Esters (FAME) WOIRHEPP B3D5.pdf  6  009796Anthrax Page 309 of 643 11 /5101 ANTHRAX MEETING GOALS : 1. Process Review: Thorough review of possible sources of Anthrax * How cultured .. How purified o What is required : Equipment I Skill 11. Attribution: Where did it cone from? .. Genetic analyses .. Physical/Chemical analyses o Contaminants It Strategic analytical plan III. Investigative Recommendations: .. Sampling locations (previous sites I blue boxes) Sampling techniques o Additional lab work (ic_ Dr. Turnbull, etc ..) * IV . Review of Epidemiology: CDC"LI_ _ _ _- ' IV. First hand Q&A with the experts. b6 b7c WOIRHEPP B3D5.pdf  7  009797Anthrax Page 310 of 643 ---b6 - J:S' - wRJ 1~<<.,7- .(jJ~cJ I G ~"" cdFO/.~ FGJ:'-w~u/-Trl~ -I.(h 7:=-, -!.J If f <; I'h11 ell]) :I" ,_[/Q.L[T5? v ~(_ ww To o .,., __ fm -ttG.!211>i-/O' ... f e, r }.Jf{ Jr~ o ..- Possible Cultivation Methods of B. Anthracis in a Powdered Form o Small scale - Petri dishes with agar media - Roux bottle/Tompson bottle with agar media - Flask culture with liquid media - Small scale (mini) fermenter o Large scale - RouX/Tompson bottle (large) - Flask culture (large number) - Fermenter (greater than 10 liters) B3D5.pdf 009804Anthrax Page 317 of 643 WOIRHEPP Possible Sources of B. anthracis Powder in Current Scenario OCONUS Grnm s - liters   Liquid Prepared Powder CONUS Less than 201 CONUS Greater than 201 15 Sma ll Scale Agar Small Scale Liquid Media Large Scale Agar Large Scale Liquid Media ~ Liquid 1/ 1 Powder (Dry form) B3D5.pdf 009805Anthrax Page 318 of 643 WOIRHEPP Possible Sources of B. anthracis Powder in Current Scenario OCONUS Grams - liters   Liquid CONUS Less than 201 Prepared Powder CONUS GreJp L6 b-'C I Drafted By ' Case ID # : 279A-~'lF-222936-LA6 l ( pending ) IL-__________~I / 3) 0 Ti t le : _~ERITHRAX ; MC 184; SCIENTIFIC ~EVIEW PANEL MEETING JUNE 11 -1 2 , 2002 Synopsis: On June 11-12, 2002 the vlashingtcn field Office convened a panel of leading chemical and biological authorities to revie',.; laboratory analyses p erformed t o dat.e by partner laboratories . The expert panelists prov ided review and comments on the data and proposed addi tional analyses . Encl osure(s ): Technical Rev iew Panel Meeting Agenda, Summary (One Document ) and List of participants Details : On June 11 - 12, 2002, the Washington Field Off i ce convened a panel of l eading che~ical and biologi c al experts to review laboratory analyses performed to t hat date . A list of participants is enc l osed . Signed n o n - disclosure agreements were submitted under EC 219A - WF- 222936-~AB Serial 178 . The Scien t if i c Review Pane l Heeting Agenda (enc!osed ) inc:'uded presentation and discussion of res ults obtained by the partner laboratories to date , possible sources of material, the expertise and equip~ent requi r ed for various production techniques, and possible chemical signatures . The expert panelists provided corrur.ents on the data, i nferences based on t heir experience, and proposed additional a n alyses . WOIRHEPP B3D6.pdf  1  009821Anthrax Page 334 of 643 ~o: Re, Washington Fi~ld F=o~: Laboratory 279A -NF-2229 36- LAB , 114/2005 Proceedings of the meeting are su~arized "Amerithrax Scient':'fic Review Panel ." i~ the e~closed Biological and genetic presentations were made on June 11 , 2002 by Dr Paul Keirn, Northe=n Arizona University (NAUl, Dr . paul Jackson, Los Alamos Nationa: Labo=atory (LANL ) ; Dr . Tim Read, The Inst itute for Genomic Research (TIGR) ; and United States Army Medical Resea=ch Institute of Infectious Disease's sceitnists (USAMRIID) Drs . Patricia Worsham, I I and Dr Tom Geisbert. On June 12, 2002 the Chemistry panel reviewec the analytical effo rts to date . Dr . Paul Kotula, Sandia National Laboratory (SNLl; SSA I I FBI Laboratory; Dr . Catherine Fense leau , Unive rsity o f Maryland; and Dr. John Hayes , Woods Hole Oceanographic Instit~te (WHO:. ) The panel recommended that AMES be produced , dried and processed with a variety of well characterized media and under co~trolled laboratory conditions . No changes were recommended to the December I, 2001 chemical and b':'ological analYSis protocols . Based upon this recomrr.encation p~x and t:"1e laboratory contracted with Dl.:gway Proving Grounds (OPG) and Southwest Foundat~on for Biomedical Research (SFBR ) to produce the AMES samples . b7c b. 2 WOIRHEPP B3D6.pdf  2  009822Anthrax Page 335 of 643 To : Re , Washington :1eld From : Labora:ory 279A-WF-222936-LAB, 114/2005 LEAD (s) , Set Lead 1 : (Adm) Washington Field (AMX- 2) For infor~ation only . 1 1 1 1 1 ~t;;~.~A ~a~m~S~~~.~ 1200' 1210 , Lab, 0 = ~d Rm R:TI . FBI FBI - SSA - SSA . QT Lab, QT . 1214 , FBI Lab, Q':' Rm 3112 , FBI Lab, QTtwo Rm 3110 , FBI Lab , QT Rm 3110, FBI Lab, QT b6 b7C oo WOIRHEPP B3D6.pdf  3  009823Anthrax Page 336 of 643 AMERJTBRAX SCIENTIFIC REVIEW PANEL June 11 - 12 , 2002 Int roductio n On June 11-12 , 2002 a scientif~c review pane: meeting was held at VWO. The purpose of the meeting was to have representatives from a:1 1abora~ories conducting examir.ations of evidence in t~e Ame ri thrax investigation present the s tat~ s of their ",ark and their ~esults. Addi tional scienti fic experts were included to provide furth er insight, review and recommenda~ ions =or potential future examinati o ns. The meeti ng was conducted in two sessions, Biological and Genetic on June 11, 2002 a nd Chemistry on June 12,2 002. Attached is a copy of the meeting agenda and at tendees list. I. Biology and Gen etics No rthern Ar i z o na University ( NAU ) In itiat ive : Dr. Paul Keim , Northern A~~ z ona University, a leading authority on the strain identif~cat ion of bacter~a, provides strain identification 0 = al l FBI anthrax samples. Dr. Keirn, NAU, described the 15 v ariable number tande m repea t (VNTR ) marker system used to identify ~he Bacillus anthracis ~~es stra i n (k~es). To cate, all collected evidentiary material has been identica l w it~ the laboratory reference strains of Ames. Dr. ~ei m is now in the process of analyzing the Ames isolates from the Gra~ d Jury Ames Repos itory . The Ames Repository contains al l known i s olates o f Ames which existed wi thin the CONUS prior to 0 9 / 11 / 2001. To dat e , all isolat e s identified as t he AMES strain i n the Ames Repository are identical using the 1 5 VNTR markers. One i solate was identified as the Stern stra in. A set of 21 new VNTR markers exists but these 2 1 markers are not ye t validated. One VNTR marker. HM-l , is a series of adenosine residues (poly A) whi ch appear to di=fe= slightly in one l aborato ry A..-nes reference sample. Howeve r , the HM- 1 ma rker app~ars to rapidly mutate , perhaps ~oo fase for forensic valipation . In addition, the current assay for r~-l appears prone " to polymerase o stutcer and requires valida c ion by independent technique(s). M Dr. Keirn is al so extracting and purifying t he PX01 and PX02 plasmids from selected AMZRITHRAX specimens, for sh i pment to The Institute f o r Genomic Research (T :GR ) . The plasmids :rom t h e original 1981 Ames isol ate have been p ur ified and are c urrently being sequenced . P1asmids from an isolate from the AMI building in Florida are being purified. Loa Alamoa Natio n a l Laboratory (LANL) WOIRHEPP B3D6.pdf  4  009824Anthrax Page 337 of 643 Initiative: Dr. Pa ul Jackson. LANL provides genetic profile analyses to test for po~ential genetic modificat~ons ~de through the use kno~ cloni~g vectors and antibiotic and vaccine resistance modifications. Dr. Jackson's analysis of the evide~ce for signs of genetic eng ineer ing (antibiotic resistance, engi~eering vectors, vaccine resistance ) have found evidence of no such attempts. Dr. Jackson will continue this work on the reposityry s a mp les An additional Israeli DNA cloning vector supplied by _ I USAMRIID is being added to the prococol. The Israeli vector was recently used to genetically modify t~e Ames strain at USAMRIID and will oe useful i~ scree~ing the numerous isolates submitted by USAMRIID. The I nst i tute for Genomic Research (TIGR ) ~~c TIGR provides DNA seqJencing of plasmid D~A isola Led :rorn the repository samples, evidentiary samples and selected refere~ce samples. Initiat~ve: Dr. Tim Read, recently completed the comparison of the genomic sequence of the Stevens (Florida ) isolate and a reference sample (NSF funded). The plasmid sequencing of the o r iginal 1981 Ames Strain is nearly complete. Future cont ractual efforts include sequencing the pXOl and pX02 plasmids from selected items o f evidence and Ames Repository samples. The res ults of those sequencing efforts will be provided upon comp letion. Uni ted States Army Medical Research Institute of Infectious Disease (USAMRIID) Initiative: USAMRIID provides primary (i nitial ) characterization o f all suspect samples and serves as the Repository for 3acillus anthracis (BA) samples returned under Subpoena from all laboratories identified to date as having 3A. US~1RIID's primary support co date has been analytical services. including microbiological, immunological and morp~ological characterization of . the evidentiary material. '. '. Dr. Patricia Worsr.arn. USAMRIID. analyzed anthrax cuI tu res derived from Leahy spore powder to assess the significance of variou~ colony morphology differences oDserved in the original evidence after culturing. This work was unde rtak en to determine if k~erithrax evidentiary samples could be compared to repository samples using standard microbiological techniques to detect the source of the anthrax. Dr. Worsham found and described differences in the phenotypic expression of various colonies grown from the -Leahy spore powder. At least two ( 2) distinct colony morphologies are observed as early as 18 hours after incubation on variuus media (i.e . sheep blood agar. congo red agar. and trypticase soy agar ) . WOIRHEPP B3D6.pdf  5  009825Anthrax Page 338 of 643 One type of colony is ~ot very efficien~ in fo~ng mature spores and is termed oligosporogenic. The other type identified was the wildtype. These differences were mo re pronou~ced at 48 hours to 72 hours growth. Future '" "ork i s focused on determining t.he stability of these d~fferent colcny types and will a l so address the issues concerning the inhibit10~ of the sporulation process. efforts aE ceve lop~ng to detect differences in anthrax batches. The experimental data varied greatly for each dilution sample (n=5 ) using USAMRI ID 's experimental methodology, d~~onstrating a lack of reproducibilit.y and utility of t he method. The use of a pesticide and food industry methodology , such as a standard decimal reduction tech:1ique, was proposed as a potential method they may be of greater utility. Dr. Tom Geisbert, USAMRI!D, presented scanni:1g electron microscope ( SEM) and transmi ss i on electron microscopy (TEM) results on Amerithrax evidentiary samples using various preparative techniques. This work was undertaken to initially characterize the Amerithrax dry spore evidence early in the investigation. It was apparent from these results that the Daschle and Leahy spores were purified prepara tio:1s devoid of vegetative cel ls. Negative stains of the Dasch l e material indicated other electron dense component s within the sample. The identity of these components is currently being addressed by scientists from Sandia National Laboratories. Conversely, the New York Post sampl e contained an unknown matrix holding a mix of unpuri:ied spores with vegetative cells. It was recommended by the panel that the supernatant from the TEM preparations also be examined microscopically. Additionally, A more extensive SEM/TEM review of the Post mater ial has been i~itiated at USAMRI ID . I USk~IID . described initial experirr.ental a the~: stabi:i~y measurement proceoure b6 b7C ., II . chemistry Sandia National Laboratory ( SNL) Initiative: Sandia National Labo~atory(SNL)provides high resolution (nanometer level ) scanning electron microscopy(SEM ) and transmission electron microscopy (TEM) elemental /chemic al mapping of samples. Dr. Pa ul Kotula, described the SNL :indings o f an amorphous silica ( Si02 )coating observed on the spore coat (a specific WOIRHEPP B3D6.pdf  6  009826Anthrax Page 339 of 643 internal structure of spores) and not on the exosporium of the evidentiary samples. No ele~ental S i licon {Si l was observed on the vegetative cells in the NY Post material. The NY Post material also contained a small isolated ~etal flake of unkno~ origin. A ~ore extensive survey of the Pos~ material is underway in an atte~pt to cr.aracterize non-spore ma~erial. Samples of Bacillus cereus grown by the Laboratory Div~sion were observed to pick up particulate silica from a Centricol: g~ad i ent column. In offline discussions. the SNL and USAMRIID rr.~croscopists noted the similarities between their observations. FBI. Laboratory Initiative: The Chenistry Unit of the FBI Labora~ory provides analysis to determine if orga~ic solvents were used to dry the evidentiary samples and elemental analysis of materials. ch e~ical Supervisory Special .lI,.genc (SSA) I I Chemistry ~nit, analyzed numerous su~rogate samples produced by various methods, as well as the Leahy and NY Post macerial. No acetone '....as observed in the NY post material. A trace level of acetone was observed in the Leahy ~aterial at leve ls not significantly diffe rent from those observed ~n irradi ated surrogates driec without the use of acetone . Wh:..le the Laboratory :Jivision desires additional samples of validation. the results indicate that irradiation with C060 does not generate significant amounts of volatile organic compounds and that the Leahy and Post samples do not contain significant concentrations of acetone. university of Maryland ( UMD ) Initiative: Dr. Catherine Fenseleau, Department of Chemis try. University of Maryland is recognized world wide as a leading authority on the analytical chernisLry of biological organi sms. Dr. Fenselau reported on her progress in the development and validation of methods for trace agar and sugar deter~ination. A new method for agar residue has been developed but requires additional optimi zation and validation studies. Future effor ts incl~de development of an assay for peptones and or other media compo~ents that may give a clue as to production methods. Dr. Fensel~u's method is being transferred to another laboratory to facilit'ate timely optimi za tion. Woods Hole Oceanographic Institute (WHOI. ) Lawrence Livermore National Labora t o ry ( LLNL ) Intitiative: Dr. John Hayes at Woods Hole Oceanographic Institute (WHOI) is recognized as a world expert on techniques of modern atmospheric radiocarbon C-l4 dating and biolipid deuterium/ hydrogen analysis (a 2 year old science) . b6 b7C WOIRHEPP B3D6.pdf  7  009827Anthrax Page 340 of 643 Dr. Hayes prese~ted data on the radiocarbo~ C-14 dating of the evidence and surrogates. D~ Hayes ' dated the Leahy material as "Modern- and also correctly dated twO ~ blind" surroga t e samples produced in 1967. The C-14 dat a 0: Dc. Hayes ag~ees with ~ndependen t resu l t s (not presented at this meeting) =rom LLNL. LL~~ dated the Leahy sample at the year 2 000 ( + /- 2 years ) . Dr. Hayes also presented data regarding the measurement of and deuteri~ ratio measurements and the i r potential forensic va:ue. Dr. Hayes stated that the hydrogen / deuterium ratio measuremen ts should be useful in characterizing the evidentiary samples. however. he indicated t~at the data may not provide useful geolocation information. Dr. Hayes indicated that add it ional isotope ratio analyses might also prove informative. hyd~ogen It was noted that work has been conducted at the University of U" tan on the isotopic ratios of bacterial growth media from different sources. Efforts are underway t o obtain information from that study and to a ccess its value to the investigation. II X . Panel Recommendati ons : The panel recommended that Ames be produced with well characterized media using a number of production. drying a~d processing steps. The Ames produced f ollowing ttose techniques should then be used as experime~tal control samples. Data collected from analysis of tho se samples .....ould be use:ul for the interpretation of data collected from the evidentiary samples to date. No c nanges were suggested to the December 7. 2001 chemical and biological protocol . Discussions with the biopesticide and particle producing industries were recommended. Future AMERITHRAX Efforts: AMERITHRAX will contract with Du~~ay Proving Grounds (DPG) to produce AMES under controlled experimental conditions for use as control samples. The agar detection method of UMD will be forwarded to a contract lab for troubleshooting a~d optimizati on. Liaison with the biopesticide and particle industry will be initiated. '. , WOIRHEPP B3D6.pdf  8  009828Anthrax Page 341 of 643 AlVIERITHRAX REVIEW PANEL At tendee s 6/11-12/02 Burans, J ames , Ph.D. Commanding Officer, ?S Navy Medica Re searc h Institute for Inf ectio s Diseases Lima, Peru Contacc: Tel: 51-1-5 61 -2733; Fax : 5 -1-56 1 - 3042 e-mai l : bu rans@namrid.sid .pe Ezzell , John, Ph. D. Chief, Spec i al Pa thogens Branch, Unit e d Sta t es Army USAMRIID 1425 Porter Stree~ Ft. Decrick , MD 217 2 Contact: Tel: ( 3 01) 619-4732; Fax: (30 ) 610 -2 492 LD e~-:a-:r" "t"m'e"n"t"-""o:-:t""""'C"""' v""~- I---'E"n-g=", "~"n"" "e:-e,: . .r., ".,~. .,n". . .g-..".n:,,. -d:,. . . .,. lJ eo:' og i cal ~p " " " " ~-="' ' " = ". a ema~l: L..-_ _ _ _ _---J Sci en c e s University of Notre Da me 1 56 Fitzpatrick Hal Notre Dame, IN r4 ~5 5 6 __________________________________~ ~6 ~~ con~aci . Tel? I On~ted States Army USAMRIID 1425 Porter Street Ft . Detrick , MD~~~1 7 ~2 ________ 2 ~~0 ~ Contact: Tel : I email: ~ I b6 b7C ________________________-JI. Fert?e au , Catherine , ph.D. Dep,irtment of Che:nistry and Bioche . stry univer sity of Maryland Colleg?~ Park, MD 20742 Contact?: Tel: (301) 405 -86 1 6; Fax: (301) 405-86 1 5; email: fenselau@wam.umd.edu United States Army USAMRIID 14 25 Porter Street Ft. Detr i ck , MD 21702 WOIRHEPP B3D6.pdf  9  009829Anthrax Page 342 of 643 Geisbert. Thomas, Ph.D. United States Army USAMRIID 1425 Porte~ St~ee~ Ft. Detrick, MD 217C2 Contact: Tel: (30 1) 619-48 03 Hayes, John P. , ph.D. ( 6 / 12 /0 2 only ) woods whole Oceanographic Institute NOSAMS, Mai: Stop 8 Woods Hole, MA 02543 Contac~: Tel: (508) 289-3345 ; fax: email: ihayes@whoi.edu 150B) 457-21B3; Ko tula, Paul G., ?h.~. Materials Characterization Dept. 1822 Sa~dia Nat ional Laborato=y Albuquerque, NM 87185 Contact: Tel: (505) 844 - 8957; Fax: (505) 844-2974; e-mail: pgko~ul@sandia.gov Jackson , Paul J., Ph . D. Biosciences Division Los Alamos National ~aboratory Mail Stop MaS8 Los Alamos, NM 87545 Contact: Te l (505) 66 7 - 2775; e-mail : Keirn, Pa ulS . , ph .D Department of Biological Sciences Northern Arizona University Flagstaff. AZ 86011 Contact; Tel; I92B) 523 - 107B; Fax; e - ~il : Paul.Keim@nau.edu , b6 b7C pijacKson@lanl. gov I92B) 523-7500; Room 3P0102 NHB Washington, DC . .2 0 5,, 5_ _ _ _- , ..= 0., Contact; Tel; I I Fax; ~I_ _ _ _ _ _ ___' WOIRHEPP B3D6.pdf  10  009830Anthrax Page 343 of 643 Martin. Dan, Ph. D . Life Sc~ences Division Dugway Proving Grounds Dugway, UT 84022 Room 3POI 02- N:;B Washington, DC Contacc: Tel : r2:5 : ===:::J :0 t_:O S Fax: Meyer, Richard F., Ph . D. Nationa l Center for Infect ious Diseases Centers for Disease Cont.rol NS-G42 Atlanta , GA 30333 Cont.act : Tel: ( 4 0 4) 639-0075; Fax ( 404) 639-4234; e-mail : rrm9@cdc.gov Hichael , Jo seph R . , ph . D. Ma teria ls Characterization Dep t . 1822 Sandia National Laboratory Albuquerque, NM 87185 Contact Tel: (505) 844 - 9115; Fax: (5051 e-mail: j rmicha@sandia.cov 844-2974; b. b7C Dugway Proving Grounds Dugway, Contact: UT Tel: 0)~2:2:-:5~O~O~O:::===:::JIFax l 84 L DC Washi~gton, Contact : _-maUl Te l : . I :>0'01 Fax: CDC - Building 17, Mailstop G34 1600 Clifton Road Atlanta, GA 30333 Con tact ? Te l l IFax : e-mail: IL-______________-' WOIRHEPP B3D6.pdf  11  009831Anthrax Page 344 of 643 Department of Juscice 95 0 Pennsy l vania Ave .. N.W. Was~ington DC 2 0 53 0 Contact: Tel:rl..:..::.::..::..:..----, Fax : Ravel. Jacques. ph.D. The Institute for Genomic Research (T IGR ) 9712 Medical Center Drive Rockville. MD 20850 Contact: Tel: (301 ) 838-5884 e-mail: jravel@tior.com b7C b' Read , Tim, ph.D. The Inst~tute for Genomic Research (TIGR) 9712 Medical Center Dr ive Rockville, MD 20850 Contact: Tel : (301 ) 838 - 0200; Fax: (301 ) 838-0208 University of Connecticut Hea lth Ce n t e.!'" 263 Farmington Avenue Farming ton, CT , " 6,, 3-"'--___--, ? " ?2 ? Contact, TeL fax, e-mail: I LI_ _ _ _ _ _ _ _ _ _ --"I ] Worsham, Patricia L.? . Ph . D. United States Army USAMRIID 1425 Porter Street Ft. Detrick, MD 21702 Contact: Tel: (301 ) 619-4937; e-~ai1: Pa c r ic ia.Worsnam@det.amedd.army . mi1 ., , WOIRHEPP B3D6.pdf  12  009832Anthrax Page 345 of 643 FBI PARTICIP~S I(NO ) I r-~I===========;---'I ,;:1=======,-'1 I I (Lab Div) (Div 5) 'r=========-----, (WFO) (Lab Div) b6 b7C ( Lab Div) ( Lab Div ) (WFO) SSA Wilson, David (WFO ) , " , WOIRHEPP B3D6.pdf  13  009833Anthrax Page 346 of 643 SCIEl\lIFIC REVIEW PA.'1EL AGENDA PRESENTATIONS I Tuesdav June 11. 2002 Welcome & Administrati ve Details 8:30am-9:00am Dr. Paul Keirn, Korthern Arizona University 9:00am-9:45am Genetic analys is of the Amerithrax Bacillus anthracis Ames isolates Dr. Paul Jackson, Los Alamos National Laboratory 9:45am-IO:30am Analysi s of the Amerithrax Bacillus anthracis Ames isolates for evidence of genetic engineering II III BREAK I 0:30am- 10:45am III Dr. Tim Read , The In stitute for Genomic Research 10:45am- II:30am Sequencing the viru lence plasmids of select Amerithrax Bacillus anthracis Ames isolates LUNCH II :30pm-l:OOpm IV Dr. Patricia Worsham, United States Anny Medical Research Institute of Infectious Diseases \. l:OOpm- 1:45pm .;Analy sis of the Amerithrax Bacillus anthracis Ames isolates for evidence of differential phenotypic colony expression AGENDA WOIRHEPP B3D6.pdf  14  009834Anthrax Page 347 of 643 PRESENTATIO S (con't) Tue day June 11 b6 v 1:45pm-2:30pm ~nited States Army Medical Research In L..-_ _ _ _ _ _ _...... Infectiou Diseases titute of b7C Thermal stability analysis of Amerithrax Bacillus anthracis Ames isolates VI Genetics General Discussion & Summary 2:30pm-5:00pm PRESENTATIONS I Wednesdav June 12.2002 Dr. Thomas Geisbert, United States Army Medical Research Institute of Infectious Diseases 8:30am-9: 15am Microscopic analysis of the Amerithrax. Bacillus anthracis Ames isolates using scanning and transmission lectron microscopy Dr. Jo eph Michael, Dr. Paul Katula, Sandia National Laboratories 9: 15am-l 0:00am Elemental analysis of the Amerithrax Bacillus anthracis Ames isolates using high resolution scanning and transmission electron microscopy BREAK lO:OOam-IO:30am II III \ Microscopy General Discus ion & Summary 10:30am-11:30am :~. LUNCH 1 I':30am-l:OOpm AGENDA PRESENTATIONS (con't) Wednesday June 12,2002 WOIRHEPP B3D6.pdf  15  009835Anthrax Page 348 of 643 IV -..rm=--r-",,;;rr;---"f ederal Bureau of Investigation Laboratory L 1:00pm-1:45pm Chemical analysis of surrogate and Amerithrax Bacillus anthracis b6 b7C Ames isolate samples V Dr. Catherine Fenselau, University of Maryland 1:45pm-2:30pm Development of mass spectrometry analytical methods for trace agar in surrogate and Amerithrax Bacillus anrhracis Ames isolate samples VI Dr. John Hayes, Woods Hole Oceanographic Institute 2:30pm-3:45pm Isotope ratio mass spectrometry of surrogate and Amerithrax Bacillus anthracis Ames isolate samples VII Final General Discussion & Closing Remarks 3:45pm-5:00pm ,, " \ WOIRHEPP B3D6.pdf  16  009836Anthrax Page 349 of 643 SCIE ITIFIC REVIEW PANEL AGE)lDA PRESENTATIONS I Tuesday June II. 2002 Welcome & Administrative Details 8:30am-9:00am Dr. Paul Keirn, Northern Arizona University 9:00am-9:45am Genetic analysis of the Amerithrax Bacillus Qlllhracis Ames isolates Dr. Paul Jackson, Los Alamos National Laboratory 9:45am- I 0:30am Analysis of the Amerithrax Bacillus anlhracis Ames isolates for evidence of genetic engineering BREAK 10:30am-10:45am 11 III III Dr. Tim Read, The Institute for Genomic Research I 0:45am-II :30am Sequencing the virulence plasmids of select Amerithrax Bacillus anthracis Ames isolates LUNCH I I :30pm-1 :OOpm IV Dr. Patricia Worsham, United States Army Medical Research Institute of Infectious Diseases 1:00pm-I :45pm Analysis of the Amerithrax Bacillus anthracis Ames iso lates for evidence of differential phenotypic colony expression WOIRHEPP B3D6.pdf  17  009837Anthrax Page 350 of 643 AGENDA PRESENTATIONS (con't) Tuesday June II b6 b7C v IUnited States Army Medical Research Institute of L-_ _ _ _ _ _- - ' lnfecti ous Diseases I :4Spm-2:30pm Thermal stability analysis of Amerithrax Bacillus anlhracis Ames isolates VI Genetics General Discussion & Summary 2:3Opm-S:OOpm PRESENTAIIO]\S Wednesday June 12,2002 Dr. Thomas Geisben, United States Army Medical Research Institute of Infectious Diseases 8:30am-9: ISam Microscopic analysis of the Ameritbrax Bacillus anthracis Ames isolates using scanning and transmission electron microscopy II Dr. Joseph Michael, Dr. Paul Katula, Sandia National Laboratories 9:1Sam-IO:00am Elemental analysis of the Amerithrax Bacillus anthracis Ames isolates using high resolution scanning and transmission electron microscopy BREAK IO:OOam-IO:30am III Microscopy General Discussion & Swnmary IO:30am-II :30am LUNCH II :30am-1 :OOpm WOIRHEPP B3D6.pdf  18  009838Anthrax Page 351 of 643 AGE~A PRESENTATIQNS (con't) Wednesday June 12,2002 IV LI_==;;:;-r.;==_ I :OOpm-1 :45pm federal Bureau of Investigation Laboratory b6 b7C Chemical analysis of surrogate and Amerithrax Bacillus anthracis Ames isolate samples V Dr. Catherine Fenselau, University of Maryland 1:45pm-2:30pm Development of mass spectrometry analytical methods for trace agar in surrogate and Amerithrax Bacillus anthracis Ames isolate samples Dr. John Hayes, Woods Hole Oceanographic Institute 2:30pm-3:45pm Isotope ratio mass spectrometry of surrogate and Amerithrax Bacillus anthracis Ames isolate samples Final General Discussion & Closing Remarks VI VII 3:45pm-5:00pm WOIRHEPP B3D6.pdf  19  009839Anthrax Page 352 of 643 Biological and Genetic Summary Paul Keirn - Northern Arizona University o o o o o 15 MLV A Markers - 21 new potentia] markers Po ly A's being validated, 50 sites, stutter observed To date all evidentiary samples are identical Future: Working on repository samples Future: Pro viding Plasmid extracts to TIGR Paul Jackson - Los Alamos National Laboratory o o No signs of genetic engineering (antibiotic resistance, engineering vectors, vaccine resistance) of evidence, environmental samples, 1981 Ames, Repository samples Future : Working on repository samples Tim Read - The Institute for Genetic Research (fIGR) o 1981 Ames Suain Plasmid Sequence nearly finished Future: complete 75 pXOl and pX02 ~uences o Pat Worsham - Phenotypic Differences o o o o Consistently observed 2 colony morphologies after 48 brs two colonies culture (plate) true NAU results indicate 2 colonies are the same Future: Repository samples L __ ---lf Thennal Stability c6 b?C o o Inconsistent resuJts at 50% and 90% kill ratio Recommendation: decimal reduction by industry standard techniques WOIRHEPP B3D6.pdf  20  009840Anthrax Page 353 of 643 b6 Chemistry Swnmary Tom Geisbert ~ b7C USAMRlID o o NY Post - Unknown matrix material holding spores Recommendation: Examine supernatant from TEM preps regarding. hairlike projections. Sandia National Lab o o o o o o Six coating observed on spore coal o f evidence No Si observed in vegetative cells (NY Post) NY Post - small, singular. isolated metal flake (composition similar but not exactly ball bearing material) P. Jackson nOled that shavings from the top bearings in ferementors often fall into broth Sample of (B.c.) througb centr'icoll picked up Si02 particles Geisbert and SNL in offline discussions were "comfortable" with each o thers resullS L -_ _---.J~ FBI Lab o Presented results of head space, IR and IAPIMS results from lab reports Catherine Fenselau - UMD o Validated method for Agar residue from BC14579 o o o Unknown sample degradation over time Future: follow on with peptide/peptone methods development Future: troubleshoot agar procedure l o hn Hayes - Woods Hole o o o o Correctly aged blind unlmowns ( 1967) Leahy dated as modem WHOl results consistent with (unpresented) LLNL results Future: Post sample should be done week of 6/17 WOIRHEPP B3D6.pdf  21  009841Anthrax Page 354 of 643 Overall Observations and Recommendations Evidentiary Profile Consensus o o o Terminal harvest Leaning towards agar growth ~lequipDnent: cennrifUge o Heavily washed (Purified) (Daschle & Leahy) PaoelReconunendations o o o o o Produce Ames by Agar & Broth with chemically well characterized media via a number of drying. sizing methods Usc AMES rather than surrogates as controls for future experiments No changes to December chemical and biological flow charts Interview biopesticide industry regarding. production Interview particle/drying industry WOIRHEPP B3D6.pdf  22  009842Anthrax Page 355 of 643 (Rev. 10-01-1999) FEDERAL BUREAU OF INVESTIGATION Precedence: To: From: ROUTINE Attn: Date: 12 /21/2001 Washington Field nashington Fiel d A-2 [ A= A =l == k I= S~=C~B =e C=Ismith Contact: Appr oved By: Drafted Case ID Title: AMERITHRAX; MC 184 ; I Wilson David ~--------------L~~ MEETING OF ANALYTICAL CHEI4:STRY EXPERTS bo Synopsis: O~ 7 December 2001, a panel of experts from the national laboratories, the Nationa: Academy of Science, and the National Science Foundatio~ met with FBI inves~igators a nd scientific perso~nel at the Washington Pield Office to develop an analytical scheme for the chemical characterization of a~thrax evidence. Details: On 7 December 2001, a pane l of 20 experts from the national l aborat ories, the National Academy of? Science, and the National Science Foundation met with 13 FBI investigators and scientific personnel at the Washington Field Office. The purpose of the meeting was to develop an analytical scheme for the chemical characteri zation of anthrax evidence. Selection of a ndees from the Depar:.ment of Ene rgy '.... as coordi nated by I I Chief Science and Techn o logy Advisor, Department o f Justl.ce _ e e c t i on of attendees f ~ Qm academia ?...'as coordinated by l lanar _ lof the National Science Foundation and National Academy of SCl.ence, resgectively. A list o f part icipants is provided at the end of this docume nt. The fl ow chart below summarizes the consensus forensic approach from the meeting. The draft approach will be reviewed by a subset of those attending and by other experts. The approach covers all four aspects of physical characterization of a sample: microscopic mo rphology, bulk analysis, elemental analysis, and molecular analys is. Minimization o f sample consumption was a prime consideratior:. The min i mum amount of sample that wo-..:.ld be required to conduct all analyses is 55 mg (45 mg consumed) . b7c WOIRHEPP B3D7.pdf  1  009843Anthrax Page 356 of 643 To: Re: i'lashington Fie l d From: Washington Piela 279A - WF -222936- LAB. 12/21/2001 L I MOiJlholo?Y I jJ1 ., LM Forensic Characterization of a Bacillus Sample J I Elemema1 .J...l;o"" Ash I I ~'., Bulk Analysis Hcadspt.ce \ ) t? Molecular ~ D .r.f IC PIMS' r..,c~ ablatiolliMS SF.MIEDX) SIMS<-' ' " Water Extr. / "'" CH ,Ch Extr / ..lJ,."?' , ~ " Aqueous phase I Jj. Centrifuge / Oc:tMS' MALm" I Optical spcctro~copy o SEMlEDX' TEM/ED' Laser .blation/MSlJ IT S("hd. pha.~~ / onC~raphy"" MAW/" Kllllosprav MS'.IO TXRF' XPS" Accch:ntlOr MS' Agatali<: cig::.itu~n ,,,,,tope nth" " Antic,pated Relult I . p I~ p"ty oo ..J'~" .k, profile 'US"'" n"S"' n All partic ipants agreed that each method should be opt imized and validated with surrogate samples prior to analys i s o f evident i ary material. Surrogate samples produced by a va riety of methods are available, or can be generated, for testing purposes. Tests with surrogate samples may reveal that som~ methods are either unsuitable or fail to provide additional probative info::tnation , in which case, e vider:ce consumpti on will be reduced accordi ngly. The effect o f various deco nta m ~ nation methods (e .g .. cobalt 60 irradiation , autoclaving, dry hea tir.g ) on subsequent determinati on o f chemical structures will also be evalua ted with surrogates. f'.1icroscopic morphology involve s the analysis of 1 mg of samples by Sca~ning Electron Microscopy (S EM) , Transnission Electron Microscopy (TEt>1) , and Light Microscopy (L:'o1 ) to provide information about the size. shape and quality of the spores . The presen ce of s il icon- based additives and /o r the lack of vegetative cells and othe r debris provides insight into the quality o f the product ion and technical skills of the producer. ~h e bulk analysis is an examination o f 22 mg of t he evidentiary material by a wide variety of chemical ana lytical 2 WOIRHEPP B3D7.pdf  2  009844Anthrax Page 357 of 643 To; Re: Washington Field From; rlashingtcn Fiela 279A-WF-222936-LAB. 12 /2 1 /2001 techniques and provides insight into general chem1cal class characteristics. the possible use of volatile organic drying solvents. and trace eleme:1tal profiles and maps. Napping shows che spatial correlation o f the elements with each other. Electron Diffraction (TEM/ED) allows deduction o f crystallographi c st~c~ure. Further elemental analysis is carried out 0:1 IOmg of sample '...'hich h as been oxygen plasma ashed :'0 remove all organic matter. Ashing reduces potentia l interferences from the organic samp:e matrix. A variety of e lemental techniques, including induct:'vely coupled plasma (rep). laser ablation and Secondary I on Voass Spectrometry (S IMS ), provides information regarding the elemental composition of the buffers and nutrients us ed in the growth process. High resolution SEM/ EDX (Energy - Dispersive X-ray microanalysis ) will provide spatially correlated identification of the elemental composition of particles present :'n the sample. The molecular analysis involves determining specific compounds present i:1 the material. Ten mg of samp:e will be extracted by water (po lar soluble ) , and anothe~ 10 mg by nonpolar (me thylene chloride, CHzCl ll extrac.t i on . The mo l ecu lar composition can provide characteristics of anthrax production and handling. Following water extracti on and centrifugation , icn chromatography can aid in the identification o f the buffer and nutrient sal~s and simple sugars used as cryoprotectant s in a freeze-drying process. Gas Chromatography (GC / MS). Matrix Assisted Laser Desorption Ionisation (MALDI) and Nanospray /MS can be used to characterize sugars, buffer/media salts and peptide sequences. The solid phase material can be subjected to accelerator mass spectrometry to determine the relative age of the material (us ing Cl2 1cl o isotope ratios 1. Agarase digestion fol lowed by chromatography can be used to identify unique sugars from an agar (plate ) medium. Heavy isotopes may also be used for geolocation if sufficient libraries of reference material exist. The o~ganic (methylene chloride) extract can be used to isolate any silicone anti foaming agents or hydroca~bon oils used. MALDI can also be used to analyze the complex polysaccharides found in agars and the bacteria itself. Attendees; Non-FBI b6 b 7C I University of Rochester Research emphasizes analytical innovation to study the geological, chem~cal and biological processes t~at shape the earth's surface environment-particularly . the abu~dances and isotopic compositions of trace elements in natural waLers and sediments. Contact information: Tel: I Fax: ~I_ _ _ _ _ _ _--, e-mail' ~I____________________~ ) J WOIRHEPP B3D7.pdf  3  009845Anthrax Page 358 of 643 To: Re , Washington Field From: vlashington Fiela 279A-WF-222936-LAB, 12 /21/2001 web page: http: //www . chern. rociester. edu / Facul tYA _____...J 1. Lawrence Livermore National Laboratory Characterize co~pour.ds for environmental , biomedical, tox~cological, and forensic research areas. Design 0= analytical equ:"pment to de:ect environmental pollutants, cw, exp:osives. DeSis: =ield-portable GC-MS instrumentation. Tel: I j e-mail: I I On~te d States Navy Commanding officer, US Na'l.'Y f'.1edical Research Inst itute for Infectious Diseases. Tel: 1 I e - mail:L_ _ _ _ _ _ _ _- ' I I Nat i onal sc i ence Foundation, I I Division of Chemistry ~-.~~s e a~~~h "~~~e R . ~~ r c ~ n t ~rests have included spectroscopic studies of molecular crystals, charge transport process in organic crystals and polymers , development of electrooptic and photorefractive polymer systems and spectroscopic investigations of slow re-o~ientational relaxation processes in polymers. r---------, Tel: I I Fax: l e-mail: 1 web page: httc : //ww w. osf,gov/moS Zd lVl. jijl.OCS / Ch (start,htm I bE b7c I Fenselau, Catherine University of Maryland Developing methods for the rapid detection and analysis of microorganisms by mass spectrometry and is exploring the interactions of drugs with proteins that involve molecular recognition and chemical reacti on. Exper:"~ental tools include cell culture, molecular modeling, ~~R and all kinds o f mass spectrometry. Tel: (301 ) 4 05 -8616 ; Fax: (301)405 - 8615 e-mail: feoselau@wa[,umd,edlJ web page: r.ttc: IIw~:w. chern . umd. eC:...I/biochem/fe:1seJ el,l,' L,~a nod'''-N"'a~t~~6 S ~n , a , ~n~a~I~Labo rat o ry I Spectroscopy and hyperspectral imaging analysis using multivariate statistics . Spectroscopi es include mid - , FT-, and near-IR, Raman, fluorescence, AES, ESCA (XPS), ICP-AES , IMS , XRF. Molecular processes at catalytic surfaces. Tel, I I e -mail . I I Hassell, Christian 4 WOIRHEPP B3D7.pdf  4  009846Anthrax Page 359 of 643 To: Re, Washington Field F:::-om: Washington Fiela 279A-I-STel: L. - - - - ,I Pax; I [====::J 5 5 WOIRHEPP B3D7.pdf   009847Anthrax Page 360 of 643 To: Re, Washington Field :rom: Nashingto:1 Fie la 279A - WF - 222936 - LAB. 12/2112001 WINPAC I r-__~ e~ T~ l~'~.======~-----J1; Fax: e - rna i ~L.________________..J Los Alamos Natio:1al Laboratory Trace inorganic analysis of environmental and biological samples. Trace analysis of bacilli, peptid e mapping using isotopes. Expertise in a wide variety oE a~alytical spect f oscOPY . Tel, . Ie-mail, WINPAC Tel: e-mail . LI_ _ __ _J C;;T e l ' ~l~;;;~~~J; ~~; "c~l:i~e t~ ~Jr'c E~ e an2 ~cG, hnology A~d~~i ~o r~ D ~p~= -~~e n t~ ~J ustice C h e f s~y 2 n c~':a d~~ a V ~5 ~ ,-"~e a r~ m ~~ o~f , e - mail: I r I b6 National Research Counci l (NRC ) Director of the Board on Chemical Sciences and Tech nology (Be ST ) nis responsibilities center on the coordination of studies on science and science policy , particularly in the area of federal policy and its interrelationships with the chemica l sciences. Before movi ng to his current position in 199 0 , Raber was a member of the chemistry faculty at the Uni'Jersity of South Florida, where his researc h program focused on organi c and comp utational chemis t= .~____- , p r~ I Fa x r e-mai l : Te l: I 'N'eb page: www.natlOOa lacede" i . org / best Pacific Northwes t Na tional Laboratory Trace organic analysis o f environmental a~d biol o gical samples. Methodological development for 5upercritica: fluid techn iques (chromatography , mass spectrometry, extrac ti on). Coupling o f supercritical fl uid ~ec~olog i es to LC, GC and d e v elop ment o f field i~struf~~n ~a : i ~Ul-________, c ~r a -~ Q O Tel : [ I e - mail: L.______________----' Onlve rS l CY of PiEE s 6 urgh A physical chemist, specializing i n ne.,." physical and chemic al measurement methods in surface science to understand b7c I 6 WOIRHEPP B3D7.pdf  6  009848Anthrax Page 361 of 643 To: Re; Wa s hington Fleld From: Washington Fielu 279A-WF-2 2 2936- AB, 12/21/20 1 struct re, dynamics, and c emistry on meta s, and insu ators. Tel: Fax: e - mai I :L________________~----~ i web page: www . dev . chern. i:: semi c onducto~s, I I I Attendees: PBI 1-_ _ _ _ _ _ _...L..IiCoI.<::l~Jing on Pield Office ~orensic Science Research Unit Quality Assurance U i race Evidence Unit hemistry Unit .; Qual'ty Assurance Unit /----------,...---1 Forensic Science Research U it Chemiscry Unit Quality Assurance Unit ......_ _ _ _ _ _ _ _...L....:..._ _--;. Chemistry Unit -b6 b7C 1.--------_---1 i e i st ry in' t Washi g~on Fie d Off ice .; Washington p ' e ld Office oo 7 WOIRHEPP B3D7.pdf  7  009849Anthrax Page 362 of 643 I~ Ji II t ~ '!I ~ f ~i ~; J C\. ~ ~ ". ! ~ I o I j ~~I _ o J ) JI d ) 1 1 J IJ ~ WOIRHEPP B3D8.pdf   009850Anthrax Page 363 of 643 {Rev. 01-31-2(03) FEDERAL BUREAU OF INVESTIGATION Pre c e d e nce : To : From : ROUTINE Date : 09/ 1 1/2006 '1vash:'ngton Field Coun t e r te rro r i s m Washingt on Fie ld AMX - 2 / Attn : Attn : AME~IT~RAX-1 WMDOU b6 b ie NVRA , -_ _ _ _ _ _ _ _ _ _ _ _ _- , Contact: Appro ved By : Draftad By : ~I:::::::::::::~~= ( Pe ndi:19) I SA l Case IO # : 279A-vlF-222936-SCI16 Ti tle : AMERIT~RAX; MAJOR CASE 184 Synop s i s : To summarize the as soc i ation of t e n Bacillus ant hracis samples acquired du ri ng searches conduct ed at the u .S. Army Med ic al Research Institute 0: Infectious Diseases (USAMRI ID) a n d Battelle with ~R - 1 029 _ Details : Phenotypic Analys is of the Bacillus anchracis ( Ba) spo r e powders used i n the a~thrax letters sent to Senators Dasch:e , Leahy , and the New York Post determined that multiple morphologica l va r iants were present . When grown on so lid media , the phenotypes (i _e ., appearances) of these va r iants differ from each other , and from the ancest ral Ames strain ; demonst r ating dif f erences :'n te x tures , colors , and growth patte rns t han colonies produced by t he 1981 ancestra~ isolat e of k~ e~ _ The five variants identified were desi g nated as mo rphs A, S , C, D, and E . Analys is of the deoxyribonucleic acid (DNA) seque nces from i s ol ates o f the fi ve morp~ological variants l ed to the discovery o f 28 u~ iq~ e genetic mutations within thes e five classes of mo rphologi cal variants _ Chara cteri zat ion o f the DNA sequ e nc e s o f three Morph A is o lat es fr om the Leahy, Post , a nd Daschl e le tt ers revealed that these th r ee isolates each have a different mutat ion in the same reg ion o f their genomes . Th e Morp h A muta t ion s f r om the Leahy , Post , and Daschle letters were named Al , A2 , and A3 , respectively . Molecula r assays with the ability to detect t r ace levels of the Al and A3 mutations in a background of pr e dominantly wi l d type Ba have been deve loped and validated by Commonwealt:'l Biotechnologies , Inc _ (C81 ) . WOIRHEPP B3D10.pdf  1  009851Anthrax Page 364 of 643 To: Re , Washingt on Field From: Washington Field 279A-WF - 222936 - SCII 8 , 09/11/2006 Similarly , characterization of the Norph D isolate from th e Leahy letter revealed the presence of a 258 base pair deletion, designated the " 0 Deletion ." Molecular assays with the ability to detect trace levels of the D deletion ~n a background of predominantly w~ld type Ba have been developed and validated by the Illinois Institute o f Technology Research Institute {IITRI} and ro1~dwest Research Institute (MRI) . Sixteen laboratories in the United States and lab oratories in three foreign cour.tries were determined to possess stocks of the Ames strain of Ea before the anthrax mailings. The FBI collected a total of 1,056 Ames :solates from these laboratories and sto re d the~ in an FBI Bacillus anthracis Repository (FBIR) . All samples s~bmitted to the FBIR have been analyzed for the p~esence of AI, A3 , and D mutations using the above described assays. Only ten samples in the FB:R have all three mutations present and are listed below. FBIR Number 005 - 016 044 - 040 049-004 049 006 049 008 049-016 052-026 053 - 070 054-076 Ori.gi.n Laboratory Location Acqui red Sample Identifier or Label Battelle USAMRIID USAMRIID USAMRIID USAMRI ID USAMRIID USAMRIID USAMRIID USAMRIID JM-1 Rrn 164 B3 Cold Room B3 Cold Room B3 Cold Room B3 Cold Room Bldg . 1412 , 1st Floor Cold Room 83 , Rm 304 Bldg. 1412 , Rrn 212 FA3 Cold Room 0114 - 5 I RMR - I 029 b6 h7e I Ames Spores 2433 CDC7738 Dugway ~~es spores ; Dugway on the cap Dugway ~rnes spores; Ixl0 1Cl/mL SOmL tube of B . anthracis Ames ; 3 xl O1 Cl /~:... 066 - 044 USAM RIID Fe bruary 2002 shipment from Ivins to Northe r n Arizona University Ames strain RMR 1029 from Dugway 1997 2 WOIRHEPP B3D10.pdf  2  009852Anthrax Page 365 of 643 To; Re: Washington Fi eld From: Washington Field 279A- WF-222936-SCI18, 09/11/2006 RMR- 1029 was a large Ea Arees spore batch produced to conduct numerous anthrax aerosol challenges . Upon its assembly, the concentration o f RMR - l029 was approximately 3 . 6xl Olo/ml , consisting of one liter in total volume, split between two one liter flasks (0 .5 liter each ) . In order to produce the quantity of spores necessary to ~ake RMR-I 02 9 , Dugway Proving Ground was contracted to produce Ea Ames spores, which were combined with spo res produced i~ - house by Bruce Ivins at USAMRIID . RMR - 1029 consisted of a combination cf 34 spore production da tes , 22 production dates at USA...\1RIIJ and 12 produc tion dates at Dug ....'ay, totaling approximately 3 . 6xl013 total spores , 85% of which were produced at Dugway . Due to the quantity o f spo=es comprising ru1R-1029 and the fact that this sample was cne of the first to be ident~ f ied as having all three morphs present, the k~ERITHRAX Task Force set out to determine if, and how , t~e othe r triply positive samples ....?ere derived from, or otherwise rela ted , to RMR1029. The results of this investigation is described below . FBIR Sample 005 016 On June 19, 2001 , Batte l le received 30 mL of B. anthracis Ames spores, with a Colony Forming Units per milliliter (CFU/ml) o f 3 . 9 x 10 l o/ml o r 1 . 17 x 10 12 total spores, fr om USAMRIID . A 3a ttelle Ma t erial ID# of ~ 011' - S~ was assigned to the material . Slan~s of 0114 - 5 submitted to the FBI Repos itory were generated on 4/01/2002 (Reference 279A-,IF-222936-BATTELLE Serial 91) . These slants were processed into the FBIR and were given FBIR sample number 005-016 . Bruce Ivins recalls sending a subsample of RNR-1029 to r----,I at Battel l e for aerosol challenges (Reference 279A-WF222936 - USAl~RIID Serial 935) . According to the Rl-1R-1029 inventory log maintained for RMR- I029 Ivins disbursed 50 ml s pores OQ May 1 , 2001 and 30 ml of spores on June IS , 2001 to _ J Battelle (Refe ren ce 279A-W F- 222936 Serial 6263). ~------~ 0 b6 b 'C O f FBIR Sample 044-040/049-006 This sample ',.,'as identified by the FBI du ring -:he December 2003 consensual search of USAMRIID and was found in the box identi fied by 3ruce Ivins as belo nging ta l I This sample entered into the FBIR on two separa t e occas~ons as FBIR numbe rs 044-040 (s ubmitted to the repository by Bruce Ivins) and 049- 006 (entered into the repository by NMRC after the samp l e was seized) . I I (Reference 279A-WF- 222936-USAMRIID Serial 1004) iden t ~ f .lea the v:lal labeled "Ames Spores 2.3x:' 0 I O /ml" as 3 WOIRHEPP B3D10.pdf  3  009853Anthrax Page 366 of 643 To: Washing ton Fi e ld From : W sh i ngt on Field a Re , 2 7 9A-WF-222 936 -SC l 1S , 09/ 11/2006 belonging La her b ased on the ~andwri~~ng on the vial. did not reca l l the circumstances surrounding the vial. FBIR samples 044-040/049-006 is likely a sub-sa~p : e o f RMR1029 based on ~he :act that the concentration 0: the sample is the same a that de r ermined for RMR- 1029 on March 16 , : 999 . Gi ven that _ . doesn I t rerr.ember t he circurr.stances surrounding this sample , two possibilities as to the ownership of II : h:: sam~ le arise. It is possible tcat chis sample was used by in her own researCQ samole was orepared by a:1d was provided t b yi l or Ivins . If the a er 1S the case, this samp e cou d be the original stock of Ames spore ~ ~ recei ved from Ivins on March 24, 1999, (see discussion b e l ow 0 FBIR semple 049 - 008) which would be :he most reasonable explanation for finding both samp l es ( paIR 049 - 006 and 049 -008 ) in the box identif~ed o.n9ing tq I Add~tionally , both and wo::-ked f o d I ~ ~ fte com eted his Postd oc at USAMRIID he sho,,",'edi j wh ere he stored his samples in case I I nee e t hem f or fu t! p rirr.ents (Referen c e I consolidated 2 19A vlF-222936 - POI Serial 1437 ) . I samples in to one box , wr ote his name on It and t a nert it shut . This bo x was moved i~to suite 84 after J group moved t here (Reference 2 79A-WF- 222936-USAMRIID Se::-ial 912) . bG b7C r :nd 1 I I I FaIR Samp le 049-004 This sample was ide~tified by the FBI during the December 2003 consensual search of USA~RIID . From a walk- in cold room (Room B311) within containment suite 83 , Ivins disclosed a one l'::"ter fl ask labeled 7737 , RI1R-1029 (Reference 279A-WF-222936 USAM RIID Serial 4 71) . This is thought to be one of the two flasks in which RMR- l029 was stored . This sample was subsequently processed into the FB:R and given FBIR sample numbe r 049-004 . {Note : When RMR - I 029 was submitted to the repository by Bruce Ivins it was n egative f or all th ree morphs . See disc~ssion below for FBIR sample 066-044.] FaIR Sample 049 - 00 8 This samp l e was identified by the FBI during the December 2003 co n sensual search 0: USAMRIID (Refere~ce 279A- WF-222936 - USAMRIID Se r ial 4 71) and was found ~n a box, wh i ch was desc ri bed as belonging ta l (Referencer-____- , 279A - WF- 222936-USAMRIID Seria l 849 ) , b y Bruce Ivins . LI-'nPnFTI (Reference 279A- W F-222936 - USAMRIID Seria l 1489} ~ aen~~fied th e v1al labeled "Ames Stock 2x10 8 No Phenol 15Jul y99 " as I I I 4 WOIRHEPP B3D10.pdf  4  009854Anthrax Page 367 of 643 To : Re: Washi ngton Fie l d From: Wash ington Fi eld 2 79A - WF - 222936-S CI 1B , 0 9/ 11/200 6 belonging to him based on the handwriting on the via l . ~ i ndicated that he obtained his spores fron 3 r uce l'/:'ns ~ p~ovided a copy of a docunent dated March 24 , 1999 showing that he obtained " Arr.es spores - From Dr . Ivins , @ 2 . 