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Microbial Forensics

200 nm

WMD Incident Threat Spectrum








Overall Threat is Low

Biological terrorist threats
Pathogenic viruses, bacteria, fungi and toxins:
– Anthrax
– Smallpox
– Plague
– Toxins
– Tularemia
– Foot & Mouth Disease
– Enterics

Pre-Event Planning


Robert Stevens. Princeton. 2 . both postal workers in Washington. NY Post (9/18/01) Tom Brokaw. often fatal Bacillus anthracis spores can live in soil for many years. Ottilie Lundgren of Oxford. Humans can become infected with anthrax by handling products from infected animals or by inhaling anthrax spores from contaminated animal products. and Thomas Morris Jr... often fatal Gastrointestinal – consumption of contaminated meat. Boca Raton. Amerithrax Five distinct anthrax attacks. a photo editor at a supermarket tabloid based in Florida. NBC (9/18/01) Senator Tom Daschle (10/09/01) Senator Patrick Leahy (10/09/01) AMI Building contamination. NJ • • • • Editor. no envelope/letter Anthrax victims Joseph Curseen Jr. and Kathy Nguyen. Fall 2001 Four letters mailed from postal collection box. Conn. a New York hospital worker. the last person to die of anthrax in 2001. FL Bob Stevens died 10/05/01.Anthrax Anthrax infection can occur in three forms: Cutaneous (skin) – 95% of infection cases Inhalation – symptoms resemble common cold.

sickened 17 others AMI Building. AMI Building. Boca Raton. Florida 2001 3 .killed five people. Florida FBI Hazardous Materials Response Teams.

staff or equipment to conduct bioforensic analysis • No methods to enable handling of biothreat agent powders • No methods to support traditional forensic analyses of evidence contaminated with a biothreat agent • No place to receive large quantities or large pieces of evidence contaminated with a biothreat agent • No established evidence handling.Post Event Investigation What kind of pie? Event! “Pre-Event” Post-Event How did they make it. peer review of methodologies or central coordination for bioforensic analyses Amerithrax • Investigation focus – Knowledge – Access – Laboratory experience – Motive 16 US Labs – 1. where was it made? Bioforensic Investigative Challenges in 2001 • No dedicated biocontainment labs. what’s in it and Is there a stockpile of pies? WHERE are they? Thethe Pieequipment Terrorist What was they used? Who made the pie.200 employees 4 . analytical methodologies. quality guidelines.

Virginia First Use of Spore Concentration to Locate Evidence Task: Search for “Daschlelike” letter among 1 million quarantined letters packed in 642 plastic bags in 230 drums (55 gal) Initial plan: Sort all letters by hand Estimated time.5 to 2 months Employed a microbiological sampling approach 5 . 1.Inside the warehouse Interior of Mail Sorting Facility Large-Scale Mail Search Springfield.

0 4:00 5:00 600 Most “pristine” source of spore powder Time (PM) All four air samplers detect spores 6 .0 S1 S2 S3 S4 8.0 6.2 m Airflow Air sampler Air handler (HEPA filter) Safety cabinet Mailbag Sampling Summary Total bags 642 ≥1 cfu 62 ≥10 cfu 20 >100 cfu 7 All bags  10 cfu sorted by hand 642 results in 3 days Detection of Airborne Spores Coincident With Finding of Leahy Letter Colony forming units Sealed envelope 10.0 2.Containment Facility Constructed Staging Sorting room: 83 x 73 x 16 ft 6060 ft2 96945 ft3 Decon 26 x 22 x 5 m 572 m2 2860 m3 50 ft 15.0 4.