3xl0 10/ml i n 1% pher:o':". Stored @4 ?C." A copy of I I laboratory notebook dated J"Jly 15 , 1999 r evealed that I Imade dill.:tions of an Ames spore stock Wilh ar: initial concentration of 2 . 3x:'0 :c/ rn l to a concentrations 2xIO' /r:ll then ,oIashed ::he spores three times with water . I l indicated that he typ=--ca::ly washed and diluted t he spores r eceived from Ivins to remove the phenol to provide a 'Horking stock . b6 b7C 0= On M rch 16 , 1999 , Bruce Ivins conducted an experi~ent to a determine the CFU/ml of RMR - 1029 as recorded on page 91 of ~is notebook (Reference Ivins' notebook number 40]0 ) . In this ent r y Ivins indicated that the CFU/~l originally determined for RMR 1029 was 3 . 6x l0 10 as determined on October 22, 1997 . Ivins determined the CFU/m1 of RMR-l029 on March 16 , 1999 to be 2 . 32 . 4x lO J') . According to the i :1Ventory log If.aintaine d for RMR- I029 I v ins disburs e d 1 ml of spores on March 23 , 1999 [Note : the inventory does not. indicated to whom the spores were given] (Re:erence 279A-WF- 222936 Serial 6263) . FSIR Samp le 0 49 016 On September 8 , 200 4 3ruce Ivins was shown a photog!"aph o f a Bacillus anthracis Ames s pore sample in a SOml conical tube labe':" e d " Ames spores 2433 CDC 7739 . " I vins indicated that r.his was a sample that h e p ro vi ded to l I an d that it was eit her a s ubsa mple o f RMR-I 029 , or ma t e r ~ a I th a t l or I I h ad ma d e fo r I It Re fer e n c e 2 7 9A W'"-='2J'2J'2J'9 J"6 ---' Ic_:;:Cl ' ' ' " " C USAMRIID Serial 935) . A r eview of Ivins notebooks revealed that he had transferred subsamples of RMR- ::029 to r - I on six d i fferent occasions , the first documented tra ~ occurred on October 4, 2001 (Reference 279A-~F - 222936-USAMRIID Serial 795) . The October 4 , 2 00 1 date for the ini~ ~ al transfer of RM~-l029 to c:~~=r.l i S i n agreement with the inven~ory log mair.tained :or RMR 1029 which ind~cates that 10m1 of ~~R - I029 was disbursed to LI_ _--' on that date (Reference 279A- WF- 222936 Serial 6263 ) . FBIR Samp l e 052 - 026 FBI R Sample 052 - 026 was seized f r om USAMRII D Build i ng 1425 containment suite B3 Roon 304 during a consent search of USAMR I ID i n J u ly of 2004 . wa s shmm photcgraphs of thi s I 5 WOIRHEPP B3D10.pdf  5  009855Anthrax Page 368 of 643 To : Re: Washington Field From: Wa shing ton Fi eld 27 9A- WF-22 2936 -SCI1 B, 09/ 11/ 2006 sample with " Dugway Ames Spores " wrlLten on the cap of the tube and indicated that the sanple and writ ing on the tube "co uld be hers " (Reference 279A- WF-222936-USAMRIID Serial 1461) . I I stated that she probably received the sample i~ t~e late 1990 ' s and that she obtained her Bacillus anthracis Atr.es samples from Bruce Iv ins or I I cou:d not recall w;,y she received this sa~ple and ind~ cated t~at she did not typically share samples with other researchers . Accor ding to the inver.tory log mai~tained for RMR-1029 Ivins d i sbursed 1 ml of RMR - 1029 ta l I on November 14 , 2001 (Reference 279A- WF-222936 - GJ Serial 129 0) . The phrase " Dugway Ames Spores " was commonly used by Ivins to describe Rt-1R- 1029 . FBIR Samole 053 - 070 Teresa Abshire was shown two photos of FBIR Sample 053 - 070 seized from USAMRIID Building 1412 , roorr. 212 by the FB I during a consent searc~ of USAMRIID during July 0= 2004 . These photos depict a 1 . 25 mL vial labeled "D..:gway Ame s Spores , " and a 50 mL conical t ube inside which the vial was found . Abshire indicated that she received this sample sometime in 2003 or 2004 from Bruce Ivins . Abshire believes that she requested the sanple labeled " Dugway A.rnes Spo r es " from Ivi::s to see if these spores loo ked like the spores from the anth r a x attack lette r s from the Fal l of 2001 ( ~eference 279A- WF- 222936-USAMRIID Serial 1418) . FBIR Samp le 054-076 FBIR Sample 05 4- 076 was seized f r om USAMRIID Building 1425 co ntainment suite AA 3 Room AA 314 during a consent sea r ch of U5AMRI I D during July o f 2004 . I I Principal Invest i gator , Diagnostic c ystemr Division (050) , was shown a photo of sample 054 - 07 6 . indicated ~hat she received ttis sample from Ivins and tha unt~ it was seized in 200 4, this sample was considered OSO ' s Bacillus anthracis sample (Reference 279A-WF- 222936- USANRIID Serial 1472) I provided agents ... ith a copy of the "Receipt for ':'ransfer 0 B. anthracis spores ," fo r this sample. The "Receipt for Transfer of B. anthracis spores, " indicates that 1m: 0: B. anthracis spores (Ames Strain) at a conce nt r a t ion of 3x lO l o/ ml was prov i ded to OSO on April 22 , 2002 (Refer ence 2 79A-WF-222936-USAMRIID Serial 1472 ) . The concentration of 3x10 1 0/ m1 is ident i cal to the concentration of RMR-I029 as it was orig i nally determined and described on the RMR - 1029 invento ry (Referen ce 279A-W F-222936-GJ Serial 1290 and I v ins' notebook number 4010 page 68 ) . b6 b7C .1 6 WOIRHEPP B3D10.pdf  6  009856Anthrax Page 369 of 643 To: Re, Washington Fie ld From: was h ington Field 279A-WF-222936 - SCI1B , 0 9 / 11/20 06 FB I R Sample 0 66- 0 44 In February of 2 002 {prior t o t h e establis hment of t he FBIR ) , Bruce Iv i ns submitted four Tryptic Soy Agar (TSA) slan ts o f his Bacillus anthraci s direct ly to Dr_ Paul Keirn of the Northern Ariz o na University (NAU ) in Fl agstaff , Ar i zona f o r genet i c typi n g _ Al l o f Ivi n s ' sample s were analy zed using Dr. Keim's M ltiple - Locus Var-iab l e Numbe r Tandem Repea t Ana l ys i s u (MLVA ) a nd dete r mined to be the Ames stra in o f Ba cillus anth ra ci s. Since Dr. Keirn requires o n l y a mi nimal amo unt of samp l e for testing , he retains the majority of each sample f o ll o wing analysis . One of the samples Ivins subm i tted to Dr. Keirn was labeled, "Ames strain RMR 1 0 29 f r om Dugway ( 199 7) ." Two months later , in April of 2 0 02, Ivins s ubmitted f our samples of Bacil lu s anthracis t o the FBIR. Of parti c ular interest , FBIR sample FBIR00 6- 00 2 , labeled , wDugway Ames spores 199 7 " is be l iev ed to be RMR- I0 29 , a lthou gh its l abel is somewhat ambiguo us. Gene tic t es ting of FBIR00 6- 002 generated negativ e results for the AI , A3 , and D mu tat ions, f urther bol stering the ambiguity of this sample. As explained above, RMR-I 029 was seized by the FBI in April o f 2004 and transpo r t ed t o NMRC . In June of 2 00 4, NMRC submitted RMR - I029 t o t h e FBIR, wh ich wa s acce s s ioned as FBIR049- 0 04. The genetic testing for sampl e FBIR049 - 004 resulted in p o s i tive results for the AI , A3, and D mutations. I n February of 2006, th e AMERITH RAX Tas k :or ce requested Dr . Keirn submi t Lo the FBI R, t;" e samples t h a t I v ins ha d sen t to h im i n February o f 2 002 . T;" e s a mples were processed into the re p o si to ry unde r FBIR acc e ssion number FBI R0 6 6- 044. Moce t han fo ur year s af t e r Ivi n s submi tt e d t h e s a mp l e to NAU , gene t ic testing o f FBI R066-044 ( labeled "Ames strain RMR 1 0 29 from Dugway (1 997 ) " ) generated pos:itive results for th e AI , A3 , and 0 mutations . Summary RMR-1029 wa s submitted to the repository as FBIR samples 04 9-0 04 and 0 66- 044 as des c r ibed above. Based on the info rmation provided above, it was determined that samples 0 0 5-0 1 6, 049-008, 049-016, and 052 - 026 are subsamples of RMR-1029. FBIR samp l es 044-040/049-006 and 054-076 are very l i kely subsamples of RMR1029 based on the fact that the concentrations of these samples are i denti c al t o t ha t described for RMR-I029. FBIR sample 05307 0 i s likely a subsample of RMR- I 029 based on t he l abeling on 7 WOIRHEPP B3D10.pdf  7  009857Anthrax Page 370 of 643 To: Re: Washington Field From: washington Fi eld 279A-WF-222936-SCI18, 09/11/2006 the vial; addi tiona lly, this sample was received by it ' s custodian from Bruce Ivins after the anthrax mailings in 2001 . Set Lead 1 : ( Info) COUNTERTERRQRISM AT WMDOU Review the p r ovided in f o~mation . oo 8 WOIRHEPP B3D10.pdf  8  009858Anthrax Page 371 of 643 FD-302 (Rev ]0-6-95) . I ? FEDERAL BUREAU OF INVESTIGATION Date or lnImaiption 12 / 12 / 2003 be b7C BRUCE EDWARDS IVINS, Ph.D., white male, DOB 4/ 22 / 46, SS# 280-44-5449, U. S. ARMY MEDICAL RESEARCH INSTITUTE OF INFECTIOUS DISEAS ES (USAMRIID ) , Ft. Dec rick, MO , telephone 3 01 - 619 - 4927, was interviewed at his place of emPloyme1t , USAMRIID. b Spec ial Agents I I and Af t er being advised of the identity of t he interviewing Agents and the purpose of the interview, I VINS provided the fo l lowing information: J In r , sPo'Qse to the SllbDoe:a issued by the FBI i~ 2002 _ and t J searc hed the f r eezer ~n 83, IVINS believes I prepared the f o ur samples of Baci llus anthracis (Sa ) Ames strain that were found during t he fre eze r search and submitted the samples to the FBI Repository ( FBIR) in April 2 0 02. I VINS provided the f o llowing labels and descriptions for each of the f our samples : 1) "Original slant Ames s pores - 1981" Th i s i s t he o riginal sample sent from Texas , whi ch was Ba Ames st r ain isolated from a cow. 2) " 7800a - primary subculture from original slant- 1985- Bruce Ivins" - This is a subculture that IVINS made in 1985 from the o r igi nal 1981 slant, 3) "780 0b - Greg Knudson Ames strain from 1985 - Multiple Passages" - This is fr om .LI_ _ _ _ _ _ collection, IV INS is unsure how this sample was made. -.lJ "7737 - Dugway Ames spores- 1997" - This is Sa Ames spores sent from DUGWAY PROVING GROUNDS f or use in aeroso l challenges. Thi s A material was used as the reference lot. o riginally 3xlO 13 (3 0 trillion) purified spores were sent, The spores were stored in two 5 0 0 mL flasks, I VI NS has approx i mately 1 0 0 mL lef t o f this s a mple. 4) IVINS believes chat tissu e samples , possibly a spleen, were als o sent from the cow from which the Ames strain wa s originally isol ated. IVINS does not know where the t i ssue samp les Investigation on File ~ _ 1 2""1"'1"'2.LI"'0"'3'--_ _ .. "1 Fort Detrick f MD Dale 279A-WF-:222936- USAMRIID dictat. ~d~===========-__, , I SA l B3D11.pdf WOIRHEPP This document contains neither recommendations nor ooncl l1$ions or the FB I. II is the property of the FBI and is loaned 1 your agency, 0 it and its contents u e not 10 ~ distributed outsi de )'OW' "ser.cy, 1   009859Anthrax Page 372 of 643 1'0-302, (Rev I0-6-9S ) 279A-W F-222936 -USAMRIID ContInuatIon offD-302 of __~B~R~U~C~E~E~D~W~ARD~~S~ I~V~I~N~SL-______________ .~ 12 / 12 / 03 would b e stored . ~I would be able to provide more information regarding additiona l tissue samples from the cow. =:;--:cc::-:I b6 b7C land IVINS searched the 8 3 freez e r again and found Lf"o ur--;; nc m;ro;;r"e"B,"a Airirn;;!es samples (l abe le d Reference Material ~ 1030 , 7 739 a,b , c, desc riptions to follow ) , a box of samples rl~a:b l j d ~e:: with I Iname , and a box of samoles l abe led wi th 1_ _ I Iname (true name may bei lbox does cont ai n 8a Ames stra in, but IV INS d oes no E know 1? somebody else has already submitted this sample to the FErR , Thrse are not sampl e s t hat anybody was t in to hide. left USAMRIID in the early to mid 1990' s . laboratory notebooks are in IVINS' o ffi ce. notebooks should be in the l ibrary at USAMRI I D. The l1bra ry usually discards notebooks afte r 20 years. I . IVINS provided the four samples of Sa Ames stra in ( l abeled Reference Material 1030, 773 9 a,b , c ) to the FBI r epos i t o ry i n October 2 00 3. I VINS provided Agents with a typewr itten descripti o n o f t h e 4 samples (The descrip tion summary will be submit ted t o the lA section o f the subf ile ) . IVINS provided the following labe l s and descripti ons for each o f the four samples: 1) "Reference Material 1030" is compri red of Ba Ames s train spores produced by I VINS and l . o n 13 different days: 20 Nov 95, 1 4 De c 9S, 8 Jan 96, 22 Jan 96, 8 Feb 96 , 12 Feb 96, 16 Feb 96, 19 Feb 96 , 18 Ma r 96 , 25 Mar 96, 1 Apr 96 , 15 Apr 96, 18 Nov 96. Batches o f spores produced on the aforement i oned dates were made in Le i ghton and Doi medium and purified on Renog raf in- 7 6 gradi ents . This was fou nd in the B- 3 cold r oom (Room B- 3ll), a 4 - 8 degree Ce l sius coole r . These spores are the leftovers from aerosol challenges . The sample is stored i n water and It phe no l. 2) 7739a was produced by 1 1- The spore~ : :o duced were made i n Le ighton and Doi media. I VINS revi ewed Inotebook fo r a description o f how 7739a was made. I . w te in the notebook that she obtained the inocu lum used to grow up 773 9a from a freezer tube in the free zer. No othe r d et ails were listed. I 3) 7739b was pro duced by i Ion 12 / 8 / 1999. The spore s pro duc ed were made 0n L~e'i~~h~~ n ~n~d ~o i~ ~, ~ g t o~~a '"D ~ media. No detai ls are known about the inoculum and methods u s ed. WOIRHEPP B3D11.pdf  2  009860Anthrax Page 373 of 643 FD- 302a(Rev 10-6-95) 279A-WF-222936-USAMRIID Continuation ofFO-302 or __~B~R~U~C~E~E~D~W~N~RD~S~I~Y~I~N~SL-______________ ,~ 12 / 12 / 03 . P", _ -,,3_ _ 4 ) 7739c was produced byl Ion 3 / 28 / 2001_ The spores produced were made ~:-'~7 g~ t~ n ~n '-D o7 media_ No details ~' L e i~ h~ o~~a ~d ~ i~ 'n are known about the inoculum and methods used . '-______~I and Iwou ld be able to provide more inf ormation regard i ng the inoculum and production method used to make 7739a, 7739 b, and 7739c. IVINS stored these 4 strains o f Sa in the 83 walk-in cold room (8311 ) . 8311 was descr i bed as a freezer room , which has a bench and 2 shelves along the wal ls. Samples a r e stored on the floor , the benches , and the she lves in the cold r oom. Anybody who has access to B3 or 84 has access to 831 w d ess to B311 include but are not limited to : PATRICIA WORSHA~, technic i an (unknown name). Sinc e B- 3 and B- 4 are :cwo~n~nle~c~t~e~d ,===n ~b o d:== h:~:a] access to 8-4 had access to 8-3. which I a :Y ::: Y W: o h d ri ncludes ;l I FNU (First Name Unknown) I I IVINS also had samples labeled 7736 and 7738, however, the entire sample has been exhausted . Therefore, he did not provide the FBIR a sample of 7736 and 7738. Sample 7738 was a d i lution of 7737. A Ba Ames strain spore age time line is as follows : (Oldest spores ) Original slant 1981, sample #78 00, sample #1030, sample #7739 (Youngest spores ) I I has the samp l e l abeled A0462 and it is being stored in B3 11 or 8313 . JOHN EZZELL and TERRY ABSHIRE should know the history of sample A0462. I I (writer believes to be I b was working at USAMRIID with EZZELL and then left to go back to Parton Down. IVINS believes that I I worked on the Ame s strain at Porto~ Down. IVINS believes that I I l at Louisiana State University (LSU) sent A0462 around 1994I 1997 to I l at USAMRIID to condUct a strain study. Idid not find out until later that A0462 was Ames. IVINS found out that A0462 was the Ames strain around 2000-2002_ Before that IVINS just knew the strain by its label, "A0462". I b6 b7e I WOIRHEPP B3D11.pdf  3  009861Anthrax Page 374 of 643 FD-302a (Rev. 10--6-95) 279A-WF-222936-USAMRIID ConllnWllion of Fo..302 of __~B~R~QC~E~E~D~W~ARD~~S~I~Y~I~NS~______________ .~ 12 112 1 Q3 ,Page _-'4'---_ PJWK HEINE (writer believes to be HE~~Y HEINE) may have worked with A0462 as part of his research project looking at antibiotic resistance i n different strains of Sa. I VINS is not familiar with the "Ames-Arkansas-Buffa!o" sample. IVINS does not know if samples labeled " Buffalo" (named after t he an imal, not the city ) , "Texas", or "Arkansas", were ever typed by PAUL KEIM's group at Northern Arizona University. IVINS does not know i f samples collec t ed from Zimbabwe, Turkey, or Namibia, were typed. IVINS provided agents with a list of strains that were typed (This list will be submitted to the IA section of bE the subfile). I l isolated the Ba sample that he b7C called Texas 2 from sheep li ver . I I traveled all around the country to different laboratories collecting different strains of Ba. IVINS read in l~"""",~I, paper that Ames a nd New Hampshire strains of Ba are conside r e d to be hott er than the other strains. The s amp 1 est ha t I ,-";;-""",,,,,,,,,,--!I brou g h t from home in a ! paint can and then returned to USAMRIID are kept i n t he cold room, B311. andl worked foJ PATRICIA W1m"RC" nr<'--;"1"'11 ha ""--'E"h"'" r "o "'EF"a crt informa ti o:~:-:a n :;-Cw = ld be ~v e e 1-;"""c '"n = n :-::Cd :-:o U~ O S HAM w able to provide more informat i on regard ing their research and samples . cloned the I Iworked with [1 protective antigen gene into E . coli. l is a mol ecular biologist, who worked for 3 years in the l, ~~~~ USAMRIID . IVINS does not know where is workin now_ an4 worked with and There was some friction between (Masters Degree ) an (Doctoral Degree) because of their degree levels. IVINS d1struste d l I a nd questioned his scientific integri ty. Many other people at USAMR II D, inc luding l I and l felt the same way toward 9 I I I ~~C==~;fT~~~~~1 I I . . I when1 ~eft USAMRIID he took the "good" protective antigen and l e t the "crappy" p r otective antigen. I~ not know if I I took Ba Ames strain from USAMRIID, bu did have access t o Ba, since he used to run the fermenter 1n t e B4 suite. I lshared Delta Sterne, Delta Ames, Delta Vollum, all of which were heat cured of the toxin plasmid with l I WOIRHEPP B3D11.pdf  4  009862Anthrax Page 375 of 643 FD-302a (Rev. 10-6-95) 279A-WF-222936-USAMRIID COI1tmuation ofFD-302 of __~B~R~U~C~E~E~D~W~ARD~~S~I~V~I~N~S~______________ .~ 12 / 12 / 03 . Pllj!e __-'5'---__ (Writer knows I to be a researcher at LOYOLA UNIVERSITY in bie Chicago, Illino~s). 1 I told l Ithat he made the Ba himself. although he did not make them h ~msel?.1 l is currently working on Anthrax research at the NATIONAL INSTITUTE OF HEALTH. IVINS did not hear of a shipment sent to r----------'I in j Russia. There were strains sent to Russia as part of an exchange program. I VINS provided Agents with documents showing the request for shipment, the import permit , and the shipping papers (These documents wi ll be included in t h~ lA section of the ti : e ). USAMRIID sent Russian Scient i s t, L J (Address l i sted on shipping documents: 142278 State Research Centre f or Applie d Microbiology, Obolensk, Serpukhov , Moscow Region, Russia ), strains V770-NPI-R , and Delta-Ames-1. V770 - NPI-R is the strain that Bioport uses for making the anthrax vaccine . V770- NPI - R i s similar to the Sterne strain and is n ot a derivative of the Ames strai n . The shipment request date was September 18, 20 00 . Delta Ames strain is the Ames strain which has been cured of the toxin plasmid. I Igenerated the "HI! or "H* " (H star) strain, which was thoug h t to be a vaccine res i stant virulent strain of Ba. I said that Ba Ames strains with the capsule plasmid only (cured of toxin plasmid) are still on the s e l e ct agent list because of research tha t has demonstrated the ability to transfer plasmids back into cured strains, thus producing a virulent s t rain from an av i rulent strain. can return the capsule plasmid to Ba ANR strain, but d oesn 't know if the toxin plasmid (the larger plasmid ) can be returned to a toxin cured strain. an Israeli scientist visiting USAMRIID from approx i mately 1998-20 00, was working in the bacterioloqy d i vision c o nduc ting research on 8a Ames s t rain. I J had access to the BSL 3 suites . I I another I sraeli scientist , was also working at USAMRIID in the bacteriology division and had access to the AR4 West in the BSL 2 su i tes , IVINS i s unsur e of wha t l I was working on a nd i t! I had access to the hot suites. I VI NS described I k s an o d d character, the type of person who wou l d "kill h i s parents and ask t~e i u ry for ID:rc y because h e 's an orphan". worked I b6 I I I I w,th ! J andj j I I ,-------"1 In late 2000 Israel asked USAMRIID througd to ship Ba Ames strain to an Israel i laboratory. During the time peri o d of 1 0/ 16 / 2000 t o 11 / 13 /2000. there was email I WOIRHEPP B3D11.pdf  5  009863Anthrax Page 376 of 643 FD-302a (Rev_ 10-6-95) 279A-WF-222936-USAMRIID bE b7C .~ Continuation or FD-)02 uf __~BUR~U~C~E~E~D~W~8RD~&S~I~VUI~N~SL-______________ 12 / 12/03 .p"" __-,6,--_ traffic be t ween I I VINS, an~. regarding the shipment _ At the request of l IYI NS reaue~t e d permission t o ship Ba Ames strain tO L~-;~o-~ I ~ n Israel . The r e quest was disco ntinued wh en the DEPARTMENT OF COMMERCE asked questions regarding the shipment and t he sample amount. The request was reopener on 2~22~2r~' and IVINS completed the necessary paperwork_ However , at the DEPARTMENT OF COMMERCE denied the requ e st. had been really pushing for the shipment t o Israel . I VINS el~eves i t is possible to have t aken a samp le o f Ba Ames strain back to Israel without anybody knowing it. IVINS h as worked on a researc h proj eet with .LI" " ,, - , - ; : : - ; : - ; ; - ' The research included inserting the protective ant i gen ( PA) into E.col i, transferring the PA into Bacillus subtilis (Bs) , transferring the PA into non-sporulating Delta Stern strain which is cured of the toxin plasmid (Delta Sterne PPA 102 CR4 ) and then using this strain to c reate a vaccine. I VINS has samples of the original Bs stain and the Bs conta ining PAl . Many USAMRIID Ba researchers are no longer working at USAMR I ID. These researchers may have had some samples rema i ning when they left emp loyment at USAMR I ID and did not name anyone "in charge" of the samples. IVINS provided the names of the following rese a rchers who may have left samples behind: I I )I====::;:::;:;:==r~-:--:---,I may I. (A USAMRI have I Isampl es ) pri nc ipa l inv est i gator molecu lar biologist , left arl y 90's ) ha v e I may h ave '--________----'1samples ) ivision Chief) IVINS agreed to allow Agent Stee l e accompany him into the bio l ogical containment s uit e B3 loc ated in bui l ding 1425 to l ocate Ba Ames samples discussed during the interview and to look for additiona l samples of Ea. Results are included in a separate FO 302. WOIRHEPP B3D11.pdf  6  009864Anthrax Page 377 of 643 f-U-J021 (Rev. 10+95) 279A-WF-222936-USAMRIID __~BuR~U~C~E~E ~W~A~RD~S~IwV~I~N~S~______________ ~D 12 / 12 / 03 Conltnu.al,on of FD-J02 of .~ The following is a list of the documents I VINS provided to the Agents that will be submitted to the 1A section of the file: 1 ) A typewr itten description of the 4 samples found in the B3 freezer that IVINS submitted t o the FBIR in October 2003 . 2 ) A list of Ba strains that have been typed by Paul Keirn at Northern Arizona University . 3 ) Ba Shipment request from Israel, email traffic regarding shipme n t, letter fr om the Department of Commerce denyi ng request. 4) Ba Shipment request from Russia , impo r t permi ts , shipping receipt. 5) Multiple Ba strain inventories . including the Select Agent Registry created by the Safety Off ice at USAMRIID and the Strain Report created f or the Medi ca l Re s earch a nd Develop~ent Board, which lists IVINS collection. 6) 2 Ba strain lists of I ______________--"1 Ba coll ec tion L 7) Application f or registration o f Etio logic Agents to USAMRIID 's safety office for I I and IVINS 8) Emails regarding request to find out if USAMRIID made dried, powdered anthrax Ba spores b6 b7C WOIRHEPP B3D11.pdf  7  009865Anthrax Page 378 of 643 FD-302 (Rcv. 10-6?95) -I- FEDERA.L BUREAU OF INVESTIGATIOJ'oi om: of trllmcl iption 10/03/2005 On September 21 , 2005, TERESA (TERRY ) ABSHIRE. white b6 -Lem4le , born I I Social Security Account Numbe~ I b7C ~ , u.S. Army Medical Research Institute of Infec tious Di sease (USAMRIID ), 1425 Porter Street, Ft. Detrick, Maryland 21702, phone number I I. was interviewed at h er place of business. After being advised of the n ature of the interv iew and the identity of the interviewing agents, she provided the f o llowing information: ABSHIRE used to get anthrax from 3RUCE IVINS, but began making her own an thrax in 1987. ABSHIRE was shown two (2 ) photos of a single 1.2S mL via l labeled "Dugway Ames Spores." and a single photo of the 5 0 mL conical tube i ns i de which the vial was found. This photo was shown to ABSHIRE by Special Agent (SA) Langham. This tube and vi al were seized from USAMRIID Bui l ding 1 412, room 212 by FBI SAs during a consent search a uthorized by the Commander of USAMRIID. ABSHIRE probably r e ceived this samp l e within the last year, and this was n ot the sample that she got from IVINS in 19B7 . The 1987 material was in a nunc tube with a round bottom. ABSHIRE believes that she requested the sample labeled "Dugway Ames Spores" from IVINS to see if this ma terial looked like the spores from the anthrax letter attacks from the Fall o f 2001. ABSHIRE indicat e d that the "Dugway Ames Spores" did not look like the spo r es from the l etters, but that some o f h e r own Bacillus anthra cis (Ba) spore prepa rat ions did resemble the materi al contained in the letters . In e xami ning t he spores from the letters ABSHIRE observed t wo (2) distinct colony morphologies; this resembles one of ABSHIRE's samples that also forms two ( 2 ) distinct colony morphologies. ABSHIRE b e lieves this sample has already been submitted to the FBI Bacillus anthracis Ames Repository ( FBIR ) . In addition to her own material, ABSHIRE has seen another sample that phenotypically resembles the anthrax contained in the letters . This was a Ba sampl e prepared bYTI~rr.~~~~~'-~~~~~~~ was tasked by: I to assess USAMRIlb ' s l.nternaI reposl.tory. Specifically, I f was to make certain that USAMRIID's Repository contained only pure cultures. ! ABSHIRE thinks that the spores contained in the letters have a morphology differen t from Ba isolated from an infected animal . Samples iso l ated from i n fected animals often have a medusa head morphology, and samples that have been passed in culture will I n~eslig!ltion 011 _0~9,- 2,,:!~ /.! 1 / 2,-,0,,0,,5,---_!lt Frederick. Hary land ' ;0 ' SAf 27 7 -WF-222936-USAMRIID J Oate diela1ed cN"/,,A"--_ _ _ __ __ " SSA J II: is tht ~rtv cI til. FBI and i, loaned to your allency: Th" occument conl.l,"s neithef rtICOfI"J1lellldalions IIOf concIuslon1 01' 1I'Ie FBI it and its contl!nts IIIl! not to be dl1tril:lu:ed 0UlIide your agl!n.ey. WOIRHEPP B3D12.pdf  1  009866Anthrax Page 379 of 643 FD-J02a (Rcv.I0-6-9S) 2 7 9A-WF-222936-USAMRIID CuntinuatiOil of FD-302 of _ _ T"e"-~..,.e".5!",--"Ab"",s,,...? r"e"--_ __ __ _ _ __ __ , On o h '," 0 9 / 21 / 2 0 05 . "age -2- have morpho l ogies that are somewhat asporogenic and lack the medusa-head appearance. ABSHIRE stated tha t IVINS uses 1% phenol as a preservative in his spore preparations. ABSHIRE no longer adds phenol to her spore preparations and stores her anthrax spores in water or partial broth. ABSHIRE has grown Ba in a fermentor, but only t he Sterne stain. ABSHIRE fermented the S t erne strain for the purpose of producing and isolating the Ba toxin. This fermentation was done by ABSHIRE while she worked in the B4 containment suite of USAMRIID Building 1425. There has never been a fermentor Building 1412 and Ba Ames has never been fermented at USAMRIID. It would have been too dangerous to have grown Ba Ames in a fermentor. ABSHIRE used to store some of her Ba in a tub in Building 1412. Attached to this tub was a white piece of paper with the words, "B. anthracis spores," wri tten on it in black writing. The tub with the piece o f paper on it was taken by the FBI to be fingerprinted. This tub, with the sign a ttached to it, was in Building 1412 while HATFILL was at USAMRIID. was ever in ABSHIRE does not believe that the Bacteriology Division at USAMR IID specifically, ABSHIRE does not believe thatl Iwas ever in the B3 and B4 containment suites located in bU1Id1ng 14 25 of USAMRI ID. rl--------'I I b6 b7C =j I , but may [=====~AB SHIRE does not know anyone referred to as recognize him if she was shown La ;-p=,~c~t~u~r=e~.-' The photos of the 1.25 mL vial ~~d 50 mL conical tube were placed in the corresponding 1A envelope. This sample is known to the writer to correspond to FBIR sample 053 - 070. WOIRHEPP B3D12.pdf  2  009867Anthrax Page 380 of 643 ~~~~;~~~MA .../ ~-I2- - q7 01701 11 " x9Y 4 x4Quad. ," 200 Sheets ? 100 Sets WOIRHEPP B3D13.pdf  1  009868Anthrax Page 381 of 643 -. 5C')?1 e (~M f( 1030" f5 . lflJ?!hlilc( '-> /l-m-Ps 5/"'W'.5, (X< poc,,K 3t S5 [I'fJ; ?r 7;)/ 7'1/ 7 ~ 8(.) r/,(>I/t77"os S(t>re s t~~re fl'~cka/ Cl Ct-.o-r.:/J7 "k S()f ({.L61iL'~ nf'! tA'H' frgclr,-r~cI/n Lejhjp" -D;'fi~'nCPIif!!er S$~ RftI~"f,j)f1- 7? fi(nc/!ca':~17 I ~ ~I"'.r<>s. tv'f}-l? ':> 'f1"7l> fl4~. Th71t~j..pr,q"rJ,,/r'/I'n /";?J~jR;lrJ/ /~ ((I F-I. (R) [)a ~j,rJZ/,pg / S;4rP5 co,.,,/JilJfb~' i 1)02t'/r~ / (; /'1 1'1~ !/'l)/rc;/' l!;;,)29( -:;/~/'1r 'J;/PI?C/ r2//~/f't? fs , ~ i - ;)J)r 1147 3/1glr~ 5,107:?d~, 4I!LU~.Jj5/i~ . - I'll;)'kl)f $Creiv~/t/:;P5 5-m/ q/lf"-;~ ~-~ ;J..Jf~];trnt;Zelir sCi~/v'crl'~~)e5 . -: ..--=: ~J11{&~ ~: /o/;?tY7 . " , WOIRHEPP B3D13.pdf --  2  009869Anthrax Page 382 of 643 fl4e Cc,,4 on .f{. H/.lo 30 5jl?'7'CS fJl(Yl "'.s.-)LiJr : ~ : Fq 7~1-i '-rafJ;/f 'J:;71f.2j;,----?-- - -..----- - - . . Aetr/-sJ",h/ ({;O ' { / 4 r-;!YJ;'~ /0 -1 ~1~~~IJ, CcwdJ t1.-#v (JJ;) /rJ/ r fkW I7nlo iSJ! q;JCl I'YlU1~ql!vn OVfynl/fkl- :~// ]5/ S g'" ?3. C7 V .,! ! I P1 f'q ri'= s". ft)f'c/t'c:rfJ:- .s f ~ . i~ .. ih-eK..M 'ffe Coo n/-~ /s j;eh/,eel7 () ,:; ilx/0"ltd 't- .:L1X10. '!)tn / . e' ~'-. ' L= \ P - . /J._ ' V'/1/ ['!-"'07 0C{1'L4.t7 . -- . !~~ ~ :~~ -- t'ip..$/f2- . _ --- _ _ ~ _ _ ... . ,----- o. --. .. - _ -. - . ._- - ---- - --- ~---- - - - -. - -- - -- .. _-_ .. - - -----.-- --.- - -.- -- -- -- ~ WOIRHEPP B3D13.pdf  3  009870Anthrax Page 383 of 643 Anthrax Spore Staining Reagents: 5% malachite green (in water) Safranin (from a Gram staining kit) Methanol water to rinse slides strips of paper towel (good quality) cut to size of slides 1. Place a 10 111 sample of speres on a slide. Spread with the end of the pipet. Allow slide to air dry. 2. When slides have dried, MeOH fix for 10 minutes. Allow slides to dry. 3. Place slides to be stained over boiling/ steaming water' for 8 minutes. Using a plastic transfer pipet, generously cover slide with malachite green . Cover slide with paper towel strip. Make sure slide does not dry out during the boi ling/steaming. Addit ional malachite green will have to be added du ri ng the boiling/steaming. J 4. Rinse slides with water. 5. min. Using a plastic transfer pipet cover slide with safranin. Rinse with water. Let stand 1 6 . Let dry and view under microscope (oil immersion) Refractile spores stain green . Non?refracfile spores and vegetative ce lls stain pink/red. '1 found the easiest way to do this was to -make" a steamer out of an old pipet tip container. I put boiling water in the bottom of the contai ner and placed the slides on the plastic that held the tips. Then I added the malachite green and closed the lid. This created a wonderful steam bath. --- -.----:----~ -- ----- -_. - .. _-- -_._---_. -" '- " ( f3./UkY~4A;~ = ~I~~~?~i - -_.. -- ..--- - --- WOIRHEPP B3D13.pdf  - ,. - .... 4  009871Anthrax Page 384 of 643 ~ r::tm ?1>;T:ft /t(!;y,!"? _~/...:s )L}r1f"lSforf>5 , 6U)}-b I!MI1/;;;; - ..-1--~~ /e~;:t-k~ - ;;;)J' j(Mfi. -10 30 (ted ,~fh PCf7-0'6 ft mEUR'> '3f0P5 r<> (;;ee Qep(j--/~ ) j--- &/Ztdj>- ~ ~- 4l'"V1~ "$//7/9"8 ) O.5Mp/q!e> 5 ---~'/~!~/OI7 ~ Gho/ 8 0JIG",k  - -'----==---......,;. Gi9 -- 009872Anthrax Page 385 of 643 - -. , .. \J /1mf> ~ .fr,?yn /If- 73 - rn'~ft:!.S~;2LC. ~/J!Lndi:z11 ",f' ;Xj1tt!f/o,x c; ' ..I i s;;e>rPs - (j f) 3-/7-'/6 J- 12-/Il/fl/ 'rrer/ f1h Ilm~5 5PY"5 (/I-3Alt Y"'~ j-J.., I:/a o O/,';,rll?~/I~ rfof!'{>//IbvsfX'r)J?I1A I(i~,,!, 011 qn Oiy"'fl'ts !3f! -~ (n,trOSCPj?f:/ !OiJoX fAils EUR, c011k5/ (OIl), I';; )'/5'c9rY1 1 ! I jl IS 3D'! / ,(1 ftcjL/ -:. C () '1.2 ~ . " it 50fiells (o/(//irenlj}e)ls)/ M'M (J j.el{5t (}o 51'~perfie/l/ So I /aAd ar /J70rf $ ..", /0(70 dij;&e>Vf ~i. r St(ovJ jook! rif r ,. o ", r. () ' [ I o le -;7 L L L 1)00 v~~;?w .qeJ!f J) Jf)qr?(,q 2J)pf}~ refoclfjp s~s f/r~o j'Z~: "claPn 5," {Ct~)IM/t:h~b/e m?/&;:;j ,\ TAt' -, O?-{>- S ,. , . ' : 0. c- I -cI. ;j,,/ N". 'fJ ,. - ' ~l -- ,WjJ::?,tI,oi M'l;X< rl?d. ?5 _ .t? - 'Ii , > I f pre.>en as J/ldVt&q[5!'<>r"s/n S4'~I1S/0"'. I . I &s~ O~>1 fftp-Cib;;Vf ~ob.<;, f.,-~/a:(j,.b/?S/ 0e-I;!!Jv-e.hy ;?M,!f/axp : r., t rcr70 _refodtYe-SfJor.: " .... ; /'10. V~?li')l,'<:::_e .c~ ~ .,' '" " ' 1';J, J~ -- . f ' ., - t "II< /Jot? :Wmclrl~ clejr/s ~ ~..s' :~ { ctp?< J AetlltjlcQAn ~ &Q(/& ~nz(/s . Jill" /I , f , f/trfs. !).I<1'f f;e/qrl~ Y1 3)11/'1'( MEDIA PREPARATION BATCH FORM? CAPSU L.E AGAR CAPSULE AGAR ?LATE SAlCH *: BHlLOT: Q9l0QJO 110 :JAGAR LOT: 4J.~ ' ( I WFlLOT: JIIOOSSD DATE PlATES PREPARED: ..u, ""f Jlllle;; FAILED _ tJ. . l ,. ~ 1 t STEAIl1TY CHECK. 24 HOURS 37 - V:--. - _0. - ' ''0/= . - t flgC/(?D~;J,~b~Z~.qL&XJ -a;:;J 8/)4k~q7! c~s;.;k Clqr. ~ - r-h~ f-l~/S-(I.IelY m CGf1Bk'..a~e.rJJ6!tfp131.?< 6i,&,;>.) - r. ~ t ~. R s.c,/f.s -e -+--.. , -. --.---- --.-..... - _.- - C!.1'1 Ji~X7 /?tf%,-~-=~~~ -J~whh,r;), ;5 3o,y--- P~.~ -~? 2?1~7Yr~---, 7 - - . . - WOIRHEPP B3D13.pdf   009874Anthrax Page 387 of 643 r 'b I - - -- -- -WOIRHEPP B3D13.pdf ---  8  009875Anthrax Page 388 of 643 --- ;@ _?At~!eqp Je~J/2~ ('bL4}V-~-7{ii"ZoC2r ~ p /q/p CD (j) #smwfn .;2.<0 #- ir7-t!p.orrr/efii?lMIi I~~ 2:. _ JJlf--ilA> .-==-_-.~ ~~ --Gn/-n/s =- S~f?(AMI( /O~S) 'f-l9r!es. (f(~l. /0,)'1) Phk cD .- @ J W (5) o (f) s __~__.__ ~() ___ _ _ o - (C) to (j) _ _ 5 ~-~_.-=- ? .-.-:-_~o~''-: ? __~_~-:-_ _ _ , ' ~=_ - - - ;:,- --- , "- -< _ .oo _ - - - - _ . _ - ---- --- - - - (f) fP _-___ - .-... .~-. ~S -~_=:~---= --.f!) __ t ~ __ -=- --.-----; -. (!) .- - - - ' " --- , - -- - ~I - - .---._--- ~- o -.---",,------~~~-;' () , ----. -. -. ,-, - =-- (f) --. -S" --- ---.----- . - Lf ( -- - ---- ~ -- - - - - ~ -0 -. -- -- ----l ,.. ._--_._. o ~ ' WOIRHEPP B3D13.pdf - ---:: ___------.--- . tJ. : "') .; __ ' =;5' -I_'1_-=tg jJ~~  10  009877Anthrax Page 390 of 643 ~ . see Fq~ c7s . ..___ __ _._. ______ ...__ . = 'o?/rroI,~hbr. J R (O')1 ~s - ().. ./~fj'fY-Jr,h>C3- ~c.4 _ MI\ , ~' Wi/t1/n/~ /,5 111/ o!J2 SfDrf'S , SAMPLES IRRADIATED FOR: IVINS RUNS PERFORMED ON : 19oMar-98 EXJI'06URE llNElMW 5.00E + 06 5030298 hinw",lminl 182 27639 IR/mi", _kpOIIlre r.u J Run Performed by: s.n.a. ItO. 552 SAMPLES MUST BE SAFETY TESTEO TO ENSURE TOTAL INACTIVATION TI I;; _ ff'Q.J, C ,1 . I) ~ Viti 'A o./_ 'I!''"'''~ . .?) ~ _ WL.t5.. - -- - . - Qt. }qb CPk/fj17 3-J~5) . . ' h p(.tfp . _ y C&[.. _ b6 _~ b7C ~ I' (-ervJoJoc!~)3....n1.If5/ ~ ~ p:1 eY/pLaf}~f~/s_,:- 7)Ee.5jJ s ~ (irr3dkl~./ ~re Jel,'~tiIj1?7- . /Qo~/- ~3-_ jd ~-r1S, X ... -,-,- e/[~~ :. - c2(j]J(ftl r ? $ =' - --c ~ Th/~ C/fhl'lll7'L [J-.JO -7'&)I. ~, , leg/Ie.!'ql?,1c9""vt')11: t~ o i(1~?Jft~n ~J'1 ~~lol).-~,i;o;;'.:, ble-;h Jf" ac 7t klcy.. - 30, ft. eW:ir;ify.,k.lin;'is/iJL aX --: WOIRHEPP B3D13.pdf  11  009878Anthrax Page 391 of 643 --- --.- ' ---' --' - NCI~CROC Test _ ' 0 _ CHEMICAL SYNTHESIS AND ANALYSIS (>O :J.I, ,oP 0.1 N 'i'eRequested CHEMICALANAlYS/S FEPORT ~ . I ~2>-.U I 7 r"t """J Sampie Identity Lot. Q.99'{ % -' I I " . " . , I o ? . -. _ ? . 7 . r , '. - .>', i b6 ---. --. b7C .-. '"'--Yallow Copy . Requestor Prrl< Copy o QA o "-T-- WOIRHEPP B3D13.pdf  13 -......,---  009880Anthrax Page 393 of 643 ~ - - - -- -_._-- b6 1">7c .-J -.. 199) From: B;~; TY' ns "' ~ To: I __ I Rece!pt Requee c Suhject: G~P Ames spores 'J5AMRlrD~ [I. FIpF7Rf"'X A / 2 / 98 9:25AM 1:'379 bytes: 25 lr. , USAMRII05_ F'TDETRCK, rat oo ------- - - - ------- ---- ---- --- - Message Contents ____________ __ ____________ __ __ _ Ames spores 130 tr~llion :otal. 1000 ml at 30 b illion per mll. They are fpresucably) to be used in experiments for suPPOrt of the A'J1!. ::-elicensure effon . We have tested them in severill ways', and they look very good! Chat E::] and I purified on Renografin? These Rerr.embe:; the lII> curatdy weighed. in 7.5 mL of me thlnol OXItaiOed iD. l~ Phenol ~- :::F/7;tn Ik!(sf: . --. volumetric flask . Add 20.0 mL o.f /lIttT1lQ/ Strwfud SoIlIl,. then add water 10 volume, and mil.. . Procedure-Usin, 3'i'l ponions of t he Stafldard PrtJHUGfl!l' and the Ttst Prtparatl()ll. record their gas chroml:togrmu.;u the appa ratus ad)uned to the parameters set fon b In the Ic:o:D; panylng table. Meuure the areas under the peaks ~ and benzyl alcohol of the chromatogram for .the Sra. , ~ aratiafl designating them P I and Pl. respeCtively, Slmil.uIy, lermin~ the corresponding values PI and PI for the !..~n(r~ ~aliOfl , Calculate the conlent. in mg per mL. of phel1Ul ..........' in each mL of the specimen taken by the formu la.: jlf~;;Z ~/5 - ,_... ~~ - 1000Cf V)fp,fp')(Pz/ f, ). in which C is the concentration. in ml pel m,L. Standard Prrparation, ;../} /2{tiP 4~ and V is the rolume. In mL. 01 or ph~iJlk: or tit II imen under '-, P" ptJ ratio". lesl used in preparing etlch 100 mL r bJ1rf( ret WOIRHEPP B3D13.pdf  14  009881Anthrax Page 394 of 643 --- -, ... . ~- . "-.- ._-- - .. -.- .. _._-. ---- - .. --J -- -' --- , ' -. -. , I ' _. , - G.~lr-efji!1?> ~/)"f?' (1:14/\ /O;2cJ jJ:sfoiC( ' . -~ -- __- . _.. ~__~P!11~r4rt~~~ -~/;2 /(~ ----< ----- ~ -- sc.r:eiiJ~ oJ ~ CC&J1/ f?5 f~ul. fJ1} f''t{{i/tt;-f, - .6/;41 /2/~:V ;'7L(J'I~tjL.? 7 :~ ~- f-~- ~'.' = _.-, ---,...'o" _ ~ ! =~-=_.- ~-. -~: - : , - iII-.i. ;-t-j. , , "'----J , ' : -....;. , I I I ~ WOIRHEPP ",. I -- ---. B3D13.pdf  15 ~  009882Anthrax Page 395 of 643 . - -:: f _. -- - . , . :>..- b6 b7C l' WOIRHEPP B3D13.pdf  16  009883Anthrax Page 396 of 643 ~ . > - -. :,"cs~f?/l!g~~~f ji1aJf:J:r~ ~_ 37'C. fJaJe/of};b -/If !AI/dell! (f:Y~- /Iv> / 1t# Were MC//bk/ tYhTI ,ene/a;; (f70fn/? ClIJ~ ) Ifmcly /YloYr!/lIJIyV/17 I fillt7) a/l'1osl- t1cJf;;p,e5_ TJJ (J cr/lenpf !O~f.I.~ 7/~ - - IUd I b~ rnc;/f -- - - - WOIRHEPP B3D13.pdf  17  009884Anthrax Page 397 of 643 \, ::iJll SlnJt)t b?!k~7 15_?..L'rieCb/ld;'t2-k.2!smi?)/-/: _ -, 0. 05 f1'/ - . - ~ . ...- ~ - ~-- ..--.-.-. . - '"' . ,. : C . -- wd-A ftt 5)4kl~ for I{'Aours.(){)() !f"1)alc2S?c..(~)ftktI'J d~ ;c69-J. );f /I/.t~, f)e! ct/f;p /llcl./l7dd iii h~ 0'; JtJ) d cur/,l'tJ./- /0 -~ cdJ,.j;;,J5, h1 t8s Mf9 - f6ieJ ()y/ ~ ;1iv/rfeJ?!(/''Plrh" 7:;e1 0/ jl;A !Ufo ~~3 7'L one >.Qf d;JohMre /~Cub;;'cIof rJpG (~k~~;p) 0 n~ f/r;h}vfre /r1w.kk t9r(j 413/"-t; ;44n &w~ '/0 J J;,;i l?J;)jj~/nlj M.c/n--EUR~v.J . .sef;t -:- o 4)1 r~ , , -_ pc!- cl~7clr; , _~ , , .- - ------; - -. --: - ~. _ . _-__. '?.~ _ . ;)pc -: ~00-...fk~i!!-';;;j?EQ"7[;d17kl';;~Lo. -'--t)~ ~-~ _ _L __ _ ~ _.. ___. _. __ _ . " ------. ----j - ,. ,  18 Ii I WOIRHEPP B3D13.pdf  009885Anthrax Page 398 of 643 - ---- -- - ---- -- -- ----- --- - -- -- . ---- ., +-- ft I , . ---- - - - - - - -.. _-.o ,._-- .. --~- . -, . _ ." _ . ." i L , -L-, -'>- ,, . " "'r . I -L-- . - - - _.- - I , -;, --,. - . - - - - , - --. --- ---'--- -- --WOIRHEPP B3D13.pdf .. _. _ _ . _~------ 0 __ - ______ _ . ,,  19  009886Anthrax Page 399 of 643 " "._... -_. -- - - - - - Making Bacillus anthr8cJs Volium 1B Spores (lol 189~) for BIOPort Reagents: , . Sterile water for injection (WFI) McGaw, tnc., Irvine, CA 92714-5895 ~ lot J7S035A Expiration dale 1211999 Catalog number: S9200-55 This product (WFI) is used wherever water is called for. It IS used with all the products below except PBS , and it is used 10 put onto nutrient aga r ClIltures of B. anthracis prior to scraping the cells and spores off 2. Hypaque-76 Nycomed , Inc . Pnnceton, NJ 08540 :\ I , "I , - lot M215RL Expiration date: 1012000 Catalog number: H-375 ThiS product is d ~ uted to 58% (" Iv> with WFI by adding 58 ml of Hypaque-76 to 42 m l of \f\IF1 It is used fo r purification of spares by centrifuging the spores . through the 58% Hypaque-76. I I 3. Ultrapure phenol GIBCOIBRl. Grand Island, NY 14072 Lot: 97N3269 Expiration date: none listed Catalog number 15509-01 1 ThIS product IS used for making 1% (vlv) phenol In 'NFl, whd'l ls the standard storage mediLrn for the B anlhracis spores. This malerlal (1% phenol) was prepared on 21 DEC 98. Dulbecco's Phosphate Buffered Saline with Calcium and Magnesium (PBS) BioV'v'hittaker, Walkersville, MO 21793 Lot: 7M1372 Expiration d ate: 7/21 / 1999 Catalog number. 17-513F This product is used to dilute the BHI broth culture of B. anthracis Vollum 1B spores UO (1ml culture into 9 ml of PBS) before spreadIng onlo pla tes of nutrient agar (NA). It (9.0 ml) was dispensed into a glass. screw-capped tube (SleriJe) Ofl 17 OEC 98. " Brain Heart Infusion (8HI) Difco Laboratories. Detroit MI48232-7058 Lot 99700JO Expiration dale: 10/2001 Catalog number: 0037-17-8 This product comes as a powder. BHl liquid IS prepared with WFI and sterilized according to the manufacturer's instructions 8HI powder (1 8.5 g) is added to 500 mI WFI, then atJ!oclaved 15 mmutes at 12 1- 124"C. Twenty-fIVe m l is aseptically added 10 a 125-m1. screw-capped, sterile. glass, Erlenmyer flask . It was prepared and autocJaved on 17 DEC 98. :1 'i .. " ,. , , , " !. ! L , , , f --"-_. -. - - -,-'------- WOIRHEPP B3D13.pdf  20 ----------  . ,\ 009887Anthrax Page 400 of 643 .... . .... . ' ~ ... ? o ~~L~ V18 -sto rP';' _ .. - ..- .... .._.-(._ .-._- -.; - - ----..--.. -- -.-y ..- 1-0 )v f / '1; 'f -rJ. ~ Id !~r-'I) OYl . ?>1!..i I / ii'l/ YI' "6 b7 C I L._ _ _ _ _ _..J ~,r jJl,d;djr;~ 1/.7 S /'1'1- I-)e (..f/4lf1m/ j ro),ale) -fj.,1'1 it-- /I] t 4. tyJJ !/;'cJ1 mc/s. ;If)' bill .be (j'kn It> () c.d- ?.-c.fi.F Jfy: !..jf C l';;,1 etid! cle../e""",hd~ ~a~ - J P9/r1 . r r-. AA.XLS SAMPLES IRRADIATED FOR: RUNS PERFORMED ON : EXPOSURE IVINS 28-Jen?99 TIMEIMIN 4 .00E + 0 6 4 012254 (time r/minl 9 162 24767 IRJmin. a,,:poSIJfa ratal , R\.W1 performed by: Sample no. 18 I 1.-_ __ SAMPLES MUST BE SAFETY TESTED TO ENSURE TOTAL INACTIVATION -. 1'--- _ -,.... ~ .~ r:t--;! ? . i : .............. I? , , .. . ----.--- - -_._- . -. W WOIRHEPP B3D13.pdf  21  009888Anthrax Page 401 of 643 HCI-FCFDC SCPO QC.OO88 Form Number: CToS " ,/ CHEMICAL SYNTHESIS AND ANAl..YSlS QUA1JTY CONTROL LABORATORY CHEMICAL ANAlYSIS REPORT ....y _-"'---'-t1:..:L =-____ Requested By/Dale ? ] '1 ('11 f t" .r1o< 1r>S g -J- rj'1) Proj.... Coot Cerfer c? % < 13 iJ Telephone 3 01/(,11 ' -... , , Sam", PreparMb> !; ,1Ird ~,th"" 0'(' 0,,,,,& P~'/'$I (Ig bl.) 5P'~> , p, 'It .>? l'ir e!,.",,} (J~ ~ - ~ ) :; LD" '...." " " " -t , .------======~=~---'-~ , '.... ,~ - b6 . b7C White Copy . CSAl Ye/Iow Copy . Requestor P;nk Copy o QA WOIRHEPP B3D13.pdf  22  009889Anthrax Page 402 of 643 - -- - -- -~ - - - --, - - ~~ - , , ---- -----,~- - --- -_ .. ----_. - .. - --~.~- ... B3D13.pdf WOIRHEPP -.- -- ."- -_ .  - ------ -23 - - - ---  009890Anthrax Page 403 of 643 ..o.. ~ _ . .NCI-FCRDC Test Reques11D Numbar. 0Zl1.7?:f SO"" 0C-0088 Form f\l..Irnber: CT-5 CHEMICAL SYNTHESIS AND ANALYSIS QUft.Ll1Y CONTROl LABORATORY CHEMICAL ANALYSIS REPORT AssayAequested Phenol Requested BytDate Project o Cost Conte< Sample ",_ation 0.0 ;'2 , Sample p~,,,,1 ( I Fhl . ) .- as to> SII' X,t1-J n nfAm Te!epI1one 'P'r'> IQ 5<1/"9 - ~ 9 iL 2 ' 1% ph?no / fif?2 -rJ or , Lot #I Pn.J!!..D rr! n d~ -J-r,~ . Identity .:aM< ~CL 5 iJF"7? 7J . b6 b'C 1 ..... YVhite Copy . CSA.L. Yellow Copy . Requestor PInk Copy . QA . - .--------" ~--- WOIRHEPP B3D13.pdf  24 "--  009891Anthrax Page 404 of 643 , USAMRIID Bacteriology Division 142SI'orter Street, Fort Detrick, MD 21702-5011 Bacillus anlhracis Vollum 18 Master Spore Suspension Lot # - 189;::-".2_ _ _ _, Originally prepared byLI-==;::;-_ _-, Preparation date - Snl80 Obtained from Michigan Department of Public Health Jon 6127/95 16 b7:: I Lot inventory - 5 mt in stoppered vaccine vial Concentration - approximately 3 X 10 6 spores per ml Storage - 2' -8' C Certificate of Analysis oo --,.-- Spore cou~t-1~~rinnm~~~~nrr.mr------------------------------' Per cent smooth (encapsulated) - >99% Retractility? >90% of spores are refractile Debris - <5% debris Per cent vegetative cells? <5% vegetative cells Phenol concentrationn o 0.813% Endotoxin concentration ? <0.1 endotoxin unitlml Prepared by Bruce Ivins, USAMRIID Bacteriology Division, 30 11619-4927, FAX 301/619-2152, email- bruce IVlns:.a:!detrick.army.mil Daie 3/;:2/ '11 l-:_ , ' --....,...-- B3D13.pdf WOIRHEPP  25 -- - --;---.,.-- -------, ------  009892Anthrax Page 405 of 643 r, . . . ~ :: <.. p. -- - - - -- - - , USAMRIID Bacteriology Division 1425 Porter Street, Fort Detrick, MD 21702-5011 Bacillus unthracis Vollum IS Challenge Spore Suspension . ., Lot # - 189-4 Derived from Master Spore Suspension 189-2 Preparation date - 116/99 Lot inventory - 81 vials, 1.2 ml per vial Concentration - 2 X 108 spores per ml Sent to BioPort - 1119/99 Remaining at USAMRIID - 12 ml Storage - 2' -8' C '. Certificate of Analysis Spore count? 2 X 108 per ml Per cent smooth (encapsulated) - >99"10 Refractility - >90% of spores are refractile Debris - <10% debris Per cent vegetative cells ? <5% vegetative cells Phenol concentrationn? 0.995% Endotoxin concentration - <0.1 endotoxin unitlrnl Prepared by Bruce Ivins, USAMRIID Bacteriology Division, 30116 19-4 927 , FAX301/619-21 52 , email - bruce ivins@detnck.army,mil .3 /J;2/f'l g nature Date , j' !I , . - , --~----~-.- ---B3D13.pdf WOIRHEPP 26 ....--- -~--  .. ---.- .. _._----  009893Anthrax Page 406 of 643 , - VJ - _ . WOIRHEPP B3D13.pdf {l !"UW-.-J2-. ~ , - 7'" 27 ~ cm CYC2 __ --- .- --- - - .:. .--: ------ - - - -   009894Anthrax Page 407 of 643 - v ". .... __ ' ''' .- RUSSIAN FEDERATION Miaistry of Haith STATE RESEARCH CENTER OF APPliED MICROBIOLOGY 142279. Obolensk. Moscow region, Tel:: Fax: (0967)36-00-01 (0967) 36-00-10 E-mail: ccnter@gncpm.scrpukhov.su info@nrciam?semukhov.su STRAIN PASSPORT 1. Microorganism species: Bacillus antluacis 2. Strain: TsiankovskH n vaccine 3. Special title: anthrax vaccine strain 4. Date and ~urce of delivery: 1996. VGNKI (State Veterinary Science and Controllnstitute) 5. Pbenotype: 6. Description: liquid of grey-yellow colour, capable of sedimenting under static conditions, easily resuspendablc sediment being formed. 7. General spore concentration: 3.5 x 109 fem 3 , J , ,. , -- 8. Biological concentration, cfu: 3.0 x I09/cm l 9. Number of pink spores (Zille-Ni lr~n staining): 9()O1. 10. Number of mucous colonies fonned on bicarbonate agar: lOOOA II . Foreign microflora: no 12. Cwture-morphology properties: chains of' immobile Gram-positive ~lIs with cut tails 1.0-1.S mkm in width. 3.0-8.0 mkm in length 13 . Capsule formation ability: forms capsule in an organism and 00 nutrient mediua. 14. Growth on nutrient media: Solid medium (tryptic fish fl our hydrolysatc, at (36?J tC for 18?24 h: forms flat dull colonies with rough surface and of R?shape with uncven endgcs ofa diameter on.5-5.0 nun. Liquid medium : in 2~3 days at (36? l tC culture in a form of". piece of cotton wool" at thc bottom of the vessel, no change of the medium transparency is observed 15. Spore formation : in 5 days o f growing on solid medium at (34? Jt C, in 80-90% of cells oval spores located in the center of the cells are fonned . The spores become pink undcr ZilieNilsen staining 16. Sensitivity to phages: sensitive to speeies specific bacteriophage " K-VIEV" 17. Biochemical properties: do not posses hemolytic properties 18. Virulence: LD ~ at subcutaneous challenge ofimbred white mice is equal 10 8 sporcs 19. Additional information: spore culture was preparaed in October, 1988 on the base of reference strain of Tsiankovskii If vaccine 20. Shelf-life: in 30010 water-glycerine solution at (2?2t C. W WOIRHEPP t .. . .- .J t -- -:, -, , I ~- . "'-~_i , '-I, o B3D13.pdf  28 -r- .. - --;---.  009895Anthrax Page 408 of 643 -. -- R USS IAN FED E RATION Minutry of llealth STATE RESEARCH CENTER OF APPLIED MIC ROBIOLOGY 142279. Obolensk. Moscow region, Tel" F.. , E.mail: (0967) 36-00-01 (0967) 36-00-10 center@gncpmserpukhov.sl info@nrciam.semukhov.su . ', STRAIN PASSPORT I. Microorganism species: Bacilhts anthmcis 2. Strain: Tsiankovskii I vaccine 3. Special title: anthrax vaccine stra.in 4. Date and source o f delivery: 1997, VGNKI (State Veterinary Science and Control Institute) S. Tox? 6. L I L . 'I ' ~. I - o static conditions, easily "::::,~~~~~~c, :~::~:"\ +bei'"g formed. 7. Genera1 spore concentration: 6.0 x 8. Biological concentration, cfu: 5.5 x I09/cm) 9. Number of pink spores (Zille-Nilsen staining): 900/, i, <, I;- 10. Number of mucous colonies fonn.:d on bicarbonate agar: 98"" 11. Foreign microflora:. no 12. Culture-morphology properties: chain.s of immobile Gram-positive cells with cut tails 1.0-1 .5 mkm in width, 3.0-8.0 mkm in leng\h 13. Capsule formation ability: forms capsule in an organism and on nutrient mediua. 14. Growth on nutrient media: Solid medium (tryp tic fish flour hydrolysate, at (36? ltC for 18-24 h): forms flat dull colonies with rough surface and of R-shape with Wleven cndges of a diameter of 2.5-5.0 nun. Uquid medium : in 2-3 days at (36?ltC culture in a form of " II. piece of cotton wool" at the bonom of the vessel, 00 change of the medium t:raru>paren.cy is observed 15. Spore fonnation : in 5 days of growing on solid medium at (34?itC. in 80-9()C'1o of cells oval spores localed. in the centcr of the cells are fonned. The spores become pink under ZilleNi lsen staining 16. Sensitivity 10 phages: seru;itive to species specific bacteriophage "K-VIEV" 17. Biochemical properties : do not posses hemolytic properties 18. Virulence: LDso at subcutaneous challenge of imbred white mice is equal to 32 spores 19. Additional information: spore culture was prepared in October, 1998 on the base o f reference strain ofTsiankovskii t vaccine 20. Shelf-life : in 30% water-glycerine solution at (2?2t C. r, I i i ~ , I :-~ li I, j-t E " '!\ WOIRHEPP B3D13.pdf ~ ~. ~-S!Jt.ua--r -o--?.  29 -,--; 009896Anthrax Page 409 of 643 ~ " --.. - o (j /Ceh - -- c).- - -- - - -- ----- --" - --- ffm.?5 O(fif-i'l}u/ skI? f ;). ?, 5 '-IN 6 f1I4kf7~/ I~ 01/10 ~ f> If- -qfor' .5c6,v> r ~--es J/er9 .5 ~ rt'surenkJ ;'fi )Z,;);'; f Iv! ,'4,[, r; /,;Jsm/ ft/y';-<-- ~7"'~Y=7k~k -;,~ II t'<.1if-/&&r' /" f:3;y.5 vtlJ.c OPJut 3tJy11~ lJl) &v/J!<;~ cI~e_,J-'tCl;r/-;.;tje qbC4,-)'-.. 10 q//Il/ - - 7h-P>~ 5fo/-!:>:7 ~a.ft!q _~/lm~7 Sqku/;il(J O-J~ ~ ong,.r; tii f _: iy,J/" o hl'Nrv.q}jf: . ::..- ~~=~:-, -- i.n : - - 'if ~~ I-I 'f'tfl c>>J , -_._, .- ---.o o +- -- - - ---------- ------- -- -'- .o. -- - ---- ---._--- ---- .... - --_._'.- - ---. ----.- ,------- . --- . - - -... -- .---.-.--~ -- -- -- .. _-- -.- _. - -- ---- . --_.. -...- -- -- -- ..;. -~ - - ----- ------~ - .. - .- - -- .30 .... - - _. -~---. -.- ..... 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"","_, Btdt!lClorr ~~ AMm ForI~ 1 1415 PcJJtef SI ~'r,!amI -. j !lPn:19_' 92t DocumetIs .1Id CMNftttC. """" 1._ -0j",.,' , "/';',' CIIent..,. ..... ~ O v. O ND ~f'.:'(J_ em~' ____ I I Ttn'f AII.'t." Dill. qf fllcllllll -- WOIRHEPP B3D14.pdf  8  009905Anthrax Page 418 of 643 1----- - DIh!IIfAeceIJII: ACUPI-.ll\' ~~....., ~ ' ... IIf~ [1l19----1 "' b7:: Rnson ICDIItIdIOnla'RRllosn:ory ..- WOIRHEPP B3D14.pdf  9  009906Anthrax Page 419 of 643 -, .. .' , L_5cyq rNawalllt8llhr1l Rnn":h Cllltlf (NIIIRC) 1 ~DI!-..n_!!u./ NIIIRC 01 Adltns SMfSpmg 1 ""'" af eMF 503 R~..t()qrt ,," 110 1i3Oii318JlG2 """' EA101 i I Documents 8IId CMWMm. n:l1IO!CI WIlli ~ Sill..",,1e ~1OrJ """-O YH 0 ... I Mi!!pr ... !Weer" I""""'" 1M!~4 _ DaI."Rtcttp! " b?C WOIRHEPP B3D14.pdf  10  009907Anthrax Page 420 of 643 -, :~ , " ,- -...... fII~ ~ '~.:..;:J I:,~. " b!C WOIRHEPP B3D14.pdf  11  009908Anthrax Page 421 of 643 '. 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'" _ ~ (ttvu SaletyOtrlce) OtAGNosnc SPECIMEN [CIIIssJlk:atlon Group _Cal (b) _ {eij (thru Safety 0fIka) CCNTROUED BIOLOGICAl OR BIOLOGICAl. PRODUCT x Human ar _ Non-Humen I _ on. . . . _ blast (hu ClInIcal Uae BIoIogIrA --;;FECTIOUS SUBSTANCE (ETlOlOGIC AGENT) AFFECTING HUMANS (1hn.I T OXIN (ETIOLOGIC AGENT) (1t1rU s.t.Iy 0fI'Ice) ~ Offiat) /f?<7 ! CootraII~}h INFECTlOUS SUBSTANCE (ET1OlOGK: AGENT) AFFECTING ANIMALS ONLY (thru Saf8tyOfftce) RADIOACTIve MATEfWIL , .....11'4 ~ Pr-=dlc1 OMQ) . BIOLOGICAl. AGENT NON-tNFECTlOUS (thru Safety Office) , " - OTHER ~ or Hlazardous (thru Safety omcrJ - - .1425 Oescnpbon 01 matenai(a) ~ of B anttv8dIl Ames sporm 39 X lD"'101m1- 1:JI8)( 10"12 spare. total, n a 12S-fN ~ate ft8I!IIk ~~. contract 0f"gIII'1Izati (BMtde) wdh matarfal to perfonn. ccnlTacted slUljl:)' -oua"ntyTrriI mg ) ~ III X"l0"9D1 ml ;[f3 X1 0"12"""'iPoru total Ship to Address (lnefude taMpt\Qna number day and emergsl"lCY of ::t;'onn ph)'SlCally manned 24 hcus a day No pagers or c:.u PhOne numbets 5 R 142 WNl.lef'fanlon OhIO 43152 Day. 61414247230 24 hour I l::===:_SBathNlelMREF JS..3 Em~f:'/ number must be Requlr.d ShIpnMnt Oat. Tuesday 8 May 2001 ChargetoAPC BGOB Melhod of Sh1Jmenc Requested \.FS ~ME&PQa__ USAMRIID Cc:u1er Other' Sp8C18l R.qurrements Nono -Y O~~ ftft1,g..c.d (wei k:e cold peck) b6 ~==--~-------------------------=~----------------------------- b7C Name Addreu Telephone Numbtw(mcbtedayanclemergency)otSenaar Date 3MAY01 Bruce tvm 1425 Porter StnIet A DatncII: tAO 21702 I certify that Ihe amtenb of thIS Signature of DMBIon ChHlf Oth..- OilY? 3011819-4921 24 haw? 30 1JfJ1 .2257 T I I])" ~ lOele .-;j/O I / I tOl'" shipment accord Ing to appl.cable lATA and DOT OELIVEF Deh.,.er8d to PAGKAGING I certrfy that thus conSolgornent oo packing II1 sb'tJd1MS Pm1ed Name and Signature of Cartrtylng Official us.oo.MRltO Forno" A (011' ..Oonoo ry 2001) Da1e WOIRHEPP B3D14.pdf  17  009914Anthrax Page 427 of 643 US ARMY MEDICAL RESEARCH INSTITUTE OF INFECTIOUS DISEASES SHIPMENT REOUEST REAGENT MATERIAL f DIAGNOSTIC SPECIMEN I CONTROLLED BIOLOGICAL PRODUCT I BIOLOGiCAl AGENT I ETIOLOG IC AGENT (INFECTIOUS sueST ANCE OR TOXIN) I RADIOACnVE MATERIAL Please check appropnate block and route through appropnate approvmg auttlonty REAGENT MATERIAl Non hazardous or (a) Hazarl10us (thru Safety Office) (b) (el] (thru Safety Offtce) DIAGNOSTIC SPECIMEN [ClasslflCabon Group CONTROLLED BIOLOGICAL OR BIOLOGICAl. PRODUCT x ~ECTIOUS SUBSTANCE (ETIOLOGIC AGENT) AFFECTING HUMANS (thru TOXIN (ETIOLOGIC AGENT) (thl\l Safety Office) Human or Non Human I Inv and be list {thru ClinICal Use BIOlogICS Control/~} Safely Office) /p.e;(7 j J r INFECTIOUS SUBSTANCE (ETIOLOGIC AGENT) AFFECTING ANIMALS ONLY (thru Safety Office) RADIOACTIVE MATERIAL (thru Rad.at.on Protecbo"l Office) BIOLOGICAL AGENT NON-INFECTIOUS (thru Safety Office) OTHER - Non hazardous 5i'J Of - Hazardous (thru Safety Office) (,.,.s Descnplloo of matenaJ(s) "1ttm1 of B anthraclS Ames spores 39 X 1D"10imi ~X 10"12 spores total In a 125 m l polycarbonate flask. to supply a contract orgamzabon (Battelle) with malenal 10 perform a contracted study Quantrty(ml mg ) 70 ml 3 9X10"101ml 273 X 10"12 spores total Ship to Address (Include telephone number day and emergency of reo~~ ,, o~ ~~ t)~E~me ~,~en? number must be ~c cy physICally manned 24 hours a day No pagers or cell ph one numbers [SattelleiMREF JS-3 1425 S R 142 We st Jefferson Ohio 43162 Day 614/424 7230 24 hour 1_ I , . , ReqUired Shipment Date Tuesday 8 May 2001 Special ReqUirements Charge to APe 6GOS Meltlod of Shipment Requested UPS y All ExPfess USAMRIID Couner None Dry Ice y.. Refngerated (wet ICe cold pack) b6 b7 C Othe< Othec Date Name Address Telephone Number (Indude day and emergency) of Sender 3 MAY 01 Bruce IVins 1425 Porter Street Ft Detrick MD 21702 I certify that the contents of thiS C Signature of DIVISion Chief Day 30116194927 24 hour 301/6192257 LOGISTICS DIVISION DELIVERY DelIVered to MATERIEL SERVICES Date PACKAGING I certify that thIS consignment IS packaged for shipment accordmg to applicable lATA and DOT oacklng instructions ~ nn teo Name and Slgll3lUre oi Cenlrymg OthCl2!1 D ate U!:,AMRIID Furm 11 R (01 FebruaryZOO1) WOIRHEPP B3D14.pdf  18  009915Anthrax Page 428 of 643 / , US ARMY MEDICAL RESEARCH INSTITUTE OF INFECTIOUS DISEASES SHIPMENT REQUEST REAGENT MATERIAL ! DIAGNOSTIC SPECIMEN I CONTROLLED BIOLOGICAL PROOUCT I BIOLOGICAL AGENT I ETIOLOGIC AGENT (INF ECTIOUS SUBSTANCE OR TOXIN) I RADIOACTIVE MATERIAl Please check appropnate block and route Itlrough appropnate approVIng authorJty REAGENT MATERIAl Non hazardous or (a) Hazardous (ttw Safety Office) DIAGNOSTIC SPECIMEN {Classificabon Group (hI (ell (lhru Safely 01f~1 CONTROLLED BiOlOGICAL OR BIOLOGICAl PRODUCT x Human or Non-Human I InV and bc:;-lIst (thru Clinical Use BiOlogiCS Cootrol OffICer) INFECTIOUS SUBSTANCE (ETIOLOGIC AGENT) AFFECTING HUMANS (thru Safety INFECTIOUS SUBSTANCE (ETIOLOGIC AGENT) AFFECTtNG ANiMAlS ONLY (thru safe;;~~'rCe1 J1n1(.b({ OfftaJ)f./lI!,r,j';/ TOXIN (ETIOLOGIC AGENT) (thru Safety OffICe) CU\C"OACT'VE ?"ATEq'Al {th.... Rad,auon Pro,&c1IOn OffICe! BIOLOGICAL AGENT NON INFECTIOUS (thru Safety Office) OTHER Non-hazardous or Hazardous (thru Safety Office) DescnptJOn of matenaf(s) 30 m[ of B anttncs Ames spores 39 X l1Y'101rni 1 17 X 10"12 spores total an 3 pOlypropylene tubes 10 ml per tube to enable contract orgamzabon (BatteHe) to perform contracted study Quantity (ml mg ) 30 ml 39 X 10" lOJml 1 17 X 10"12 total Ship to Address (lOclude telephone number day and emergency of reel lent Erne ency number must be phYSICally m anned 24 hours a day No pagers or ceU phone numbers BattellelMREF JS 3 1425 S R 142 West Jefferson Oh.o43162 Day 614/4247230 24 hour L _ _ _- l ReqUired ShIPment Date 18 June 200 1 Special ReqUIrements None "'Y Ice y. Refngerated (wet OIhec Charge to APC 6GDS Method of ShIPment Requested x.. UPS AJr Express USAMRIID Couner Other b6 b7C ICe cold pack) Name Addres s Telephone Number (mclude day and emergency) of Sender Bruce Ivlns 1425 Poner Street Ft Detnck MD 21702 Date 15JUNOl Day 301/619-4927 24 hour? 301 /6192257 bove Date 15 JUN 01 I certify that the contents of thiS Signature of DIVISion Chief DELIVERY Delivered L O , Dare- PACKAGING I cenlfy thaI this conSignment IS packaged for si'lLpmenl according to applicable lATA and DOT packing ,nstructIOns Pnmed Name Gno StgrlOlure of Cendylng OfflCl3l Dale WOIRHEPP B3D14.pdf  19  009916Anthrax Page 429 of 643 ,.. UNITED STATES DISTRICT COURT FOR THE DlSTRlCT OF COLUMBIA FILED OCT3 12007 In the Malter of the Search of IMHCY IllYER _TO~ C ERK l U.s.1>SIIiICT COURT ResideoC'e at Frederick, owned by Bruce Edwards ]vins, DOB SSN. . . . APPLICA nON AND AFFrDAVlT FORSEARCH\VARRANT CASE NUMBER: 0 7 - 5 2 4 - AI - 0 1 __.... o"m"'."sLF"'-.D e!!I1".!lrc"r.. ____ belIl(!: duly sworn depose and say: T h"' ... a~ I am a(n) that [J rostal Insp< dctennined thai the strain of Bacillus anrhracis found in each of the decedents' bodies is the same strain of anthrax found in the threat letters mentioned above. This partieuhlr strain is commonly known as the Ames strain of Bacillus anthracis. Law enforcement officials have spoken to biological expertS who have knowledge and training in the field of infectious diseases including anthrax . According to these individuals quantities of Bacillus anthracis can be produced in a scientific laboratory, such as those found at universities, military research facilities, or other research institutions. Culturing anthrax and working safely with dried anthrax spores requires specifIC training and expertise in technical fields such as biochemistry or microbiology. It also requires particular laboratory equipment such as a Iyophilizer or other drying device, biological safety cabinet or other containment device. incubator, centrifuge, fennentor. and various personal protective gear, described more fully in the Attachment to this affidavit. r>etennination of Origin of Anthrax Contained in 2001 Mailings Th:: mail attacks arc believed to have occurred on two separate occasions, which are delineated by the postmark dales orthe recovered leiters. Letters used in the fi rsl attack were postmarked on 0911812001 and were focused to media establishments located in the New York 3 WOIRHEPP B3D15A.pdf  4  009920Anthrax Page 433 of 643 area. Three weeks later, leiters postmarked 10109/2001 were sent to two U. " Senators ill Washington, OC, In order to fully characterize the threat letters and their contents, the Task Force has conducled numerous physical (phenOlypic) and genetic (genclypic) analyses. Physical comparison of the spore powders taken from the Post and Brokaw letters versus the Senator Leahy and Senator Dascble letters reveals obvious differences. Thc spore powders recovered from the POSI aDd Brokaw letters were granular and multicolored in consistency, while the Senator Leahy and Senator Daschle letten contained fine spore powders that were unifonn in color. Microscopic examination of the evidentiary spore powders recovered from all four letters identified an clementalsignalure oC8ilioon within the spores. This Silicon signature had not been previously described for Bacillus anthracu organisms. Genetic Analysis ofthc Anthrax Used in thc Attacks Upon visual inspection of the Bacillur anthracis organisms used in the mati attacks, resean:hen; have discovered numerous phenotypic variations, which distinguish it from the original Ames anthrax isolated in 1981, which is the earliest known sample, The phenotypic variations were identified as differences in morphology (i.e. shape, color, texture) from the original Ames anthrax. Changes in morphology can be attributed to individual genetic mutations within the &cil/ws allllrracis DNA. As a whole, the collection of all of the genetic mutations found in the anthrax used in the 2001 mailings, serve to provide a "ONA fingerprint" which can, and has been used to investigate other Ames isolates collected from laboratories possessing the Ames strain, Four individual, highly sensitive, and specific molecular assays capable of detecting four of the genetic mutations discovered in the Bacillus anlhracis used in tbe mail attacks have been de ... elopcd and validated. Following tbe mai lings, sixteen domestic government, commercial, and ul1Jiv~rsily laboro.tories thai had ' Ames strain Bacillus anthracu in their inventories Bacillw isolates all sixteen domestic received laboratories, as well as. from laboratories in Canada. Sweden, Md the United Kingdom. All total, the Task Force bas obtained mace than t 000 isolates of the Ames strain of Bacillus Qnlhrocis from these laboratories and archived these isolates in the FBI Boell/w onlhracis Repositol) (hereinafter "FBIR"). The four aforementioned molecular assays have been applied to each of the more than 1000 Bacillus anlJrracis samples contained within the FBIR. Of the more than 1000 FBIR samples, omy eight were determined to contain all four genetic mutations. The Task Force in ...estigation has delennined that each of the eight isolates in the FBIR is directly related to a single Bacillw QJlllrracis Ames strain spore batch, idcnuficd as RMR?l029. 4 WOIRHEPP B3D15A.pdf  5  009921Anthrax Page 434 of 643 o RMR?1029 was stored. in the B3 biocontainment suite within Building 1425 of the United States Army Medicalln'tione of Infectious Diseases (USAMRIID), Fort Detrick, Maryland. Access to the suite is afforded only 10 those personnel who arc approved by the USAMRDD Security, Safety, and SpeciaIlmmWlizations Program to have the required background check. training, and medical proteclion (vaccination or personal protective equipment (PPE>> . Dr. Bruce Ivins has unrestricted access to the suite and bas been the sole custodian of RMR-I029 since it was first grown in 1997. Bacterial contaminant found in attack letters Both afthe anthrax spore powders recovered from the Post and Brokaw icncrs contain low levels of a bacterial contaminant identified as a strain of Bacilleu subtilis. The &leillus subtilu contaminant has not been detected in the anthrax spore powders recovered from the envelopes mailed to either Senator Leahy or Senator Dascblc. Bacillus subtilis is a nonpathogenic bacterium found ubiquitously in the environment. However, genomic DNA sequencing of the specific isolate of Bacil/us sub/iii! discovered within the Po..rt and Brokaw powders reveals that it is genetically distinct from other known isolates of Bacillus sublilis. Analysis o f the Bacillus sublilis from the POSI and Brokaw envelopes revealed that these two isolates arc identical. . Phenctypic and genotypic analyses demonstnue that the RMR ~ 1029 does not have the Baeil/us subtl/is contaminant found in the evidentiary spore powders, which suggests that the anthrax used in the letter attacks was grown from the material contained in RMR- I 029 und not taken directly from the flask and placed in lhe envelopes. Since RMR-1029 is the genetic parent to the evidentiary spore powders~ and it is not known how the Bocil/us J;ubrilis contaminan t came to be in the POSl and Brokaw spore powders, the contaminant must have been introduced during the production of Lbe Post and Brokaw spores. Taken together, the postmark dates, the Silicon signature, the Baeil/us sublills contaminant, the phenotypic, and the genotypic comparisons, it can be concluded that, on at least two separate occasions, a sample ofR..MR-1029 was used to grow spores, dried to a powder, packaged in an envelope with a threat lecter, and mailed to the victims. I Envelopes used in the anthrax attacks In the 2001 anthrax attacks, four envelopes were recovered. The four envelopes were all 6 ?.. inch federal eagle envelopes. The "federal eagle" designation is derived fro m the postage frank in the upper right-hand comer on the envdope which consists of the image of an eagle perched 011 a bar bearing the initials "USA." Underneath the lenering is the number "34," which denotes the postage value of 34?. The eagle, leuering, and denomination are referred to as the I As tbe Court may searcb lime to timt: obtainedrecall ~rrants, fro:m~;~~~;;~~~,~oun~'~O;f~lh~i'~'~iX~Y'~'G'~in~V~"~ri~'~"~io~n~th< the ~k~ hasa.nd , ~; " FOf<< , for T~ residences from searches in geoetic assays that }cd to the tdentifacatiO of RMR D parent ohhe a.nthrax wed in the 2001 letter attacks. Fi ii!~~~~ the ~h~iS~~;!. ;"?,,oncluc.,m. to 10:43 p.m. 7:24j1.m. to 9:39 p.m. 7:25p,m. to 10:55 pm. 6:10 p.m. to 10:12 p.m. 7:40 p.m. to 12:43 a.m. Total Time iD B3 t hour 42 minutes 1 hour 20 minutes t hour J 8 minutes 20 minutes 23 minutes 2 hours S9 minutes 3 hours 33 minutes 3 hours 42 minutes After October S, Dr. Ivins did not enter Suite B3 in the evening again until October 9, for 15 minutes, and then October 14, for 1 hour and 26 minutes. The investigation has also analyzed the daily schedules, work areas accessed. and the number of oours worlccd per week of all other researchers assigned to the Bacteriology Division who had ae?cess to Suite 83 during the monlhs ofSeptcmber and October 2001 . When these records are compared to Dr. Ivins for the same period, Dr. Ivins's habits arc significantly difTerent than those of the other reseacchcrs. in that he was frequently in Suite B3, where RMR1029 was kept. late at night and on weekends when no other researchers were present in Suite B3. Additionally, while Dr. Ivins was in Suite B3 at night. no other USAMRJID employee was present. On March 31. 2OOS. Dr. Ivins was asked by Task Force investigators about his access to B3 and could provide no legitimate reason for the extended hours, other than "home was not good" and he went there"to escape" from his life at home. A review of Dr. Ivins's and coworkers's laboratory notebooks and projects at the times in question was conducted. The investigation determined that Dr. Ivins', role in the experiments were mini,mal. and did not j ustify the lime be spent in B3. Dr. Ivins has admitted to investigators that the research he was conducting in 200 I did not require, and does nol explain. his late night hours in the B-3 laboratory aroWld the time period of me anthrax mailings. Since producing anthrax spore preparations was one of Dr. Ivins's principal responsibihties al USAMRlID. he had multiple and unfettered opportunities to produce or divert Ames strain spores for illegitimate purposes. His acces.~ to Suite B3 and USAMRUD afforded all of the C was interviewed by the Task Force, and deny disc Ie sing such infonnation to Dr. Ivins. Over the course of this investigation, Dr. Ivins bas been repeatedly interviewed and had open access to the law enforcement personnel responsible for investigating the anthrax attacks. During none of these internctions, did Dr, Ivins ever indicate he had knowledge that RMR-I029 had phenotypic similarities to the material used in the anthrax anacks, nor did he suggest that investigato!S analY.l.e RMR-I029. To the contrary. Dr. Ivins repeatedly claimed that the anthrax used in the attacks resembled that of anotller researcher at USA.MRlID and were dissimi lar to the Bacillus anthracis Ames organisms maintained in his laboratory, which included RMR- 1029. Mental health issues and possible motive The investigation has shown that in 2000 and through the mailings in 2OCH. Dr. Ivins had mental health issues. Dr. Ivins's mental bealth issues came 10 the attention of invC$tigatO!1; while reviewing e-mails ofUSAMRIID rc:scan:.hcrs. Through the c-mails it was determined that Dr. Ivins was undergoing significant stress in both his home and work life. The mental health issues and stress were significant to the extent that Dr. Ivms sought professional help from a psychiatrist and wu immediately prescribed medication that started in February 2000, The following an: excCIpts from e-mails dated April 2000 through December 2001. from Dr. Ivins to a friend regarding work. home, state of mental health, and usc of med ication (emphasis are as they appeared in the e-mails): April 3, 2000, ~Occasional ly I get this tingling that goes down both arms. At the same time I get a bit dizzy and get this unidentifiable ~rnctallic? tastc in my mouth. (I'm not trying to be funny . . . It actually scares me a bit.) Other . times tI's like I'm n01 only sitting at my desk doing work, I'm also a few feet away II WOIRHEPP B3D15A.pdf  12  009928Anthrax Page 441 of 643 watching me do it There's nothing like living in both the fim penon ,ingular AND the third penon singulu!" June 27, 2000, -Even with..tbc eelaa and the counseling. the depression episodes still come and go. That's unpleasant enough. What is REALLY scary is the paranoia .... Remember when I told )'Ou about the -metallic? taste in my mouth that I gol periodically? Irs when I get these "paranoid" episodes. Of course 1 regret them thoroughly when they arc over, bUI when I'm going through them, it's as if I'm on a passenger on a ridc.... Ominously. a lot of the feel ings of isolation - and desolation - that I went through before college are returning. I don't want to relive those years again .. . . I've been seeing the counselor once a week.- June 28, 2000, "Apparently Gort: (and maybe even Bush) i. considering making the anthrax vaccine for the military voluntary. or even stopping the program. Unfortunately, since the BioPort people aren't scientists, the task of solving their problem has fallen on us. .. .. Believe me, with aU the stress of home and work, your cmailldters to me are valuable beyond what you would ever imagine - and they help me keep my sanity ...... June 29, 2000, "BioPortjust tested il& final"'t of A VA [anthrax vaccine) In a potency test. If it doesn't pass, then there arc no more lots to test, and the program wiU come to a hall. That's bad for everyone concerned, including us. ('m sure that blame will be spread around." July 4, 2000, 'The thinking now by the psychiatrist and counselor is that my symptoms may noC be those of a depression or bipolar disorder, they may be that of a ?Paranoid Personality Disorder.July 6, 2000, ( I think the oooo is about (0 hit the fan ... bigtime. The final 101 of A VA, lot 22, isn't passing the potency test, and now there's no,th in.g.to ;. . back. it up. Plus. the control vaccine isn't worlcing. It's just a fmc mess. l are spending probably 95~o of our lime on this." July 7, 2000, in an e-mail. Dr.lvins offered to be interviewed as a case study, as long as it remained anonymous. Dr. Ivins indicated that he did not want to see a headline in the National Enquirer that read, ?PARANOID MAN WORKS WITH DEADLY ANTHRAX!!! " July 23 , 2000, -It's been a really stressful week , from all stand points. Home, work, and it's not going well with the counselor I'm going to. (She said she thinks I'm going 10 have to ask to get put with another counselor or into a group session.... Sometimes I think thaI it's all just too much.? 12 WOIRHEPP B3D15A.pdf  13  009929Anthrax Page 442 of 643 o August 12,2000. 'Last Saturday, as you probably guessed from my email. was oce of my worst days in months. J wish I could conlroJ the thoughts in my mind. It's hard enough sometimes controlling my behavior. When J'm being eaten alive inside. I always try to put on a good front here at work and at home, so I don't spread the pestilence. _. .I get incredible paranoid. delusional thoughts at times, and there's nothing I can do until they go away. either by themselves or with drugs.' August 29, 2000, ~ are 10% aflhe Bacteriology Division. If we quit, the anthrax program and BioPort would go down the drain. I'm not boasting. ~ but the three orus have 8 combined tota] 0(52 yean ofres~h experience with anthrax. You just can't go out and find someone lik~ with their knowledge, skill and abilitics. Ain't golUll happen." March 4, 2001, -The people in my group just don't pick up on wbat 1 try to say. They are not into the kinds ofproblcms I bring up, so it's hard for them to deal wilh them. The psychiatrisl is helpful only beeause he prac;ribes !he Celexa. He', not that easy to talk 10, and be doesn'l really pick up on my problems. The wor.anl saw before I went mto group wanted to get me put in jail. That wasn't very helpful either, I'm down to a point wbere there are SOme things lhat are ealing away that J fee) I cao'llell ANYONE.... " September 7. 2001 , "r was taken off the Special Immunization Program because of what happened last spring, and I've just gotten back on it, geUing my Inthn.x and Yellow fever shots. We are currently fmishing up the last of the AVA, and when that is gone, there's nothing to replace it wiLh. I don', know what will happen to the research programs and hot su ite work until we get a Dew 101. There are no approved lOIS currently available at BioPort. . . . o has been havUlg us have biweekly meetings on the rPA vaccine progress, and on August 29 I we:n! to the Pentagon - first time there - to go to a meeting in his place on the vaccine. There is a real bag of worms with a new lot of rPA produced by tlle BDP (8 private company) for NCl. who is under contract to USAMRlID. BOP signed a sub-contract with to produce the rPA for a human use vaccine Phase I trial. They were paid and they produced it. Now they arc refusing to n::lease it unless tbe Amy pays some incredible sum of money fOT lawsuit indemnification (about S200,OOO per year for the nCltt 50 yean). The Amty refuses to do that of COUi-se, and everything is in Limbo:' September IS, 2001. "I am incredibly sad and angry at whal happenod, now that it bas sunk in. Sad for all of the victims, their families., their mends. And angry. Very angry. Angry at those who did this, who support them, who cod:lle them, and who excuse them.- September 26, 2001, "Of the people in my "group,- everyone but me is in the depression/sadness/flight mode for stress. I'm really the only scary one in the group. Others are talking about how sad they are or seared they are. but my tJ WOIRHEPP B3D15A.pdf  14  009930Anthrax Page 443 of 643 reaction to the WTClPenlagon events is far di fferent. Of coun;e. l don't talk about how I really feel.with them - it would just make them worse, Seeing how differently I reacted than did to the recent events makes me really lhink about mY.ielf a lot. I' and sarin gas. heard that Bin Laden terrorists for sure have anthrax In that same September 26,2001 emai l, Dr, Ivins states "Osama Bin Laden has just decreed death to all Jews and all Americans" - language similar to the lexl oflbe anthrax letters posnnarlced Iwo weeks laler warning "DEATH TO AMERICA," ' 'DEATH TO ISRAEL." On October 16, 2001, in an e-mail.Dr. lvins.s coworkerconununicated the following to a former coworker, "Bruce has been an absolute manic basket case the last few days." December 1 S, 2001, -I made up some poems about having two people in one (me + the person in my dreams): . .. I'm a little dream-self, short and stout. I'm the other hal f of Bruce - when he lets me out. When I get all steamed up, I don't pout. I push Bruce aside. them I'm Free to run about! Hickory dickory Doc - Doc Bruce ran up the clock. But something then happened in very strange rhytrun . Hi!';. other self went and exchanged places with him. So now, please guess who Is conversing with you', Hickory dickory Doc! Bruce and this other guy, siUing by some trees, Exchanging personalities. It's like having (wo in one. Actually it's rather fun'- The investigation has revealed that Dr. Ivins was prescribed various psychotropic medications including antidepressants, antipsychotics, and anti-anxiety, for his mental health issues from 2000 through 2006. On July 18,2007, a forensic psychiatrist completed a detailed review of Dr. Ivins insunmce billing records for medical appointments Bnd prescriptions, Additionally, this psychiatrist was provided with an overview of Dr. Ivins's social habits, interests, and obsessions. The forens ic psychiatrist stated (hat based on his experience, if Dr. Ivins was themailer. it is quite possible that Dr. Ivins retained some kind of souvenir or references to the mailing events. 14 WOIRHEPP B3D15A.pdf  15  009931Anthrax Page 444 of 643 Controversy concqninK the anthru vacx:inlS Beginning shortly after the first Gulf War and throogh 2001, USAMRlID and Dr. IV ins was the focus of public criticism concerning their introduction of a squalene adjuvant (or additive) to the AVA anthrax vaccine, which was blamed for the Gulf War Syndrome. In 2000 and 2001 , as evident by the e-mails above, that same anthrax vaccine was having problems in the production phase at Bioport, a private company in Michigan responsible for manufacturing the vaccine. The Food and Drug Administration (FDA) had suspended further production at Bioport, and the U.S. government. specifically the Department of Defense, was running out of approved lets of the vaccine. The situation placed pressure 00 select staff members at USAMRllD, including Dr. Ivins, who were part of the Anthrax Potency Integrated Product Team (IPT). The purpose of the 1FT was (0 assist in the resolution of technical issues that was plaguing Bioport's production of approved lots of the vaccines. In the weeks immediately prior to the attacks, Dr. rvins became aware that an IOvestigative journalist who worted for NBC ews had submitted a Freedom of information Act (FOIA) requests on USAMRIID seeking detailed information from Dr. Ivins's laborlltory noteOOoks as they related to the AVA vaccine and the use of adjuvants. On August 28. 2001 , Dr. Ivins appeared angry about the request providing the following response in an e-mail: '"Tell Matsumoto to kiss my ass. We've got bcctet things to do than shine hi! shoes and pee on command. Hc's golten cverything from me he will get ." In early 2002, shortly after the anthrax letter attacks, the FDA re-approved the AVA vaccine for human use, production at Bioport resumed, and anthrax research at USAMRllD conlinued without interruption. As mentioned previously, one of the anthrax letters post marked on September 18, 2001 . was addressed to Tom Brokaw, NBC News in New York. Dr. Ivins thereafter received ''the highcst honor given to Defense Department civilians at a Pentagon ceremony on March 14.2003" for his work in "getting the anthrax vaccine back: into production ... Use of false namcs and fascination with sorority group linked 10 location oCanthrax mai lings The investigation has shown that over the past twenty-four years, Dr. Ivins was known to have utilized at least two Post Office Boxes to communi cate with members of the publ ic, to pursue obsessions, and possibly engage in the unauthorized use of another person's name. The use of the P.O. Boxes is significant because the post office where Dr. Ivins frequented and maintained. a PIO. Box, CoUege Estates Station. Frederick, Maryland 21702. was serviced. by the Frederick Main post office. Frederick. Mary1and 21701. Analysis of the USPS shipping records indicate the Frederick Main received lOOO 6 3/4" Federal Eagle banded envelopes on the same day as post offices known to have received envelopes with identical print defects as those seen on the envelopes used in the mailings. On May 14, 2007. identified fo ur communications that it had previously received from Dr. Ivins. does not personally know Dr. Ivins but has repeatedly corresponded with him by mail]. In Dr. Ivins's fin' letter to 2 da,ed May 8, 1982, he used the return address of P.O. Box 1265, Frederick, Maryland 21702. In the letlers, Dr. Ivins expressed hIS support '0 ,s WOIRHEPP B3D15A.pdf  16  009932Anthrax Page 445 of 643 o work and an interest in sorority hazing. One or the sororities with which he 4 interest was identified as Kappa K~~:.,~ Dr. Ivins enclosed a " leuer to the On July 9, 2007, the Post Office Box application (PS Fonn 1093) at the College Estates 51'a lion, Frederick, Maryland 21702, for P.O. Box 1103, in the name of Bruce E.lvins was obtained. A review of the application card revealed that Dr. Ivins rented P.O. Box 1103 on November 12, 1993, providing a military identification and using his Maryland Motor Vehicle Administra:ion Drivers 1-152-098-188-301 as proof of identification. Dr. Ivins an Frederick, MD 21702 and telephone numbers of listed OIl the P.O. Box application card to receive mail The last payment on Ihe P.O. Box was made on November 13.2004. A review of Address records revealed that P.O. Box 1103 was closed 011 December 30, 2005 and no forwarding address was provided. On July 30, 2007, was interviewed regarding P.O. Box 1103. personally know Dr. Ivins but has histOrically been familiar with his n..me o o as shov.-n the P.O. Box application and stated that it had never rented a P.O. and had never met. nor had any social or professional association with 8 Dr. Ivins . However. ~.lI recognized the name Bruce Ivins because had previously mentioned his name. - ' -indicated ld & that Dr. Ivins lw-assed it in the past, and believed lha~ ~as frightened ofDr. lvios, resulting from unspccified incidents that occUlTed prior meeting _ also recaJJed a vandalism incident thai occurred in tbe months prior to leaving the Maryland area wi~ They had awoken onc morning in their Gaithersburg apartment to find Greek letter! spray painted on the fence, sidewalk, and vehicle outside their apanmenl. to" A copy ofth' -Police R..",,,_4 o o o was obtained from the Records Division and prc.vi rrtf"~ ~ -Po"""b"J) ,. ~~ .ll-tftf,fJ~" -, ~/"r, t.<-//~-'-- C<. -r C-~1 -r....t, d?"'r ,t;,,, 00 50. ,4 rtf)~"" 7C. AvrA":r .,., e,"'4<-= ~i~ .vj "" (~ ~-"Io(." I ja7 20 WOIRHEPP B3D15A.pdf  21  009937Anthrax Page 450 of 643 The statemenlS contained in this Affidavit are based in part OU infonnation provided by FBI Special Agents and U.S. Postallnspectors. on observations made by law enforcement agents, and on my experience and background as an FBI Special Agent. I have not included each and evcry fact known to me concerning this investigation. I have set fOM only the facts that Jbeliev.e are necessary to establish the necessary foundation for the search warrant. lirOMAS F. DELlAFERA Postal Inspector United States POSlallnspection Service OCTu lOOT Sworn to before me this __ day of , 2007 ,/ ~/~t?y.. GISTRATE JUDGE United States District Coun for the District of Columbia u.a~.lIlGE DEBORAH A ROBINSON N, Clol1< 21 WOIRHEPP B3D15A.pdf  22  009938Anthrax Page 451 of 643 ATTACHMENT TO AFFIDAVIT The search of the Subject Property and Premises will include a physical search and forensic swabbing for the items mentioned in the text of the search warrant. including more specifically the following: 1. Any assembled or unassembled Biological Threat Agent (BT A), or trace thereof, and any BTA weapon, and/or any incendiary. radiological, nuclear, chemical or booby trap device. 2. Laboratory equipment used in the production/replication of biological threat agents including. but not limited to agar or liqUid growth media.,. media components, anti-foaming agents, animal droppings or carcasses, autoclaves, bleach, blenders, live animals and materials for their care, cylinder of nitrogen or oxygen. soil, dimethylsulfoxide (DMSO). fermentors, grinders, home brewing kits, incubators, inoculating loops, mechanical slirrerslpaint stirrers, microscopes, microwave for making homemade agar plates, mortar/ pestle. milling devices, ovens, Petri dishcslimprovised glass/plastic cook dishes, pipettes, pressure cookm, Q?tips, roller flasks, rubber gaskets/seals. screens, sieves. sodium hydroxide, stains/inks/dyes used to identi fy BTAs, sterilization equipment (Bunsen or alcohol bumerslbutanc torches). swabs, lest tubes, tissue culture flasks. virus flasks, and other materials. 3. Personal Protective Equipment/Safety Equipment including, but not li mited 10, biosafety hoods and cabinets (commercial or homemade), masks (surgicaVdust/HEPA), exhaust vents, filters (!{EPA). gloves. lab coals, mechanical fans, safety glasses/goggles, shoe covers. soap, tubing pipes, and vinegar. 4. Items used in the weaponizationldelivery of biological weapons including, but 110t limited to pens, tape. paper. scissors, envelopes. target addresses, aerosol canisters. agricultural or garden sprayers. acetone or other drying agents, baby powder, baking powder. baking soda, boxes, commerciaUindustrial powders, copier toner/printer andlor toner/inks, compressed air or other gas systems, conuneal. cornstarch, explosive. hypodermic syringes/needles, jars or flasks with gauze on top, oatmeal, powdered milk, saltlpep~r. spray bottles, squirt guns, stamps, sugar. talcum powder, and other materials. 5. Documents, memoranda, notes or other writings and equipment which include but are not limited to schematics, diagrams, blueprints, surveillance equipment or its manuals and other related paperwork. receipt for purchase of items, how? to magazines, books and pamphlets which describe production. weaponization, and delivery techniques, directions, maps, diagrams. and blueprints of delivery or target locations. communication which would include but not be limited to handwritten notes and drawings. memos and photographs, which would include descriptive in rormation such as telephone numbers, addresses, affiliations and contact points of individuals involved in a conspiracy to acquire, refine, alter, transport, andlor usc a biological weapon. Note that such documentary material described herein may be in printed or electronic fonn such as, but not limited to, computer disks. Such computerized and other electronic malerial would constitute contraband, fruits, instmmentaJities or evidence of the offenses enumerated herei n and in the attached affidavit for search warrant. III WOIRHEPP B3D15A.pdf  23  009939Anthrax Page 452 of 643 6. Publications or other written materials evidencing knowledge of federal laws including those related 10 biological weapons and other weapons of mass destruction, 7. Pholographs, newspapers, or magazine clippings, travel infomlation, brochures, maps, hotcl receipts, rental receipts, notes, ledgers, phone numbers, toll records, address books (which may be in printed or electronic [onnat), hand-held electronic memo-type notebooks. scanners, commercial transponarion receipts, and firearms, ammunition, iireanns owners identi ficati on doc\lments andlor pennits and licenses, digital or other types of pagers and all alpha and/or nwncric data/message stored therein, cellular phones, cordless telephones and th(.i.r base units, caller identification units and all alpha-numeric infonnation and data stored therein. biological weapons/agent fonnulas, biological weaponslagents, andlor recipes, all of which related to the construction of biological weapons, or plans to produce, conceal, or usc such -q(D",_) ; I I (not 10 exceed 10 V1' Ul.Ll TRt\lUIIQI WOIRHEPP B3D15A.pdf  27  009943Anthrax Page 456 of 643 ? . RETURN VA TE A~I) AO 109 (V90) Scit.w'( WW1lnt DATf WAIUtANT Rl:CEIVt:D nw.f. WAPJlA"''' fXfDlTED (..1)PY OF W"'RRA~T AND RECEIPT FOR ITEMS LEFT ?l1Tt! >> 131/ ""Do7 lNVEl'O'OIlY MAllE IN THf. PRESF"iCE OF .. r- p .; II 11;f' I D' Mr: <,..I!L INVENTORY OF 'ROf'~ITY SEIZED P URS UANT1'O THE WAlUl.Atrr 7~ ex" 'r~ C-AQ fl- CERTIFIC ATlON I swear !hllt this Inventory is 10 true and detailed account or the perty seized by me 00 the warnnt. FILED I<<)V 0 9 2007 Sub.; itcm(s) listed below were: Rece ived From Returned To ~el rned To o o ized (Name) (Street Adld,,:s.s:I_ _ (City)----E ~~> < Descriptio n of Hem(s): _~M=(2"-t<<=(f_""''_~.s''''-'''/H'"''--!:.J''4C"='--'-,_-'-_'''''='''X7=~~'==-::V"'- __________ _ A < __ It' - 5 -/ f,IIJ,A'V'-~ SWA4 JUt 111 /-1-.5 -.;{ v'Aat-A .&;!Ti?-< ,(" tJ /I-5-j' I/ -.f-? 11-.5 6 11..??;7 #-? - 9 d-S-t! #-[-42 /I -I-I{' //-5-/S / 7 /I'S-& II ?J -H' N-S -/f" H -.f-Cl ff~ @O,4. 4AM en ,[J/Ao I J _~()" ( 1_ 4,.t1r#,efJoA'\ /JV'A.d lI?r -.If!' t,/_,r - $()" ,tI.4?'if7('OA" A~q-,.. t(c/6;? Y.;fc ~ JJ;f/fA? "/If _ ,.t'''Ar~;, " .. ~ JOy' J;/,;o,tJ ~ ,2/ <;+1 _ ~4A'4'~ < 8(Jo,,EUR. L..J.~...r;tJ;QA. ;~~ Recei.cd By: --'4!::';;;l1~~::g;:;;::=:::::;=:;;=(Signature) ......... Recei ved ~ From : ( ignalure) 33 WOIRHEPP B3D15A.pdf   009949Anthrax Page 462 of 643 o UNITED STATES DISTRICT COURT FOR THE DISTRICT OF COLUMBIA In the Maner o flh e Search of FILED Den 1 2007 APPLICATIOl\ AND AFFIDAVIT FOR SEARCH WARRANT CASE NUM BER: 0 7?525? "'?01 believe _---"T"'b"'owm"'.,.'u:FL D"".!!II.. a!lr.,-,r"a~____ being duly ~\\'Ortl depose and say: o I am I ' D) fOllaJ lospttlor witb lbe U.s. PoSCtllnsDtttion f,yttr (0fJ'd1i TIlle) and have r~uon 10 thai C 00 lbc person or or II on the: property or premiSl:$ Imown as (r.lI'I'It, deKlip'ion lAd IX 2 ~ 4 door-sedaD, burinl VLV e, o _ _ Frede:rkk, MO the DIstrict orMuyland, Ihere I~ now concealed I cenam pc.""::;:n~.~o:,. ~;:';:~ Irate qUJlnlitia of Bacillus anthratis or slmulants Ihu'tOf, h used in preparatLon of seltct .,ents. p.pus, tape, pens. DotU. books, manuals. receipt.. r<gicaJ expertS who have knowledge and training in the field of infectious diseases including antluu: . According to these individuals quantities of BocUlus anthracis can be produced in a scientific laboratory, such as those found at universities, military research facilities, or other research institutions. Culturing anthrax and working safely with dried anthrax spores requires specific training and expertise in technical fields such as biochemistry or microbiology, It also requires particular laboratory equipment such as 3 lyophilizer or other drying device, biological safety cabinet or other contairunent device, incubator, centrifuge, fermentor, and various personal protective gear, described more full y in the Attachment to this affidavit Determination of Origin of Anthrax Contained in 2001 Mailings The mail anacks are believed to have occurred on ty.'o separate occasions, which are delineated by the postmark dates of the recovered letters. Letters used in the first attack were postmarked on 09/1812001 and were focused to media establishments located in the New York 3 4 WOIRHEPP B3D15B.pdf   009953Anthrax Page 466 of 643 , . area. Three weeks later, letters postmarked 10109/2001 were sent to two U.S. Senators Washington, DC. In In order 10 fully characterize the threat leners and their contents, the Task Force has conducted numerous ph)'5ical (phenotypic) and genetic (geootypic) analyses. Ph)'5ical comparison of the spore powders taken from the POSI and Brokaw letters versus the Senator Leahy and Senator Daschle leuers reveals obvious diIf~. The spore powders recovered from the POSI and Brokaw letters were granular and multicolored in consistency, while the Senator Leahy and Senator Daschle leeters contained fine spore powders that W~ unifonn in color. Microscopic examination oflhe evidentiary spore powders recovered from all four IcHers identified an elemental signature of Silicon within the spore.", This Silicon signature had not been previously described for Bacillus anthracLs organisms. Genetic Analysis of the AntluM Used in the Attacks Upon visual inspection of the Bacillus anthracu organisms used in the mail attacks, researchers have discovered numerous phenotypic variations, which distinguish it from the original Ames anthrax isolated in 1981, which is the earliest known sample. The phenotypic variations were identified as differences in morpbology (i.e. shape, color, texture) from the originaJ Ames anthrax. Olanges in morphology can be attributed to individual genetic mutations within the Bacillus anlhracis DNA. As a whole. the collection of all ofthc genet ic mutations found in the M.thrax used in the 2001 mailings, serve to provide a "DNA fingerprint" whjch can, and has been used to investigate other Ames isolates collected from laboratories possessing tin: Ames strain. Four individual, highly sensitive, and specific molecular assays capable of detecting four oflhe genelic mutations discovered in the Bacillus anlhmcis used in the mail attacks have been developed and validated. Following the mailings, sixteen domestic government. conunercial. and uni versity laboratories that had virulent Ames strain Bacillus antltracis in their inventories prior to the .