 For whatever reason. However. The envelopes are of the pre‐stamped variety. 3. the first letter of the  first word of each sentence is written in slightly larger upper case lettering. of forensic value were found on the letters to Senators Daschle and Leahy. The author uses the words "can not. 5. They are not the  traditional business size envelopes. The author uses dashes ("‐") in the writing of the date "09‐11‐01. more detailed  version. and the letters to Senators Daschle and Leahy likewise contain the same handprinted text. The letters to the New York Post and Brokaw contain identical handwritten text. The names and address on each envelope are noticeably tilted on a downward slant  from left to right. The author writes in all upper case block‐style letters. No matches were found.Letters to “Tom Brokaw NBC TV” and “Editor New York Post” 09-11-01 THIS IS NEXT TAKE PENACILIN NOW DEATH TO AMERICA DEATH TO ISRAEL ALLAH IS GREAT Photocopying Machine Comparisons with Trash Marks All four of the recovered anthrax envelopes contained a white. Handprinting characteristics 1. "cannot. These trash markings were compared to letters maintained in the FBI Anonymous Threat Letter File and to 1.  2." Many people use  the slash ("/") to separate the day/month/year. In writing the number one. This may be a characteristic seen on other envelopes he has  sent." when many people prefer to spell it as one  word. the first letter of all proper nouns (like names) is slightly larger. the stamps denoting 34 cents. but the smaller size measuring  approximately 6 1/4" x 3 ½". This is  apparently the author's way of indicating a word should be capitalized in  upper case lettering. but not on the letters to the New York Post and Brokaw.  which  are normally available directly from the post office. photocopied letter on paper cut to irregular size by trimming one to three edges of the page.014 photocopier exemplar sets collected from copy machines located inside or near the vicinity of every known biological laboratory that possessed virulent Ames anthrax in 2001. He writes it as "1" instead of the simple vertical line.  Also."  4. the author chooses to use a formalized.” or copy imperfections.  6. he may not be comfortable or  practiced in writing in lower case lettering. Three “trash marks. 8 .

C.200 8.900 9 .300 cfu after 11 swabbings 328.000 08 1.100 18 429. Sorting machine 1 Letter Time (DC) 17 05 09 14 06 11 03 12 02 15 10 04 20 07 08 16 Daschle Leahy 18 19 07:55 07:56 07:56 08:09 08:11 08:14 08:14 08:23 08:24 08:28 08:30 10:35 10:40 10:41 10:41 10:41 10:41 10:41 10:54 11:15 D.300 Daschle Leahy 197.700 7.400 1.300 16 7.200 01 800 4.000 382.100 8.200 300 6.026.300 2.200 1.300 117.400 29.600 115.787.100 07 336.800 19 175.731.000 13 200 1.Protocol Requirements Sample Analyses Flow Chart Open Letter Powder Bacteria ID Issues: Control charge Control Humidity Maximize yield Classic bacteriology Particle sizing Strain ID Genetic Engineering Spore density Percentage viability Cfu/ml Dead vs live spores Electron Microscopy (EM) Analytical chemistry Transmission EM Inorganic Silica / Silicates Cations and anions Elemental Analysis Isotope Analyses Organic Carbohydrates Agar (Agarobiose) Heme Headspace Scanning EM EDX Analysis Twenty Letters With Trenton Postmarks D. Sorting machine 2 cfu (6 x) cfu Letter 165.937.C.300 6.

ID & Analysis of Contaminated Collection Box 628 sampled: 1 contaminated All sampling at night Princeton. NJ Contact Sampling: Different Surfaces Walls Low in spores & competitors Floor High in spores & competitors 10 .

anthracis Data Recording Data Recording B. cereus Amerithrax Exosporium 11 . anthracis B.Top lighting captures texture B. cereus Bottom lighting captures hemolysis B.

elemental mapping – Dugway Proving Ground – production methods – Northern Arizona University – anthrax isolates – University of Maryland – agar vs broth – University of Utah – oxygen/hydrogen isotope ratios in growth media – The Institute for Genomic Research – DNA sequencing – Commonwealth Biotechnologies – genetic assays – Battelle Memorial Institute – particle size – AFTAC – trace analysis NBFAC Hub Laboratories at USAMRIID • BSL-3 Suite USAMRIID WHAT DO WE KNOW? • • • • • • Virulent Bacillus anthracis (Ames strain) Antibiotic and vaccine sensitive No known genetic engineering or modifications Concentrations and purity known Age known Manufacturing process known 12 . vaccine resistance – NSF – funded genetic sequencing – Woods Hole Oceanographic Institute – radiocarbon dating – Lawrence Livermore National Lab – radiocarbon dating – CDC – protective antigens in blood samples – Sandia National Lab – EM.Amerithrax • Scientific support – USAMRIID – repository for Ames strain – Los Alamos National Lab – genetic engineering.