- - .. -- received Ames strain BacillUJ 'vllhraci~ isolates or samples from aU sixteen domestic laboratories, as well as, from laboratories in Canada, Sweden. and the nited Kingdom . All total, the Task Force ha.'" obtained more than 1000 isolates of the Ames strain of Bacillw Q11Ihraci.J from these laboratories and archived these isolates in the FBI BacIllw anthr aCLf Repository (hereinafter "FBIR."). The four aforementioned molecular assays have been applied to each of the more than 1000 BaciJIus anlhracis samples contained within the FBIR. Oflhe more than 1000 FBIR samples, only eight were delCrmined to contain all four genetic mutations. The Task Force investigation has dctennincd that each ofthe eight isolates in the FBIR is din.. -ctly related to a single Bacillus anlhracis Ames strain spore batch. identified as RMR? 1029. WOIRHEPP B3D15B.pdf  5  009954Anthrax Page 467 of 643 RMR?1029 was stored in the B3 biocontainment suite within Building 1425 ofthe United States Anny Medical Institute of Infectious Diseases (USAMRIID). Fon Detrick. Maryland. Access 1 0 the suite is afforded only 10 those pclWnnel who are approved by the USAMRDD Security, Safety, and Special Immunizations Program to have the required backgrQund check, training. and medical protection (vaccination or per.K>Dal protective equipment (PPE)). Dr. Bruce Ivins bas unrestricled access to the suite and has been the sole custodian of RMR?I 029 since it was first grown in 1997. Bacterial contaminant fQuod in attack leoers Both of the anthrax spore powders recovered from the Post and Brokaw letters contain low levels of a bacterial contaminant identified as a strain of Bacillus subtilis. The Bacilll4S subtUis contaminanl has not been detected in the anthrax spore powders recovered from the envelopes mailed to either Senator Leahy or Senator Daschle. Bacillus subtilis is a nonpathogeoic bacterium found ubiquitously in the enviromneol. However, genomic DNA sequencing of the specific isolate of Bacillws subtilis discovered within the Post and Brokaw powders reveaJs that it is genetically distinct from other known isolates of Bacillus subtilis. Analysis oftbe Bacillu.s sub/ills from the Post and Brokaw envelopes revealed thaI these two isolates arc identical. Phenotypic and genotypic analyses demonstrate: that the RMR-I029 does not have the BaCil/U3 ntbliliJ contaminant found in the evidentiary spore powders, which suggests that the anthrax used in the lener anacks was grown from the material contained in RMR-1029 and nO( taken directly from the flask and placed in the envelopes. Since RMR-l029 is the genetic parent to the evidentiary spore powders, and it is not known how the Bucillus sublili.s contaminant came to be in the Pml and Brokaw spore powders. the contaminant must have been introduced during the production of the Posl and Brokaw spores. Taken together, the postmark dates, the Silicon signature, the Bacillus sl4btilis contaminant. the phenotypic. and the genotypic comparisons, it can be conclUded that. on at least two separate occasions, a sample of RMR- I 029 was used to grow spores, dried to a powder, packaged in an envelope with a threat letter, and mailed to the victims.' Envelopes used in the anthrax attacks In the 2001 anthrax attacks, four envelopes were recovered. The four envelopes were all 6 Y. inch federal eagle envelopes. The -federal eagle" designation is derived from thc postage frank in the upper right-hand comer on the envelope which consists of the image of an eagle perched on a bar beari ng the initials "USA." Underneath the lettering is the number "34," which denotes the postage value of34?. The eagle, lettering, and denomination arc referred to as the I As the Court may recall from Its review, durin? the COlU'SC of this silt year iDvestigatiOQ, the Task Force has from ril1'le to time ubtained search warrants, supported by affidavib establishina probable residences and or ,,,.... individuals searches in this investigation wereoo;d..,"~d w;;;;;iho idenlific.atmn ofR.MR 1029 at USAMRJID as the genetic paTen, of the anrhrax u5ed in the 2001 ~ner attacks. WOIRHEPP B3D15B.pdf  6  009955Anthrax Page 468 of 643 o indicia. The eagle and the bar are stamped in blue ink, while the denomination is stamped in grey ink. Approximately 4S miUion federal eagle 6 y." envelopes were manufactured by Wesrvaco Corporation (now known as McadWestvaco Corporation) of Williamsburg, Pennsylvania, between December 6, 2000 and March 2002. These Federal eagle 6 .y:' envelopes were manuractured exclusively for and sold solely by the U.S. Postal Service between January 8, 2001 and June 2002. Subsequent to the attacks, an effort was made to collect all such envelopes for possible forensic examination. including the identification of defects that occur during the envelope i manufacturing process. As a result of this collection, envelopes with printing defects "dentical 10 printing defec" identified on the envelopes utilized in the anthrax attach during the fall of2001 were collected from the Fairfax Main post office in Fairfax, Virginia and the Cumberland and Elkton post offices in Maryland. The fairfax Main, Cumberland, Maryland, and Elkton, Maryland post offices arc supplied by the Dulles Stamp Distribution Office (SOO). located in Dulles.. Virginia. The DuJIes SDO distributed. "fedcraJ eagle" envelopes to post offices throughout Maryland and Virginia. Given that the printing defects identified on the envelopes used in the anacks arc transient, thereby being present on only a small population of the federal eagle envelopes produced, and that envelopes with identical printing defects to those identified on the envelopes used in lhe aHacks were recovered from post offices serviced by the Dulles SOO, it is reasonable to conclude that the federal eagle envelopes utilized in the attacks were purchased from a post office in Maryland or Virginia. Of the siltteen domestic government, commercial, and university laboratories that had virulent RlI.'IR?I029 Ames strain Bacillus Dn(hroci$ material in their inventory pri or to the attacks, only one Jab was located in Maryland or Virginia, where the relevant fed eral eagle envelopes were distributed and sold by the U.S. Postal Service: the USAMRHn facility at Fort Detrick, MD. Tape, Ink. and Fiber Trace Evidence All four of the envelopes containing these letters were taped along the seams. with transparent tape. Due to striation patterns placed on transparent tape as part of the manufacturi ng process, it is .sometimes possible to match a piece of suspect tape with the tape roll from which it originated, According to FBI Laboratory experts. the envelopes were addressed with a pen which dispenses fluid-l ike ink, n.ther than the ink typically found in a ball point pen. Due to the distinguish:ng characteristics of ink, which vary by manufacturer, it is sometimes possible to match ink writing with the pen or brand ofpen used to apply it. Forcosic analysis oftlle tape attached to the four envelope5 has identified eight different types of fiber attached to the tape: black cotton, black wool, black nylon, brown polyester, blue wool. yellow acrylic, red calion, and red acrylic. Identification of Collection Box at 10 Nassau Street Infonnation gathered to date suggests that aU of the lethal anthrax letters were mailed from a single street collection box located at 10 Nassau Street, in Princeton, New Jersey. The Ictters were postmarked on either September 18, 200 1 or October 9, 200 1 at the same HamiIton o WOIRHEPP B3D15B.pdf  7  009956Anthrax Page 469 of 643 Township Regional Postal Facility in Hamilton, New Jersey. The collection box. on Nassau Street was identified lhrough forensic biological swabbing of every U.S. Postal Service drop box thai collects mail to be processed at tbe Hamilton facility. Funhcr fomISic examination of the contaminaled mailbox recovered a number of Caucasian human hairs from inside the box, which are suitable for comparison. Dr. Bruce Edwards Ivins Dr. Bruce Edwards Ivins is a senior microbiologist who has worked for 27 years in the Bacteriology Division at USAMRJID. The Task Force investigation has revealed that Dr. Ivins is considered an expert in the growth, sporulation, and purification of Baal/WI a1Jlhracis. Dr. Ivins obtained a Bachelor of Science degree in Bacteriology in 1968, a Master ofScicncc degree in Microbiology in 1971, and a Doc,ora,e of Philosophy (ph.D.) degrmails above, that same anthrax vaccine was having problems in the production phase at Bioport. a private company in Michigan responsible for manufacturing the: vaccine. The Food and Drug Administration (FDA) had suspended further production at Bioport, and Ihe U.S. goverrunen~ specifically the Department of Defense, was running out of approved lots of the vaccine. The situation placed pressure on select staff members at USAMRlID, including Dr. Ivins, who were part of the Anthrax Potency Integrated Product Team (IPT). The purpose of the lPT was to assist in the resolution of technical issues that was plaguing Bioport' s production of approved lots of the v&ccinc&. In the weeks immediately prior to the attacks, Dr. Ivins became aware that an investigative journalist who worked for NBC News had submitted a Freedom of InfonnatlOn Act (FOIA) requests on USAMRIID scclting detailed information from Dr. Ivins's laboratory noteboolc:! as they related 10 the AVA vaccine and the use of adjuvants_ On August 28, 2001 , Dr. Ivins appeued angry about the request providing the following response in an e-mail: ''Tell Matsumoto to kiss my ass. We' ve got better things to do than shine his shoes and pee on command. He's gotten everything from me he will gel" In early 2002, shortly after the anthrax letter attacks, the FDA re-approved the AV A vaccine [01 human usc, production at Biopon resumed, and anthrax research at USAMRITO continued without interruplion. As mentioned previously. one of the anthrax letters post marked on September 18, 2001, was addressed to Tom Brokaw, NBC News in New YOlk. Dr. Ivins thereafter received "the: highest honor given to Defense Department civilians at a Pentagon ceremony on March 14, 2003" for his work in "getting the anthrax vaccine back into production." Usc offnlse Dames and fascination with sorority groUP linked to location of anthrax mailings The in vestigation has shown that over the past twenty-four years, Dr. Ivins was known to have utilized at least two Post Office Boxes to conununicate with members of the public, to pursue obsessions, and poSSibly engage in the unauthorized use of another person' s name. The use of the P.O. Boxes is signiftcant because the POSt office where Dr. Ivins freq uented and maintained a P.O. Box, College Estates St.tion, Frederick, Maryland 21702, was servieed by the Frederick Main post office, frederick, Maryland 21701. Analysis of the USPS shipping records indicate the Frederick Main received 1000 6 3/4'" Federal Eagle banded envelopes on the sante day as post offices known to have nx:eived envelopes with identical print defects as those seen on the envelopes used in the mailings. On May 14 2007, _ idenlificd four communications that it had previously received from Dr. Ivins. ~oes not peJSonal~w Dr. Ivins but has repeatedly corresponded with him by mai l]. In Dr. Ivins's first letter to~ dated May 8, 1982, he used the return address ofP .0. Box 1265. Frederick, Maryland 21702. tn the letters, Dr. IVlns expressed his support 10 15 16 WOIRHEPP B3D15B.pdf   009965Anthrax Page 478 of 643 _ work and an interest in sorority hazing. interest was identified as Kappa Kappa Dr. sorority, (KK(;). On July 9. 2007, the Post Office Box application (PS Fonn 1093) at the College Estates Slalien, Frederick, Maryland 21702, for P.O. Box 1103, in the name or Bruce E. Ivins was obtained. A review oflhe application card revealed that Dr. Ivins rented P.O. Box 1103 on November 12, 1993, providing . identification and using his Maryland Motor Vehic le Administration Drivers as proof of identification. Dr. Ivins Frlation of 18 U.S.C., Sections 23323 and 1114. Specifically, there is probable cause to believe that a search and forensic swabbing of the Subject Residence and Subject Vehicles as described in the Attachment to this affidavit, may fC\'ca1labomtory, makeshift, and dual purpose equipment, or materials, supplies, and protocols used in preparation of the deadly anthrax contained in the letters, or may reco\'er trace amounts of the non-anlhracis Bacillus or the powdered anthrax material remaining in the Subject Residences and Vehicles, or may reoover textile fibers that match those recovered from the tape on the envelopes used in the anthrax mailings. or may recover hairs that match the Caucasian hairs recovtred from inside the contaminated mail drop box in Princeton. New Jersey, Or nlay produce papers.. tape, pens. pencils. noles, books, manuals, receipts. financial records. correspondence, address books, maps, handwriting samples, photocopy devices, scanners, printen., toner or printer cartridges. photocopy exemplars, envelope bands. New Jersey addresses, global positioning devices., zip-code infonnation, photographs, computer files, cellular phones, phone bills, electronic pager devices. other digital devices. or other documentary evidence thlt may link Dr. Bruce Edwards Ivins 10 the anthrax mailings and identify conspirators involved in the attacks. Based upon my knowledge, training and experience, my consultations with other criminal investig-cltors, persons engaged in criminal activity frequently store, or inadvertently deposit, these kinds of items in their homes or automobiles and leave them on the premises for many years after a crime, failing to recognize their forensic value and incriminating nature. This conclusion is supported by trash covers that have been found in material recently discarded from the house. On October 15 and October 22, 2007, trash was rocovered from the front of 622 Military R~ Froderick, Maryland, including receipts from a 1995 purchase as well as other documents related to KKG sorority. In my experience, vehicles are often used to store documents, maps, receipts, and other paper! related to travel that may be relevant to this in\'estigalion. Because this affidavit is part of an ongoing investigation that would be jeopardized by premalure disclosure ofinfonnation. 1 further request that this Affidavit, the accompanying Order, and other related documents be filed under seal until further order of the Coun. :;;. I.) t:,....- ~-( ~'- 1<I lrace quantities o(Baclllu$ anlhr.d~ p-0pC01y) or simuhimlslhert'ur, h)lirs. teJ:lile fibers, lab equipment or OJaterials used in p"paration of select agen":, papers, tape, pens, nOIfS, books. manuals, receipts. flnandaJ r<l wood siding. with a whiLe front door that is located on the right side of the front a fme home. The front door has an exterior stonn door. Left of the front door are two windows cased with red shuttm. A black nuling leads up sever,,) stairs to the front door where the nwnbcrs~c indicated in black above the door. The search will include a large white shedi with peeling paint., that is located on the rear of the property. Bruce Edwards Ivins purchased the Investigation has disclosed the property has been his principle residence September. 2001. (2) Automobile #1 : a .,'chicle with Maryland registr.tic,"' 0211112009. and Vehicle Identification The vehicle is described as a blue, 4 door Edwards Ivins. Frederick, MD. Bruce Edwards Ivins purcbased the vehicle the sole owner, and i~ the primary user. (3) Automobile #2: searcb a vehicle with Maryland 0612512008, and Vehicle Identification veh icle is described as a Honda blue, 4 door sedan. to Bruce Edwards MD. Bruce Edwards Ivins purchased the sole owner. The vehicle is primarily used. by Ivins's and is driven on occasion by Bruce Edwards Ivins. (4) Automobile 113: A warrant to search a velUcle with Maryland registration 88S437M. expiration 0710112009. and Vehicle Identification Number 2B4HBI 13. a 1996 Dodge and is MD. is driven onoccasi,m (5) Safe Deposit Box #48, located at Farmers &. Mechanics 7~ S'reet. Frederick, Maryland 21702. and is subscribed to Bruce E. Ivins Maryland 21702. Avail.ble records indie.'. Branch #1. 1305 West 0611211990, WOIRHEPP B3D15C.pdf  2  009978Anthrax Page 491 of 643 the box during the period 1210712004 - 07n OnOO7. Access n.:conls previolls (0 12107/2004 no longer exist As discussed below, there is probable cause to believe that a search of the aforementioned premises may result in the collection of evidence relevant to an ongoing criminal investigation into the dissemination oCa Weapon of Mass Destruction (anthrax) through the U.s . mail system in September 2001 and October 2001 in violation of Title 18, United States Code, Sections 2332(a) and I I 14, which killed five people and infected at least 17 oth e~. Overview The Federal Bureau of Investigation and the U.S. Postal Inspection Service (hereinafter "Task Force'') investigation of the anthrax attacks has ted to the identification of Dr. Bruce Edward Ivins, an anthrax resean:her at the U.S. Anny Medical Research Institute for Infectious Diseases. Fort Detrick. MD, as a person necessitating further investigation for several reasons: (1) At the lime of the attacks, he was the custodian ofa large flask oChighly purified anthrax spores that possess cenain genetic mutations identical 10 the: anthrax used in the auacks; (2) Ivins has been unable to give investigators an adequate explanation for his lale night laboratory work hours around the time of both anthrax mailings; (3) Ivins has claimed that he was sutTering serious mental health issues in the months preceding the attacks. and told a coworker that he had "i ncredible paranoid, delusional thoughts at times" and feared thal he might not be able to control his behavior; (4) Ivins is believed to have submitted false samples of anthrax from his lab to the FBI for forensic analysis in order to mislead investigators; (5) at tbe time of the attacks. Ivins was under pressure at work to assist a private company that had lost its FDA approval to produce an anthrax vaccine the Army needed for U.S. troops, and which Ivins believed was essential for the anthrax program at USAMRllD; and (6) Ivins sent an email to o o o o o o o o o a few days before the anthrax attacks warning her that "Bin Laden terrorists for sure have anthrax and sarin gas" and have "'just decreed death to all Jews and all Americans," language similar to the anthrax letters warning "WE HAVE THIS ANTHRAX ... DEATH TO AMERICA ... DEATIl TO ISRAEL." The Task Force submits that a search and forensic swabbing of the Subject Residence and Subject Vehicles may reveal forensic or documentary evidence that will assist the investigation by linking Dr. Bruce Edwards Ivins to the anthrax mailings and identify conspirators involved in the anacks. The search is for laboratory equipment, tape, ink, paper, textile fibers, Caucasian hair, trace bacillus spores, handwriting samples, photocopy samples, and all relevant documents, notes and records in various formats, as more fully described in the Attachment to this affidavit. Factual Background In September and October, 2001, at least five envelopes containing significant quantities of Bacillus an/hracis, were mailed to persons in thc District of Columbia, New York CitY, :md Boca Raton, Florida, in violation of Title 18, United States Code, Section 2332(a), which prohibits the use ofa Weapon of Mass Destruction. Bacillus anthracis is a Weapon of Mass destruction because it is a bacteria and "biological agent" capable of causing death, disease, and other biological malfunction in humans and certain other animals as defined in Tille 18, United 2 3 WOIRHEPP B3D15C.pdf   009979Anthrax Page 492 of 643 States Code, Section 178. Bacillllj QlJlhra(is is classified as a Seleci Agent, which requ ires special handling and conlainment protocols as ~overned by the Centers for Disease Control and Prevention (CDC) in Atlanta. The disease caused by Bacillus anlhrads is known as anthrax, subseqoently these terms will be used interchangeably. The letters were addressed to members of the national media and to the Capilol Hill omces oflwo United Slates Senators located in lhe District of Columbia. Each of the envelopes contained a handwritten note referencing "9-1 1-0 ' " and warning "WE HAVE THIS AA'THRAX. YOU DrE NOW. ARE YOU AFRAID? DEATil TO AMERICA. DEATH TO ISRAEL. ALLAH IS GREAT." The two lelten addr> . Dr. Bruce Ivins has unrestrict~ access to the suite and has been the sole custodian of RMR-IO:l9 since it was fi rst grown in 1997. Bac;tcrial contaminant found in attack !tUrn Bolh of the anthrax spore powders recovered from the Post and Brolcaw letters contain low levels of a bacterial contaminant identified as a strain of Bacillus 3ubtilis. The BacilhLf sublilis contaminant has not been detected in the anthrax spore powders recovered from the envelopes mailed to either Senator Leahy or Senator Daschle, Baci/L,a subti/iJ is 3 nonpathogenic bacterium found ubiquitously in the environment. However. genomic DNA sequencing of the specific i~lalc of Bacillus sublilis discovered within the Pmt and Brokaw powders reveals that it is genetically distinct from other known isolates of Bacillus subtillS. Analysis o-r the &u::illus subtiJu from the POSI and Brokaw envelopes revea1ed that these two oolates UC' identical. Phenotypic and genotypic analyses demonstrate that the RMR? I029 does not have the Bacil/us sublilis contaminant found in the evidentiary spore powderS, which suggests that the anthrax used m the letter attacks was grown from the materia1 contained in RMR-I029 and nOl taken directly from the flask and placed in the envelopes, Since RMR? I029 is the genetic parent to the evidentiuy spore powders, a.nd it is not known how the BacUlu3 3ubillis contammant came to be in Ule Pust and BJOkaw spore powdru. lhe contaminant must have been inuoouccd duri ng the production of the Post and Brokaw spores. Taken together, the pOstmark dates. the Silicon signature, the Bacillus Jublills contaminant, the phenotypic. and the genotypic comparisons, it can be concluded that. on at least two separate occasions, a sample ofRMR-I029 was used to grow spores, dried to a powder, packaged in an envelope with a threat letter, 8l1d mailed to the victims. I envelopes used in the anthrax attacks In the 200 I anthrax attacks, four envelopes were recovem::l. The four envelopes were . all 6 V inch fadera!. eagle envelopes. The -redel'lll cagJc" dcsisnation i& derived from thc postage franlc in the upper right?hand corner on the envelope which consists of the image of an eagJe perched on a bar bearing the initials "USA.- Underneath thc lettering is the number "34," which denotes the postage value of34?. The eagle. lettering, and denomination are referred to as the time to time oblllined search ""arrants, supported by .ffidavlts ~~~~'::':;~~:~~':~.~ .. t:llicla. uf uillel ilklivkJutd~ I As the Court may recall from hs review, during the COW-Ie ofthiJ .ix ..... ho ha\'c t..'OIlJ':: 1I1.:k, illlo"cstig.livc investig.tlOn, the Task For<< bas from residences and All prior searches in this o Task F~ ihCiC,ocUc UIoIy, tmt to lbc. iIkoIiflC.l.tioo. o(R.~ 1029 11 USAM1UlD as the ~nctic ' palenco(l:helnthcaxused 1be2001 tenerlnacb. irlvesti&ation ~.,. =~. ~:::~~ l WOIRHEPP B3D15C.pdf  6  009982Anthrax Page 495 of 643 indicia. The eagle and the bar are stamped in blue ink. whi le the denomination is stamped in grey ink. Approximately 45 million Federal eagle 6 y..oo envelopes were manufactured by Westvaco Corporation (now known as MeadWest\'3CO Corporation) of Williamsburg, Pennsylvania. between December 6. 2000 and March 2002. These Federal eagle 6 W' envelopes were manufactured exclusively for and sold solely by the U_S. Postal Service between January 8, 2001 and June 2002. Subsequent to the attacks, an efTort was made to collect all such envelopes for possible forensic examination., including the identification of defects that occur during the envelope manufacturing process. As a result of this collection, envelopes with printing defects identical to printing defects identified on the envclopes utilized in [he anthrax attacks during the fall of 2001 were collected from the Fairfax Main post office in Fairfax. Virginia and the Cumberland and Elkton post offices in Maryland. The Fairfax Main, Cumberland, Maryland, and Elkton, Maryland post offices are supplied by the Dulles Stamp Distribution Office (500), locatoo in Dulles, Virginia. The Dulles SOO distributed "feder1ll cagle" envelopes to post offices throughout Maryland and Virginia. Given that the printing defects identified on the envelopes used in the attatks are transj~ thereby being present on only a small population ofille federal eagle envelopes produced, and that envelopes with identical printing defects to those identified on the envelopes used in the attacks were recovered from post offices serviced by the Dulles SOO, it is reasonable to concl"ude that the federal cagle envelopes utilized in the attacks were purchased from a post office in Maryland or Virginia. Of the sixteen domestic government, commercial, and university laboratories that had virulent RMR-I 029 Ames strain Bacillus anrllrac(s materiaJ in their inventory prior to the attacks, only one lab was located in Maryland or Virginia, where the relevant federal eagle envelopes were distributed and sold by the U.S. Postal Service: the USAMRIID facility at Fort Detrick, MD. Tape. Ink. and Fiber Trace Evidence All four oCthe envelopes containing these letters were taped along the seams with transparen: tape. Due to striation panems placed on transparent tape as part ofthc manufacturing process, it is sometimes possible to match a piece of suspect tape with the tape roll from which it originated. According to FBI Laboratory experts, the envelopes were addressed with a pen which dispenses fluid-like ink. rather than the ink typically found in a ball point pen. Due to the distinguishing characteristics of ink, which vary by manufacturer, it is sometimes possible to match ink writing with the pen or brand of pen used to apply it Forensic analysis ofthc tape attached to the four envelopes has identified eight difTerent types of fiber attached to the tape: black cotton, black wool, black nylon, brown polyester, blue wool, yellow acrylic, red cotton, and red acrylic. Ident' fical,on of Collection Box at 10 Nassay Street Infonnation gathered to date suggests that all of the lethal antJuax leiters were mailed from a single street collection box located at 10 Nassau Street, in Princeton, New Jersey. The letters were postmarked on either September 18, 2001 or October 9, 2001 at the same Hamilton 6 WOIRHEPP B3D15C.pdf  7  009983Anthrax Page 496 of 643 Township Regional Postal Facility in Hamilton. New Jersey. The collection box on Nassau Street was identified through forensic biological swabbing of every U.S. Postal Service drop box that collects mail 10 be processed at the Hamilton facility. Further forensic examination of the cont.amjnatcd mailbox recovered a number of Callcasian human hairs from inside the bo'(, whi ch are suitable for comparison. Dr. Bruce Edwards Ivins Dr. Bruce Edwards jvins is a senior microbiologist who has worked for 27 years in the Bacteriology Division at USAMRlID. The Task Force investigation has revealed that Dr. Ivins is considered an ex~ in the growth, sporulation, and purification of BacillU.J anthrari$. Dr. Ivins obtamed a Bachelor of Science degree in Bacteriology in 1968, a Master of Science degree in Microb:ology in 1971, and a Doctorate of Philosophy (phD.) degree in Microbiology in 1976, al1 from tte University of Cincinnati. Dr. Ivins then completed a two year Post-doctonll Fellowship at the University of North Carolina (UNC), Chapel Hill. A review of USAMRllD records. laboratory notebooks, written protocols, arxl professional publications has shown that Dr. Ivins has worked with Bacillus anthraci.t at USAMRIID since 1980. He has pcrsonaUy conducted and supervised Ames anthrax spore productions for over two decades. At the lime oflhc anthrax mailings, Dr. Ivins possessed extensive knowledge of various anthrax production protocols. Dr. Ivins was adept at manipulating anthrax production and purification variables to maximize sporulation and Improve the quality of anthrax spore preparations. He also understood anthrax aerosolization dosage rates and the importance of purity, consistency. and spore particle size due to his responsibility for providing liquid anthrax SpOre preparations for animal aerosol challenges. Dr. Ivins produced large batches of Bacil/w: anthracis which were required for such challenges - tests in which v8e<:inatcd animals inhale pre-defined doses of anthrax spores to assess the efficacy orthe anthrax vaccine. Dr. Ivins's 20 years ofwodcing in the laboratories ofUSAMRIID provided him personal, hands-on laboratory experience in the production of liquid spore preparations of Badllu.s anthracis. He has used lyophilizers, biologicaJ safety cabinets, incUbators, and centrifuges in vaccine research. Such devices are considered essential for the production of the highly purifi ed, powdered anthrax used in the Fall 200] mailings. Dr. Ivins was also veT)' experienced in conducting laboratory work in a containment area, and well versed in decontamination procedures specifically for Bacillw antirrocis. Dr. Ivinsts employment at USAMRJID also provided protection against anthrax infection at the time of the mailings due to his extensive and current anthrax vaccination history. Unexplained late night laboratory access USAMRIlD containment Suite B3 is a Biological Safety Level-3 (BSL-3) suite of laboratories used by USAMRlID Bacteriology personnel for research on dangerous animal and human pa:hogens. The flask. identified as RMR-1029 was scored in Suite BJ at the time of the letter attacks. Suite access is obtained via a Security Access Control (SAC) badge reader at the 7 8 WOIRHEPP B3D15C.pdf   009984Anthrax Page 497 of 643 door to the cold-side change room. which is secured by a magnetic-lock. A cenlral security system monitorS and records a time stamp for each SAC badge and keypad request. A review of Dr. Ivins's laboratory access records for Building 1425 was assessed to dctcnninc: trends in 'WOrking hours and evening times (after 6 p.m.) spent in Suite B3 . Hi s regular work:ng hours on average consisted of a 7:30 a.m. to 4:45 pm. shift. but he would periodically return in the evenings, presumably to check on the status of various experiments. Beginning in mid-August 2001; however, there was a noticeable spike in Dr. Ivins's evening access to De BJ hot Suite. The following depicts the comparison of Dr. Ivins's total hours spent at night, after 6 p.m., in Suite BJ during the yean 2000 and 2001 : ight Hours in Suite B3 [112000. 2001 1 )S )0 2S = ~ IS ~ 20 10 5 o Jan Feb. Mar. Apr. May Jooe July Aug. Sept Oct Nov. Dec. Mouths The investigation examined Dr. Ivins's laboratory activity immediately before and after the window ofoppornmity for the mailing of the Post and Brokaw letters to New York which began at 5:00 p.m. Monday. September 17, 2001 and ended at noon on Tuesday. September 18, 2001 . Beginning on Friday. September 14. Dr. Ivins 'WOrked the following three consecutive evening shifts prior to the mailings with time spent in Suite B3: ooy Fri day Saturday Sunday o. 1e September 14 September 15 September 16 T?lDlelD B ?Id? 1425 . m2 ., T 0 lal T uno . B3 III 8:54 p.m. to 12:22 a.m. 8:05 p.m. 10 II :59 p.m. 6:38 p.m. to 9:52 p.m. 2 hoW'S 15 minutes 2 hours IS minutes 2 hours 15 minutes After September 16, Dr. Ivins did not enter Suite 83 in the evening again until September 25. 8 WOIRHEPP B3D15C.pdf  9  009985Anthrax Page 498 of 643 The investigation further examined Dr. Ivins's laboratory activity before and after the window of opponunity for the mailing of the Daschle and Leahy letters to Washington, D.C. which began 3:00 p.m. Saturday, October 6,2001, and ended at noon on Tuesday, October 9, 2001. Beginning on September 28, Dr. Ivins worked eight consecutive nights which consisted of the following times in buikling 1425 with time spent in Suite B3: D.y Friday Saluro.y Sunday Mond.y Tuesday Wednesday Thwsday Friday D t Seplember 28 Seplemb..- 29 September 30 October 1 Oclober 2 October 3 Oclober4 October 5 oo TI m... B 'Id' o 1425 o lB 7:16 p.m.1o 10:59 p.m. 8:02 p.m. 10 II : 18 p.m. 9:53 p.m. 10 12:04 a.m. 9:14 p.m. 10 10:43 p.m. 7:24 p.m. 109:39 o .m. 7:25 p.m. 10 10:55 p.m. 6:10 p.m. 10 10:1 2 p.m. 7:40 p.m. 10 12:43 o.m. ., Tota! T' lme 1ft B3 1 bour 42 minutes ' t hour 20 minutes 1 hour 18 minutes 20 minutcs 23 minutes 2 hours 59 minutes 3 bours 33 minutes 3 hours 42 minutes After October 5, Dr. Ivins did oot enter Suite B3 in the evening again until October 9. for 15 minutes, and then October 14. for 1 hour and 26 minutes. The investigation has also analyzed the daily schedules, work areas accessed, and the number or hours worked per week of all other researchers assigned to the Bacteriology Division who had access to Suite B3 during the months ofScptembcr and October 2001. When these records are compared to Dr. Ivins (or the same period, Dr. Ivins's habits are significantl y different 6an those of the other researchers, in that he was frequently in Suile 83, where RMR1029 was kept, late at night and on weekends when no other researchers were present in Suite B3. Additionally, while Dr. Ivins was in Suite 83 at night, no other USAMRJID employee was present On March 31, 2005, Dr. Ivins was asked by Task Force investigators about his ac:ces.o; to B3 and could provide no legitimate reason for the cxtended hours, other than "home was not good" and be went there ""to escape" from his life at home. A rt:view of Or. Ivins' s and c0workers's laboratory notebooks and projects at the times in question was conducted. The investigation determined that Dr. Ivins's role in the experiments were minimal, and did not justify the time he spent in B3. Dr. Ivins bas admined to investigaton: that the rt:Se&rCh he was conductim, in 2001 did not require, and does not explain, his late night houn in the B-3 labontory around the time period of the anthrax maitin~ . Since producing anthrax spore preparations was one of Dr. Ivins's principal responsibilities at USAMRllD, he had multiple and unfettered opportunities to produce or divert Ames strain spores fo r illegitimate purposes. His access to Suite B3 and USAMRllD afforded all of the equipment and containment facilities which would have been needed 10 prepare the anthrax and letters used in the Fall 200 I attacks. 9 WOIRHEPP B3D15C.pdf  10  009986Anthrax Page 499 of 643 Failure to cooperate in providing laboratory samples Dr. Jvins provided two sets of samples of Ames anthrax 10 the he represented to the FBr were drawn In the first submission in February 2002, Dr. Ivins failed to follow the protocol subsequently notified that the February 2002 samples were to to follow the protocol. A second set of samples, labeled "Dugway Ames spores - 1997" was provided to the FBIR by Dr. Ivins in April of 2002. Dr. Ivins dcclares that he uses the tenns "Dugway Ames spores - 1997" and RMR?1029 interchangeably, as theyaze the same flask of material. Both of the April RMR-I029 samples were submitted for genetic testing and found to be negative for the presence of tile four mutations found in the anthrax used in the attacks. On December 12. 2003, an FBI Special Agent accompanied Dr. Ivins inlo Suite B3 at USAMRIID and identified additional Ames samples ofOr-. Ivins's and others that had not been submined as part orthe above mcntioned ~se. Dr. Ivins submitted slants prepared fIom the newly identified Saloples to the FBlR OD April 7, 2004. On the afternoon of April 7, 2004, an FBI Special Agent accompanied Dr. Ivins into Suite 83, and seized the original samples Or. Ivins had. used to prepare the slants submitted to the FBlR earlier that day. Additionally, the Agent seized the RMR?I029 flask itself. All of the samples were secured in the B3 walk-in cold room within a double-lockcd safe, and sealed with evidence tape until such time that they could be transported to the avy Medical Research Center (NMRC), which was under conlrdCt by the FBl. On June) 7. 2004, RMR.-I 029 and the addi tional Ames samples were submitted to the FBlR, by the NMRC, under the direction oflhe FBI. Samples of these FBlR submission were sent to the same contracting laboratories for genetic testing. The sample labeled RMR.-1029 tested positive for aU four genetic marlcers, therefore, manifesting the genetic characteristics common to the evidence. On March 31. 2005, Dr. Ivins was ? be provided to the FBlR on April 10, found to be genetically distinct from the anthrax and anlhra"( material recovered by the FBI from the RMR? I029 flask seized from Dr. Ivins' lab on April 7, 2004. Dr. Ivins was confronted with this and was asked did not submit the genetically positive sample which was clearly Dr. Ivins was adamant in his response that there had been no omission from and he ofsamplcs in April insisted thut he had provided RMR?I029 to the FBI in his second 2002. 10 WOIRHEPP B3D15C.pdf  11  009987Anthrax Page 500 of 643 Knowledge that his stock ofanthrax matched the anthrax used in theatlack On March 31 , 2005, when Dr. Ivins was informed by FBI agents that RMR-I 029 shared genetic similarities with the Ames strain used in the mailings. he indicated that he was already aware oflhis infonnation. Dr. Ivins explained that many months to a year ago, FBI Special Agent Darin Steele told him that RMR-1029 matched the evidence. SA Steele denied having the conversation described by Dr. h'ins above. As lOdicated In previous paragraphs, tho RMR- I029 submission provided by Dr. Ivins in April 2002 did not match geneticall y or phenotypically. Therefore, neither SA Steele nor any other member of the Task Force could make iii. comparison between the Ames strain used in the mailing and RM:R 1029, until after the June 17, 2004 submission and subsequent laboratory analysis. When interviewed again on May 7, 2007, Dr. Ivins told investigators that, within three months after the leiter attacks, he was aware that his stock of anthrax, RMR?I029, exhibIted unique morphological similarities to the anthrax used in the attacks, and that be allegedly learned this infonnation from three cowork.ers at USAMRJID who participated in the forensic analysis of the anthrax in the letters. Each oftbo.se three coworkers was interviewed by the Task Force, and deny disclosing such infol111ation to Or. Jvins. Over the coune ofthis investigation, Dr. Ivins has been repeatedly interviewed and had open access to the law enforcement personnel responsible for investigating the anthrax attacks. During nOlle ofthcsc interactions, did Dr. Ivins ever indicate he had knowledge that RMR- I029 had phenotypic similarities to the material usct.l in the anthrax attacks, nor did he suggest that investigators analyze R..M:R-I029. To the contrary, Dr. Ivins repeated ly claimed that the anthrax used in the attacks resembled that of another researcher at USAMRIID and were dissimi lar to the BacilllLJ amhracis Ames organisms maintained in his laboratory, which included RMR?I029. Menta! health issues and possible motive The investigation has shown that in 2000 and through the mailings in 200 1, Dr. Ivins had mental health issues. Dr. Ivins's mental health issues came to the attention of investigators while reviewing c-mails ofUSAMRlID researchers. Through the e-mails 11 was detennined that Dr. Ivins was undergoing significant stress in both his home and work life. The mental health issues and stress were significant to the extent that Dr. Ivins sought professional help from a psychiatrist and was immediately prescribed medication that started in February 2000. The following arc excerpts from e-mails dated April 2000 through December 2001, from Dr. Ivins to a friend regarding work, home, state of mental health. and use ofmedic:ation (emphasis are as they appeared in the e~mails): April 3, 2000, "Occasionally I get this tingling that goes down both anns. At the same time 1 get a bit dizzy and get tltis unidentifiable -metallic" taste in my It actually scares me a bit.) Other mouth. (I 'm not trying to be funny, times it's like I'm not only sitting at my desk doing work, I'm also a few feet away tI 12 WOIRHEPP B3D15C.pdf   009988Anthrax Page 501 of 643 wa1ching me do it. There's nothing like living in both the first peROn singular AND Ihe third person singular'June 27.2000, "Even with the telexa and the counseling. the depression episodes still Come and go. That's unpleasant enough. What is REALLY scary is the paranoia .. ..Remember when I told you aboullhe -metallic? tasle In my mouth that I gOI periodically? It's when I gel these "paranoid- episodes. Of cot.rs~ I regret them thoroughly when they are over, but when I'm going through the.)'}. it's as ifJ'm on a passcngeron a ride .... Ominously, a lot of the feelings of isolal ion - and desolation -that 1 wenl through before coUege are returning. I don't wanl to relive those years again. ... I've been seeing the counselor once a week. " June 28, 2000, "Apparently Gore (and maybe even Bush) is considering making the anthrax vaccine for the military voluntary, or even stopping the program. Unfonunately, since the BioPon people aren't scientists. the task of solving their problem has fallen 00 us..... Believe me, with all the stress of home and wone. your email letters to me arc valuable beyond what you would evtr imagine - and they help me keep my sanity ...... June 29, 2000, MBioPort just tested its final lot of AVA [anthrax vaccine] in a potency test If it doesn't ~s., then there are no more lots to tC$t,. and the program will come Co a halt. That's bad for everyone concerned, including us . I'm sure that blame will be spread around." July 4, 2000, -The thiziling now by the psychiatrist and counsetor is that my symptoms may not be those of a depression or bipolar disorder, they may be that of a ? Paranoid Personality Disorder: July 6, think the oooo is about to hit the fan ... bigtime. The final lot of AVA, lot 22, isn't passing the potency test, and now there's nothing to back it up. Plus, the control vaccine isn't working. It's j ust a fmc mess. ~ an. spending probably 9S% of our time on this." July 7. 2000, in an e-mail. Dr. Ivins offered to be interviewed as a case study. as long as it remained anoDymous. Dr. Ivins indicated that be did not want to see a headline in the National Enqu;~r that read, "PARANOID MAN WORKS WITH DEADLY ANTHRAX!!!" July 23,2000, -It'$ been a really stressful week, from a ll stand points. and it's not well with the counselor I'm going to. (She said she going to have to ask to get put sessTOiii.... Sometimes I think thal it's all just too much.Home, 12 WOIRHEPP B3D15C.pdf  13  009989Anthrax Page 502 of 643 August 12, 2000, -Last Saturday, as you probably guessed from my ematJ , was one of my worst days in months. I wish I could control the thoughts in m y mind. It's hard enough sometimes controlling my behavior. When I'm being ealen alive inside, I always try to put on a good front here at work and al home, so I don't spread the pestilence... .J get incredible paranoid, delusional thoughts at times, and there's nothing I can do until they go away, either by themselves or with drugs.August 29, 2000, ~ 10% of the Bacteriology Division . If we quit, the anthrax pro~ would go down the drain. I' m not boasting. ? b but the three of us have a combined total of 52 years of research experience with anthrax. You just can 't go out and find someone Iike_ with their knowledgc, skill and abilities, Ain't gonna happen." Mareh 4, 2001 , -rhc people in my group just don't pick up on what I try to say. They are nOl into the kinds of problems 1 bring up, so it's hard for them to deal with tbem. The psychiatrist is helpful only because he prescribes the Celexa He's not that easy to talk to, and he doesn't really piclc up on my problems. The woman I saw before I went into group wanted to get me put in jail. lbat wasn't very helpful either. J'm down 10 a point where there are some things that an: ealing away that I fcel l can't tell ANYONE .... " September 7,2001 , "I was taken o!ftbe Speciallmmuniution Program because of what happened last spring, and I've just gotten back on it, getting my anthrax and Yellow fever shots. We are currently finishing up the last oCthe AVA>and when that is gone, there' s nothing to replace it with. I don> know , what will happen to the research programs and hot suite work until we get a new lot. There are no approved lots currently available at Bio,P ort., .._ has been having us have biweekly meetings on the rPA vaccine progress, and on August 29 I went to the Pentagon - first time there - to go to a meeting in his place on the vaccine. There is a real bag of worms with a new 101 ofrPA produced by the BDP (a private company) for NCI. who i5 under contract to USAMRIID. BDP signed a sub-contract with to produce the rPA for a hwnan use vaccine Phase I trial. They were paid and they produced il Now they are refusing to release it unless the Anny pays some incredible sum of money for lawsuit mdemnification (about $200,000 per year for the next SO years). The Army refuses to do that of course, and everything is in Limbo." September 15, 2001 , "I am incredibly sad and angry III what happened, now that it has sunk in. Sad for all of the victims, their families, their friends . And angry, Very angry. Angry at those who did this> who support thcm, who codd le them, and who excuse thcm. September 26, 2001, -Of the people in my -group,? everyone but me is In the depression/sadness/flight mode for stress. I'm really the only scary one in the group. Othcn are talking about how sad the)' are or scared they are., but my 13 14 WOIRHEPP B3D15C.pdf   009990Anthrax Page 503 of 643 reaction to the WTClPentagon events is far different. or course, I don', talk about how I really feel with them o it would just make them worse. Seeing how differently Treacted than did to the rocent events makes me really think about myselr a lot. beard that Bin Laden terrorists for sure have anthrax and sarin gas. In that same September 26. 2001 email. Dr. Ivins states ''Osama Bin Laden has just decreed death to all Jews and all Americans" - language similar to the lexi of the anlhr.!x lett.,. posunarlced Iwo weeks later warning "DEATH TO AMERlCA," " DEATH TO ISRAEL." On October 16,2001, in an e~mail. Dr. Ivins's coworker communicated the foHowing to a former coworker, "Bruce has been an absolute manic basket case the last few days." December IS, 2001, ;II made up some poems about having two people m one (me + the person in my dreams): ... rm a little ~am-5Clf, short and stout. rmlhe other halr of Bruce - when be leIS me out. When r get all steamed up, I don't POUI. I push Bruce aside, them I'm Free to run about! Hickory dickory Doc - Doc Bruce ran up the clock. But something then happened in very strange rhythm . His other self went and exchanged places with him. So now, please guess who Is conversing with you. Hickory dickory Doc! Bruce and this other guy, sitting by some trees, Exchanging personalities. It's like having two in onc. Actually it's rather fun!- The investigation has revealed that Dr. Ivins was prescribed various psychotropic medications including antidepressants, antipsycbotics, and anti-anxiety, for his mental health issues from 2(0) through 2006. On July 18. 2007, a forensic psychiatrist completed a detailed review of Dr. Ivins insurance billing records for medical appointments and prescriptions. Addi tionally, this psychiatrist was provided with an ovel'\'iew of Dr. Ivins'S social habil.;;, interests, and obsessions. The forensjc psychiatrist stated thai based on hi.. experience, if Dr. Ivins was the mail er, it is quite possible that Dr. Iv1ns retained some kind of souvenir or references to the mailing events. 14 15 WOIRHEPP B3D15C.pdf   009991Anthrax Page 504 of 643 Controversy concerning the anthrax vaccine Beginning shortly after the fint Gulf War and through 2001. USAMRnO and Dr. Ivi ns was the focus of public criticism concerning their introduction of a squalene adjuvant (or additive) to the AVA anthrax vaccine, whieh was blamed for the Gulf War Syndrome. In 2000 and 2001, as evident by the e-mails above, that same anthrax vaccine was having problems in the production phase at Bioper!. a private company in Michigan responsible for manufacruring the vaccine. The Food and Drug Administration (FDA) had suspended further produetion at BioPOlt, aJw tJle U.S. Kuvt..'IllJleul. ~~ i(it.:Kt ly tilt: Dt:partJllt:ftl ufDt::rt:llS4::, WiI.S JUIUliu!: uut uf approved lots or the vaccine. The situation placed pressure on select staff members at USAMR.lID, including Dr. ivins, who were part of the Anthrax Potency Integrated Product Team (.IPT). The purpose of the lPT was to /Wist in the resolution oftcchnical issues th:lt was plaguing Bioport's production of approved lots of the vaccines. In the weeks immedialely prior to the attacks, Dr. Iviru became aware that an investigative journalist who worked for NBC News had submiued a Freedom of informatIOn Act (FOlA) requests on USAMRIlD seelcing detailed information from Dr. Ivins ', laboralory notebooks as they related to the AVA vaccine and the use of adjuvant.. On Auguat 28. 200 I. Dr. Ivins appcart:d angry about the rcquC$t providing thc following raponsc in an e-mail: "Tell Matsumoto to kiss my ass. We've got benet things to do than shine his shoes and pee on command. He's gotten everything from me be will get.... In early 2002, shortly after the anthrax letter attacks. the FDA re-approved the AVA vaccine for human use, production at Bioport resumed, and anthrax research at USAMRJ ID continued without intClTUption. As mentioned previously, one oflhc anthmx letters post marked on September 18,200 1, was addressed to Tom Brokaw, ~rac News in New York:. Dr. l~n ~ thcreafter received "the highest honor gjven 10 Defense Department civilians at a Pentagon ceremony on March 14,2003" for h.is work in "getting the anthrax vaccine back into produ<:tion," Use offalse names and fass:inationytith sorority grpupJink;cd to location QCpnlhrp mai lings The investigation has shown that over the past lwenty.four years, Dr. Ivins was known to have utilized at least two Post Office Boxes to communicate with members of the public, to pursue obJCSSions, and possibly engagc in the unauthorized usc of another person ' s name. The use ufthe P.O. Boxes is siguifiQUu bt:Caus~ th~ post uffiw wheu; 01 . Ivills fra:t UCliled and mainlained a P.O. Box. College Estates Station, Frederick, Maryland 2 I 702, was serviced by the Frederiek Main post office. Fnaderiek, Maryland 21701. AnaI)'Ol1,. . . . . . . . was obtained from the Records Division and provided the following information: ~ 111 ~=:::: filed a report of vandalism with 1 2 advised that between 2330 and 0830 bours, unknown 0 cmthemOmingO~:=~~~~~~~~==:::::: sprayed the Greek: symbols for KKG on its front fence. three (3) areas of sidewalk adjacent to its residence. and on , was a the rear wmdow of its 1981 HondaAccord. ~dviscd that its roonunale, member of the sorority KKG in North Carolina four (4) years ago. 16 B3D15C.pdf WOIRHEPP  17 o  009993Anthrax Page 506 of 643 his Post DoctOOli F This incideeln~tl;i~S,!~~~~ta~ in Ule 19805. community the same town home o resided and experienced the case ofKKG related vandalism == .9!;!?!:! ~ldd.ress, after completing to moving to Frederick before moving out of the state in 1983. . . . . . . . . . . . . . _ recalled that Dr. Ivins had told it that he bad mainlained a P.O. Box for the purpose of corresponding with individuals who bad responded to advertisements Dr. Ivins had placed in possibly a newspaper or magazine. Dr. he advenlsed that he possessed a K..KG sorority handbook which contained cherished infonnation solely reserved for KKG members. CW-4 believed that Dr. Ivins was advertising thaI he had access to privileged infonnation and would be willing to share the secrets with those responding to the advertisements. Dr. Ivins admiued to CW-4 that he had broken into aKKG sorority house to steal a secret KKG handbook. CW-4 belic't'ed that Dr. Ivins had committed this alleged crime On AprilS, 2001, 7 2 , during his Post Doctorate Fellowship at the UNC. Chapel Hill. Dr. Ivins provided CW-4 one of his alternate e?mail addresses as goJdenphoenixlll@hotmail ,wm. A search of the internet for postings under goldenphoenix 111 identified the following posting dated February 20,2007, on a website at YIWW.abovetopesecret.com: "Wildswan. you are quite right about what you said about KKG. lfpeople look hard enough and dig hard enough, have mends, relatives, perhaps financ ial resources, etc., then they can pretty much find out about whatever GLO they want. Kappas are noted for being lovely, highly intelligent campus leaders. Unfortunately, they labeled me as an enemy decades ago, and I can only abide by thejr tlFatwah" on me. I like individual Kappas enonnously, and love being around them. I never choose an enemy. but they've been after me since the 19605, and REALLY after me since the late 1970s. At one time in my life, I more about KKG than any non-Kappa that had ever lived. Unfortunately I've forgotten a lot rYe read the history ofnG that was writtcm several decades ago about its founding. Question for you: Did your chapter use the combined service, or did yoll separate your services into the "RedRoom and WhiteRoom"? did you use special blue or white blindfolds? You can reach me at goldenphoenix 111@hotmail.com ... as a phoenix rises from its ashes...tt mev.' This posting is significant in that in his own words, Dr. Ivins defines the depths of his obsession and knowledge in the sorority KKG. Additionally, as previously described above, the letters used in the 2001 anthrax mailings were mailed from a blue collection box located at 10 Nassau Streett Princeton, New Jersey. The sorority, KKG t has an office at 20 Nassau Street, Princeton, New Jersey, located on the same side of the street and 60 feet to the right from the blue collection bo't. 17 WOIRHEPP B3D15C.pdf  18  009994Anthrax Page 507 of 643 Connection to "4;11 Grade. Greendale School" The investigation into the fictitious return address on envelopes used for the second round of anthrax mailings, "4~ GRADE," "GREENDALE SCHOOL," has established a possible link to the American Family Association (AfA) headquartered in Tupelo, Mississippi. In October 1999. AFAo a Christian organization. published an article entitled "AFA /akeJ Wisconsin to court. .. The article describes a lawsuit filed in federal court, by the AFA Center for Law and Policy (eLF). on behalf of the parents of students at Greendale Baptist Academy. The article focuses on an incident that occurred on December 16, 1998, in which case workers of the Wisconsin Department of Human Services went to the Greendale Baptist Academy in order to interview ~ fourth-grade student. The case workers, acting on an anonymous tip tbat Greendale Baptist Academy administered corporal punishment as part of its discipline policy, did not disclose to the slatTwhy they wanted to interview the student. The case workers interviewed the student in the absence ofthc student 's parents and informed the school statTthat the parents were not to be contacted. The AFA eLP filed suit against the Wiscomin Department ofHwn an Services, citing a violation of the parents' Fourth Amendment rights. ....~..........~~~. .~. .~. . . . . .~............ donatioruwe~ made to the AFA in the name ofuMr. and Mrs. Bruce Ivins" on eleven separate occasions beginning on December 31, 1993. After an approximate two year break in donations, the next donation oceurred on November 11. 1999, one month after the initial article referencing Greendale Baptist Academy was published in the AFA Journal. It was also discovered thal the subscription to the AFA Journal. in the name of"Mr, & Mrs. Bruce Ivins," at. . . . . ... Frederick, Maryland 21702, was active ~til March 2005. Frustrations with Members of the United States Senate L are practicing The investigation has shown that Dr. Ivins and his wife, Catholics. Their children are graduates or Saint John's at Prospect Hall Catholic Hjgh School located in Frederick, Maryland. In an e.-mail dated, September 26.2001 , Dr. Ivins states that both be and 7 are actively involved in the church choir. On July 10,2002, in an e-mail to a friend, Dr. Ivins identified his wife, b as the President of the Frederick County Right to Life, as weU as having connections to many other pro-Iifelanti-abonion groups. Dr. Ivins later states in the same e-mail, "I'm not pro-abonion, I'm pro-life, but I want my position to be one consistent with a Christian." In 2001, members of the catholic pro-life movement were known to be high1y critical of Catholic Congressional members who voted pro-choice in opposition to the bel iefs of the Catholic Church. Two of the more prominent members of Congress who fell in this category were Senator Tom Daschle, then Senate Majority Leader; and Senator Patrick Leahy, Chainnan of the Senate Judiciary Committee, both recipients of the 2001 anthrax mailings. In a September/October 2001 newsletter from the organization Right to Life of Greater Cincinnati, Senator Leahy and Senalor Daschle were featured in an anic le entitled: Pro-Abor/ion "ea/ltolic" Senaiors?, wherein a statement declares that these men should no longer be labeled Catholic and should be excommunicated for aiding and abetting abortion. During Task Force interviews, 12 \. WOIRHEPP B3D15C.pdf  19  009995Anthrax Page 508 of 643 staff members related that Senator Leahy is pro-choice on the issue of abortion. Three staff members stated that Senator Leahy was on a pro-life -bit list" because of his stance on abonion. On September 26, 200 I, in an e?mail to a friend, Dr. Ivins writes "The news media has been saying that some members of Congress and members ofille ACLU oppose many of the Justice Depanmcnt proposals for combating terrorism, saying that they are unconstitutional and infringe too much on civillibertics. Many people don't know it but the official ACLU posi tion IS to oppose aU metal detecto.. in airports and schools and other public buildings. It~ interesting rbi'll we may now be living in a time when our biggcs:llhreallo eivilliberties and freedom doesn~ come from the government but from enemies of the govenunent. Osama Bin Laden bas Just decreed death to all Jews and all Americans, but I guess that doesn't mean a lot to the ACLU. Maybe I should move to Canada ....o On November 17, 2001, Senator Leahy's Chief of StatT, was interviewed by Task Force investigators. The staffer stated that Senator Leahy has drawn fire from some conservali\'e prolife supporteR. The staffer advised that Senalor Leahy and Senator Daschle were seen by some mernhe!1 of the pubUc as slowing the passage of the P.A.T.RI.O.T. Act. Senator Leahy rcponedly was critical of the P.A. T.R.LO.T. Act because he believed it was too far reaching and threatened civil liberties. The slafTer also related that Senatot Leahy had been criticized by conservatives and Republicans for resisting the appointment of Republican appointed Judici al nommees. Bacillus Trace Evidence Experts at tbe FBi's Hazardous Materials Response Unit have found that trace quantities of Bacillus anlllrads and Bacillus $ublilus can be detected using biological swabbing techniqUC5 similar to those used by United Nations weapons inspectors. Anthrax and Sublilus spores are like seeds, which arc surrounded by a dense shell capable of enduring extreme cnvirorunental conditions and tempel1ltures remaining donnant for decades, until such lime that nutrients are again available and the organism returns to its vegetative cycle ofreplitation. Even when the ... iability of an anthrax or subtilus spore is destroyed by envirorunental or other factors, the genetic material of the organism will remain preserved within the dense shelt of the spore. and can he detected by DNA analysiS using the Polymerase Chain Reaction (PCR). The investigation to date has yielded a diverse array of items contaminated with anthrax spores from the letters, such as the previously mention mailbox at 10 Nassau Street in Pnncct.on. New Jersey. 1be contaminated items tenaciowly retain anthrax spores on their surfaces, even when subjected to the highs and lows of outdoor temperatures, various environmental conditions, and elements of the weather. The United States Postal Service had great difficulty cleaning the two regioJlal postal facilities that processed the anthrax letters. Trace quantities of anthrax spores were found to persist in those facilities nearly two years after the attacks, despite repeated efforts to eliminate those spores using chlorine dioxide gas and other remediation techniques. Taken together, it is reasonable to believe that bacillus anlllracis and JUblilu,y spores residual from the September and October 2001 mailings may still pttsist in trace amounts inside the horne or vehicle oftbe penon who mailed them, even six years after the crime. Regardless 19 WOIRHEPP B3D15C.pdf  20  009996Anthrax Page 509 of 643 of their viability, the mechanjcs Qfthe spore structure protects the DNA of the organism, which can be detected using PCR techniques. ConctusiQn Based on the foregoing, 1 submit that there is probable cause to believe that a search and forensic swabbing oftbe Subject Residence and Subject Vehicles may result in collection of evidence relevant to the dissemination ofa weapon of mass destruction (anthra.~) through the U.S. mail SystClll in September and October 2001 in violation 18 U.S.C., Sections 2332. and 1114. Specifically, there is probable cause to believe that a search and fon:nsic swabbing of the Subject R~idencc and Subject Vehicles as described in the Attachment to this affidavit, may reve.il1laborntory, mnkeshift, and duol purpose equipment, or matcriai!, supplies, and protocols used in preparation oflhe deadly anthrax contained in the letters, or may recover trace amounts ofthc non-anthraci..s Bacillus or the powden:d anthrax material remaining in the Subject Residences and Vehicles. or may recover textile fibers that match those recovered from the tape on the envelopes used in the anthrax mailings, O( may recover hairs that match the Caucasian hairs recovered from inside the contaminated mail drop box in Priocelon, ew Jersey, or may produce papers, tape, pens, pencils, notes, books. manuals, receipts. financial re<:ords, or correspondence, address book" map', handwriting ,anlpl.. , photocopy device.. scanners, printers, toner or printer cartridges, photocopy exemplars, envelope bands, New Jersey addresses. global positioning devices, zip-code infonnation. photographs, computer files. cellular phones, phone bills, e-lectronic. page-( devic-C'S, Clthn- digit&l devic-C5, or other d(X;umentary evidence tl:at may link Dr. Bruce Edwards Ivins to the anthrax mailings and identify conspirators involved in the attacks. Based upon my knowledge. training and experience, my eOllsuitations with other criminal investigators, persons engaged in criminalattivity frequently store, or inadvertently deposit, these kinds of items in their homes or automobiles and leave them on the premises for many years after a crime, failing to rccognizc their forcn3ic value And incrimimll ing nature. This conclusion is supported by trash covers that have been found in material recentl y discarded from the house. On October IS and Octoher 22, 2007, trash was recovered from the front of 622 Military Road.. Frederick. Maryland, includin~ receipts from a 1995 purchase as well as other docwnents related to KKG sorority. In my experience, vehicles are often used to store documen~, maps, receipts, and other papers related to travellhat may be relevant to this investigation. Because this affidavit is part of an ongoing investigation that would be jeopardized by premature disclosure of information. I further request that this Affidavit, the accompanying Order, and other related documents be filed Wlder 5eal \UlljJ further order of the Court. ;;. I.). ~,.0-( I" ~'- I"'~~.,c.. '::>Z. Ttl t..<.N~":'- "'-''''"'7 -r.....,6/" ""'}!'~ If)f"/O? -;t;,. , 20 WOIRHEPP B3D15C.pdf  21  009997Anthrax Page 510 of 643 The stalemenLs contained in this Affidavit are based in part on infonnation provided by FBI Special Agenu and U.S. Postal Inspectors. on ob!CfVatioDs made by law cnforccmml 'gents, and on my experience and background as an FBI Special Agent. I have not included each and every fact known to me concerning this investigation. I have set forth only the facts (hal I believe are necessary to establish the necessary foundation for the search warrant moMAS F. DELl.AFERA Postallnspec:tor United StatC6 Postallnspccrion Service OCT 312001 Sworn to before me this __ day of ,2007 /' /~~~ ~TE1UDGE United States District Court for the DistriC't of Colwobia IlEBORAHA ROBINSON u.&WlllMiE ..... , CIeri< 21 WOIRHEPP B3D15C.pdf  22  009998Anthrax Page 511 of 643 o ATI ACIIMENT TO AF'FIDA VIT The search of the Subject Property and PrCJnlses will include a physical search and forensic swabbin)! for the items mentioned in the rexi of lhe search warrant incJudini more specifically the following: 1. Any assembled or unassemhled Biological Threat Agent ( BTA), or trnce thereof, and any HI A weapon. andlor any incendIary. rachoJoglcal. nuclear, chemIcal or booby trap device. 2. Laborafory equipment used in (he production/ replication of biologJcalthreal agents including, but not hmited to ngllr or liquid growth media. media components. Inll-foaming agents, animal droppings or carcasses, autoclaves. bleach, blenders, live animals and materials for their care, cylinder of nillogen or oxygen. soil. dimethylsulfoxide (DMSO). famcntors, grinders, home brcwin,lil kits, incubators, inoc:ulalin,l:t loops, mechanical stirrers/pamt sliJn'r5. nucroscopes. microwave for making homemade agar plates, mortar/pestle,. milling devices, ovens, Petri dishes/improvised glass/plastic cook dishes. pipettes. pressure cookers, Q-tips, roller flasks, rubber gaskets/seals, screens. sie\'cs, sodium hydroxide, stains/inks/dyes used to identify STAs. sterilization equipment (Bunsch 01' alcohol bumetSlbuu.ne torches). ~'ab', test tubes, tissue cultcre flas.lc.s, virus flasks, and other materials. 3. Personal Protective Equipment/Safety Equipment including, but not limited to, biosafcty hoods and cabinets (commercial or homemade), masks (surgicaVdusVHEPA). exhaust vents. filters (HEPA). gloves. lab coalS. mechanica l fans, safelY glasses/goggles, shoe covers, soap. tubing pipes, and vinegar. 4. Items used in the weaponi7.ationldclivery of biological weaporu including, but not limited to pens, tape, paper, scissors. envelopes, target addresses, aerosol canisters, agriCUltural or garden sprayers. acetone or other dJ ying agents, baby powder, baking powder, baking soda, boxes, corruncrciaVindustrial powders, copier toner/printer andIor tonerlinks, compressed air Or other gas systems, cornmeal, cornstarch, explosive, hypodcnnic syringeslnecdlt.-s, jars or flasks with gauze on top, oatmeal, powdered milk. saltlpcpper. spray bottles. squirt I\.lDs. stamps. sugar, talcum powder, and other materials. 5. Documents, memoranda. nOles or other writings and equipment which include but are not umned to schematJcs, diagrams, bluepnnts, survetlLancc equipment o r Its manuals and other related paperwork , receipt for purchase of items, how-to magazines, books and pamphlets which describe ~roduclion, weaponiution, and delivery techniques, directions, maps, diagrams, and blueprints of delivery or target locations, communication which would include but not be limIted to handwritten notes and drawings, memos and photographs, which wou ld include descriptive infonnation such as telephone numbers, addresses, affiliations and contact points of individuals involved in a conspiracy to acquire, refine, alter, transport, andlor use a biological weapon. Note that such documentary material described herein may be in printed or electronic form such as, bUI nol limjted 10, computer disks. Such computerized and other eJectronic material would constitute contraband. fruits , instrumentalities or evidence of the offenses enumerated herein and in the atta--...bcd affidavit for search warrant. WOIRHEPP B3D15C.pdf  23  009999Anthrax Page 512 of 643 .. , 6. Publications or other wrinen material s evidencing knowledge of federal laws including those related to biological weapons and other weapons of mass destruction. 7. Photographs, newspapers, or magazine clippings, travel information, brochures. maps, hotel receipts. rental receipts, notes. ledgers. phone nwnbers, toll records. address books (which may be in printed or electronic format), hand-held clectroruc memo-type notebooks, scanners, commercial transportation receipts, and firearms, ammunition, fireanns owners identification docwnents andlor permits and licenses, digital or other types of pageTN and all alpha andlor numeric data/message stored rherein, cellular phones, cordless [clcphortcs and their base units, caller identification units and all alpha-numeric information and data stored therein, biological weapons/agent (onnulas, biological weapons/agents, andlor recipes, all of which re1ated to the construction of biological weapons, or plans 10 produce, conceal, or use such devices or agents or indicated po)ssible confederates involved or possible targets of such weapons. 8. Any notes, correspondence, memoranda, \\Tilings or other documents, regardless of storage media, which relate to the production or refinement of biologlcal weapons (including anthrax bacteria/spores), or plans to produce. conceal or usc such weapons, which items concern, among other things, rental agencies. storage facilities. bioiogicaVchemical suppliers or outlets for necessary biological eqUipment. Examples of such writings include, but are not limited to, those that evidence biological weapons production or predisposition such as sales rec:eipts. invoices, shipping records. literature that relatcs to biological weapons?making or the production of biological materials, electronic addresses of sites with biological agentlweapons?making information and stored ('leclronic communications, computer passwords or codes, delivery devices. and material from which biological weapons can be made from related chem icals and malerials. 9. Bank documents and other records and documents pertaining to the expenditure of funds for illegal activities, assets and funds used to facilitate illegal activities and assets and funds obtained from the conduct of illegal activities. 10. Cellular and telephone records. 11. Keys, records, or other information pertaining to storage facili ties, safely deposit boxes or oilier locations where assets or contraband may be stored. 12. Books, videos, pamphlets and any other written, audio, video media advocating production, refinement. distribution of biological weapons or other acts of violence or criminality. 13. Destructive devices, weapons of mass destruction or their components or parts and any written medium pertaining to their design. use, operation. functi on, capabilities or olher attributes. 14. Records, documents, notes, or other material indicating the use of aliases or false identities including birth certificates, driver's licenses, photographs. travel documents or passports and miscellaneous items such as pennits, registrations. certificates, licenses. and badges of all types . (2) WOIRHEPP B3D15C.pdf  24  010000Anthrax Page 513 of 643 ? .. , J 5. Computer hardware. computer software. computer-related documentation, computer passwords and data security devices. records, documcnts, photos, and materials usc to communic.Hc, both writtcn. eleClronic mail. and by any orher means directed either tolfrom subject. a. Computer hardware is defined as any computer equipment including any e1cctron ic devices which are capable of analyzing. caiJecling, converting, creating. displaying, or transmitting electronic, magnetic, optical compuler impulses or data. These devices include. bUI are not limited (0, compule~ , computer components, computer peripherals, data-processing hardware (such as CPUs, memory typewriters. and self?containcd laptop or notebook computers), encryption circuit boards, internal and peripheral storage devices (such as fixed disks, external hard disks. fl oppy disk drives and diskettes, tape drives and tapes and optical storage devices), peripheral inpuUoutput devices (such as keyboards, printers, scanners, p lotters, and video display monitors). related communications devices (such as modems, cables and connections, recording equipment, Random Access memory (RA.\1) or Read-Only memory (ROM) units, aUiomatic dialers. speed dialers, programmable telephone dialing or signal ing devices, and electronic tone gathering devices), as well as devices, mechanisms, or parts that can be used to restrict access to such hardware (such as physical keys and locks.) b. Computer software IS defined as any instructions or programs stored in the [onn of electronic, magnetic, or optical media which are capable of being interpreted by n computer or its related components. These items includc, but are not limited to, operating systems, application softwwt, utility programs, compilers, interpreters, and communications software, as well as software used to communicate with computer hardware periphcrals, either din.'Ctly or mdirectly by telephone lines. radio, or other mcans of transmission. Computer software may also include certain data, data fragment s, or control characters integral 10 the operation of the computer software. c. ComputcHclated docwnentation is defined as any written, recorded, printed, or electronically?stored material which provides instructions or cxamples concern ing the operating of a computcr system, computer software, and/or any related device, including but not limited 10, user agreements, software manuals, and operdting procedure guidelines. Such documentation may also include material which explains or illustrates the configuration or use of any seized hardware, software. or related item. d . ComputCf. passwords and data security devices are dermes as all those devices. programs, or data? whether themselves o r in the nature of hardware or software? that can be used, or is desi gned for use to restrict access to, or facilitate concealment of, any computer hardware, software, computer? related documentation, t:Iectronic data. records, or materials within the scope of this application. These items include, but are not limitoo to, any data security software or infomlation (such as lest keys and encryption codes), similar informluon that is required 10 access computer programs or data or to otherwisc render Prob '18mS or data mto usable form. e. Records. documents. and materials. as used above, includes all the forgoin g items of evidence in whatever form and by whatever means such records, documents., or material, their drafts, or their modifications may have been created or stored, including, but not hmited to, any ill WOIRHEPP B3D15C.pdf  25  010001Anthrax Page 514 of 643 o o o j handmade ionn (such wriling or drawing) , any photographs or negatives, any mechanical fonn (such as printing or typing) or any electrical, electromc, magnetic, or optical form (such as floppy diskettes, hard disks. CD? Roms, optical disks. printer bu ffers , smart cards, thumb drives, memory calculators. electronic dialers. or electronic notebooks). as well as printouts or readouts from any storage devices. f. The tenn electronic mail is defined as any communications, including, but not limited to, the transfer of signs, signals, writing. images, sounds, data, or inlclligmce. previously received, transmitted. or sto~d , or prepared 10 contemplatton or transmission, or any corrununications in the process of being received or lransmiued , whcthcr stored on any electronic media named abo\'c or held in temporary, intermediate storagt: incidental to transmission . 16. Any notes, correspondence, mcmoranda, writings, address books. documents or other materials, reganlless of storage media, which establish an evidentiary link to (I) the particular envelopes and pholo-(;opied leners used in the crime, including samples of documents photocopied around that time period, (2) thc locatit>n from which the attack letten were mailed in Princeton. New Jersey. including but not limited to materials relating to the KKG soronty a.~iatcd with the particu lar mailbox from which the anthrax letters were sent. and any other materials I. rueing Ivins or his family or associates to the Princeton area, (3) the return address on the envelop es used in the attacks. including any materials relating to "Greendale" schools. "4111 grade" , " Frankli n Park," and postal zip code "08852" and zip code directories; (4) the victims to whom each of the attack letters were mai led, including but not limited to materials relatmg to NBC, NeK' York Post. reporters Gary Matsumoto and Tom Brokaw, the National Enquirer ond other publications of American Media Incorporated,letters to the editor, and any member of the United States Congress; (5) a possible motive to commit the attacKS in 2001, inCluding but not limited to materials relating to the anthrax vaccine, Gulf War Syndrome, bioterrorism issues, the fonner bio?weapon program at Fe Detrick, the bio-defensc program at USAMRHO. any vendetta against the KKG sorority or SOroritIes in general , dislike for members ofthc press or members of Congress, and (6) plans or attempts to obstruct justice or otherwl sc mislead Task Force investigators on this case, including matcrials relating to the submission of false evidentiary samples., how to defeat a polygraph. the purchase: or use of any electronic eavesdropping or counter~surveiJlancc devices including computeT softwart', spyware or other services, investigative coWlter~measure materials and detection devices, materials relating to the cooperating FBI sources mentioned in this affidavit., and the purchase of weapons. stun-guns, taser-gWlS. and baHistic vests . "1 WOIRHEPP B3D15C.pdf  26  010002Anthrax Page 515 of 643 UNITED STATES DISTRICT COURT FOR TilE DISTRICT m' COLUM BIA In the Matter orthe Search of 1993 DODd. beariD, reeistered to Brute Edwards 1vlDs, oo _ rederick, MD SEARClI WAR.RA.I';T CASE NUMBE~ 7 - 5 2 6 - iii o 0 1 TO: ~aJ Inspector Tboml5 E. Dell.fc[J 1I:xi any Authorized Officer offh.e l'ruled SUlle! Affidavil(S) ta"Ul& been made before me by Postal laocctQ[ Iltpma f. DcUlfcn thai 0 on the person or a on the pmnsscs mown as (MN, dac:tq!oontaCI tlllocItIoIIJ 199) Hood. Civk ~~ ill cotor, bcariOC VIJ'II' _ Edwards Ivins, I , _ fndcrick, MD who has ruson to benn'C ttb;ltrtd 10 Bryn i. the ... Di:I~ o( Maryland then: is no.,.. concelll!'J t1l,...{jb.,R( q W I I . _I co.w} , r (not 10 exceed 10~) the penon or place Di:mc:d above for the ~Of' property spc:clfied. scrvinl thi:! ~.. rnot and makina the lCarc.hU (in the c1aytiJ'nt. 6:00 A.M. 10 10:00 P.M.) 19(at any lime in the day 011\1gb.1as I find rcuoJUlble cause bas bttnwablisbcd) and iftbcpcnoo 0 1 propcrtybe found thereto seizcsame. 1elvin&a copy orCllis warRnt and receipt for the penoIl 01" pro~ taken, aTld prepue a \Wi~ iovutoty of tbe person Ck propet1rft!i~~lnc' Court rclUm dua wana.,tlO the undcnlancd U.S. JudgelU.S. Ma,lStrItc Jqe. u requued by law, For \he District of Columbia OCT 812007 e.5: N 1AV ATA - IHITT GT N, Clerk py WOIRHEPP B3D15C.pdf  27  010003Anthrax Page 516 of 643 AO 109(l19O)s.z.n WPT*II RETUR"i DATE W .... !UlA NT f.ECEIVED DATE AND TIME W.... Rl.ANT EXEC\JTED copy OFWAIUlANT AND lECI!IPTFOR IT!MSLEFT WITH 1/ f L/ Coi17 I ~ r 1'00'" INVf.NTO~~ MADP. IN THE 1'!tES{~~ If) (~ ?001',... ~'r-~~ P,,-U I 14:,.,jJ I /1 17'-/ .;/~ r ' ,. i'<.. INVENTORY OF rROPEII.TY SElUD PVII.SU.... l'lTiOnfE WAARANl cJ 0 )I" ~ - 7:7<..? ..,.; -;-.,. I::., ~ Ih(~ "';c,. { .f~",Je) / .-- . J " .... e" V .'(t) # k<.r I"- . CERTIF1CA nON I swear that this mVCfMory is a true and detailed atWWlt orthe property seized by me 011 the w acTVll. I / , C / ,#("6 .1 . / ,. , j: FILED t<>. Dr. Bruce Ivins has unrestricted access to the suite and has been the sole custodian ofRMR-1029 since it was first grown in 1997. Bacterial contaminant found in attack letters Both afthe anthrax spore powders recovered from the Post and Brokaw letters contain low levels of a bacteria) contaminant identified as a strain of Bacillus subliJis. The Bacillus subtilis contaminant has not been detected in the anthrax spore powders recovered from the envelopes mailed to either Senator Leahy or Senator Daschle. Badl/us subtilis is a nonpathogenic bacterium found ubiquitously in the environment. However, genomic DNA sequencing ofebe specific isolate of Bacillus subtHis discovered within the POOl and Brokaw powders reveals that it is genetically distinct from other Icnown isolates of Bacillus subtilis. Analysis of the Bacillus subtilis from the Post and Brokaw envelopes revealed that these two isolates are identical. Phenotypic and genotypic analyses demonstrate that the RMR-J 029 does not have the Bacillus subtilis contaminant found in the evidentiary spore powders, which suggests that the anthrax used in the letter attacks was grown from the material contained in RMR-I029 and not taken directly from the flask and placed in the envelopes. Since RMR-I029 is the genetic parent to the evidentiary spore powders, and it is not known how the Bacillus suhtilis contaminant came to be in the Post and Brokaw spore powders, the contaminant must have been introduced during the production of the Post and Brokaw spores. Taken together, the postmark dates, the Silicon signature, the Bacillus subtilis contaminant, the phenotypic, and the genotypic comparisons, it can be concluc.ed that, on at least two separate occasions, a sample ofRMR-1029 was used to grow spores, dried to a powder, packaged in an envelope with a threat letter, and mailed to the victims.1 Envelopes used in the anthrax attacks In the 200t anthrax attacks, four envelopes were recovered. The four envelopes were all 6 ~ inch federal eagle envelopes. The "federal eagle designation is derived from the postage frank in the upper right-hand comer on the envelope which consists of the image of an eagle perched on a bar bearing the initials "USA." Underneath the lettering is the number "34," which denotes the postage value of 34?. The eagle, lettering, and denomination are referred to as the M I As the Court OlaYrecall from its review, dunng the course of this six year investigation, the Task F')f(~e has from time to lime obtained search warrants, supported by affidavits e.stablishing probable for the residences and All prior searches in this investigation wcrc";O;;du;:.;d to the K1entification ofRMR 1029 al USAM RllD II the genetic parent of the anthrax used in the 200 I letter attacks. 0; "f!~!!=! iliO-...;etiC assays 5 WOIRHEPP B3D15D.pdf  6  010010Anthrax Page 523 of 643 indicia. The eagle and the har:ue stamped in blue ink, while the denomination is stamped in grey ink. Approximately 45 million Federal eagle 6 ';." envelopes were manufactured by Westvaco Corporation (now known as MeadWestvaco Corporation) of Williamsburg, PeMSylvania. between December 6. 2000 and March 2002. These Federal eagle 6 ",." envelopes were manufactured exclusively for and sold solely by the U.S. Postal Service between January 8, 2001 and June 2002. Subsequent to the attacks, an effon was made to collect all such envelopes for possible forensic examination, including the identification of defects that occur during the envelope manufacturing process. As a result of this collection, envelopes with printing defects identical to printing defects identified on the envelopes utilized in the anthrax anacks during the fall of2001 were collected from the Fairfax Main post office in Fairfax, Virginia and the Cumberland and Elkton post omcos in Maryland. The Fairfax Main, Cumberland, Maryland, and Elkton, Maryland post offices are supplied by the Dulles Stamp Distribution Offiee (SOO), located in Dulles, Virginia. The Dulles SDO distributed "federal eagle" envelopes to post offices tluoughout Maryland and Virginia. Given that the printing defects identified on the envelopes used in the attach are transient, thereby being present on only a small population of the federal eagle envelopes produced, and that envelopes with identical printing defects to those identified on the envelopes used in the attacks were recovered from post offic~ serviced hy the Dulles SOO, it is reasonable to conclude that the federal eagle envelopes utilized in the attacks were purchased from a post office in Maryland or Virginia. Of the sixteen domestic government, conunercial, and university laboratories that had virulent RMR?l 029 Ames strain Bacillus anthracis material in their inventory prior to the attacks, only one lab was located in Maryland or Virginia, where the relevant federal eagle envelopes were distributed and sold by the U.S. Postal Service: the USAMRIID facility al Fort Detrick, MD. Tape. Ink. and Fiber Trace Evidence All four of the envelopes containing these letters were taped along the seams with transparent tape. Due [0 striation patterns placed on transparent tape as pan of the manufacturing process, it is sometimes possible to match a piece of suspect tape with the tape roll from which it originated. According to FBI Laboratory experts, the envelopes were addressed with a pen which djspenseq fluid-like ink, rather than the ink typically found in a ball point pen. Due to the distinguishing characteristics of ink, which vary by manufacturer. it is sometimes possible to match ink writing with the pen or brand o( pen used to apply it. Forensic analysis of the tape attached to thc four envclopes has identificd eight different types of fibcr attached to thc tape: black cotton. black wool, black nylon, brown polyester, blue wool, yellow acrylic, red cotton, and red acrylic. Identification of Collection Box at IO Nassau Street Information gathered to date suggests that all of the lethal anthrax letters were mailed from a singJe street collection box located at to Nassau Street, in Princeton , New Jersey. The letters were postmarked on either September 18. 200 I or October 9. 200 I at the same HamIlton 6 WOIRHEPP B3D15D.pdf  7  010011Anthrax Page 524 of 643 , . Township Regional Postal Facility in Hamilton, New Jersey. The collection box on Nassau S_t was identified through forensic biological swabbing of every U .S. Postal Service drop box that coUects mail to be processed at the Hamilton facility. Further forensic examination of the contaminated mailbox recovered a number of Caucasian hwnan hairs from inside the box, which arc suitable for comparison. Dr. Bruce Edwards Jvins Dr. Bruce Edwards Ivins is a senior microbiologist who has worked for 27 years in the Bacteriology Division at USAMRllD. The Task Force investigation has revealed that Dr. Ivins is considered an expert in the growth. sporulation, and purification of Bacillus (lnlhraciJ. Dr. Ivins obtained a Bachelor of Science degree in Bacteriology in 1968, a Ma..teT of Science degree in Microbiology in 1971, and a Doctorate of Philosophy (ph.D .) degree in Microbiology in 1976, all from the University of Cincinnati. Dr. Ivins then completed a two year Post-doctoral Fellowship at the University of North Carolina (UNC), Chapel Hill. A review ofUSAMRIID records, laboratory notebooks. written protocols. and professional publications has shown that Dr. Ivins has worked with JkJcillus anthracu at USAMRIID ",inee 1980. He ha.~ personally condueted aod ~uperviscd Ames anthrax spore productions for over two decades. At the time of the anthrax mailings. Dr. Ivins possessed extensive knowledge of various anthrax production protocols. Dr. Ivins was adept at mnnipulating a..,thrax production and purification variables to maximize sporulation and. improve the quality of anthrax spore preparations. He also understood anthrax aerosolization dosage rates and the importance ofpurily. consistency, and spore particle size due to his responsibi lity for providing liquid anthrax spore preparations for animal aerosol challenges. Dr. Ivins produced large batches o f Bacillus anthracis which were required for such challenges? tests in which vaccinated animals inhale pre--defined doses of anthrax spores to assess the efficacy of the anthrax vaccine. Dr. Ivins's 20 years of working in the laboratories ofUSAMRIID provided him personal, hands?on laboratory experience in the production of liquid spore preparutions of Bacillus anthracis. He has used lyophilizers, biological safety cabinets, incubators, and centrifuges in vaccine research. Such devices arc considered essential for the production of the highly purified, powdered anthrax used in the FalJ 2001 mailings. Dr. Ivins was also very experienced in conducting laboratory work. in a containment area. and well versed in decontamination procedures specifically for BaciJ/us an/Macis. Dr. Ivins's employment al USAMRTID also provided protection against anthrax infection at the time of the mailings due to his extensive and current anthrax vaccination history. Unexplained late night laboratory access USAMRIID contairunent Suite B3 is a Biological Safety Level?3 (BSL-3) suite of laboratories used by USAMRIID Bacteriology personnel for research on dangerous animal and human pathogens. The flask identified as RMR?I029 was stored in Suite 83 at the time of the letter attacks. Suite access is obtained via a Security Access Control (SAC) badge reader al the 7 WOIRHEPP B3D15D.pdf  8  010012Anthrax Page 525 of 643 door to the cold-side change room, which is secured by a magnetic-lock. A central security system monitors and records a time stamp for each SAC badge and keypad request. A review of Dr. Ivins' s laboratory access records for Building 1425 was assessed to determine trends in working hours and evening times (after 6 p.m.) spent in Suite B3. His regular worJeing hours on average consisted of a 7:30 a.m. to 4:45 p.m. shift. but he would periodically return in the evenings, presumably to check on the status of various experiments. Beginning in mid-August 2001 ; however, there was a noticeable spilce in Dr. Ivins's evening access to the B3 hot Suite. The following depicts the comparison of Dr. Ivins's total hours spent at night, after 6 p.m ., in Suite 83 during the years 2000 and 2001 : Night Hours in Suite B3 ja 2000 a 2001 j 35 30 25 20 = :l! 15 f 10 5 o Jan Feb. Mar. Apr. May Jm: July Aug. Sept. Oct Nov. Dec. Mouths The investigation examined Dr. Ivins's laboratory activity immediately before and after the window of opportunity for the mailing of the Post and Brokaw letters to New York which began at 5:00 p.m. Monday, September 17,2001 and ended at noon on Tuesday, September 18, 200 1. Beginning on Friday, September 14, Dr. Ivins worked the following three consecutive evening shifts prior to the mailings with time spent in Suite B3: Day Friday Saturday Sunday Date September 14 Septemher 15 Septemher 16 Time in B? ulldine 1425 8:54 p.m. to 12:22 a.m. 8:05 p.m. to 11 :59 p.m. 6:38 p .m . to 9:52 p .m . T ot. IT" . Ime ID B3 2 hours 15 minutes 2 hours 15 minutes 2 homs 15 minutes A fter September 16. Dr. Ivins did not enter Suite B3 in the evening again until September 25 . 8 WOIRHEPP B3D15D.pdf  9  010013Anthrax Page 526 of 643 ,. The investigation further examined Dr. Ivins's liOOratory activity before and after the window of opportunity for the mailing of the Daschle and Leahy letters to Washington, D.C. which began 3:00 p.m. Saturday, October 6,200 1, and ended at noon on Tuesday, October 9, 2001. Beginning on Seplember 28, Dr. Ivins worked eighl consecutive nights which consiSled of the following times in building 1425 with time spent in Suite B3: Da. Friday Saturday Suoday Monday Tuesday Wednesday Thursday Date Time fa B.UdiD1141S Friday Seplember 28 Seplember 29 September 30 OclOber 1 Oclober 2 Oclober3 OclOber4 Oclober 5 7:16 p.m. 10 10:59 p.m. 8:02 p.m. 10 11: 18 p.m. 9:53 p.m. 10 12:04 a.m. I hour 18 minutes 9:14 p.m. 10 10:43 p.m. 20 minutes 7:24 p.m. 10 9:39 p.m. 23 minutes 7:25 p.m. 10 10:55 p.m. 2 bours 59 minutes 6:10p.m. 10 10:12 p.m. 3 bours 33 minutes 7:40 p.m. 10 12:43 a.m. 3 houn 42 minutes Total Time in B3 1 hour 42 minules t hour 20 minutes After October 5, Dr. Ivins did not enter Suite B3 in the evening again until October 9, for 15 minutes, and then October 14, for I hour and 26 minutes. The investigation has also analyzed the daily schedules, work areas accessed, and the number of hours worked per week of all other researchers assigned to the Bacteriology Division who bad access to Suite B3 during the months of September and October 2001 . When these records are compared to Dr. Ivins for the same period, Dr. Ivins's babits are significantly different than those of the other researchers, in that he was frequently in Suite B3, where RMR1029 was kept, late at night and on weekends when no other researchers were present in Suite 83. Additionally. while Dr. Ivins. was in Suite 83 at night, no other USAMRllD employee was present. On March 31, 2005, Dr. Ivins was asked by Task Force investigators about his access to 8 3 and could provide no legitimate reason for the extended hours, other than "home was not good" and he went there "to escape" from his life at home. A review of Dr. Ivins 's and coworkers 's laboratory notebooks and projects at the times in question was conducted. The investigation determined that Dr. Ivins's role in the experiments were minimal, and did not justify the time be spent in B3. Dr. Ivins has admitted to investigators that the research he was conducting in 2001 did not require, and does not explain, his late night hours in the B-3 laboratory arotmd the time period of the anthrax mailings. Since producing anthrax spore preparations was one of Dr. Ivins's principal responsibilities at USAMRIID. he had multiple and unfettered opportunities to produce or divert Ames strain spores for illegitimate purposes. His access to Suite 83 and USAMRJID afford ed all of the equipment and containment facilities which would have been needed to prepare the anthrax and letters used in the Fall 2001 attacks. 9 WOIRHEPP B3D15D.pdf  10  010014Anthrax Page 527 of 643 Failure to cooperate in providing laboratory samples - -- - - - - -- -- - - -- Ivins provided two sets of samples of Ames anthrax to the to were drawn from RMR-I029. In the first submission in February 2002, Dr. Ivins failed to follow the Dr. Ivins was to . failure to follow subsequently notified that the February 2002 samples were the protocol. A second set of samples, labeled "Dugway Ames spores - 1997" was provided to the FBIR by Dr.. Ivins in April of2002_ Dr. Ivins declares that be uses the tenns "Dugway Ames spores - 1997' and RMR-I 029 interchangeably, as they are the same flask of material. Both of the April RMR-I029 samples were submitted for genetic testing and found to be negative for the presence of the four mutations found in the anthrax used in the attacks. On December 12,2003, an FBI Special Agent accompanied Dr. Ivins into Suite B3 at USAMRIID and identified additional Ames samples of Dr. Ivins' s and others that bad not been submitted as part of the above mentioned ~nse . Dr. Ivins submitted slants prepared from the newly identified sampl~IR on April 7, 2004. On the afternoon of April 7,2004, an FBI Special Agent accompanied Dr. Ivins into Suite B3 , and seized the original samples Dr. Ivins had used to prepare the slants submitted to the FBIR earlier that day. Additionally, the Agent seized the RMR-I029 flask itself. All of the samples were secured in the B3 walk-in cold room within a doub le-locked safe, and sealed with evidence tape until such time that they could be transported to the Navy Medical Research Center (NMRC), whieh was under contract by the FBI. On June 17,2004, RMR-1029 and the additional Ames samples were submitted to the FBIR, by the NMRC, under the direction of the FBI. Samples of these FBfR submission were sent to the same contracting laboratories for genetic testing. The sample labe led RMR-1029 tested positive for all four genetic markers, therefore, manifesting the genetic characteristics common to the evidence. he On March 31. 2005 , Dr. Ivins was provided to the FBlR on April 10. found to be genetically distinct from the anthrax anthrax material recovered by the FBI from the RMR-I029 flask seized from Dr. Ivins ' lab on April 7, 2004. Dr. Ivins was confronted with this and was asked to explain why he did not submit the genetically positive sample which was clearly responsive Dr. Ivins was adamant in his response that there had been no omission from his submission, and he insisted that he had provided RMR-t029 to the FBI in his second submission of samples in Ap ri l 2002. JO WOIRHEPP B3D15D.pdf  11  010015Anthrax Page 528 of 643 o Knowledge that his stock of anthrax matched the anthrax used in the attack On March 31, 2005, when Dr. Ivins was informed by FBI agents that RMR-I029 shared genetic similarities with the Ames strain used in the mailings. he indicated that he was already aware of this information. Dr. Ivins explained that many months to a year ago, FBI Special Agent Darin Steele told him that RMR~ 1029 matched the evidence. SA Steele denied having the conversation described by Dr. Ivins above. As indicated in previous paragraphs, the RMR-I029 submission provided by Dr. Ivins in April 2002 did not match genetically or phenotypically. Therefore, neither SA Steele nor any other member of the Task Force could make a comparison between the Ames strain used in the mailing and RMR 1029, until after the June 17,2004 submission and subsequent laboratory analysis. When interviewed again on May 7, 2007, Dr. Ivins told investigators that, within three months after the letter attacks, he was aware that his stock of anthrax, RMR-l 029, exhibited unique morphological similarities to the anthrax used in the anacks, and that he allegedly learned this infonnation from three coworkers at USAMRIID who participated in the forensic analysis of the anthrax in the leners. Each of those three coworkers was interviewed by the Task Force, and deny disclosing such infonnation to Dr. Ivins. Over the course of this investigation, Dr. Ivins has been repeatedly interviewed and had open access to the law enforcement personnel responsible for investigating the anthrax attacks. During none of these interactions, did Dr. Ivins ever indicate he had knowledge that RMR~ I 029 had phenotypic similarities to the material used in the anthrax attacks, nor did he suggest that investigators analyze RMR-I029. To the contrary, Dr. Ivins repeatedly claimed that the anthrax used in the attacks resembled that of another researcher at USAMRlID and were dissimilar to the Bacillus anlhracis Ames organisms maintained in his laboratory, which included RMR- 1029. Mental health issues and possible motive The im:estigation has shown that in 2000 and through the mailings in 2001, Dr. Ivins had mental health issues. Dr. Ivins 's mental bealth issues came to the attention of investigators while reviewing e-mai ls ofUSAMRIID researchers . TIuough the e-mails it was detennined that Dr. Ivins was undergoing significant stress in both his home and work life. The mental health issues and stress were significant to the extent that Dr. Ivins sought professional help from a psychiatrist and was irrunediately prescribed medication thai started in February 2000. The following are exce'l'ts from e-mails dated April 2000 through December 2001, from Dr. Ivins to a friend regarding work, home, state of mental heallh, and use of medication (emphasis are as they appeared in the e-mails): April 3, 2000, ?Occasionally 1 get this tingling that goes down both anns. At the 3ame time I get a bit dizzy and get this unidentifiable W metallic? taste in my mouth. (I'm not trying to be fuMY~: It actually scares me a bit.) Other times it's like I'm not only sitting at my desk doing work, I'm also a few feet away 11 WOIRHEPP B3D15D.pdf  12  010016Anthrax Page 529 of 643 watching me do it. There's nothing like living in both the fust person singular AND the third person singular!JUDe 27, 2000. -Even with the Celexa and the counseling. the depression episodes still come and go. That's unpleasant enough. What is REAlLY scary is the paranoia ....Remember when I told you about the -metallic- taste in my mouth that I got periodically? It's when I get these "pannoid" episodes. Of coune I regret them thoroughly when they arc over, but when I'm going through them, it' s as if I'm on a passenger on a ride. .. ' Ominously, a lot of the feelings of isolation - and desolation - that I went through before college are returning. I don't wlmt to relive those years again.... I've been seeing the counselor once a week.- June 28, 2000, "Apparently Gore (and maybe even Bush) is considering making lhe anthrax vaccine for the military volwllary, or C'Ven stopping lhe program . Unfortunately. since the BioPort people aren' t scientists, the task of solving their problem bas fallen on us.. . .. Believe me, with all the stress of home and work, your email letters to me are valuable beyond whal you would ever imagine - and they help me keep my sanity ...... June 29, 2000, "BioPon just tested its final lot of A V A (anthnlx vaccine] in a potency test. Ifit doesn 't pass, then there are no more lots to test. and the program will come to a halt. That's bad for everyone concerned, including us. I'm su~ that blame will be spread around." July 4, 2000, -The thinking now by the psychiatrist and counselor is that my symptoms may not be those of a depression or bipolar disorder, they may be that of a -Paranoid Personality Disorder.I think the .... is about to hit the fan ... bigtime. The July 6, 2000. 