tape. copying process • Isolate collection from all known labs – 16 US Labs.WHAT DO WE KNOW? • Manufacturing process known Agar grown (traces found in particulate) Water washed (no solvents) Freeze dried (not spray dried) – low moisture content.8 million base pairs) • Morphological polymorphism effort • Isotope ratio – inconclusive? • Handprinting . O. ink. absence of additives Low energy milled – absence of sheared or masticated spores No additives to enhance aerosolizability (fumed silica or bentonite) Automated X-ray spectral image analysis Elemental profile of rehydrated spore Red = Spore C. Fe … Blue = Carbon support WHAT DO WE KNOW? • Forensic evidence .copy 13 . P … Green = Si. Ca. fibers. multiple other countries • DNA sequencing (5.

Isolate colonies 3. Develop tests 6. Sequence DNA 5. Find samples with same 4 mutants 8. Screen 1013 samples 7. • Developed strain-specific markers. • Most important evidence linking anthrax to lab 1. Identify flask 14 . Find rare mutants (4) 4. • Identified DNA-based signatures for the variants. Grow spores 2.2H and 18O Isotopic Distribution Whole Genome Sequencing • Sequenced the “wild type” as well as the Morphological variants – Over 5 million DNA base pairs.

Last June. the Justice Department agreed to pay $4. Hatfill.6 million to settle a lawsuit. declared his innocence at a news conference outside of his lawyer's office in Virginia. Stephen J.Physical / Chemical Signatures Dr. was the focus of intensive FBI and news media attention in the case beginning in mid-2002. 15 .

had spent his career waiting for a biological attack. Suddenly. rational thought process in furtherance of his criminal behavior •has a familiarity. direct or indirect. Ivins still carried resentment from four decades earlier at Lebanon High School in Ohio.” Scott Shane. Bruce E. awkward teenager devoted to photography and. They may have been the focus of previous expressions of contempt  which may have been communicated to others.” 16 . even then. NY Times. January 3. metropolitan area •did not select victims randomly.  •possesses or has access to some laboratory equipment •has exhibited an organized. or observed by others. NJ. These targets are probably very  important to the offender. and the office of Senator Tom  Daschle as the targeted victims (and possibly AMI in Florida). who had helped develop an anthrax vaccine to protect American troops. to the study of bacteria. the New York Post. said Rick Sams. He lacks the personal skills necessary  to confront others. of each victim and used sufficient postage to ensure proper delivery of the letters. he was advising the FBI and regaling friends with scary descriptions of the deadly powder.“Dr.   He may hold grudges for a long time Dr. Ivins “shared with me feelings about how he’d been treated in high school. In recent years. where he had been a nerdy. the offender: •is likely an adult male •if employed. or other  employees. He is apparently comfortable working with an  extremely hazardous material •has likely taken appropriate protective steps to ensure his own safety •has access to a source of Anthrax and possesses knowledge and expertise to refine it. Ivins. He chooses to confront his problems "long distance" and not face‐to‐face. with the Trenton. 2009 Dr. He may work in a laboratory. He made an effort to identify the correct address. Ivins BEHAVIORAL ASSESSMENT Based on the selection of Anthrax as the "weapon" of choice by this individual. Dr. The  offender deliberately "selected" NBC News. a pharmacologist who had been among Bruce Ivins’s few school friends. including zip  code.  •is a non‐confrontational person. his expertise in demand. He was bitter about being excluded. is likely to be in a position requiring little contact with the public. at least in his public life. at 55.

shifting blame) • History of disguising identity (pseudonyms) • Obsessive behavior (KKG sorority) • Inability to describe/explain his own behavior By late 2006.Summary of investigative evidence implicating Dr. trash.750 grand jury subpoenas • 5. Ivins • Opportunity (RMR1029.000 person hours • 10.000 witness interviews • 6. sent out 6. academic and commercial) 17 . 400 in-depth • Cooperation of 29 labs (gov’t.000 items of potential evidence • 5. investigators had conducted 9.730 environmental samples from 60 sites • More than 1.040 individuals scrutinized.000 grand jury subpoenas and conducted 67 searches. alone in lab) • Motive (failing vaccine program) • Mental health struggle (“homicidal””sociopath”) • Proximity to source of envelopes (local batch) • Language used in the letters (emails) • Guilty conscious (emails.100 interviews. 7 years • 600.