7 finallo ! of A VA, lot 22, isn't passing the potency test, and now there's n-:;0 !hing to. ii. ?iiiiii back it up . Plus, the control vaccine isn't working. It's just a fine mess .o are spending probably 95% of our time on this." July 7, 2000, in an e-mail. Dr. Ivins offered to be interviewed as a case study, as long as it remained anonymous. Dr. Ivins indicated that he did not want to see a headline in the National Enquirer that read. "PARANOID MAN WORKS WITH DEADLY ANTHRAX!! !" July 23 , 2000. -It's been a really stressful week, from all stand points. and it's not wen wilh the counselor I'm going 10 . (She said she I'm going to have to ask to get put with another counselor or into a group .... Sometimes I think that it's all j ust too much: Home, 12 WOIRHEPP B3D15D.pdf  13  010017Anthrax Page 530 of 643 ,. August 12, 2000, "Last Saturtlay, as you probably guessed from my email, was onc ermy worst days in months. I wish I could control the thoughts in my mind. It's hard enough sometimes controlling my behavior. When I'm being eaten alive tnsidc, 1 always try to put on a good front here at work and at home, so I don't spr=i the pestilence. .. .1 get incrt Gulf War and through 2001, USAMRllD and Dr. Ivins was the focus of public criticism concerning their introduction of a squalene adjuvant (or additive) to the AVA anthrax vaccine, which was blamed for the Gulf War Syndrome. ID 2000 and 200 I, as evident by the e-mails above, that same anthrax vaccine was baving problems in the production phase at Biopon. a private company in Michigan responsible for manufacturing the vaccine. The Food and Drug Administration (FDA) had suspended further production at Bioport. and the U.S. government, specUicalJy the Department of Dcfeose, was running out of approved lots of the vaccine. The situation placed pressure on select staff members at USAMRnD, including Or. Ivins, who were part oflhe Anthru. Potency Integrated Product Team (lPT) . The purpose oftbe IPT was to assist in the resolution of technical issues that was plaguing Bioport's production of approved lots of the vaccines. In the weeks immediately prior to the attacks. Dr. Ivins became aware that an investigative journalist who worked for NBC News had submitted a Freedom of Information Act (FOIA) requests on USAMRIID seeking detailed infonnation from Dr. Ivins'. laboratory notebooks as they related to the AVA vaccine and the use of adjuvants. On August 28, 2001 , Dr. Ivins appeared angry about the request providing the foUowing response in an e-mail : "Tell Matsumoto to kiss my ass. We' ve got better things to do than shine his shoes and pee on conunand. He 's gotten everything from me he will get." In early 2002, Shortly after the anthrax letter attacks, the FDA re-approved the A V A vaccine for human use, production at Dioport resumed, and anthrax research at USAMRlID continued without interruption. As mentioned previously. one of the anthrax letlers post marked on September 18, 2001, was addressed to Tom Brokaw, NBC News in New York. Or. Ivins thereafter received ''the highest honor given to Defense Department civilians at a Pentagon ceremony on March 14, 2003 " for his work in "getting the anthrax vaccine back into production ." Use offalse names and fascination with sorority group linked to location ofantbrax mailings The investigation has shown that over the past twenty-four years. Dr. Ivins was known to have utilized at least two Post Office Boxes to communicate with members of the public, to pursue obsessions, and possibly engage in the unauthorized.use of another penon 's name. The use of the P.O. Boxes is significant because the post office where Dr. Ivins frequented and maintained a P.O. Box, College Estates Station, Frederick, Maryland 21702, was serviced by the Frederick Main post office, Frederick, Maryland 21701. Analysis of the USPS shipping records indicate the Frederick Main received 1000 6 3/4" Federal Eagle banded envelopes 011 the same day as post offices known to have received envelopes with identical print defects as those seen on the envelopes used in the mailings. On May 14, 2007, identified four communications that it had prev iously received from Or. Ivins. ~oes not personall y know Dr. Ivins but has repeatedly corresponded with h im by mail). In Dr. Ivins's lirstlt:tltr to ' " elated May 8,1982. he used the return address of PO, Box 1265, Frederick, Maryland 21702 . In the letters, Dr. Ivins expressed hi S support to 15 WOIRHEPP B3D15D.pdf  16  010020Anthrax Page 533 of 643 . . work and an interest in sorority hazing. interest was identified as Kappa Dr. Ivins enclmed a "letter to the sorority, On July 9, 2007, the Post Office Box application (pS Fonn 1093) at the College Estates Station, Frederick. Maryland 21702. for P.O. Box t 103, in the name of Bruce E. Ivins was obtained. A review .o fthe application card revealed that Dr. Ivins rented P.O. Bo;r 11 03 on November 12, 1993. providing a military identification and using his Maryland Motor V chicle Administration Driven License. 1? 152-098?188?301 as prooCofidentification. Dr. Ivins an nddress MD 21702 and telephone numbers of on the P.O. Box application card to receive mail last payment OD the P.O. Box was made on November 13,2004. A review of Change of Address records revealed that P.O. Box 1103 was closed on December 30, 2005 and no forwarding address was provided. On July 30, 2007, was interviewed regarding P.O. Box 1103. not personally know Dr. Ivins but has historically been familiar with his name .. " was shown the P.O. Box application and stated that it bad never rented a P.O. Box anywhere and had never met. nor had any social or professional association with a Dr. Ivins. However, the name Bruce Ivins because" bad previously mentioned his name. q 7 indicated to that Dr. Ivins harassed it in the past, and believed that _ was frighttmed of Dr. Ivins, resulting from unspecified incidents that occurred prior to '- m~linK o. . . . . . also recalled a vandalism incident that occurred in the months prior to leaving the Maryland area with 7 7 They had awoken one morning in their apartment to fi nd Greek letters spray painted on the fence, sidewalk, and vehIcle outside their apartment. 1:S:recogruZed ~ as A obtained from the Records Division and provided Rcport,, ~~~. .~~. . . . the following information: On the morning 01'o o 1=:::~~, fiJed a report of with Police Department. ~ advised that between 2330 and 0830 bours, unknown subjcct(s) sprayed the Greek symbols for [(KG on its front fence, three (3) areas of sidewalk adjacent 10 its residence, and on the rear window of its 1981 Honda Accord. ~ advised ~was a member of the SOrorilY KKG in North Carolina four (4) years ago. t. WOIRHEPP B3D15D.pdf  17  010021Anthrax Page 534 of 643 This incident is significant because Dr. Ivins's address, after completing Hill in 1978 and prior?to moving to Frederick his Post Doctoral Fellowship at the in the 19805, the same town home community and experienced the case oflCKG related vandalism before moving out in 1983. On AprilS, 2007, ,!Called that Dr. Ivins had told it that he had maintained a P.O. of corresponding with individuals who had Box for Ivins had in tnat nc ..dvertisec that 0011ta].nec cherished information solely reserved that Dr. Ivins was advertising that he had access to for members. CW-4 privileged information and would be willing to share the secrets with those responding to the advertisements. Dr. Ivim admitted to CW-4 that be had broken into a KKG sorority house to steal a secret KKG handbook.. CW-4 believed that Dr. Ivins had committed this alleged crime during his Post Doctorate Fellowship at the UNC, Chapel Hill. Dr. Ivins provided CW -4 one of his alternate e-mail addresses as goldenphoenix 111@hotmail.com. A search of the internet for postings under goldenphoenix II I identified the foUowing posting dated February 20,2007, on a website at www.abovetopesecret.com: "Wildswan, you are quite right about what you said about K.K.G. If people look hard enough and dig hard enough, have friends, relatives, perhaps financial resources, etc., then they can pretty much find out about whatever GLO they want. Kappas are noted for being lovely. highly intelligent campus leaders. Unfortunately, they labeled me as an enemy decades ago, and I can only abide by their "Fatwah" on me. I like individual Kappas enormously. and love being around them. I never choose an enemy, but they've been after me since the 1960s, and REALLY after me since the late 1970s. At one time in my life, 1 knew more about KKG than any non-Kappa that had ever lived. Unfortunately I've forgotten a lot. rve read the history of KKG that was written several decades ago about its founding. Question for you; Did your chapter use the combined service, or did you separate your services into the "RedRoom and WhiteRoom"? did you use special blue or white blindfolds? You can reach me at goidenphoenixlll@honnail.com ... asaphoenixrisesfromitsashes ... " This posting is significant in that in his own words, Dr. Ivins defines the depths ofms obsession and knowledge in the sorority KKG. Additionally. as previously described above, the letters used in lbe 2001 anthrax mailings were mailed from a blue collection box located at 10 Nassau Street, Princeton, New Jersey. The sorority, KKG, has an office at 20 Nassau Street, Princeton, New Jersey, located on the same side of the street and 60 feet to the right from the blue collection box . 17 WOIRHEPP B3D15D.pdf  18  010022Anthrax Page 535 of 643 Connection to "4th Grade, Greendale School" The investigation into the fictitious return address on envelopes used for the second round of anthrax mailings, "4~ GRADE," "GREENDALE SCHOOL," has established a possible link to the American Family Association (AFA) headquartered in Tupelo, Mississippi. In October 1999o o A.FA, a Christian organization, published an article entitled "AFA takes WISconsin to court." The article describes a lawsuit filed in federal court. by the At'A Center for Law and Policy (eLP), on behalf of the parents ofstudents at Greendale Baptist Academy. The article focuses on an incident that occurred on December 16. 1998, in which case workers of the Wisconsin Department of Human Services went to the Greendale Baptist Academy in order to interview a fourth-grade student. The case workers, acting on an anonymous tip ilial Greendale Baptist Academy administered corporal punislunent as part of its discipline policy, did not disclose to the staff why they wanted to interview the student. The case workers interviewed the student in the absence of the student's parents and infonned the school staff that the parents were not to be contacted. The AFA CLP filed suit against the Wisconsin Department of Human Services. citing a violation of the parents ' Fourth Amendment rights. _I o b , donations were made to the AFA in the name of "Mr. and Mrs. Bruce Ivins" on eleven separate occa..c;ions begilU1ing on December 31, 1993 . After an approximate two year break in donations, the next donation occurred on November II. 1999. one month after the initial article referencing Greendale Baptist Academy was published in the AFA Journal. It was also d. sco ver. i t IIi e. i . . iied th,ai thii subscription to the AFA Journal, in the name of "Mr. & Mrs. Bruce Ivins." at -. Frederick. Maryland 21702. was active unti l M arch 2005. Frustrations with Members of the United States Senate The imr e5tigation has shown that Dr. Ivins and his wife, are practicing Catholics. Their children are graduates of Saint John's at Prospect Hall Cathol ic High School located in Frederick, Maryland. In an e?mail dated, September 26. 2001, Dr. Ivins states that 2 are actively involved in the church choir. On July 10, 2002. in an e-mail to a both he and as the President of the Frederick County Right to friend, Dr. Ivir.:S identified his wife. Life, as well as having connections to many other pro-l ifelanri-abortion groups. Dr. Ivins later states in the same e-mail. "I'm nOl pro-abortion. I'm pro-life. but I want my position to bt: om: con~i~tent with a Christian." In 2001. members of the Catholic pro-lite movement were known to be highly critical of Catholic Congressional members who voted pro?choice in opposition to the beliefs of the Catholic Church. Two of the more prominent members of Congress who fell in this category were Senator Tom DaschJe. then Senate Majority Leader; and Senator Patrick Leahy. Chainnan of the Senate j udiciary Committee, both recipients of the 2001 anthrax mail ings. In a Septemher/October 2001 newsletter from the organization Right to Life of Greater Cincinnati, Senator Leahy and Senator Dasehle were featured in an article entitled: Pro?Abortion "Catholic" Senators?, wherein a statement declares that these men should no longer be labeled Catholic and should be ex.communicated for aiding and abetting aborti on. During Task Force interviews, 12 18 WOIRHEPP B3D15D.pdf  19  010023Anthrax Page 536 of 643 staff members related that Senator Leahy is pro-choice on the issue of abortion. lbree staff members stated that Senator Leahy was on a pro-life "hit list" because of his stance on abortion. On September 26, 200 J, in an e-mail toafriend.Dr. Ivins writes "The news media bas been saying that some members of Congress and members of the ACLU oppose many of the Justice Department proposals for combating terrorism, saying that they are unconstitutional and infringe too much on civil liberties. Many people don't know it but the official ACLU position is to oppose all metal detectol'3 in airports and schools and other public buildings. It's interesting that we may now be living in a time when our biggest threat to cjvilli~es and freedom doesn't come from the government but from enemies of the government. Osama Bin Laden has just decreed death to all Jews and all Americans, but I guess that doesn't mean a lot to the ACLU. Maybe I should move to Canada .... It On November 17,2001, Senator Leahy's ChiefofStaff, was interviewed by Task Force investigators. The staffer stated that Senator Leahy has drawn fire from some conservative prelife supporters. The staffer advised that Senator Leahy and Senator DaschJe were seen by some members of the public as slowing the passage of the P.A.T.R.1.0.T . Act. Senator Leahy reportedly was critical of the P.A.T .R.LO.T. Act because he believed it was too far reaching and threatened civil liberties. The staffer also related that Senator Leahy had been criticized by conservatives and Republicans for resisting the appointment of Republican appointed judicial nommees. Bacillus Trace Evidence Experts at the FBI's Hazardous Materials Response Unit have found that trace quantities of Bacillus anlhracis and Bacillus subtilus can be detected using biological swabbing techniques similar to those used by United Nations weapons inspectors. Anthrax and Subtilus spores are like seeds, which are surroWlded by a dense shell capable of enduring extreme environmental conditions and temperatures remaining dormant for decades, until such time that nutrients are again available and the organism returns to its vegetative cycle of replication . Even when the viability of an anthrax or subtilus spore is destroyed by environmental or other factors, the genetic material of the organism will remain preserved within the dense shell of the spore, and can be detected by DNA analysis using the Polymerase Chain Reaction (PCR). The investigation to date has yielded a diverse array of items contaminated with anthrax spores from the letters, such as the previously mention mailbox at 10 Nassau Street in Princeton, New Jersey. The contaminated items tenaciously retain anthrax spores on their surfaces, even when subjected to the highs and lows of outdoor temperatures, various environmenta l conditions, and elements o f the weather. The United States Postal Service had great difficulty cleaning the two regional postal facilities that processed the anthrax letters. Trace quantities of anthrax spores were found to persist in those facilities nearly two years after the attacks, despite repeated efforts to eliminate those spores using chlorine dioxide gas and other remediation techniques. Taken together, it is reasonable to believe that baci llus anlhracis and sublilus spores residual from tbe September and October 2001 mailings may still persist in trace amounts inside the home or vehicle of the person who mailed them, even six years after the crime. Regardless 19 WOIRHEPP B3D15D.pdf  20  010024Anthrax Page 537 of 643 of their viability. the mechanics of the-spore structure protects the DNA of the organism, which can be detected using peR techniques. Conclusion Based on the foregoing. I submit that there is probable cause to believe that a search and forensic swabbh"'lg of the Subject Residence and Subjet:t Vehicles may result in collection of evidence relevant to the ctissemination of a weapon of mass destruction (anthrax) through the U.S. mail system in September and October 2001 in violation of 18 U.S.C., Sections 2332a and 1114. Specifically, there is probable cause to believe that a search and forensic swabbing of the Subject Resider..ce and Subject Vchicles as described in thc Attachment to thls affidavit, may reveal laboratory, makeshift, and dual purpose equipment, or materials, supplies. and protocols used in preparation of the deadly anthrax contained in the letters, or may recover trace amounts oflhe non-anthracis Bacillus or the powdered anthrax material remaining in the Subject Residences and Vehicles. or may recover textile fibers that match those recovered from the tape on the envelopes used in the anthrax mailings, or may recover hairs that match the Caucasian hairs recovered from inside the contaminated mail drop box in Princeton. New Jersey, or may produce papers, tape, pens, pencils, notes, books, mnnuals, receipts, financial records, correspondence, address books, maps, handwriting samples, photocopy devices, scanners, printers, toner or printer cartridges, photocopy exemplars, envelope bands, New Jersey addresses, global positioning devices, zip-code information, photogr.iphs, computer files, cellular phone.o;, phone hills, electJnnic pager devices, other digital devices, or other documentary evidence that may link Dr. Bruce Edwards Ivins to the anthrax mailings and identify conspirators involved in the attacks. Based upon my knowledge, training and experience, my consultations with other crimmal investigators, persons engaged in criminal activity frequently store. or inadvertently deposit, these kinds of items in their homes or automobiles and leave them on the premises for many years after a crime, failing to recognize their forensi c value and incriminating nature. This conclusion is sUPP0l1ed by trash covers that have been found in material recently discarded from the house. On October 15 and October 22. 2007. trash was recovered from the front of 622 Military Road, Frederick, Maryland, including receipts from a 1995 purchase as wen as other documents related to KKG sorority. In my experience, vehicles are often used to store documents, maps, receipts, and other papers related to travellhat may be relevant to this investigation. Because this affidavit is part of au ongoing investigation that would be jeopardized by premature disclosure of information, 1 further request that this Affidavit, the accompanying -2 Order, and other related docwnents be filed under seal until further order of the Court. :;:;. 1 ?...,- ,,-C 7t;__ /""5t<;. /. -;e;~ ~,fI.1?~??. ~, ---tf"'";-t!!.... "YJ-,'-7C.~J' :Y "'''''~-= >t1;~ M- N"-'- ':" "';"1 r. ...... ,"O"7' "'" .vj!~. 10/ ">; aD ~"''' ra~~;",J11>> hllM"rUlnl nmpIu, phot.copy Ilmpl., pbOlocnphs, computeT files. ulhdlr plaollU, pbone bUls. eledl"Oolc Pllu devices. olber dIJltal devices, or olbtr documentary evidence. I am satisfied b t b affidavits(s) and any recorded IUtimoDy establish problble cause to believe thai the person or is DOW concealed on the persoa or premises above.described and e:stabli5h grounds Cor the issuance of this warrant. property so described YOUA.R!HER?BYCOMMA.L"'DED!05elrc.hOAorbefo~ t;tJ"~L ( 10~) I the pcBOn or place named above: for the n o r property specified, letvin, this wamnt and making tt-.e searchU (m the daytime. 6:00 A.M. to 10:00 P.M.) (at any time in the day or night IS Trtnd reasonable cause has been established) and iCthe pcnonor property be Cound there to seize same, leaving a copy O{1hi5 watnIlt and receipt (or the person or property taken, and pt'epare I written invcatory oftbe person or temd and ~mplly rerum this warrant to the undcrsig U.s. ~.S. Mqiatrate Judge, as re quired by laW;:~;iea Stat9s District /1) , or \h. Dls!rfct ot Columbia (noiIO exceed ~ (Ilotk) '1 .~t~ prm """T 31 - , L\Ill' lW ~1~Xl) Court . AT 'Ory o.lt...d TNl'C lUlled in Washington, DC pwsuanl. t the domestic ttr:roriIm searcla wa(T'lot provisions N AV WH GTON, Clerk of RulC! ~ Jo.. j\!I.wesrwruq fIOl>. .. _ $l~ of .IudIaaI otraoer ~ A. R()SiN:iUh U8.1IAIlISTRA'It .lDJ: B3D15D.pdf WOIRHEPP  27  010031Anthrax Page 544 of 643 AD 109 tV90l Seilure WAmlI! RETURN DATE WARAArfT R?CEIVEO DATE AND TIME WARllANT EXEC\1TEO COPY Of WARRANT AN D RECI!lI"T FDa m!MS LEFT wml ,{}J 11I uD_7 I~V?NTORY 1,./z./lJ)<'7 $)17. f1 (J()bt\ j'","~1 IL ,D. MADE IN THE rRESENCE Of j)" r:.v C;le~ A~ / It:. I~VENTORY OF rROf'ERTY SEIZED PURSUANT 10 THE WARRANT c...~ )VO e> [lee.-" e. ~. ;,t:!'~' 1<:::- ~- ~.O, (, E. . " ., tJ '" flt'C V'\. il:A./ :; c;. f"Ir/..s t.;< l"<- CERTIFICATION I swear lhat this inventory is. true and detailed account of the " rperty seized by me on the warrant. I /, . , ( .' J' /CC'r ,. . ( Su"""Oed, .worn I.,.n>. Dr. Bruce Ivins has unrestricted access to the suite and has been the sole custodian ofRMR-1029 since it was first grown in 1997. Bacterial contaminant found in attack leners Both of the anthrax spore powders recovered from the Post and Brokaw letters contain low levels ofa bacterial contaminant identified as a strain of Bacillus subtilis. The Bacillus subtili!; contaminant has not been detected in the anthrax spore powders recovered from the envelopes mailed to either Senator Leahy or Senator Daschle. Bacillus sublilis is a nonpathogenic bacteriwn found ubiquitously in the environment However, genomic DNA sequencing oftbe specific isolate of Bacillus subtilis discovered within the Post and Brokaw powders reveals that it is genetically distinct from other known isolates of Bacillus subtili!;. Analysis of the Bacillus subtilis from the Post and Brokaw envelopes revealed that these two isolates are identical. Phenotypic and genotypic analyses demonstrate that the RMR.-l 029 does not have the Bacillus subtili.! contamjnant found in the evidentiary spore powders, which suggests that the anthrax used in the letter attacks was grown from the material contained in RMR-I029 and not taken directly from the flask and placed in the envelopes. Since RMR-I029 is the genetic parent to the evidentiary spore powders, and it is not known how the Bacillus sub/iIi!; contaminant came to be in the Post and Brokaw spore powders, the contaminant must have been introduced during the production of the Post and Brokaw spores. Taken together, the postmark dates, the Silicon signature, the Bacillus subtilis contaminant, the phenotypic, and the genotypic comparisons, it can be concluded that, on at least two separate occasions, a sample ofRMR?1029 was used to grow spores, dried to a powder, packaged in an envelope wltb a threat letter, and mailed to the victims.I Envelopes used in the anthrax attacks In the 200t anthrax attacks, four envelopes were recovered. The four envelopes were all 6 0/.. inch federal eagle envelopes. The "federal eagle" designation is derived from the postage frank in the upper right-hand comer on the envelope which consists of the image of an eagle perched on a bar bearing the initials "USA." Underneath the lettering is the number "34," which denotes the postage value of 34?. The eagle, lettering, and denomination are referred to as the I As the Court may recall from its review, during the course of this six year investigation, the Task Force has from rime to time Gbtained search wamnts, supported by affidavits establishing probable cause, residences and ""'o hm come under in the case, WOIRHEPP B3D15E.pdf  6  010038Anthrax Page 551 of 643 indicia. The eagle and the bar are stamped in blue ink, while the denomination is stamped in grey ink. Approximately 45 million Federal eagle 6 Y4"' envelopes were manufactured by Westvaco Corporation (now known as MeadWestvaco Corporation) of Williamsburg, Pennsylvania, between December 6, 2000 and Man:h 2002 . These Federal eagle 6 Yo" envelopes were manufactured exclusively for and sold solely by the U.S. Postal Serlice between January 8, 2001 and June 2002. Subsequent to the anacks, an effort was made to collect all such envelopes for possible forensic examination, including the identi fication of defects that occur during the envelope manufacturing process. As a result of this collection. envelopes with printing defects identical to printing defects identified on the envelopes utilized in the anthrax attacks during the fall of2001 were collected from the Fairfax Main poSl office in Fairfax, Virginia and the Cumberland and Elkton post offices in Maryland. The Fairfax Main, Cumberland. Maryland, and Elkton, Maryland post offices are supplied by the Dulles Stamp Distribution Office (500), located in Dulles. Virginia. The Dulles SOO distributed "federal eagle" envelopes to post offices throughout Maryland and Virginia. Given that the printing defects identified on the envelopes used in the attacks are transient. thereby being present on only a small population of the federal eagle envelopes produced, and that envelopes with identical printing defects to those identified on the envelopes used in the attacks were recovered from post offices serviced by the Dulles SOO. it is reasonable to conclude that the fed era1 eagle envelopes utilized in the attacks were purchased from a post office in Maryland or Virginia. Of the sixteen domestic government. commercial. and university laboratories that had virulent RMR- I029 Ames strain Bacillus anthracis material in their inventory prior to the attacks, only one lab was located in Maryland or Virginia, where the relevant federal eagle envelopes were distributed and sold by the U.S. Postal Service: the USAMRIID facility at Fort Detrick, MD. Tape. Ink, and Fiber Trace Evidence All four of the envelopes containing these letters were taped along the seams with transparent tape. Due to striation patterns placed on transparent tape as part of the manufacruring process, it is sometimes possible to match a piece of suspect tape with the tape roll from which it originated. According to FBI Laboratory experts. the envelopes were addressed with a pen which dispenses fluid-like ink. rather than the ink typically found in a ball point pen. Due to the distinguishing characteristics of ink, which vary by manufacturer, it is sometimes possible to match ink writing with the pen or brand of pen used to apply it. Forensic analysis of the tape attached to the four envelopes has identified eight different types of fiber attached to the tape : black cotton, black wool, b lack nylon, brown polyester, blue wool, yellow acrylic, red cotton, and red acrylic. Identification of ollection B x at 10 Nassau Street lnfonnation gathered to date suggests that all of the lethal anthrax letters were mailed from a single street collection box located at 10 Nassau Street, in Princeton, New Jersey. The letters were postmarked on either September 18, 200 1 or October 9, 2001 at the same Hamilton 6 WOIRHEPP B3D15E.pdf  7  010039Anthrax Page 552 of 643 Townsh-ip Regional Postal Facility in Hamilton, New Jersey. The collection box on Nassau Street was identified through forensic biological swabbing of every U.S. Postal Service drop box that collects mail to be processed at the Hamilton facility. Further forensic examination of the contaminated mailoox recovered a number of Caucasian human bairs from inside the oox, which are suitable for comparison. Dr. Bruce Edwards Ivjns Dr. Bruce Edwards lvins is a senior microbiologist who has worked for 27 years in the Bacteriology Division at USAMRIID. The Task Force investigation has revealed that Dr. Ivins is considered an expert in the growth. sporulation, and purification of Bacillus anthrads. Dr. Ivins obtained a Bachelor of Science degree in Bacteriology in 1968, a Master of Science degree in Microbiology in 1971 , and a Doctorate of Philosophy (Ph.D.) degree in Microbiology in 1976, all from the University of Cincinnati. Or. Ivins then completed a two year Post-doctoral Fellowship at the University of North Carolina (UNC), Chapel Hill. A review ofUSAMRllD records, Jaboratory notebooks, written protocols, and professional publications has shown that Dr. IVlns has worked with Bacillus anlhracis at USAMRDD since 1980. He has personally conducted and supervised Ames anthrax spore productions for over two decades. At the time of the anthrax mailings, Dr. Ivins possessed extensive knowledge of various anthrax production protocols. Dr. Ivins was adept at manipulating anthrax production and purification variables to maximize sporulation and improve the quality of anthrax spore preparations. He also understood anthrax aerosolization dosage rates and the importance of purity, consistency, and spore particle size due to his responsibility for providing liquid anthrax spore preparations for animal aerosol challenges. Dr. Ivins produced large batches of Bacillus anthracis which were required for such challenges? tests in which vaccinated animals inhale pre-defined doses of anthrax spores to assess the efficacy of the anthrax vaccine. Dr. Ivins's 20 years of working in the laboratories ofUSAMRIID provided him personal, ban(fs.on laboratory experience in the production of liquid spore preparations of Bacillus anthracis. He has used lyophilizer'S, biological safety cabinets, incubators, and centrifuges in vaccine research. Such devices are considered essential for the production of the highly purified, powdered anthrax used in the Fall 2001 mailings. Dr. Ivins was also very experienced in conducting laboratory work in a containment area, and well versed in decontamination procedures specifically for Bacillus anthracis. Dr. Ivins's employment at USAMRIID also provided protection against anthrax infection at the time of the mai lings due to his extensive and current anthrax vaccination history. Unexplained laty night laboratory access USAMRIID contairunent Suite B3 is a Biological Safety Level-3 (BSL-3) suite of laboratories used by USAMRlID Bacteriology personnel for research on dangerous animal and human pathogens. The flask identified as RMR?l 029 was stored in Suite 83 at the time of the letter attacks. Suite access is obtained via a Security Access Control (SAC) badge reader at the WOIRHEPP B3D15E.pdf  8  010040Anthrax Page 553 of 643 door to the cold-side change room, which is secured by a magnetic-lock. A central security system monitors and records a time stamp fo r each SAC badge and keypad request. A review of Dr. Ivins's laboratory access records for Building 1425 was assessed to detennine trends in working hours and evening times (after 6 p.m.) spent in Suite B3. His regular working hours on average consisted of a 7:30 a.m. to 4 :45 p.m. shift, but he would periodically return in the evenings, presumably to check on the status ofvanous experiments. Beginning in mid-August 2001; however, there was a noticeable spike in Dr. Ivins's evening access to the B3 hot Suite. . The following depicts the comparison of Dr. Ivins's total hours spent at night, after 6 p.m., in Suite B3 during the years 2000 and 2001: Night Hours in Suite 8 3 [. 2000 . 2001 [ 35 30 25 = = ~ 20 15 10 5 o Jan Feb. Mar. Apr. May June July Aug. Sept. OCl Nov. Dec. Montm - - - -- ----- - The investigation examined Dr. Ivins's laboratory activity immediately before and after the window of opportunity for the mailing of the Post and Brokaw letters to New York which began at 5:00 p.m. Monday, September 17,2001 and ended at noon on Tuesday, September 18. 2001. Beginning on Friday, September 14, Dr. Ivins worked the following three consecutive evening shifts prior to the mailings with time spent in Suite 83: Day Friday Saturday SWlday Date September 14 September 15 September 16 T ime in Buildin2.1425 8:54 p.m . to 12:22 am. 8:05 p.m. to II :59 p.m. 6:38 p.m. to 9:52 p.m. . T ota l Tim e ID B3 2 hours 15 minutes 2 hours 15 minutes 2 hours 15 minutes . After September 16, Dr. Ivins did not enter Suite B3 in the evening again until September 25. WOIRHEPP B3D15E.pdf  9  010041Anthrax Page 554 of 643 The investigation further examined Dr. Ivins's laboratory activity before and after the window of opportunity for the mailing of the Daschle and Leahy letters to Washington, D.C. which began 3:00 p.m. Saturday, October 6, 2001, and ended at noon on Tuesday, October 9, 2001 . Beginning on September 28, Dr. Ivins worlced eight con?eCutive nights which consisted of the following times in building 1425 with time spent in Suite B3: Day Friday Saturday Sunday Monday Tuesday Wednesday Thursday Friday TlmelB BuildlBg 1425 September 28 7:16 p.m. to 10:59 p.m. September 29 8:02 p.m. to 11 :18 p.m. September 30 9:53 p.m. to 12:04 a.m. October I 9:14 p.m. to 10:43 p.m. October 2 7:24 p.m. to 9:39 p.m. 7:25 p.m. to 10:55 p.m. October 3 6:10 p.m. to 10:12 p.m. October 4 7:40 p.m. to 12:43 a.m. October 5 Date Total Time 1B B3 1 hour 42 minutes t hour 20 minutes t hour 18 minutes 20 minutes 23 minutes 2 hours: 59 minutes 3 hours 33 minutes 3 hours 42 minutes After October 5, Dr. Ivins did Dot enter Suite B3 in the evening again until October 9, for 15 minutes, and then October 14, for I hour and 26 minutes. The investigation has also analyzed the daily schedules, work areas accessed, and the number afhours worked per week of all other researchers assigned to the Bacteriology Division who had access to Suite B3 during the months ofSeptcmber and October 2001. When these records are compared to Dr. Ivins for the same period, Dr. Ivins's habits are significantly different than those of the other researchers, in that he was frequently in Suite B3, where RMR? 1029 was kept, late at night and on weekends when no other researchers were present in Suite B3. Additionally, while Dr. Ivins was in Suite B3 at night, no other USAMRIID employee was present. On March 31, 2005, Dr. Ivins was asked by Task Force investigators about his access to B3 and could provide no legitimate reason for the extended hours, other than "home was not good" and he went there "to escape" from his life at home. A review of Dr. Ivins's and coworkers's laboratory notebooks and projects at the limes in question was conducted. The investigation determined that Dr. Ivins's role in the experiments were minimal, and did not j ustify the time he spent in B3. Dr. Ivins has admitted to investigators that the research be was conducting in 2001 did not require, and does not explain. his late night hours in the B-3 laboratory around the time period of the anthrax mailings. Since producing anthrax spore preparations was one of Dr. Ivins's principal responsibilities at USAMRlID, he had multiple and unfettered opportunities to produce or divert Ames strain spores for illegitimate purposes. His access to Suite 83 and USAMRIID afforded all of the equipment and containment faci lities which would have been needed to prepare the anthrax and letters used in the Fall 2001 attacks. WOIRHEPP B3D15E.pdf  10  010042Anthrax Page 555 of 643 Failure to cooperate in providing laboratory samples - -- ~ ---- -~ -- o Ji ~ L Dr. Ivins provided two sets of samples of Ames anthrax to the FBI which he represented to :he FBI were drawn from RMR-I029. In the first submission in February 2002, Dr. Ivins failed to follow the Dr. Ivins was subsequently notified that the February 2002 samples were unusable due to failure to follow the protocol. A second set of samples, labeled "Dugway Ames spores - 1997" was provided to the FBIR by Dr. Ivins in April of2oo2. Dr. Ivins declares that he uses the terms "Dugway Ames spores - 1997" and RMR-I029 interchangeably, as they are the same flask of material. Both of " April RMR-I 029 samples were submitted for genetic testing and found to be negative for the the presence of the four mutations found in the anthrax used in the attacks. On December 12,2003, an FBI Special Agent accompanied Dr. Ivins into Suite B3 at USAMRIID and identified additional Ames samples of Dr. Ivins' s and others that had not been submitted as part of the above mentioned ? o esponse. Dr. Ivins submitted slants prepared from the newly identified samples to the FBIR on April 7. 2004. On the afternoon of April 7, 2004, an FBI Special Agent accompanied Dr. Ivins into Suite B3, and seized the original samples Dr. Ivins had used to prepare the slants submitted to the FBIR earlier that day. Additionally, the Agent seized the RMR-1029 flask itself All of the samples were secured in the B3 walk-in cold room within a double-locked safe, and sealed with evidence tape until such time that they could be transported to the Navy Medical Research Center (NMRC). which was under contract by the FBI. On June 17,2004. RMR-I029 and the additional Ames samples were submitted to the FBIR, by the NMRC. under the direction of the FBI. Samples of these FBIR submission were sent to the same contracting laboratories for genetic testing. The sample labeled RMR-I029 tested positive for all four genetic markers. therefore. manifesting the genetic characteristics common to the evidence. On March 31, 2005, Dr. Ivins was informed that the slants he provided to the FBIR on April 10, 2002 found to be genetically distinct from the anthrax anthrax material recovered by the FBI from the RMR-I029 flask seized from Dr. Ivins' lab on April 7. 2004. Dr. Ivins was confronted with this and was asked to explain why he did not submit the genetically positive sample which was clearly responsiv~ Dr. Tvins was adamant in his response that there had been no omission from his i submission, and he insisted that he had provided RMR-1029 to the FBI in his second submiss ion of samples in April 2002. WOIRHEPP B3D15E.pdf  11  010043Anthrax Page 556 of 643 Knowledge that his stock of anthrax marched the anM'S used in the attack On March 31 , 2005. when Dr. Ivins was informed. by FBI agents that RMR-I029 shared genetic sinUlarities with the Ames strain wed in the mailings. he indicated that he was already aware of this information. Dr. Ivins explained that many months to a year ago, FBI Special Agent Darin Steele told him that RMR-I029 matched the evidence. SA Steele denied having the conversation described by Dr. Ivins above. As indicated in previous paragraphs. the RMR-I 029 submission provided by Dr. Ivins in April 2002 did not match gmctically or phenotypically. Therefore, neither SA Steele nor any other member of the Task Force could make a comparison between the Ames strain used in the mailing and RMR 1029, until after the June 17,2004 submissi on and subsequent laboratory analysis. When interviewed again on May 7. 2007. Dr. Ivins told investigators that, within three months after the letter attacks, he was aware that his stock of an~ RMR- l029. exhibited unique morphological similarities to the anthrax used in the anacks, and that he allegedly learned this infonnation from three coworkers at USAMR1ID who participated in the forensic analysis of the anthrax in the letters. Each of those three coworkers was interviewed by the Task Force, and deny disclosing such infonnation to Dr. rvios. Over the course of thi s investigation, Dr. Ivins has been repeatedly interviewed and had open access to the law enforcement persoMel responsible for investigating the anthrax attacks. During none of these interactions. did Dr. rvins ever indicate he had knowledge that RMR-I029 had phenotypic similarities to the material used in the anthrax attacks, nor did he suggest that investigators analyze RMR-I 029. To the contrary, Dr. Ivins repeatedly claimed that the anthrax used in the attacks resembled that of another researcher at USAMRlID and were dissimilar to the Bacillus anlhracis Ames organisms maintained in his laboratory, which included RMR-I029. Mental heahh issues and possible motive The investigation has shown that in 2000 and through the mailings in 200 1. Dr. lvins had mental health issues. Dr. Ivins's mental health issues came to the attention of investigators while reviewing e-mails ofUSAMRIID researchers. Through the e-mai ls it was detennined that Dr. lvins was undergoing significant stress in both his home and work life. The mental health issues and stress were significant to the extent that Dr. Ivins sought professional help from a psychiatrist and was immediately prescn"bed medication that started in February 2000. The following are excerpts from e-mails dated April 2000 through December 2001. from Dr. Ivins to a m end regarding work. home. state of mental health, and use of medication (emphasis are as they appeared in the e-mails): April 3. 2000. "Occasionally I get thi s tingling that goes down both anns. At the same time I get a bit dizzy and get thi s unidentifiable ~ m e talJic" taste in my mouth. (I'm not trying to be funny .. . . It actually scares me a bit.) Other times it's like I'm not only sitting at my desk doing work. I'm also a few feet away WOIRHEPP B3D15E.pdf  12  010044Anthrax Page 557 of 643 watching me do it. There's nothing like living in both the first person singular AND the third person singular! ' June 27. 2000, -Even with the Celexa and the counseling, the depression episodes still come and go. That's unpleasant enough. What is REALLY scary is the paranoia . . .. Remember when [ told you about the -metallic' taste in my moulh that I gOI periodically? Ifs when I gel these "par.moid" episodes. Of course I regrellhem thoroughly when they are over, but when I'm going Ihrough them, it's as if I'm on a passenger on a ride. .. . Ominously, a lot of the feelings of isolation - and desolation - that I went through before college arc returning. I don't want to relive those years again . . .. I've been seeing the counselor once a week.June 28, 2000, "Apparently Gore (and maybe even Bush) is considering making the anthrax vaccine for the military voluntary, or even stopping the program. Unfortunately. since. the BioPort. people aren't scientists, the task of solving their problem has faUen on us.. . . . Believe me, with all the stress of home and wade, your email letters to me are valuable beyond what you would ever imagine - and they help me keep my sanity .... " June 29, 2000, "BioPortjusllesled its final 101 of AVA [anthrax vaccine] in a potency test. Ifit doesn't pass, then there are 00 more lots to test, and the program will come to a halt. TIlat's bad for everyone concerned, including us. I' m sure that blame wlll be spread around .'" July 4, 2000, "The thinking now by the psychiatrist and counselor is that my symptoms may not be those of a depression or bipolar disorder, they may be that of a "Paranoid Personality Disorder.July 6,2000, 1 think the o ooo is about to hit the fan ... bigtime. The final lot of AVA, lot 22. isn't passing the polency test, and now there's nothing to back it up. Plus, the control vaccine isn't working. ]t's just a fine mess .. . . .. are spending probably 95 % of our lime on this." July 7, 2000, in an e-mail. Dr. Ivins offered to be interviewed as a case study, as long as it remained anonymous. Dr. Ivins indicated that he did not want to see a headline in the National Enquirer that read, "PARANOID MAN WORKS WITH DEADLY ANTIlRAX!!!" July 23, 2000, "II's been a really stressfu l week, from all stand points. Home, work, and it's not well with the counselor I'm going to. (She said she going to have to ask to get put . .. Sometimes r think: that it's all with another counselor or into a groul) j ust too much." WOIRHEPP B3D15E.pdf  13  010045Anthrax Page 558 of 643 August 12,2000, MLast Saturday, as you probably guessed from my email, was one of my worst days in months. I wish I could control the thoughts in my mind. It's hard enough sometimes controlling my behavior. When I'm being eaten alive inside, I always try to put on a good front here at work and at home, so I don't spread the pestilence... .I get incredible paranoid, delusional thoughts at times, and there's nothing I can do Wltil they go away, either by themselves or with drugs. ~ August 29,2000, e 10% of the Bacteriology Division. If we quit, the anthrax program and BioPort would go down the drain. I'm not boasting,~, but the three ofus have a combined total of 52 years of research o : experience with anthrax. You just can't go out and find someone like~7 oo o with their knowledge, skill and abilities. Ain't goona happen" 2. March 4, 2001, liThe people in my group just don't pick up on what 1 try to say. They are not into the kinds of problems 1 bring up, so it's hard for them to deal with them. The psychiatrist is helpful only because he prescribes the Celexa He's not that easy to talk to, and he doesn't really pick up on my problems. The woman I saw before 1 went into group wanted to get me put in jail. That wasn't very helpful either. I'm down to a point where there are some things that are eating away that I feel I can't tell ANYONE.... " September 7,2001, "I was taken off the Special lnununization Program because of what happened last spring, and I've just gotten back on it, getting my anthrax and Yellow fever shots. We are currently finishing up the last of the AVA. and when that is gone, there's nothing to replace it with. I don't know what will happen to the research programs and hot suite work until we get a new J has been lot. There are no approved lots currently available at BioPort. . . . having us have biweekly meetings on the rPA vaccine progress, and on August 29 I went to the Pentagon - first time there - to go to a meeting in his place on the vaccine. There is a real bag of worms with a new lot of rP A produced by the BDP (a private company) for NCI, who is under contract to USAMRllD. BOP signed a sub-contract with to produce the rPA for a human use vaccine Phase I tria1. They were paid and they produced it. Now they are refusing to release it unJess the Army pays some incredible sum of money for lawsuit indemnification (about $200,000 per year for the next 50 years). The Army refuses to do that of course, and everything is in Limbo." September 15,2001, "1 am incredibly sad and angry at what happened, now:that it has sunk in. Sad for all of the victims, their families, their friends. And angry. Very angry. Angry at those who did this, who support them, who coddle them. and who excuse them." September 26, 2001, "Of the people in my IIgroup," everyone but me is in the depression/sadness/flight mode for stress. I'm really the only scary one in the group. Others are talking about how sad they are or scared they are, but my WOIRHEPP B3D15E.pdf  14  010046Anthrax Page 559 of 643 reaction to the WTClPentagon events is far different. Of course, I don' t talk about how I really feel with them - it would just make them worse. Seeing how di fferently I reacted than they did to the recent events makes me really think about myself a lot. I just heard that Bin Laden terrorists for sure have anthrax and sarin gas. In that same September 26, 200t email. Dr. Ivins states "Osama Bin Laden has just decreed death to all Jews and all Americans" -language similar to the text of the anthrax letters postmarked two weeks later warning "DEATH TO AMERICA, " ''DEATH TO ISRAEL." On October 16, 200t. in an e-mai l.Dr. Ivins ?s coworker communicated the following to a fonner coworker, "Bruce has been an absolute manic basket case the last few days." December 15, 200t , "I made up some poems about having two people in one (me + the person in my dreams); . : . I'm a little dream-self. short and stout. I'm the other half of Bruce - when he lets me out. When r get all steamed up, I don't pout. I push Bruce aside, them I'm Free to run about! Hickory dickory Doc - Doc Bruce ran up the clock. But something then happened in very strange rhythm. His other self went and exchanged places with him. So now, please guess who Is conversing with you. Hickory dickory Doel Bruce and this other guy, sitting by some trees, Exchanging personalities. It's like having two in one. Actually it's rather fun !" The investigation has revealed that Dr. Ivins was prescribed various psychotropic medications including antidepressants, antipsychotics, and anti-anxiety, for rus mental health issues from 2000 through 2006. On July 18,2007, a forensic psychiatrist completed a detailed review of Dr. Ivins insurance hilling records for medical appointments and prescriptions. Additionally, this psychiatrist was provided with an overview of Dr. Ivins's social habits, interests, and obsessions. The forensic psychiatrist stated that based on his experience, if Dr. Ivins was the mailer, it is quite possible that Dr. Ivins retained some kind of souvenir or references to the mailing events. WOIRHEPP B3D15E.pdf  15  010047Anthrax Page 560 of 643 Controversy concerning the anthrax vaccine Beginning shortly after the first Gulf War and through 2001, USAMRIID and Dr. Ivins was the focus of public criticism concerning their introduction ofa squalene adjuvant (or additive) to tbe AVA anthrax vaccine, which was blamed for the Gulf War Syndrome. In 2000 and 2001 , as evident by the e-mails above, that same anthrax vaccine was having problems in the production phase at Bioport, a private company in Michigan responsible for manufacturing the vaccine. The Food and Drug Administration (FDA) had suspended further production at Bioport, and the u.s. government, specifically the Department of Defense, was rurming out of approved lots of the vaccine. The situation placed pressure on select staff members at USAMRIID, including Dr. lvins, who were part of the Anthra.'( Potency lntegratcd Product Team (lPT). The purpose afthe IPT was to assist in the resolution o f technical issues that was plaguing Bioport's production of approved Jots afthe vaccines. In the weeks immediately prior to the attacks, Dr. Ivins became aware that an investigative journalist who worked for NBC News had submitted a Freedom of1nformation Act (FOIA) requests on USAMRlID seeking detailed information from Dr. Ivins's laboratory notebooks as they related to the AVA vaccine and the use of adjuvants. On August 28, 2001, Dr. Ivins appeared angry about the request providing the following response in an e-mail: ''Tell Matsumoto to Idss my ass. We've got better things to do than shine his shoes and pee on command. He's gotten everything from me he wilt get." In early 2002, shortly after the anthrax letter attacks, the rnA re-approved the A VA vaccine for human use, production at Bioport resumed, and anthrax research at USAMRlID continued without intemlption. As mentioned previously. one of the anthrax letters post marked on September 18, 2001 , was addressed to Tom Brokaw, NBC News in New York. Dr. Ivins thereafter re<:eived "the highest honor given to Defense Department civi lians at a Pentagon ceremony on March 14,2003" fo r his work in "getting the anthrax vaccine back into production." Use. of false names and fascination with sororitv group linked to location of anthrax mai lings The investigation has shown that over the past twenty-four years, Dr. Ivins was known to have utilized at least two Post Office Boxes to communicate with members of the public, to pursue obsessions, and possibly engage in the unauthorized use of another person's name. The use of the P.O. Boxes is significant because the post office where Dr. Ivins frequented and maintained a P.O. Box, Coll ege Estates Station, Frederick, Maryland 21702, was serviced by the Frederick Main post office, Frederick, Maryland 2 1701 . Analysis of the USPS shipping tecords indicate the Frederick Main reccived 1000 6 3/4" Federal Eagle banded envelopes on the same day as post offices known to have received envelopes with identical print defects as those seen on the envelopes used in the mailings. On May 14, 2007, identified four communications that it had previously re<:eived from Dr. Ivins. , T does not personally know Dr. Ivins but has repeatedly corresponded with him by mail]. In Dr. Ivins's firm letter to dated May 8, 1982, he used the return address of P.O . Box 1265, Frederick, Maryland 21 702. In the lctters, Or. Ivins expressed his support to WOIRHEPP B3D15E.pdf  16  010048Anthrax Page 561 of 643 . . . . work and an interest in sorority hazing. One of the sororities with which he interest was identified as Kappa Kappa Gamma (KKG). In a ed Dr. Ivins enclosed a "letter to the editor' o~:::::~~~~ o lhe practice of hazing hy o sorority, (KKG). On July 9, 2007, the Post Office Box application (PS Fonn 1093) at the College Estates Station. Frederick, Maryland 21702, for P.O. Box 1103, in the name ofSruee E. Ivins was obtained A review orthe application card revealed that Dr. Ivins rented P.O. Box 1103 on November 12, 1993, providing a military identification and using his Maryland Motor Vehicle 1-152-098-188-301 as proof of identification. Dr. Ivins Administration Drivers Frederick, MD 21702 and telephone numbers of Also listed on the P.O. Box application card to receive mail The last payment on the P.O. Box was made on November 13,2004. A review of Change of Address records revealed that P.O. Box t 103 was closed on December 30, 2005 and no forwarding address was provided. ""as On July 30, 2007, I was interviewed regarding P.O. Box 11 personally know Dr. Ivins but has historically been familiar with his name shown the P.O. Box application and stated that it had never rented a P.O. and had never met, nor had any social or professional association with a Dr. Ivins. However, had previously mentiOned hiS name. _ recognized the name Bruce Ivins because 2 ~ ndicated to 7 2 that Dr. Ivins harassed it in the past, and believed that was frightened of Dr. Ivins, resulting from unspecified incidents that occurred prior to meeting ~so recalled a vandalism incident that occurred in the months prior to leaving the They had awoken one morning in to Maryland area with find Greek letters spray painted the fence, sidewalk, and vehicle apartment. 7 on ACOpyOfthe . . . . . . ~~~~:P~o~li;ceR:~~~~~~~~~~!!~~ ~as obtained from the R and provided the o~:~~:~~~:::=;:=: filed a report of vandalism with Department. . . . . Idvised that between 2330 and 0830 hours, unknown subject(s) sprayed the Greek symbols for KKG on its front fence, three (3) areas of sidewalk adjacent to its residence, and on advised that was a the rear window ofilS 1981 Honda Accord. member of the sorority K.KG in North Carolina four (4) years ago. On r.norning t!!~ WOIRHEPP B3D15E.pdf  17  010049Anthrax Page 562 of 643 his Post Doctoral in the 1 ~ ~!~!~Th~iS~i~nc~i~en ~s~~~bec~a~use~ Dr. Ivins's in 1978 and to moving to Frederick d st~is ~ !S.~'Pel Hill firl:'.. !I!III!.~address, after completing resided :: ~~~~;s~ame town home related vandalism before moving out of the state in 1983. On AprilS, 2007. _ recalled that Dr. Ivins had told it that he had maintained a P.O. Box for the purpose of corresponding with individuals WhO~~~:===to~a~ v~rt ~ ad ~ d~ e ~''sements Dr. he advertised that he possessed a which cherished information solely reserved for KKG members. CW-4 believed that Dr. Ivins was advertising that he had access to privileged information and would be willing to share the secrets with those responding to the advertisements. Dr. Ivins admitted to CW-4 that he had broken into a KKG sorority house to steal a secret KKG handbook. CW-4 believed that Dr. Ivins had committed this alleged crime during his Post Doctorate Fellowship at the UNC. Chapel Hill. Dr. Ivins provided CW-4 one of his alternate e-mail addresses as gQldenohoenixllI@hobnail.com. A search of the internet for postings under goldenphoenix III identified the following posting dated February 20, 2007, on a website at www.abovetopesecret.com: "Wildswan, you are quite right about what you said about KKG. Ifpeople look hard enough and dig hard enough, have friends, relatives, perhaps financial resources, etc., then they can pretty much find out about whatever GLO they want. Kappas are noted for being lovely, highly intelligent campus leaders. Unfortunately, they labeled me as an enemy decades ago, and I can only abide by their "Fatwah" on me. I like individual Kappas enonnously, and love being around them. I never choose an enemy, but they've been after me since the 196Os, and REALLY after me since the late 19705. At one time in my life,l knew more about KKG than any non-Kappa that had ever lived. Unfortunately I've forgotten a lot rve read the history ofKK.G that was written several decades ago about its founding. Question for you : Did your chapter use the combined service, or did you separate your services into the "RedRoom and WhiteRoom"? did you use special blue or white blindfolds? You can reach me at goldenphoenixll1@hotmail.com ...as a phoenix rises from its ashes ..... This posting is significant in that in his own words, Dr. Ivins defines the depths of his obsession and knowledge in the sorority KKG. Additionally, as previously described above, the letters used in the 2001 anthrax mailings were mailed from a blue collection box located at 10 Nassau Street, Princeton, New Jersey. The sorority, KKG, has an office at 20 Nassau Street, Princeton, New Jersey, located on the same side of the street and 60 feet to the right from the blue collection box. Ivins had placed in ~~Ir ~~~~:~:~~~ WOIRHEPP B3D15E.pdf  18  010050Anthrax Page 563 of 643 Connection to "4th Grade. Greendale Schoo1" The investigation into the fictitious return address on envelopes used for the second round of anthrax mailings, "4" GRADE," "GREENDALE SCHOOL," has established a possible link to the American Family Association (AEA) headquartered in Tupelo, Mississippi. In October 1999, AFA, a Christian organization, published an article entitled "AFA takes WISconsin to court. .. The article describes a lawsuit filed in federal court, by the AF A Center for Law and Policy (CLP), on behalf of the parents of students at G=ndale Baptist Academy. The article focuses on an incident that occurred on December 16, 1998, in which case workers of the Wisconsin Department ofHurnan Services went to the Greendale Baptist Academy in order to interview a fourth-grade student. The case workers, acting on an anonymous tip that Greendale Baptist Academy administered corporal punishment as part of its discipline policy, did not disclose to the statTwhy they wanted to interview the student. The case workers interviewed the student in the absence o f the student' s parents and informed the school staff that the parents were not to be contacted. The AFA elP filed suit against the Wisconsin Department of Human Services, citing a violation o f the parents' Fourth Amendment rights . o o o o o o o o_ _donations were made to the AFA in the name ofhMe. and Mrs. Bruce Ivins" on eleven separate occasions beginning on December 31, 1993. After an approximate two year break in donations, the next donation occurred on November II, 1999, one month after the initial article referencing Greendale Baptist Academy was published in the AFA Journal. It was also di",clo v . th at th e . i il.e.red ili... SUbscription to the AFA Journal, in the name of "Mr. & Mrs. Bruce Ivins," at ,. Frederick, Maryland 21702, was active until March 2005. frustrations with Members of the United States Senate The investigation has shown that Dr. Ivins and his wife, o are practicing Catholics. Their children are graduates of Saint John's at Prospect Hall Catholic High School located in Frederick. Maryland. In an e-mail dated.September26. 2001.Dr.lvins states that both he an< I are actively involved in the church choir. On July 10, 2002, in an e-mail to a friend, Dr. Ivins identified his wife. & as the President of the Frederick County Right to Life, as well as having connections to many other pro-lifelanti-abortion groups. Dr. Ivins later states in the same e-mail, "I'm not pro-aix>rtion, I'm pro-life, but I want my position to be one consistent with a Christian." In 2001 , members of the Catholic pro-life movement were known to be highly critical of Catholic Congressional members who voted pro-choice in opposition to the beliefs of the Catholic Church. Two of the more prominent members of Congress who fell in this category were Senator Tom Daschle, then Senate Majority Leader; and Senator Patrick Leahy, Chairman ofthe Senate Judiciary Committee, both recipients of the 2001 anthrax mailings. In a September/October 200 1 newsletter from the organization Right to Life of Greater Cincinnati, Senator Leahy and Senator Daschle were featured in an article entitled: Pro-Abortion "Catholic" Senators?, wherein a statement declares that these men should no longer be labeled Catholic and should be excommunicated for aiding and abetting abortion. During Task Force interviews, 12 WOIRHEPP B3D15E.pdf  19  010051Anthrax Page 564 of 643 staff members related that Senator Leahy is pro-choice OD the issue of abortion. TIlree staff members stated that Senator Leahy was on a pro-life "hit list" because of his stance on abortion. On September 26, 2001 , in an e-mail to a friend,Dr. Ivins writes "The news media has been saying that some members of Congress and members of the ACLU oppose many of the Justice Department proposals for combating terrorism, saying that they are unconstitutional and infringe too much OD civil liberties. Many people don't know it but the official ACLU position is to oppose all metal detectors in airports and schools and other public buildings. It's interesting that we may now be living in a time when our biggest threat to civil liberties and freedom doesn't come from the government but from enemies of the govenunent. Osarna Bin Laden has just decreed death to all Jews and all Americans, but [ guess that doesn't mean a lot ~o the ACLU. Maybe [ should move to Canada .... " On November 17,2001. Senator Leahy's ChiefofStaff. was interviewed by Task Force investigators. The staffer stated that Senator Leahy has drawn fire from some conservative prolife supporters. The staffer advised that Senator Leahy and Senator Daschle were seen by some members of the public as slowing the passage of the P.A.T.R.I.O.T. Act. Senator Leahy reportedly was critical of the P.A.T.R.I.O.T. Act because he believed it was too far reaching and threatened civil1iberties. The staffer also related that Senator Leahy had been criticized by conservatives and Republicans for resisting the appointment of Republican appointed judicial nominees. Bacil1us Trace Evidence Experts at the FBrs Hazardous Materials Response Unit have found that trace quantities of Bacillus anthracis and Bacillus sublifus can be detected using biological swabbing techniques similar to those used by United Nations weapons inspectors. Anthrax and Subtilus spores are like seeds, which are surrounded by a dense shell capable of enduring extreme environmental conditions and temperatures remaining donnant for decades, until such time that nutrients are again available and the organism returns to its vegetative cycle of replication. Even when the viability of an anthrax or subtilus-spore is destroyed by environmental or other factors, the genetic material ofthe organism will remain preserved within the dense shell of the spore, and can be detected by DNA analysis using the Polymerase Chain Reaction (peR). The investigation to date has yielded a diverse array of items contaminated with anthrax spores from the letters, such as the previously mention mailbox at 10 Nassau Street in Princeton, New Jersey. The contaminated items tenaciously retain anthrax spores on their surfaces. even when subjected to the highs and lows of outdoor temperatures, various environmental conditions, and elements of the weather. The United States Posta1 Service had great difficulty cleaning the two regional postal facilities that processed the anthrax letters. Trace quantities of anthrax spores were found to persist in those facilities nearl y two years after the attacks, despite repeated efforts to eliminate those spores using chlorine dioxide gas and other remediation techniques. Taken together, it is reasonable to believe that bacillus anlhracis and subtilus spores residual from the September and October 200 1 mailings may still persist in trace amounts inside the home or vehicle of the person who mailed them, even six years after the crime. Regardless WOIRHEPP B3D15E.pdf  20  010052Anthrax Page 565 of 643 of their viability, the mechanics of the spore structure protects the DNA of the organism, which can be detected using peR techniques. Conclusion Based on the foregoing. I submit that there is probable cause to believe that a search and forensic swabbing of the Subject Residence and Subject Vehicles may result in cancetion of evidence relevant to the dissemination of a weapon of mass destruction (anthrax) through the U.S. mail system in September and October 2001 in violation of 18 U.S .C., Sections 2332a and 1114. Specifically, there is probable cause to believe that a search and forensic swabbing of the Subject Residence and Subject Vehicles as described in the Attacrunent to this affidavit, may reveal laboratory, makeshift, and dual purpose equipment, or materials, supplies. and protocols used in preparation of the deadly anthrax contained in the letters, or may recover trace amounts of the non-anthracis Bacillus or the powdered anthrax material remaining in the Subject Residences and Vehicles, or may recover textile fibers that match those recovered from the tape on the envelopes usod in the anthrax mailings, or may recover hairs that match the Caucasian hairs recovered from inside the contaminated mail drop box in Princeton, New Jersey, or may produce papers, tape, pens, pencils, notes. books, manuals, receipts, financial records, correspondence, address books, maps, handwriting samples, photocopy devices. scanners, printers, toner or printer cartridges, photocopy exemplars, envelope bands, New Jersey addresses, global positioning devices. zip-code information, photographs, computer files, cellular phones, phone bills, electronic pager devices, other digital devices, or other documentary evidence that may link Dr. Bruce Edwards Ivins to the anthrax mailings and identify conspirators involved in the attacks. Based upon my knowledge, training and experience, my consultations with other criminal investigators, persons engaged in criminal activity frequently store, or inadvertently deposit, these kinds of items in their homes or automobiles and leave them on the premises for many years after a crime, failing to recognize their forensic value and incriminating nature. This conclusion is supported by trash covers that have been found in material recently discarded from the house. On October 15 and October 22, 2007, trash was recovered from the front of 622 Military Road, Frederick, Maryland, including receipts from a 1995 purchase as well as other documents related to KKG sorority. In my experience, vehicles are often used to store documents, maps, receipts, and other papers related to travel that may be relevant to this investigation. Because this affidavit is part of an ongoing investigation that would be jeopardized by premature disclosure of information, I further request that this Affidavit, the accompanying Order, and other related documents be fi led under seal unti l further order o f the Court. ~ /.j 4r 0-( I" ler~rC... otf ~ "- ra~~:", Jl TU r 0''''' ~ ,e... SQ~(4 J?,,,,, , .b.::9 7t; .... A-orf.o",-~.-r-, .+- rr~ /lLO v.&. . -:' 7C.. ~.J'-;y 'R> <~y~'7I:".a ~/~ ,e__ M-II" .."" "'-''-''7 -r..... , P'7 ~ A/j ~-;-.g;J/o/., rjo 7 WOIRHEPP B3D15E.pdf  21  010053Anthrax Page 566 of 643 The statements contajned in this Affidavit are based in part on information provided by FBI Special Agents and U.S. Postal Inspectors, on observations made by law enforcement agents, and on my experience and background as an FBI Special Agent. I have not included each and every fact known to me concerning this investigation. I have set forth only the facts that I believe are necessary to establish the necessary foundation for the search warrant. THOMAS F. DELLAFERA Postal Inspector United States Postal Inspection Service OCT Sl ZOO, Sworn to before me this __ day of , 2007 /ti~ United States District Court for the District ofColwnbia Stat.. DIstrIct au DIstrict of C TRUE COP mbr DEBORAH A.AOBINSON u.s. ftIAGIIIIMTE JUOIII WH ITTI GTO ,Clerk WOIRHEPP B3D15E.pdf  22  010054Anthrax Page 567 of 643 ATIACHMENTTO AFFlDAVIT The search of the SUbject Property and Premises will include a physical search and forensic swabbing for the items mentioned in the text of the search warrant, including more specifically the following: I. Any assembled or unassembled Biological Threat Agent (BTA), or trace thereof, and any BTA weapon, andIor any incendiary, radiological, nuclear, chemical or booby trap device. 2. Laboratory equipment used in the production/replication of biological threat agents including, but not limited to agar or liquid growth media, media components, anti-foaming agents, animal droppings or carcasses, autoclaves. bleach, blenders, live animals and materials for their care, cylinder of nitrogen or oxygen. soil, dimethylsulfoxide (DMSO), fennentors, grinders, home brewing kits. incubators, inoculating loops, mechanical stirrers/paint stirrers, microscopes, microwave for making homemade agar plates, mortar/pestle, mining devices, ovens. Petri dishes/improvised glasslpLastic cook dishes. pipettes. pressure cookers. Q-tips. roller flasks, rubber gaskets/seals, screens, sieves, sodium hydroxide, stains/inks/dyes used to identify BTAs. sterilization equipment (Bunsen or alcohol burnerslbutane torches), swabs, test tubes, tissue culture flasks, virus flasks , and other materials. 3. Personal Protective Equipment/Safety Equipment including, but not limited to, biosafety hoods and cabinets (commercial or homemade), masks (surgicalldustIHEPA), exhaust vents, filters (HEPA), gloves, lab coats, mechanical fans, safety glasses/goggles, shoe covers, soap, tubing pipes, and vinegar. 4. Items used in the weaponizationldelivery of biological weapons including. but not limited to pens, tape, paper, scissors, envelopes, target addresses. aerosol canisters, agricultural or garden sprayers, acetone or other drying agents, baby powder, baking powder, baking soda, boxes, commercial/industrial powders. copier toner/printer andlor toner/inks, compressed air or other gas systems, conuneal. cornstarch, explosive, hypodennic syringes/needles, jars or flasks with gauze on top, oatmeal, powdered milk. saltlpepper, spray bottles, squirt guns, stamps, sugar, talcum powder, and other materials. 5. Documents, memoranda, notes or other writings and equipment which include but are not limited to schematics, diagrams, blueprints, surveillance equipment or its manuals and other related paperwork, receipt for purchase of items, how-to magazines, books and pamphlets which describe production, weaponization, and delivery techniques, directions, maps, diagrams, and blueprints of delivery or target locations, communication which would include but not be limited to handwritten notes and drawings, memos and photographs, which would include descriptive infonnation such as telephone numbers, addresses, affiliations and contact points of individuals involved in a conspiracy to acquire, refine, alter, transport, andlor use a biological weapon . Note that such documentary material described herein may be in printed or electronic form such as, but not limited to, computer disks. Such computerized and other electronic material would constinue contraband, fruits, instrumentalities or evidence of the offenses enumerated herein and in the attached affidavit for search warrant. II) WOIRHEPP B3D15E.pdf  23  010055Anthrax Page 568 of 643 6. Publications or other written materials evidencing knowledge of federal laws including those related to biological weapons and other weapons of mass destruction. 7. Photographs, newspapers. or magazine clippings, travel infonnation, brochures, maps, hotel receipts, rental receipts. notes, ledgers, phone numbers, toll records. address books (which may be in printed or electronic format), hand-held electronic memo-type notebooks, scannm, commercial transportation receipts, and firearms. amnnmirion, firearms owners identification docmnents andlor pennits and licenses. digital or other types of pagers and all alpha and/or numeric data/message stored therein, cellular phones. cordJess telephones and their base units. caller identification units and aU alpha-numeric information and data stored therein, biological weapons/agent fonnulas, biological weapons/agents, andlor recipes.. all of which related to the construction of biological weapons, or plans to produce, conceal, or use s uch devices or agents or indicated possible confederates involved or possible targets of such weapons. 8. Any notes, correspondence, memoranda., writings or other documents, regardless of storage media. which relate to the production or refinement of .biological weapons (including anthrax bacteria/spores), or plans to produce. conceal or use such weapons, which items concern, among other things, rental agencies. storage facili ties, biologicaVchemical suppliers or outlets for necessary biological equipment. Examples of such writings include, but are not limited to, those that evidence biological weapons production or predisposition such as sales receipts. invoices, shipping records. literature that relates to biological weapons-making or the production of biological materials. electronic addresses of sites with biological agent/weapons-making information and stored electronic communications, computer passwords or codes, delivery devices, and material from which biological weapons can be made from related chemicals and material s. 9. Bank documents and other records and documents pertaining to the expenditure of funds for illegal activities. assets and funds used to facilitate illegal activities and assets and funds obtained from the conduct of illegal activities. 10. Cellular and telephone records. 11 . Keys, records, or other infonnation pertaining to storage faci lities, safety deposit boxes or other locations where assets or contraband may be stored. 12, Books, videos, pamphlets and any other written, audio, video media advocating production, refinement. distribution of biological weapons or other acts of violence or criminality. 13. Destructive devices, weapons of mass destruction or their components or parts and any written medium pertaining to their design. use, operation, function, capabilities or other attributes. 14. Records, documents, notes, or other material indicating the use o f aliases or false identities including binh certi ficates, driver?s licenses, photographs. travel documents or passports and miscellaneous items such as pennits. registrations, certificates, licenses, and badges of all types. WOIRHEPP B3D15E.pdf  24  010056Anthrax Page 569 of 643 15. Computer hardware, computer software, computer-related documentation. computer passwords and data securi ty devices, records, documents. photos, and materials use to communicate, both written, electronic mail, and by any other means directed either to/from subject. a. Computer hardware is defined as any computer equipment including any electronic devices which are capable of analyzing. coUeeting, converting, creating, displaying, or transmitting electronic, magnetic, optical computer impulses or data. These devices include, but are not limited to, computers. computer components, computer peripherals, data-processing hardware (such as CPUs. memory typewriters. and self-contained laptop or notebook computers), encryption circuit boards. internal and peripheral storage devices (such as fixed disks, external hard disks, floppy disk drives and diskettes, tape drives and tapes and optical storage devices), peripheral input/output devices (such as keyboards, printers, scanners, plotters, and video display monitors), related communications devices (such as modems, cables and connections, recording equipment, Random Access memory (RAM) or Read-Only memory (ROM) units. automatic dialers, speed dialers, progranunable telephone dialing or signaling devices, and electronic tone gathering devices), as well as devices, mechanisms, or parts that can be used to restrict access to such hardware (such as physical keys and locks.) h. Computer software is defined as any instructions or programs stored in the fonn of electronic, magnetic, or optical media which are capable of being interpreted by a computer or its related components. These items include., but are not limited to, operating systems. application software, utility programs, compilers, interpreters, and communications software, as weB as software used to communicate with computer hardware peripherals, either directly or indirectly by telephone lines, radio, or other means o f transmission. Computer software may al so include certain data, data fragments, or control characters integral to the operation of the computer software. c. Computer-related docwnentation is defined as any written, recarded, printed, or electronically-stored material which provides instructions or examples concerning the operating of a computer system, computer software, anellor any related device. including but not limited to, user agreements, software manuals, and operating procedure guidelines. Such documentation may also include material which explains or illustrates the configuration or use of any seized hardware, software, or related item. d. Computer passwords and data security devices are defines as aU those devices, programs, or data - whether themselves or in the nature of hardware or .software - that can be used, or is designed for use to restrict access to, or facilitate concealment of. any computer hardware, software, computer-related documentation, electronic data, records, or materials within the scope or this application. These items include, but are not limited 10, any data security software or infonnation (sucb as test keys and encryption codes), similar infonnation that is required to access computer programs or data or to otherwise render programs or data into usable fonn. e. Records, documents, and materials. as used above, includes all the forgoing items of evidence in whatever form and by whatever means such records, documents, or material, their drafts, or their modifications may have been created or stored, including, but not limited to, any WOIRHEPP B3D15E.pdf  25  010057Anthrax Page 570 of 643 handmade form (such writing or drawing), any photographs or negatives, any mechanical form (such as printing or typing) or any electrical, electronic. magnetic, or optical form (such as floppy diskettes. hard disks, CD-Roms, optical disks, printer buffers, smart cards, thumb drives. memory calculators, electronic dialers, or electronic notebooks), as well as printouts or readouts from any storage devices. f. The term electronic mail is defined as any communications, including, but not limited to, the transfer of signs, signals. writing, images, sounds, data, or intelligence, previously received, transmitted, or stored, or prepared in contemplation of transmission, or any communications in the process of being received or transmitted, whether stored on any electronic media named above or held in temporary, intennediate storage incidental to transmission . 16. Any notes, correspondence, memoranda., writings, address books, documents or other materials, regardless of storage med.ia., which establish an evidentiary link to (1) the particular envelopes and photo-copied letters used in the crime, including samples of documents photocopied. around that time period, (2) the location from which the attack letters were mailed in Princeton, New Jersey. including but not limited to materials relating to the KKG sorority associated with the particular mailbox from which the anthrax letters were sent, and any other materials linking Ivins or his family or associates to the Princeton area, (3) the return address on the envelopes used in the attacks, including any materials relating to "Greendale" schools, "4th grade", "Franklin Park," and postal zip code "08852" and zip code " directories; (4) the victims to whom each of the attack letters were mailed, including but not limited to materials relating to NBC, Ne'N York Post, reporters Gary Matsumoto and Tom Brokaw, the National Enquirer and other publications of American Media Incorporated, letters to the editor, and any member of the United States Congress; (5) a possible motive to commit the attacks in 2001, including but not limited to materials relating to the anthrax vaccine. Gulf War Syndrome, bioterrorism issues, the fonner bio-weapon program at Ft. Detrick, the bio-defense program at USAMRllD, any vendetta against the KKG sorority or sororities in general, dislike for members of the press or members of Congress, and (6) plans or attempts to obstruct justice or otherwise mislead Task Force investigators on this case, including materials relating to the submission of false evidentiary samples. how to defeat a polygraph, the purchase or use of any electronic eaVesdropping or counter-surveillance devices including computer software, spyware or other services, investigative counter-measure materials and detection devices, materials relating to the cooperating FBI sources mentioned in this affidavit, and the purchase of weapons. smn-guns, taser-guns. and ballistic vests. [4J WOIRHEPP B3D15E.pdf  26  010058Anthrax Page 571 of 643 A09J(Rev..5l8S)5arch Wmtnt UNITED STATES DISTRICT COURT FOR THE DISTRICf OF COLUMBIA In the Matter of the Search of Sate Deposit BOI #48, located at Farmers & Mechanics Balik, BraDch #1, 1305 West 7th Street, Frederick, MD, rented .b.Y.B.ru.e.<.E Ivins and o. Frederick, MD SEARCH WARRANT CASE NUMBERO 7 - 5 2 8 - M- 0 , TO: Postal Inspector Thomas F. Dellafera and any Authorized Officer orme United States who bas reasoo to believe A?rKlavit(s) having been made before me by Post.llrupestor Thomas F.Dell.Cu. thai c 00 the person or o on the ptanises known as (~~ and or Ioc:ation) Safe Deposit Bo1148, MD, ren tt-d by Bru<< E. l IOC".~ti:"'~ :"~F " iin & ~~"" ~~:; oo !roDe' N , 1305 West 7th Street. Frederick. t Frederkk, Maryland in the District of Maryland there is now concealed a certain person or propeny. namely (describe the person orptaperty) true qUlntities oCBacOl us anthrads or similiants thereof, hairs, tutile nix", lab eq uipment or m. terlals used In prep.ration orsel~t .gen ls., papers, tape, pens, notes, books, manu.ls., receipt" finandal records of any type, correspondence., .dd ress books, maps., hand writing samples, phototOpy samples, pbotocraphs, computer files , eeUular p hon es, phone bUIs, electronic: p.ger devices, otber digital devices, or other do~umenury f-Ylden~e. I am satisfied that the affidavits(s) and any recon;led testimony establish probable cause to believe that the penon or property so described is now concealed 00 the person or above-described and establish grounds Cor the premises issum'" . fm;, wamnL YO U ARE HEREBY C OMMANDED 10 search on or beCore ~ ! \ 0 z,r;D (Dale) 1- (not to exceed 10~) the person or place earned above for the n or property specified, serving this warrant and making the search U (in the daytime ? 6:00A.M. to 10:00 P.M.) (It any time in the day ornigbt as l find reasonable cause has been established) and if the person or property be found there to seize same, leaving a copy of this WIIrrant and receipt Cor the person or property taken, and prepare a written inventory oCthe person or ~~~ourt to the undersigned U S JudgclU.S. Magistrate Judg~ . as reqUired by l?Or t.h; Ois!nct c.1 ColumbIa reluoetl1nt l007 C!.- L-/: .fR. le 41f11!BORAH A. WJ:;lVV\ I otrleef A? TR HIT/ 1 ON, Clerk Date ItId Tllm Issued in Washington. DC punuant to the domesne terrorism search warrant provisions ROBJNSON "N=.~=-=""::CT;;;~"~o~r~J"':;;: ;.",Offi~ i'O: ,"==---'=--/ --:; . U&~~ ~~~ of JudICIal / -~~~~~~~~~L-~__ SIJIII 27 WOIRHEPP B3D15E.pdf  oe:::1I:~ .  010059Anthrax Page 572 of 643 o . ~' AO 109 2190) SeiZlI1'tI Warrant RETURN DATE WARRANT RECEIVED OA TE AND TQ.1E WARRANT EXEctrrEO COPY OF WAlAAl'IT AND lfCElPT FOR rreMS LEFT WITH /II-r... IIJ /3 11 z..OO7 lNVBNTOR~ MADE IN THE PRESENCE OF J 2:'007 ,-,,,,,, . to CERTIFICAnON I swelr that this inventory is I true and detailed account of the property seized by me on the wamnt. ('ir~ /1 Subsaibed, ,worn to, and ",turned beJ *Cc-~_" / / FILED lilY 0 9 2007 IWI:YIlAVER _ u.s.OIS11llCT COURT CIEIIK :d.~ us. 'lldae Of u.s. .,.-- .. 11- 0 9 ;".?7. WOIRHEPP B3D15E.pdf  28  010060Anthrax Page 573 of 643 AOI06(Rno.SfSS) A.m~ vil for ~h , . manl UNITED STATES DISTRICT COURF/LE FOR THE DISTRICT OF COLUMBIA 0 OCT 3 , 2007 In the Maner of the Search of IWIcr ~I'ER...",._ V.s.~=CWrr Office, Wall Lockers and Laboratory Space of Broce Edwards Ivins, inside Buildings 1412 and 1425 oftbe U.S. Army Medical Researcb Institute of Infectious Dbeases, 00 Porter Street, Fort Detrick, M.D APPLICATION AND AFFIDAVIT FOR SEARCH WARRANT i~lI biB 01 __Tb"o!]m"."'uF",uD""eU l!Jf,,."'r"._____ being duly s,",,'Om depose and ~C .... ... B l am a(n) los(.1 Inspector with the u.s. PostallnsPKtlon ServIce (OffICi.1 Tide) "*'TnN, Clar1< that 0 00 the person of or l1li on the property or premises known as (name, dt:sc""", .... ~"f" ippt ,\ the omu., "aU totkers .nd laboratory sp.ce orRnlce Edwards lviru Inside Buildlogs l411and 14150rthe United Statts Anny Medical Research Institute of Infectiow Distases ("VSAMRIID"), louted on Porter Street. Fort Decrkk, Maryland, including (I ) "aillocer Dumber 35 in Room 127 ofBuildinl1411, (1) ",allloc.ker number 10 in Room 8301 ofOuUdioll"25, (3) ofJke number 19 ofBuiklinl141S, and (4) the biotGnta.intnent laboratary arUs tdenlifitd liS Rooms 8303, B313 and BS0510cated in,ide Building 1425. m the DiJtricl of Maryland, there is DOW concealed a certain person or property, namely crate quantities of B.cillus anthrads or simulaau thereof, haJrs, teltlle nbers, lab equlpmtnt or materials used In preparation of select ae:enu, papers, tape, pens., notes, books, manuals, receipts, nnantlll records or Iny type, car respondence, address books, maps, handwrltln& Ilmples, photocopy samples, pbotographs. computer files, ~eUular phones, phane billA, eletlronic pager devices, otber dlglta' devices, ar olher doc:umentary evidence. which is (.Ui De _ or _ bucs fOf ICCdI and umne KI ronh Itnder P.u~ 4\(b) oCIbe Ft4ml R..t1u orCrimtnal Proeedu.re) evidente rclennt 10 tbe commi.5sion of In Ict of terr-orism, to Include the we of a weapon of mllss destructiaD (anthrax) and the murder and attempt to murder officers and employees of the United States in violation ofTitle...ll.. United States Code, SectioD(s) 2332 oond 1114 . The fa cts to llupport a finding of Probable Cause art as follows: SEEATTAOIEO AFFIDA VITHEREIN INCORPORATED BY REFERENCE AS (f FUllY REST A'ITO HEREIN ContinuOe=~ Kenneth C. Kohl, AUSA u .S. Attorney's Office, Washington. DC (202) 616-2139 ..,:<-...Siprure or AlrIl1lI S 2007 \\OOTai Date Thomas F. Oellafen, Postal lnspector United States P0511J Inspection Service ~b5Cribed in my presence at Washinaton. D.C. (pursuant t6 the domestic terrorism seareh . wamnt proviliOllJo Rule4 1{bX3>> I)EBOIWIA. ROBINSON .....II/IIISIRA1EMlOE Name and Tille of Jl.Id icia! OffIUl" WOIRHEPP B3D15F.pdf  1  010061Anthrax Page 574 of 643 AFFIDAVIT IN SUPPORT OF SEARCH WARRANT I, Thomas F. Dellafera, being duly sworn, depose and say: I have been a Postal Inspector of the United States Posta1 Inspection Service (USPIS) for approximately twenty-one years. 1 am authorized to investigate crimes inVOlving violations of Title 18 U.S.C. Section 2332(.) and 1114. This affidavit is respectfully submitted, pursuant to the terrorism search warrant provision found at Rule 41 (bX3) of the Federal Rules of Criminal Procedure, in support of an application for a warrant to search the following premises: (1) A warrant to search wall locker number 55, located in Room 121, Building 1412, United States Army Medical Research Institute of Infectious Diseases (USAMRDD), Fort Detrick, Maryland . Wall locker number 55 is labeled "Bruce Ivins", and is secured by a combination lock. (2) A warrant to search wall locker number IO,located in Room 8301, Building 1425, USAMRTID, Fort Detrick, Maryland. Wall locker number 10 is labeled "Bruce Ivins X34927", and is secured by a combination lock. (3) A warrant to search the office area of Or. Bruce Edwards Ivins, office number 19, loc.ted in Building 1425, USAMRIID, Fort Detrick, Maryland. (4) A warrant to search Dr. Bruce Edwards Ivins's laboratory space in Room B303 , B313 and B505, Building 1425, USAMRIID, Fort Detrick, Maryland. As discussed below, there is probable cause to believe that a search of the aforementioned locations may result in the collection of evidence relevant to an ongoing criminal investigation into the dissemination of a Weapon of Mass Destruction (anthrax) through the U.S. mail system in September 2001 and October 200} in violation of Title 18, United States Code, Sections 2332(a) and 1114, which killed five people and infected at least 17 others. Overview The FBI and the U.S. Postal Inspection Service (hereinafter "Task Force") investigation of the anthrax attacks has led to the identification of Dr. Bruce Edward Ivins, an anthrax researcher at the U.S . Anny Medical Research Institute for Infectious Diseases, Fort Detrick, MD, as a person necessitating further investigati on for several reasons: (1) At the time of the attacks, he was the custodian of a large flask of highly purified anthrax spores that possess certain genetic mutations identical to the anthrax used in the attacks; (2) Ivins has been unable to give investigators an adequate explanation for his latc night laboratory work hours around the time of both anthrax mailings; (3) Ivins has claimed that he was suffering serious mental health issues in the months preceding the attacks, and told a coworker that he had "incredible paranoid, delusional thoughts at times" and feared that he might not be able to control his behavior; (4) WOIRHEPP B3D15F.pdf  2  010062Anthrax Page 575 of 643 Ivins is believed to have submitted false samples of anthrax from his lab to the FBI for forensic analysis in order to mislead investigators; (5) at the time of the attacks, Ivins was under pressure at worle: to assist a private company that had lost its FDA approval to produce an anthrax vaccine the AnDy needed for U.S. troops, and which Ivins believed anthrax program at USAMRlID; and (6) ~ sent an email to few days before the anthrax attacks warning ~at "Bin Laden and sarin gas" and have "just decreed death to all Jews and all Americans," language similar to the anthrax letters warning "WE HAVE THIS ANTIIRAX ... DEATH TO AMERICA .. . DEATH TO ISRAEL." The Task Force submits that a search and forensic swabbing of the Subject office and work spaces may reveal forensic or documentary evidence that will assist the investigation by linking Dr. Bruce Edwards Ivins to the anthrax mailings and identify conspirators involved in the attacks. The search is for laboratory equipment, tape, ink, paper, textile fibers, Caucasian hair, trace bacillus spores, handwriting samples. photocopy samples, and a11 relevant docwnents, notes and records in various formats, as more fully described in the Attachment to this affidavit. Factual Background In September and October, 200t, at least five envelopes containing significant quantities of Bacillus onthraciJ, were mailed to persons in the District ofColwnbia, New York City, and Boca Raton. Florida, in violation of Tide 18, United States Code, Section 2332(a), which prohibits the use ofa Weapon of Mass Destruction. Bacil/us anthracis is a Weapon of Mass destruction because it is a bacteria and "biological agent" capable of causing death, disease, and other biological malfunction in humans and certain other animals as defined in Title 18, United States Code, Section 178. Bacillus anthracis is classified as a Select Agent, which requires special handling and containment protocols as governed by the Centers for Disease Control and Prevention (CDC) in At1anta. The disease caused by Bacillus anthracis is known as anthrax, subsequently these terms will be used interchangeably. The letters were addressed to members of the national media and to the Capitol Hill offices of two United States Senators located in the District of Columbia. Each of the envelopes contained a handwritten note referencing "9-11-0 I" and warning "WE HAVE THIS ANTIIRAX. YOU DIE NOW. ARE YOU AFRAID? DEATH TO AMERICA. DEATH TO ISRAEL. ALLAH IS GREAT." The two letters addressed to Capitol Hill had fictitious return addresses. The return address read, "4th GRADE," "GREENDALE SCHOOL," "FRANKLIN PARK NJ 08852." At least 22 victims contracted anthrax as a result of the mailings. Eleven individuals contracted. inhalational anthrax (developed from inhaling Bacillus anthracis spores) and the other eleven people suffered cutaneous anthrax (contracted through the skin). Five of the inhalational victims eventually died from their infection: (I) Robert Stevenson, 63, photo editor, American Media Inc., Boca Raton, Florida, died on IO/ 51200t; (2) Thomas L, Morris, Jr., 55, postal worker, Brentwood Post Office, Washington, D.C., died on 1012112001 ; (3) Joseph P. Curseen, Jr" 47, postal worker. Brentwood Post Office, Washington, D.C., died on 10/2212001; (4) Kathy T. Nguyen, 61, hospital employee, New York City, died on 10/3112001; and (5) Ottilie Lundgren, 94, Oxford, CT, died on 1112112001. Another J I persons tested positive fo r exposure to anthrax spores. All of the exposures and cases of infection are attributed to the anthrax mailings, based on their timing, their location, the place of employment of each individual WOIRHEPP B3D15F.pdf  3  010063Anthrax Page 576 of 643 infected, and the identity of the strain of anthrax found in each letter and in the bodies of the five deceased victims. The CDC examined isolates of the Bacillus anthracis extracted from the powder contained in the letters and compared it to body fluid samples collected from the five decedents. The CDC has detennined that the strain of Bacillus anlhracis found in each of the decedents' bodies is the same strain of anthrax found in the threat letters mentioned above. nus particular strain is commonly known as the Ames strain of Bacillus anlhracis. Law enforcement officials have spoken to biological experts who have knowledge and training in the field of infectious diseases including anthrax. According to these individuals quantities of Bacillus antkracis can be produced in a scientific laboratory, such as those found at universities, military research facilities, or other research institutions. Culturing anthrax and working safely with dried anthrax spores requires specific training and expertise in technical fields such as biochemistry or microbiology. It also requires particular laboratory equiprnent such as a lyophilizer or other drying device, biological safety cabinet or other containment device. incubator, centrifuge, fconentor. and various personal protective gear, described more fully in the Attachment to this affidavit. Determination of Origin of Anthrax Contained in 2001 Mailings The mail attacks are believed to have occurred on two separate occasions, which are delineated by the postmark dates of the recovered letters. Letters used in the first attack were postmarked on 09/181200 1 and were focused to media establishments located in the New York area. Three weeks later, letters postmarked 10/09/2001 were sent to two U.S. Senators in Washington, DC. In order to fulty characterize the threat letters and their contents. the Task Force has conducted numerous physical (phenotypic) and geneti c (genotypic) analyses. Physical comparison of the spore powders taken from the Post and Brokaw letters versus the Senator Leahy and Senator Daschle letters reveals obvious differences. The spore powders recovered from the Post and Brokaw letters were granular and multicolored in consistency, while the Senator Leahy and Senator Daschle letters contained fine spore powders that were unifonn in color. Microscopic examination of the evidentiary spore powders recovered frorn all four leners identified an elemental signature of Silicon within the spores. This Silicon signature had not been previously described for Bacillus anthracis organisms. WOIRHEPP B3D15F.pdf  4  010064Anthrax Page 577 of 643 Genetic Analysis of the Anthrax Used in the Attacks Upon visual inspection of the Bacillus anthracis organisms used in the mail attacks, researchers have discovered numerous phenotypic variations, which distinguish it from the original Ames anthrax isolated in 198 1, which is the earliest known sample. The phenotypic variations were identified as differences in morphology (i.e. shape, color, texture) from the original Ames anthrax . Changes in morphology can be attributed to individual genetic mutations within the Bacillus anJhracis DNA. As a whole, the collection of all of the genetic mutations found in the anthrax used in the 2001 mailings, serve to provide a "DNA fingerprint" which can, and has been used to investigate other Ames isolates collected from laboratories possessing the Ames strain. Four individual, highly sensitive, and specific molecular assays capable of detecting four of the genetic mutations discovered in the Bacillus anthracis used in the mail attacks have been developed and validated. Following the mailings, sixteen domestic government. commercial, and university anthracis in their inventories ? the laboratories that had . . strain Bacillus onthracis isolates or samples laboratories, as well as, fro m laboratories in Canada, Sweden, and the United Kingdom. All total, the Task Force has obtained more than 1000 isolates of the Ames strain of Bacillus anthracis from these laboratories and archived these isolates in the FBI Bacillus anthracis Repository (hereinafter "FBIR "). The four aforementioned molecular assays have been applied to each of the more than 1000 Bacillus anlhracis samples contained within the FBIR. Of the more than 1000 FBIR samples. only eight were determined to contain all four genetic mutations. The Task Force investigation has determined that each of the eight isolates in the FBIR is directly related to a single Bacillus anlnracis Ames strain spore batch, identified as RMR-I 029. RMR-I029 was stored in the B3 biocontainment suite within Building 1425 of the United States Anny Medical Institute of Infectious Diseases (USAMRIID), Fort Detrick. Maryland. Access to the suite is afforded only to those personnel who are approved by the USAMRIID Security, Safety, and Special Inununizations Program to have the required background check, training, and medical protection {vaccination or penonal protective equipment (PPE>>. Dr. Bruce Ivins has unrestricted access to the suite and has been the sole custodian ofRMR-1029 since it was fi rst grown in 1997. Bacterial contaminant found in attack letters Both of the anthrax spore powders recovered from the Post and Brokaw letters contain low levels of a bacterial contaminant identified as a strain of Bacillus SUblilis. The Bacillus subtilis contaminant has not been detected in the anUuax spore powders recovered from the envelopes mailed to either Senator Leahy or Senator Daschle. Bacillus subtilis is a nonpathogenic bacterium found ubiquitously in the environment. However. genomic DNA WOIRHEPP B3D15F.pdf  5  010065Anthrax Page 578 of 643 sequencing of the specific isolate of Bacillus subtilLs discovered within the Post and Brokaw powders reveals that it is geneticruly distinct from other known isolates of Bacillus subtilLs. AnaJysis of the Bacillus subtilLs from the Post and Brokaw envelopes revea1ed that these two isolates are identicaJ. Phenotypic and genotypic anaJyses demonstrate that the RMR-l 029 does not have the Bacillus subtilis contaminant found in the evidentiary spore powders, which suggests that the anthrax used in the letter attacks was grown from the material contained in RMR-l 029 and not taken directly from the flask and placed in the envelopes. Since RMR-I029 is the genetic parent to the evidentiary spore powden, and it is not known how the Bacillus subtilLs contaminant came to be in the Post and Brokaw spore powders, the contaminant must have been introduced during the production of the Post and Brokaw spores. Taken together, the postmark dates, the Silicon signature. the Bacillus subtilLs contaminant, the phenotypic, and the genotypic comparisons, it can be concluded that, on at least two separate occasions, a sample ofRMR-1029 was used to grow spores, dried to a powder, packaged in an envelope with a threat letter, and mailed to the victims.1 Envelopes used in the anthrax attacks In the 200 1 anthrax attacks, four envelopes were recovered. The four envelopes were all 6 % inch federal eagle envelopes. The "federal eagle" designation is derived from the postage frank in the upper right-hand comer on the envelope which consists of the image of an eagle perched on a bar bearing the initials "USA." Underneath the lettering is the number "34," which denotes the postage value of 34?. The cagle, lettering, and denomination are referred to as the indicia. The cagle and the bar are stamped in blue ink, while the denomination is stamped in grey ink:. Approximately 45 million Federal eagle 6 W' envelopes were manufactured by Westvaco Corporation (now known as MeadWestvaco Corporation) ofWiHiamsburg, Pennsylvania, between December 6,2000 and March 2002. These Federal eagle 6 y." envelopes were manufactured exclusively for and sold solely by the U.S. Postal Service between January 8, 2001 and lune 2002. Subsequent to the attacks. an effort was made to collect all such envelopes for possible forensic examination, including the identification of defects that occur during the envelope manufacturing process. As a result of this col lection, envelopes with printing defects identical to printing defects identified on the envelopes utilized in the anthrax attacks during the fall of2001 were collected from the Fairfax Main post office in Fairfax. Virginia and the Cumberland and Elkton post offices in Maryland. The Fairfax Main, Cumberland, Maryland, and Elkton, Maryland post ollices are supplied by the Dulles Stamp Distribution Ollice (SDO). located in Dulles, Virginia. The Dulles SOO distributed "federal eagle" envelopes to post offices I As the Court may recall from its review, during the course of this six year investigation, the Task Force has from time to time obtained search warrants, supported by Iffidavits est"'b~l~i,~ru~nlg~~~;~ ~!.'!~~!!!!~~ " ,,,,,,," 0' ?olh,,, individuals who have come under investigative ~ . ... Ci<;eJ<""d tI~ Bcn<,tk assays that . ? prior scarc.bes in thiJ Ulvcstigation w,,~ <... 10 the idenuftCltion ofRMR 1029 at USAMRIID as the ~netlC I " oc,' the 200 1 letter atlKu. WOIRHEPP B3D15F.pdf  6  010066Anthrax Page 579 of 643 throughout Maryland and V.irginia. Given that the printing defects identified on the envelopes used in the attacks are transient, thereby being present on only a small population of the federal eagle envelopes produced, and that envelopes with identical printing defects to those identified on the envelopes used in the attacks were recovered from post offices serviced by the Dulles SOO, it is reasonable to conclude that the federal eagle envelopes utilized in the attacks were purchased from a post office in Maryland or Virginia. Of the sixteen domestic govenunent, commercial, and university laboratories that had virulent RMR.-I029 Ames strain Bacillus aruhracis material in their inventory prior to the attacks, only one lab was located in Maryland or Virginia., where the relevant federal eagle envelopes were distributed and sold by the U.S. Postal Service: the USAMRIID facility at Fort Detrick. MD. Tape, Ink. and Fiber Trace Evidence All four of the envelopes containing these letters were taped along the seams with transparent tape. Due to striation patterns placed on transparent tape as part of the manufacturing process, it is sometimes possible to match a piece of suspect tape with the tape roll from which it originated. According to FBI Laboratory experts, the envelopes were addressed with a pen which dispenses fluid-like ink, rather than the ink typically found in a ball point pen. Due to the distinguishing characteristics of ink, which vary by manufacturer, it is sometimes possible to match ink writing with the pen or brand of pen used to apply it Forensic analysis of the tape attached to the four envelopes has identified eight different types of fiber attached to the tape: black cotton, black wool, black nylon, brown polyester, blue wool, yellow acrylic, red cotton, and red acrylic. Identification of Collection Box at 10 Nassau Street Infonnation gathered to date suggests that all of the lethal anthrax letters were mailed from a single street collection box located at 10 Nassau Street, in Princeton, New Jersey. The letters were postmarked on either September 18, 2001 or October 9, 2001 at the same Hamilton Township Regional Postal Facility in Hamilton, New Jersey. The collection box on Nassau Street was identified through forensic biological swabbing of every U.S. Postal Service drop box tbat collects mail to be processed at the Hamilton facility. Further forensic examination of the contaminated. mailbox recovered a number of Caucasian human hairs from inside the box, which are suitable for comparison. Dr. Bruce Edwards Ivins Dr. Bruce Edwards Ivins is a senior microbio logist who has worked for 27 years in the Bacteriology Division at USAMRIID. The Task Force investigation has revealed that Dr. Ivins is considered an expert in the growth, sporulation, and purification of Bacillus anthracis. Dr. Ivins obtained a Bachelor of Science degree in Bacteriology in 1968, a Master of Science degree in Microbiology in 1971, and a Doctorate of Philosophy (Ph.D.) degree in Microbiology in 1976, all from the University of Cincinnati. Dr. Ivins then completed a two year Post-doctoral Fellowship at the University of North Carolina (UNC), Chapel Hill. 6 WOIRHEPP B3D15F.pdf  7  010067Anthrax Page 580 of 643 A review ofUSAMR.lID records, laboratory notebooks, written protocols. and professional publications has shown that Dr. Ivins has worked with Bacillus anthracis at USAMRIID since 1980. He has personally conducted and supervised Ames anthrax spore productions for over two decades. At the time of the anthrax mailings, Dr. Ivins possessed extensive knowledge of various anthrax production protocols. Dr. Ivins was adept at manipulating anthrax production and purification variables to maximize sporulation and improve the quality of anthrax spore preparations. He also understood anthrax aerosolization dosage rates and the importance of purity, consistency, and spore particle size due to his responsibility for providing liquid anthrax spore preparations for animal aerosol challenges. Dr. Ivins produced large batches of Bacillus anthracis which were required for such challenges - tests in which vaccinated animals inhale pre-defined doses of anthrax spores to assess the efficacy of the anthrax vaccine. Dr. Ivins's 20 years of working in the laboratories ofUSAMRllD provided him personal, hands-on laboratory experience in the production of liquid spore preparations of Bacillus anthracis. He has used lyophilizers, biological safety cabinets, incubators, and centrifuges in vaccine research. Such devices are considered essential for the production of the highly purified, powdered anthrax used in the Fall 2001 mailings. Dr. Ivins was also very experienced in conducting laboratory work in a containment area, and well versed in decontamination procedures specifically for Bacillus anthracis. Dr. Ivins's employment at USAMRllD also provided protection against anthrax infection at the time of the mailings due to his extensive and current anthrax vaccination history. Unexplained late night laboratory access USAMRIID containment Suite B3 is a Biological Safety Level-3 (BSL-3) suite of laboratories used by USAMRIID Bacteriology personnel for research on dangerous animal and human pathogens. The flask identified as RMR-I029 was stored in Suite B3 at the time of the letter attacks. Suite access is obtained via a Security Access Control (SAC) badge reader at the door to the cold-side change room, which is secured by a magnetic-lock. A central security system monitors and records a time stamp for each SAC badge and keypad request. A review of Dr. Ivins' s laboratory access records for Building 1425 was assessed to detennine trends in working hours and evening times (after 6 p.m.) spent in Suite B3 . His regular working hours on average consisted of a 7:30 a.m . to 4 :45 p.m. shift. but he would periodically return in the evenings, presumably to check on the status of various experiments. Beginning in mid-August 2001; however, there was a noticeable spike in Dr. Ivins' s evening access to the B3 hot Suite. WOIRHEPP B3D15F.pdf  8  010068Anthrax Page 581 of 643 The following depicts the comparison of Dr. Ivins's total hours spent at night. after 6 p.m., in Suite B3 during the yean 2000 and 2001 : Night Hours in Suite B3 I_2000. 2001 1 35 l Sept Oct Nov. e :i: ~ 30 25 20 15 10 5 o Jan Feb. Mar. Apr. May June July A~ Dec. MODths The investigation examined Dr. Ivins's laboratory activity immediately before and after the window ofopportunl ty for the mailing oC the Post and Brokaw letters to New York wh.ich began at 5:00 p.m. Monday, September 17,2001 and ended at noon on Tuesday, September 18, 2001. Beginniog on Friday, September 14, Dr. Ivins worked the following tluee consecutive evening shifts prior to the mailings with time spent in Suite 83: Dav Friday Sarurdav Sunday T unelD BUI "E 1425 o ?Id? September 14 I 8:54 p.m. to 12:22 a.m. September 15 8:05 p.m. to II :59 p.m. .m. SeDtember 16 6:38 D.m. to 9:52 D Dat. TIT . ot. Imem B3 2 hours 15 minutes 2 hours 15 minutes 2 hours 15 minutes After September 16, Dr. Ivios did not enter Suite B3 in the evening again until September 25. The investigation further examined Dr. Ivins's laboratory activity before and after the window of opportunity for the mailing of the Daschle and Leahy letten; to Washington, D.C. which began 3:00 p.m. Saturday, October 6, 2001, and ended at noon on Tuesday, October 9, 2001 . Beginning on September 28, Dr. Ivins worked eight consecutive nights which consisted of the following times in building 1425 with time spent in Suite B3: . Dav Friday Saturday T"(me . BUI?Id? 1425 ID Date September 28 7:16 D to 10:59 D .m. .m. 8:02 D to 11:18 D .m. .m. I September 29 m. TIT . at. Imem B3 I hour 42 minutes 1 hour 20 minutes WOIRHEPP B3D15F.pdf  9  010069Anthrax Page 582 of 643 Sunday September 30 Mondav October I October 2 Tuesdav Wednesday October 3 Th=day October 4 Friday October 5 9:53 p.m. to 12:04 a.m. 9:14 p.m. to 10:43 D .m. 7:24 p.m. to 9:39 p.m. 7:25 p.m. to 10:55 p.rn. 6:10p.m. to 10:12 p.m. 7:40 p.m. to 12:43 a.m. t hour 18 minutes 20 minutes 23 minutes 2 bours 59 minutes 3 hours 33 minutes 3 bours 42 minutes After October 5, Dr. Ivins did not enter Suite B3 in the evening again until October 9, for 15 minutes, and then October 14, for I hour and 26 minutes. The investigation has also analyzed the daily schedules, work areas accessed, and the number afhours worked per week of all other researchers assigned to the Bacteriology Division who had access to Suite B3 during the months of September and October 200 1. When these records are compared to Dr. Ivins for the same period, Dr. Ivins's habits are significantly different than those afthe other researchers, in that he was frequentl y in Suite B3, wbere RMR1029 was kept, late at night and on weekends when no other researchers were present in Suite 83. Additionally, while Dr. Ivins was in Suite B3 at night, no other USAMRDD employee was present. On March 31, 2005, Dr. Ivins was asked by Task Force investigators about his access to 83 and could provide no legitimate reason for the extended hours, other than "home was not good" and he went there "to escape" from his life at home. A review of Dr. Ivins's and c0workers's laboratory notebooks and projects at the times in question was conducted. The investigation detennined that Dr. Ivins's role in the experiments were minimal, and did not justify the time he spent in B3. Dr. Ivins has admitted to investigators that the research he was conducting in 2001 did not require, and does not explain, his late night hours in the B?3 laboratory around the time period of the anthrax mailings. Since producing anthrax spore preparations was one of Dr. Ivins's principal responsibilities at USAMRIID, he had multiple and unfettered opportunities to produce or divert Ames strain spores for illegitimate pwposes. His access to Suite B3 and USAMRlID afforded all oflhe equipment and containment facilities which would have been needed to prepare the anthrax and letters used in the Fall 2001 attacks. Failure 10 cOODernte in providing laboratory samples In response to that subpoena, Dr. Ivins provided two sets of samples of Ames anthrax to the FBI which he represented to the FBI were drawn ~rst submission in February 2002, Dr. Ivins failed to follow the protocol _ Dr, Ivins was WOIRHEPP B3D15F.pdf  9 10  010070Anthrax Page 583 of 643 subsequently notified that the February 2002 samples were unusable due to his failure to follow the protocol. A second set of samples, labeled "Dugway Ames spores - 1997" was provided to the FBIR by Dr. Ivins ;n April of2002 . Dr. Ivins declares that he uses the terms "Dugway Ames spores -1997' and RMR-I029 ;nten:hangeably. as they are the same flask ofmatenal. Both of the April RMR-l 029 samples were submitted for genetic testing and found to be negative for the presence of the four mutations found in the anthrax used in the anacks. On December 12,2003, an FBI Special Agent accompanied Dr. Ivins into Suite B3 at USAMRIID and identified additional ~es of Dr. Ivins's and others that had not been submitted as part of the above mention~ response. Dr. Ivins submitted slants prepared from the newly identified samples to the FBlR on April 7. 2004. On the afternoon of April 7, 2004, an FBI Special Agent accompanied Dr. Ivins into Suite B3, and seized the original samples Dr. Ivins had used to prepare the slants submitted to the FBIR earlier that day. AdditionalJy, the Agent seized the RMR-I029 flask itself. All of the samples were secured in the B3 walk-in cold room within a double-locked safe, and sealed with evidence tape until such time that they could be transported to the Navy Medica] Research . Center (NMRC), which was under contract by the FBI. On June 17,2004. RMR-I029 and the additional Ames samples were submitted to the FBIR., by the NMRC, under the direction of the FBI. Samples of these FBIR submission were sent to the same contracting laboratories for genetic testing. The sample labeled R.t\1R.-I 029 tested positive for all four genetic markers, therefore, manifesting the genetic characteristics common to the evidence. he On March 31. 2005, Dr. Ivins found provided to the FBIR on April 10, to be genetically distinct from the anth .., material recovered by the FBI from the RMR-I029 flask seized from Dr. Ivins ' lab on April 7. did not submit the 2004. Dr. Ivins was confronted with this and was asked genetically positive sample which was el"arly "'spom,;v, adamant in his response that there had been no omission in April insisted that he had provided RMR-I029 to the FBI in his second 2002 . Knowledge that his stock of anthrax matched the anthrax used in the attack On March 31. 2005 , when Dr. Ivins was infonned by FBI agents that RMR-I 029 shared genetic similarities with the Ames strain used in the mailings, he indicated that he was already aware of this information. Dr. Ivins explained that many months to a year ago, FBI Special Agent Darin Steele told him that RMR-I 029 matched the evidence. SA Steele denied having the conversation described by Dr. Ivins above. As indicated in previous paragraphs, the RMR-I029 submission provided by Dr. Ivins in April 2002 did not match genetically or phenotypically. Therefore, neither SA Steele nor an y other member of the WOIRHEPP B3D15F.pdf  11  010071Anthrax Page 584 of 643 Task Force could make a comparison between the Ames strain used in the mailing and R..'1R. 1029, until after the June 17, 2004 submission and subsequent laboratory analysis. When interviewed again on May 7, 2007, Dr. Ivins told investigators that, within three months after the letter attacks, he was aware that his stock of anthrax, RMR-I029, exhibited unique morphological similarities to the anthrax used in the anacks, and that he allegedly learned this infonnation from three coworkers at USAMRllD who participated in the forensic analysis of the anthrax in the letters. Each ofthosc three coworkers was interviewed by the Task Force. and deny disclosing such infonnation to Dr. Ivins. Over the course of this investigation, Dr. Ivins has been repeatedly interviewed and had open access to the law enforcement personnel responsible for investigating the anthrax attacks. During DOne of these interactions, did Dr. Ivins ever indicate he had knowledge that RMR-l 029 had phenotypic similarities to the material used in the anthrax attacks, nor did he suggest that investigators analyze RMR-I029. To the contrary, Dr. Ivins repeatedly claimed that the anthrax used in the attacks resembled that of another researcher at USAMRIID and were dissimilar to the Bacillus anthracis Ames organisms maintained in his laboratory, which included RMR-I029. Mental health issues and possible motive The investigation has shown that in 2000 and through the mailings in 2001, Dr. Ivins had mental health issues. Dr. Ivins's mental health issues came to the attention of investigators while reviewing e-mails ofUSAMRIID researchers. Through the e-mails it was detennined that Dr. Ivins was undergoing significant stress in both his home and work life. The mental health issues and stress were significant to the extent that Dr. Ivins sought professional help from a psychiatrist and was immediately prescribed medication that started in February 2000. The following are excerpts from e-mails dated Apri12000 through December 2001, from Dr. Ivins to a friend regarding work, home, state of mental health, and use of medication (emphasis are as they appeared in the e-mails): April 3, 2000, ?Occasionally I get this tingling that goes down both arms. At the same time I get a bit dizzy anl e this unidentifiable -metallic? taste in my mouth. (I'm not trying to be funny, It actually scares me a bit.) Other times it's like I'm not only sitting at my esk doing work, I'm also a few feet away watching me do it. There's nothing like Jiving in both the first person singular AND the third person singular!June 27,2000, -Even with the Celexa and the counseling, the depression episodes still come and go. That's Wlpleasant enough. What is REALLY scary is the paranoia .... Remember when I told you about the "metallic? taste in my mouth that I got periodically? It's when I get these "paranoid" episodes. Of course I regret them thoroughly when they aTe over, but when I'm going through them. it's as if I'm on a passenger on a ride.... Ominously, a lot oftbe feelings of isolation - and desolation - that I went through before college are returning. I WOIRHEPP B3D15F.pdf  12  010072Anthrax Page 585 of 643 don't want to relive those years again. . . . I've been seeing the counselor once a week." lune 28, 2000, "Apparently Gore (and maybe even Bush) is considering making the anthrax vaccine for the military voluntary, or even stopping the program. Unfortunately. since the BioPort people aren't scientists, the task of solving their problem has fallen on us..... Believe me, with all the stress of home and work, your email letters to me arc valuable beyond what you would ever imagine - and they help me keep my sanity ...... June 29, 2000. uBioPonjust tested its final lot of AVA [anthrax vaccine] in a potency test. [fit doesn ' t pass, then there are no more lots to test.. and the program will come to a halt. That's bad for everyone concerned, including us. l ' m sure that blame will be spread around." July 4, 2000, "The thinking DOW by the psychiatrist and counselor is that my s)lllptoms may not be those of a depression or bipolar disorder, they may be that of a -Paranoid Personality Disorder. July 6, 2000, ... ] think the .... is about to hit the fan ... bigtime. The final lot of AVA, lot 22 . isn't passing the potency test, and now there's back it up. Plus, the control vaccine isn' t working. It's just a fine mess. are spending probably 95% of our time on this." July 7,2000, in an e-mail.Dr. Ivins offered to be interviewed as a case study, as long as it remained anon)lllOUS. Dr. Ivins indicated that he did not want to see a headline in the National Enquirer that read. "PARANOID MAN WORKS WITH DEADLY ANTHRAX!! '? July 23, 2000, -It's been a really stressful week, from all stand points. Home, ' with the counselor I'm going to. (She said she thinks I'm going to have to ask to get put ... Sometimes I think that it's all with counselor or j ust too much." August 12,2000, "Last Saturday, as you probably guessed from my email, was one ormy worst days in months. I wish I could control the thoughts in my mind. It's hard enough sometimes controlling my behavior. When I'm being eaten alive inside, I always try to put on a good front here at work and at home, so I don't spread the pestilence . . . .I get incredible paranoid, delusional thoughts at times, and there's nothing I can do until they go away, either by themselves or with drugs.10010 of the Bacteriology Division. August 29, 2000, Ifwe qui~thrax and BioPort would go down the drain. I' m not boasting, _but the three of us have a combined total of 52 years of research WOIRHEPP B3D15F.pdf  13  010073Anthrax Page 586 of 643 experience with anthrax. Youjust can't go out and find someone like_ with their knowledge, skill and abilities. Ain' t gonna happen." March 4, 2001. -The people in my group just don't pick up on what I try to say. They are not into the kinds of problems I bring up, SO it's hard for them to deal with them. The psychiatrist is helpful only because he prescn"bes the Celexa. He's not that easy to talk: to, and he doesn't really pick up on my problems. The woman I saw before I went into group wanted to get me put in jail. That wasn't very helpful either. I'm down to a point where there are some things that are eating away that I feel I can't tell ANYONE .... " September 7, 2001, "I was taken off the Special Immunization Program because of what happened last spring, and I've just gotten back on it. getting my anthrax and Yellow fever shots. We are currently finishing up the last of the A VA. and when that is gone, there 's nothing to replace it with. I don't know what will happen to the research programs and hot suite work. untiI..li.iat a new lot. There are no approved lots currently available at BioPort . . . . ~as been having us have biweekly meetings on the rPA vaccine progress, and on August 29 I went to the Pentagon - first time there - to go to a meeting in his place on the vaccine. There is a real bag of worms with a new lot ofrPA produced by the BOP (a private company) for NCI, who is under contract to USAMRIID. BOP signed a sub-contract with to produce the rPA for a human use vaccine Phase I trial. They were paid and they produced it. Now they are refusing to release it unless the Annypays some incredible sum of money for lawsuit indemnification (about S2oo,OOO per year for the next 50 years) . The Anny refuses to do that of course, and everything is in Limbo." September 15, 2001 , -I am incredibly sad and angry at what happened, now that it has sunk in. Sad for all of the victims, their families, their friends. And angry. Very angry. Angry at those who did this, who support them, who coddle them, and who excuse them.? September 26, 2001, ?Ofthe people in my - group,? everyone but me is in the depression/sadness/flight mode for stress. I'm really the only scary one in the group. Others are talking about how sad they are or scared they are, but my reaction to the WTCJPentagon events is far different. Of course, I don't talk about how t really feel with them - it would just make them worse. Seeing how differently 1 reacted than they did to the recent events makes me really think about myself a lot. I just tonight that Bin Laden terrorists for sure have anthrax and sarin gas. You In that same September 26, 2001 email.Or. Ivins states "Osama Bin Laden has just decreed death to all Jews and all Americans" - language similar to the text of the anthrax letters postmarked two weeks later warning "DEATH TO AMERICA," " DEATH TO ISRAEL." WOIRHEPP B3D15F.pdf  14  010074Anthrax Page 587 of 643 On October 16, 2001, in an e?mail, Dr. Ivins 's coworker communicated the following to a fonner coworker, "Bruce has been an absolute manic basket case the last few days." December 15, 2001, -I made up some poems about having two people in one (me + the person in my dreams): ... I'm a little dream?self, short and stout. I'm the other half of Bruce o when he lets me out. When I get all steamed up, I don't pout. I push Bruce aside, them I'm Free to run about! Hickory dickory Doc - Doc Bruce ran up the clock. But something then happened in very strange rhythm . His other self went and exchanged places with him. So now, please guess who Is conversing with you. Hickory dickory Doc! Bruce and this other guy, sitting by some trees, Exchanging personalities. It's like having two in one. Actually it's rather fun' The investigation has revealed that Dr, Ivins was prescribed various psychotropic medications including antidepressants, antipsychotics, and anti-anxiety, for his mental health issues from 2000 through 2006. On July 18, 2007, a forensic psychiatrist completed a detailed review of Dr. Ivins insurance billing records for medical appointments and prescriptions, Additionally, this psychiatrist was provided with an overview of Dr. Ivins's socia1 habits, interests. and obsessions. The forensic psychiatrist stated that based on his experience, if Dr. Ivins was themailer. itis quite possible that Dr. Ivins retained some kind of souvenir or references to the mailing events. Controversy concerning the anthrax vaccine BegiJUling shortly after the first Gulf War and through 2001 , USAMRlID and Dr. Ivins was the focus of public criticism concerning their introduction of a squal ene adjuvant (or additive) to the AVA anthrax vaccine, which was blamed for the Gulf War Syndrome, In 2000 and 2001 , as evident by the e-mails above, that same anthrax vaccine was having problems in the production phase at Bioport, a private company in Michigan responsible for manufacturing the vaccine. The Food and Drug Administration (FDA) had suspended further production at Bioport, and the U.S, government, specificall y the Department of Defense, was running out of approved lots of the vaccine. The situation placed pressure on select staff members at USAMRIID, including Dr. Ivins, who were part of the Anthrax Potency Integrated Product Team WOIRHEPP B3D15F.pdf  15  010075Anthrax Page 588 of 643 (IPT). The purpose or Ute IPT was to assist in the resolution oftechnica1 issues that was plaguing Biopon's production of approved lots of the vaccines. In the weeks inunediately prior to the attacks, Dr. Ivins became aware that an investigative journalist who worked for NBC News had submitted a Freedom of Information Act (FOIA) requests on USAMRllD seeking detailed infonnation from Dr. Ivins's laboratory notebooks as they related to the AVA vaccine and the use of adjuvants. On August 28, 2001, Dr. Ivins appeared angry about the request providing the following response in an e-mail: '>Yell Matsumoto to kiss my ass. We'vc got better things to do than shine his shoes and pee on command. Hc's gotten everything from me he will get." In early 2002, shortly after the anthrax letter attacks, the FDA re-approved the AVA vaccine for hwnan use, production at Biopon resumed, and anthrax research at USAMRlID continued without intetTUption. As mentioned previowly. one of the anthrax letters post marked on September 18,2001, was addressed to Tom Brokaw, NBC News in New York. Dr. Ivins thereafter receivcd "the highest honor given to Dcfensc Department civilians at 8 Pentagon ceremony on March 14, 2003" for his work in "getting the anthrax vaccine back into production." Use Drials, Dames and fascination with sorority group linked to location of anthrax mailings The investigation has shown that over the past twenty-four years, Dr. Ivins was known to have utilized at least two Post Office Boxes to communicate with members of the public, to pursue obsessions, and possibly engage in the unauthorized use of another person's name. The use of the P.O. Boxes is significant because the post office where Dr. Ivins frequented and maintained a P.O. Box, College Estates Station. Frederick, Maryland 21702, was serviced by the Frederick Main post office. Frederick. Maryland 21701. Analysis of the USPS shipping records indicate the Frederick Main received 10006 3/4" Federal Eagle banded envelopes on the same day as post offices known to have received envelopes with identical print defects as those secn on the envelopes used in the mailings. On May ~,_identified four communications that it had previously received from Dr. Ivins. _ docs nOl Dr. Ivins but has repeatedly corresponded with him by mail]. In Dr. Ivins's first letter to dated May 8, 1982, he used the return address ofP.D. Box 1265, Frederick, Maryland 21 . In the letter.i, Dr. Ivins expressed his support to ~ork. and an interest in sorority hazing. One afthe sororities . he expressed an II1terest was identified as Kappa Kappa Gamma PCIWnall! i W Dr. . WOIRHEPP B3D15F.pdf  1l 16  010076Anthrax Page 589 of 643 On July 9, 2007, the Post Office Box application (PS Form 1093) at the College Estates Station, Frederick, Maryland 21702, for P.O. Box 1103, in the name of Bruce E. Ivins was obtained. A review of the application card revealed that Dr. Ivins rented P.o. Box 1103 on November 12, 1993, providing a military identification and USing his Maryland Motor Vehicle Administration Drivers ' 1-152-098-188-301 as proof of identification. Dr. Ivins MD 21702 and telephone number.; of on the P.O. Box application card to receive mail pi!:yment on the P.O. Box was made on November 13.2004. A Ad,lr",.. records revealed that P.O. Box 1103 was closed on December 30, 2005 and no forwarding address was provided. I Frt, unknown SUl!jCCi(Sj sp",yed of sidewalk symbols for KKG on its front fence, three the rear window of its 1981 Honda A~~~!1! member of the sorority KKG in North I'll rou, his Po,,: Doctoral, in the 19805, community that before mOl, 'i,19 oUI addl,.." after completing to moving to Frederick th,,,,,,," town home case of KKG related vandalism On April 5, 2007, . recalled that Dr. lvin, had told it that he had maintainoo a P.O. . Dr. Box for the purpose of corresponding with individuals , . . . . . . . ... . . , oo 111 . ". he possessed a KKG sorority handbook which contained cherished WOIRHEPP B3D15F.pdf  17  010077Anthrax Page 590 of 643 ,for KKG members. CW-4 believed that Dr. Ivins was advertising that he bad access to priviJeged infonnation and would be willing to share the secrets with those responding to the advertisements. Dr. Ivins admined to CW-4 that he had broken into a](KG sorority house to stcal a secret KKG handbook. CW-4 believed that Dr. Ivins had committed this alleged crime during his Post Doctorate Fel10wship at the UNC. Chapel Hill. Dr. Ivins provided CW-4 one orhis alternate e-mail addresses as goldcnphocnjxlll@bQtmail,com. A search of the internet for postings under goJdenphoerUx 111 identified the following posting dated February 20. 2007, on a website at www.abovetopesecret.com: " WildSO ,..alliocker number SS in RGOm 117 of Building 1412, (1) wan locker number 10 in Room 8301 of Building 1425, (3) office number 19 or Buildinel415,.nd (4) tbe btocontalnment laboratory areas ktenUfled as Rooms B303, B313 and BS05louted Inside Bailding 14ZS. in the District of Maryland there is now concealed a certain penon or property, namdy (4cso;ri~ the peIWIl or JIIl)ptrt)') traee quantities of Bacillus anth ratis or simuJants thereof, hairs, textile fibers, lab rquipment or materials used in preparation of select agen ts, papers, tape, pens, notes, books, manuals, receipts, financial ~ any type, c:orrespondence. address books, maps, handwriting samples, photocopy samples. photographs, computer files, cellular phones, phone bills, electronic pager devices, other digital devices. or other documentary evldenc:e. I am satisfied that the afJidavits(s) and any recorded testimony establish probable cause to believe that the person or property so described is now conc~led on the person or premises above-described .and establish grounds for the issuance oflhis warant. YOU ARE HEREBY COMMANDED to seareh on or before (Date) :;;; (not to exceed 10~) the person or place narned above for the n or property specified, serving this warrallt and (at any time in the day or night as I fmd reasonable malcing the searchU (in the daytime? 6:00 A.M. to 10:00 P.M.) cause: has been established) and if the person or property be found there to seize same, leaving a copY(lfthis warrant and receipt for the person or prope~ take n, and prepare a writt~n inventory of the person or $~2',td and p~qm.ptl!, .... re turn this warrant to the undersigned U.S. JudgelU.S. Maglstnte Judge. as reqUIred by la.W the ::it.ate.s U,strrcl 'vourf F ~ "-DJLMAfI2.R Cq 7-C5D I I j r proURit or IfWYf ~I S1 1110i ~ :L' :2 .L, h'~ l)Qlear.4Timc: Isswd in Washington, DC pw-suant 10 the domestic terrorism .search warrant provisions ofRul'4 1 (~A.ROBINSON Nlme ancI Tille or~ti~1E.IUDQE 's=, . . :::~::"~.~r~J ___":""'::'--"<:--f--'.,L."<~'"~Offi,""i_:::~ DEBORAH A. ROBINSON IJ.I.IIo\GIS1RAlE JIIlClE 26 ' fl(J 11 ~ 0 Dlstnc! cf Columbia A TR py A? Hlnl G"t N. Cler~ WOIRHEPP B3D15F.pdf   4- 010086Anthrax Page 599 of 643 , .AO 109 (2I'XlHeimR Wamnl RE11JRN DATE WARRANT Rf!CEJVED DATE AND T1ME WARRAl'lf EXEO.J'n.D COPY OF WARRANT AND RECEIPT FOR Il1'MS U!FT WITH If) 13) Izoo7 1111 ) =7 :r (;,,00 0 (J) (, Vorc h INVENTORY MADE IN THE PRESENCI! OF;9- /1,1/ J-IIA,=/) INVENTORY Of' PROPERTY SEIZE{) I'VRSVANTTOTHI! WARRANT See C< -r-r~ <- I,q l j . CERTIFICATION I swear that this inventory is o true and detailed account of the property seized by me on the WlIlTBl\l :~jJc~L S"bscribed. oworn to, .00 """"nd ~ this ..t U;:': , FILED 09 2007 _lOAmi_lOll, CUJII( JIIIIIt' or u.s r 27 ~ u.s. DlSTRltT COURT ,.- .< 1 1 -"''1. 0.. ' -1- r WOIRHEPP B3D15F.pdf   010087Anthrax Page 600 of 643 UNl1_J STATES DEPARTMENT OF JU~ .CE FEDERAL BUREAU OF INVESTlGATlON R ecejpt for Property Received / Retufned/Released/Seized File # z..7 ~ I~ ~ t......r=. 7,.""1...' - ~"'"1 C. On (date) _ - " " ,-",' _::..:.::c:... bI",o .:.: b. r _ "'2.:.....-z.....v=7 =, 1-_ _ _ _ _ __ item" listed below were: (s) f!:j Received From Retu rned To Re leased To Seized o o o (Name) __V>~r~v~~~L=-_~ ~~V~~~,,~~_________________________ (Street Add",,) ~..., 1,\ 13.,.;I,J,J") i<.('l..5,VSAM~!> ,"''' I:I..J.,.. ~d_. MO .:Ci.y) E~ cr ;,,L -.1.. I",\~~ R , ...,L.,.. I..-kl"; Desedp.ion of Item(s), .. I : J....., 13E'J:. ~ z.. n ......... ; "-'L,f S C-.-J. s , ~ {. P:;J o .... ~ ~ ........ -l.:.,....... . . . . J. c.",, ~ - I'L "" ; o f' ~;>t.... ~b,.. '-IMI\ ~ SIS').'> I J 1.7 C..-h,_', ') ,7.-" ~ Q. b~ ,_?'- r () _ r u.,~r~ t':>:: \'.\-~ J.l\)i> i. Received By: Received tram: 28 ~~.J.r. c-Ir-t\.J1..,.. ~,.- ~~)--+ (Signature) . . .-- WOIRHEPP B3D15F.pdf   010088Anthrax Page 601 of 643 WOIRHEPP   29 Model"SY160P004221 1, Serial' images of Q1NF4-1 B3D15F.pdf 010089Anthrax Page 602 of 643 u.s. DEPARTMENT or JUSTICE FED ERAL BUREAU OF INVESTIGATION Receipt for Proptrty RectivedfReturn edfReleasedlSeized On (dale) _ _ _ __ At(lime) _ _ _ __ (Name) (L...cx.ation) Suite B31B41BS Building 1425 USAMRIID Ft. Detrick, MD DESCRIP110N Item(s) listed below were: : Received From -: Returned To =Released To =Seized ITEM" .::;::;:;:~===-~ 2 ) VI ?S2 Vflll:UI1m Filter ofVehiek I VloS3 Vaewm filler ohdlide I VI?S4 VKVIn filler ofVeb I , , , , , o 10 11 v l oSS Swab o('ldlid c VI-56 S.....b o(Vdlk:k V20S I V8C1IUrTI liktt ohch 2 Vl?S2 Swab o(Vdl 2 V2?S3 SWIIb ofVeh 2 V2.S4 Vacuum filter of VcIIl Vl-SS VllCUum filter ofVch 2 ?l-sl VlCllum lillCJC ofvdlldc 3 1l I) v3-s2 vacuum fillCf ofveniclc 1 .. 3-s3 VICUUm filler ohdlieJc 1 ?l-s4 fWlIb of -..dridc 1 " " l' - - --- v3--s5 swab ohehk:k 1 ---------(END OF LIST) Total of [6 Item(s) Listed Received from: _ _ _ _ _ _ _ _ _ __ _ _ __ Re('tived by: (5i,TIIlUre) (Sitnaturel ""II! I of f WOIRHEPP B3D15F.pdf  30  010090Anthrax Page 603 of 643 u.s. DEPARTMENT OF JUSTICE FEDERAL BUREA U OF INVESTIGATION Recei pt for 'Property ReceivedIReturn edlReJeased/Seized On (date) At (time) _ {Name) (Location) = J Suite B3fB418 5 Building 1425 Item{s) listed below were: Received From Rerumed To ,-' Released To :: Seized USAMRIID Ft. Oettick, MD IT ..., 17 Dl:SCRrmON 83lJ-$1 83 lJ-s2 8)lJ-u B3lJ~ " " ---20 " l2 B}Il-sS D)L3~ ---- -- 13 8313-s7 B404-sL " " " " B404?s2 """-oJ " ,. JO ....., 8 404-16 8404057 -~. II J2 )] ..0<4 8<04-.8404,, 10 840)-11 B40J-s2 " JS J6 8403053 PRg~ I 0/9 WOIRHEPP B3D15F.pdf  31  010091Anthrax Page 604 of 643 ITEM... DESCRIPTION ~) .s4 ==---: - - )7 31 8.0)-1 5 840 j ?s6 B4{l ) ?s 7 ----- -._------ ---4l ., ., . . " J9 B40) -18 8 106-11 inu,riot swab BlOW interior SWIIb B10fH.) interior SWlb offull'le hood 8306-$.4 ifl~r swab of fume hood ., oo B106-l5 inicrior swab .. . 50 ..,..., 8<06-<2 --------- ........ '_3 ....." ------ " " " ,. " ..,.... . ,..., b4H -s1 b4 H-t2 b4H-t3 btH-1. bt H-1S ",...7 ....... S406-s10 S406-s11 " " " " 60 61 62 ---Pagd Qf9 WOIRHEPP B3D15F.pdf  32  010092Anthrax Page 605 of 643 ~ rrEM/t -=~ DESCRIPTION 8)03-sl B}OJ-s2 S-OS-sl - - - - -- . OJ ._-" 66 B40S-s2 121-51 .. " 10 11 61 ----111-s2 B]OI-sI O}OI-u - ----- ,-, 8309-52 J2 1] B3Q9..s) B)09~ " " BJ09-sS " 77 71 8309-$6 8309-51 8J09-d " 80 81 19-c' ".., ,9-<) " IJ 19-s1 .. " ,9-<, "." 19-$4 . 17 81 B312-sl 8312-52 B312-d Palt J 0/9 WOIRHEPP B3D15F.pdf  33  010093Anthrax Page 606 of 643 ITEMIt OESeRlmo,.. B31H4 Blll?d Bl12-s6 89 ----" --- -- --- 90 - ----<---83011407?sl B1O&/f07?s2 - ----- -- --------- " ." " " " B10ll407-sJ B10ll407-s4 B)II-s1 8]1I-s2 B11I -sJ 8111-.4 811l-sS B111-so 811l-s7 B1II-sl B1II-s9 Bl ll -s lO " 99 ----------- '00 10. 102 103 ." ." '06 107 B11I-III 811l-s1l 831 1-s1l B)II _ sI4 B1 HaJ l\oVa)'""$l 83 Hanway- 51 81 HgJ1_y - S3 .01 10' 110 - ----- II. 112 III B411-s1 84114 ", Pagl! l ()f~ WOIRHEPP B3D15F.pdf  34  010094Anthrax Page 607 of 643 - - --- -. . ITEI\'III DESCRIPTION BI ,,, ' 60 16' '62 '63 , . "S 16' PIIgt 60[9 WOIRHEPP B3D15F.pdf  36  010096Anthrax Page 609 of 643 o ITUIJj DI:SCRJPTION ..IA S t~ me L... O 6B Sterne NSM 167 16. I.' 110 11 1 58 SlemeSBA sc Delta ~me SBA. 9C Delt. SIeme NSM 1C Ddt. Sterne Modihtd a Medi. I7l 17< &9 0cltI Steme S8A. 18 Ddt, Sterne Modified G SA DdlaS[I:I'l'Ir;S8A. 7A DelLi Sterne Modified G III 9A Deltl Sterne NSM 171 179 180 SIeTnCSlftlin SpoRl 4C Sterne Strain Spon:s 4A 181 IS' I&J Delli! Sterne $pores 3A 114 Stt:m~ Strain Spores 4B "' 116 117 DelLi Sterne Sporu 99 SA 79) Sterne ~ Purrified 12 Oct 07 Pucirted Brom Delt. Sterne Dirt)' BoIIOI1I l..a~ C IS' "' "" 191 Stenw: NSM Pre PurifiealiOll Of;ltll. Sterne Spores S8A E Purifoed 2) Oct 06 Unpurified Delli. Slane F From i"I&les ------ ------rm:r 7 11/9 192 Cleff fOP layer Ocltl Sttmt Spores G 2.3 Ott 06 WOIRHEPP B3D15F.pdf  37  010097Anthrax Page 610 of 643 =~ .- -. rr EM. '91 DESCRIPTION .Sterne SBA A ." 195 Steme L'tO PuriticMiOl1 H Sterne}4 F2 o PuriOtd 14 Nowmba' 06 Ames SA4!? Not Pwirltd '" '91 .9o o99 200 2C Ames NSM 201 202 IC Ames SBA lB Ames L+D 3A Alncs 1..+0 ,OJ , . 2B AmesNSM 2A AInu NSM III. Alnes SBA 20' 206 ,., 208 IB Ames SBA S..... b ohink, 9409 S.....b of o o o c o o ~ c = III E ~ U~! .. pi J~ ~'I!" ;'1 l .p U~~ 1" f o f 11~~i U'~~ 11;~i .? ~ R.i:i j ~ ~!Ih ~!iu ~j 0 ~" .... F ~;3b~ n p"" ~~ 0 ~M ~~ 0 ~. O- liM li M "" ij u liM ~. liM ~~ w. g.., ~~ .- "" ~ aj liM C OM "" ~j liM "0 ~M a ~~ ~~ .- ~~ ". 'N liM P(HJ B3D16.pdf WOIRHEPP - - ~. - ~ - ~) ~. y 1 "- - - co i ~~ J rP 1   010102Anthrax Page 615 of 643 I ., t , i o 0 . - "". .. ~ c " ~ ., l o ')1 o ~i; ~ I' h .' " H tl H n ;~ ~ n i" " I t ! f o I ~i oo Ij ~. o n J11 ,1 1 If. j,!f o t i o t , I o I ! t ! ! I? J i' o I 3 < .1 e:?lc ~ o E E 0 E ~ )11 dr Jlj H M n H h U tr H ~i n ; "IIo U Hi o i I H U I ,UI W N: i!1; Ui- o - ~I; i!1; l o 'I~ i!'Z o ))1 Ilj " .. ;~ .t h H ~.! "t i o ~. J ., t ! t t t ! a: o ;; "i ~o t, .j ~r ~o I " , t ~ ! ?~ oo Ij !j n . o. io o t I t o i o ! t I ~i ~i ~ ? o ! , o o i j ~ o ! o I ,I j ~ Ii j~ ! Ii ~, ;~ UJ iii j,!f Jlj iiI kJ }fi , iti kf itl )11 k! Ul .. - '" '" 0 ~ 1- - - - - .. .~ ** kl kf - Jlj III )11 itl kl - * kl iIi iii ki N c: ~ 0 0 Ul .J IUl D.. Ul .. . "" p" n o . jhi !i ~ .. I'o o .f! ;r' E 1;; 'c. " III "- G~ j ! < If o ~ !. c u 0 1i k ~ 0 I~: ~ ~. oi ~t>: E ;i 0 o ; d~ o oi ~15 li'~ I~~ !i? ~ r 11~ ON n il f~' ~dh ~15 nIl . lid, ! " ~15 i! r' ~hn ?!, ~15 ~~ ~~ u ~o i.., ~(") i~ .' ,.'. Iun jlq!;! ~M ~15 < " f~? ' ~ n ,' . q ~ 1~ fW ? 1 li~~ t ~ Jii~\ IHI '0, jIly !d~ n. !ifi !h o f! ~. 0 f~? ?~ ON ~ 15 i..., ill 15 ?~ i~ "15 00 ~M ON ON 8 ~~ ON ~~ u ON ~~ ~i ~~ ~~ u i~ u ~~ u ON ~~ ~~ '" M i 1 - ~ . . . . ! '" - :s- ~ i x cS f I ~ .- 0> . . 0 . I~ ~. . ~ I 1 '" I ~ ; ; \,of ~ B3D16.pdf WOIRHEPP   010103Anthrax Page 616 of 643 1 .1 H !}' ~i; II d ;e j" " ij ;~ o i io o ! 1 ! I t i t o t ! .'." 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I N 1 " <> - Ol 1 1 i 1 " " " 1 " " 1 B3D16.pdf WOIRHEPP   010104Anthrax Page 617 of 643 WOIRHEPP   SPSTL Sample Set CU5tomM"' .ntory Form Amount Comments I Dale : ~12002 Orlglnalor : Sample Description (Matrix) _34 1 eu:IUI'e Slane 8011:3013 _35 _3. _ 37 1 Cutture Slanl Box3013 1 Culture SLant Box 3 013 ,.- _,.,.. .... ,.-...._...,,0..0' .(1:2 _ -"':0'.(11-<<2 -------.. _ : 1)1.(1'.0::1 1'*_ ....... : 001?0 1.(12 FBIROOS ;!'!':0I'IgiNiI__ _ _ .... __ . .. "1nIWocI1I ........ ~lM "8' 00I.'=':_0I.~1.G! _ro-.aa..._oo .... _ . . MIlff, 8AAO, 22CZ" 1 ;r:ooIgNI _ _ _ 119101:. __ 1"SA.loI _ 0I"0;l _._WMled .. .,. ..... .=-_OMIoOl. .... FIlAXl5 CD5...-.I _ dlwr _ _ EWIoIo MAEl' :O I S?18iT 1 ;,:.....,.,.,_ ' _ _ rd, ~_O!L"'7nJ. ......... _ _ ....... _ _ SAI..oI " g,OOI _r8nXl6.03II, a-1oIAEI': OIS? lecT """"-~ ............ 1 Cult"'"' Slant Box3 013 _3. -----... ,.Q2 ... _ :OO-o 1 'lI': ...... _>el_ ..-..Iord _ TSAI.,.oI 'nm.:)l!,~ ..... ovO!l~ _ " ' _ _ pIa,-. """ flMflUOei.03I. ~ _ , " " ' _ . , IN ....... 1 CultUl'o Slant Box 30t 3 ,.- _,.... 1III_ loIfICI' : O' S?2oo e..n.t. ~ EI' : O I 8 ? 243' 1 O -.--.....i!': ~ "'nl l ~ l!I.AlM 1'9 10011.~_(\fI .. A005.03I,.,.,....... .... _ _ _ _ ord ....... FB 0IIt.!,m. ,..... Page4 9/Septembcrl2002 B3D16.pdf 010105Anthrax Page 618 of 643 WOIRHEPP   Activity and C munication Log Sampl es Dale o f Entry S hipp e d Initia ls FBIA005 Dat e 01 Act ion Ac tlvltyCommunlca tionOu tc omeNot es containers and stored in the Building 1412, Room 223 refrigerator. Ready for shipment to Dr. Paul Keirn Odd sample numbers (Samples FBIAOOS.OO110 FBIROO5.031) were packaged In Saf?T-Pac 5/April12002 SlApril12002 os os 19 5/April/2002 Each of the even sample numbers (Sample FBIR005.002 to FBIROOS.038) were resuspended in cryoproleClent (HIB with 12.5% glycerol). 8 drops each wore added to 12 cryov;als (A through L) and frozon in the Building 1412, Room 223 freezer. 5/April/2002 8/Aprll12002 Odd sample numbers (Samples FBIROOS.OOI 1 FBIAOO5.037) shipped to Dr. Paul Keirn via 121April/2002 0 FedEx. Tracking numbers: 477516735189, 477516735190, 477516735204 and 4775 1673 5215 Frozen 'l ' cryovial 01 sets FBIR005.002 to FBIROD5.038 tested lor viabllty via culturing on 50/0SBA. 9/Septomber/2002 os TA 22/July/2002 23/July/2002 All samples viable and sensitive to gamma phage except lor 005.026. This sample appear to 9/September/2002 be contaminated. It was gamma phage resistant ; questionable B. 8nhtracis presence. TA Page 1 9/19/2002 B3D16.pdf 79 Em owuil Sou/uq010107Anthrax Page 620 of 643 ( , ,", ~ , \; .~ ) : TESTMATE~EIP .. ~ -.- .Q.Q TIINVENTORY LOG FORM ,,~ u ( 1<1 II- Q)a.Y 7'1'11"-' .0.0 ()Ba1telle LotIBatchllD num r:_. .oo Pllffkw T<. 't ~: " '\ ...a~J . . 0.... Pi! ?'>>"'p<>)(1hN: Safety 0ftIce) DIAGNOSnC SPECIMEN (ctaS5ification Group (b) _ b7C CONTROLLED BtolOGICAl OR BiOlOGICAl.. PRODUCT X - Human or INFECTIOUS SUBSTANCE (ETIOLOGIC AGENT). AFFECTlNG HUMANS (1hnJ: SefelyOftice INFECTIOUS SUBSTANCE (ETIOLOGIC AGENT). AFFECnNG ANlt.lALS 0"-Y (thru : TOXIN ( ETIOLOGIC AGENT) (IIVU: - Non-Human J Inv. - and bc-Ilst -. (thru: Clinical Use SIoIogIcs eon1q.-L.CIIIoo O I/ f U.S. ARMY MEDICAL RESEARCH INSTITUTE OF INFECTIOUS DISEASESQ SHIPMENT REQUEST REAGENT MATERIAl J DIAGNOSTIC SPEC I ~EN I CONTROLLED BIOLOGICAL PRODUCT J BIOLOGiCAl AGENT I ETIOLOGIC AGENT (INFECTIOUS SUBSTANCE OR TOXIN ) I RADIOACTIVE MATERIAl. Please ch8Cl< appropriate bIoc:tt and routelt1rough appropriate approving bE b7C ai.rthoftty, ? x REAGENT MATERIAl. Non-ha:zartIous or (a) ~OLOGICAL Hazanlous (lh",: Sahlty 0Iftc0) (b) DIAGNOSTIC SPECIMEN loas,.- Group CONTROLLED BIOlOGlCAl OR Human or Non-Human I (e)) (lhru: SahltyOftiee) PRODUCT t)c.list INFECTIOUS S~TANCE (ET10L~IC AGENT). AFFECTING HUMANS (thru: (thru: Radiation Prolectioll Office) . ......-.. (thnI: Safety omee) inv. and (ttvu: Clnc:al Use BIoIogic:s Safety INFECTIOUS SUBSTANCE (ETIOLOGIC AGENT), AFFECTING MlIMAlS ONLY (ttvu: TOXIN (ETIOlOGIC AGEND (O"\J: Safoly omee). R.6DIOACTIVE",~ ~0I'fie8 : ~ otrtal ~ I Control;; . BIOLOGICAL AGENT, NON~NF ECTJOUS {ttvu: Safety omce} OTHER: O~tion _ . '" polyp~ .. - .._- of materiai(a): 30 ml of Bo .,lhracll Ames spores, 3.9 X 10"'101ml. 1.17 X 10"'12 spor. . total, In 3 tubes, 10 ml per tube. 10 en~ a:nIrBct agantzation (Battaile ) 10 perform COi,b~ study, Quantity (mi. mg. ,.. ): 30 ml, 3.9 X 10"'101ml, 1.17 X 10"12 kltal _.__ .o.. _--_._- ;.._ . - _ .__._-.- ._- -' -_....... . _ Ship 10 Address (indude telephone number. day and reqo:~: physically manned 24 hOurs a day, No ~ or cell phone numbers.1 emergency,of I :m:F number must be ~REF .JS--3, 5 1425 S,R. 142, West Jefferson. Ohio 43182 . Day 'i Raqu,,"ed Shipment Os 24 hour? d 18 June, 2001 _ Requirements: None Charga to PPC: SGOB Method of Shipment :x... UPS p.Jr Express USAMRIIOCo~ :x... Dfylee Refrigerated (_ lee. 6 o Freezer. ID# o Other AMOUNT DATE CONTAINER AMOUNT USED REMAINING PURPOSE OF USE STUDY PERFORMED OF USE (UNITS ..... ( BY ID NO. ) (UNITS ~ ) (type of ""4 etc.) ,//d / :z J.J..R ~V .~ ",. . J 41, .;.I~ ,011"(""1 ,~ /w Y'ft<) ~;6) 'f& .'ft (' "l/.., I-I-- .<'._. /J. u""C < Page 632 ffl{yi-ai. gr 5` T. QL B3D16.pdf 010120Anthrax Page 633 of 643 , " . , o , ., . o B3D16.pdf WOIRHEPP   010121Amhra>< Page 634 of 643 . @4 fe ?jx' 1 if P1 2 . 32B3D16.pdf 010122Anthrax Page 635 of 643 .;: , .....,. .. ~~. ~ I B3D16.pdf WOIRHEPP   010123Anthrax Page 636 of 643 (" , . ... ~. " o o o o. 0 -"" .H' --, >v o "- ~ ..... r-.. '< ~ ~ <;;:, ~ ~ .:J.. <;:) o .1 .... . . . . ;.. .: fir o""lIi''' '1II a; ~ ~ o 0 o -- _. _.- - ~ : ; ~ ;;- ;. . ,.. _. 'r. !:. . ..:::.. I.i... ,t , .. - _.. ........ - -. . _--f1I , "'It (~ o B3D16.pdf WOIRHEPP   O10124Anthra>< Page 637 ??-sqgne B3D16.pdf O1 O125Anthra>< Page 638 uf 643 I #277* vy nf'irlnHTFuwmxlu 2 Limw 3 . wmmnB3D16.pdf O10126Anthra>< Page 639 B3D16.pdf 010127Anthrax Page 640 of 643 , i. .- o .1 .! "" -" , , ~ 0 -, o J '\ ,~ \ o , , " B3D16.pdf WOIRHEPP   010128Amhra>< Page 641 ?f643 B3D16.pdf O1 O129Anthra>< Page 642 uf 643 v. Mile q-B3D16.pdf 010130Anthrax Page 643 of 643 o 147 . (1) overpack containing (1) Falcon tube (50mL I in (2) Ziplocks ~arked B. anlhracls Ane s 3xlOlOspores/mL overpack :'abeled " Do :1Ot open! Anthrax-AIDes - Lots!" lrr,L in 2mL lube 1425 Room Ivins P~314 co!d room B3D16.pdf WOIRHEPP