Review of the Scientific Approaches Used During the FBI's Investigation of the 2001 Anthrax Letters

ReviewoftheScientificApproachesUsedDuringtheFBI'sInvestigationofthe2001AnthraxLettersCOMMITTEEONREVIEWOFTHESCIENTIFICAPPROACHESBOARDONLIFESCIENCESCOMMITTEEONSCIENCE,TECHNOLOGY,ANDLAWPrefaceAcknowledgmentsContentsTables,Boxes,FiguresSummary1Introduction2BiologyandHistoryofBacillusanthracis3ScientificInvestigationinaLawEnforcementCaseandDescriptionandTimelineoftheFBIScientificInvestigation4PhysicalandChemicalAnalyses5MicrobiologicalandGeneticAnalysesofMaterialintheLetters6ComparisonoftheMaterialintheLetterswithSamplesintheFBIRepositoryBibliographyIndexofDocumentsProvidedbytheFederalBureauofInvestigationAppendixA:RadiocarbonDatingAppendixB:TheForensicsPotentialofStableIsotopeAnalysisAppendixC:CommitteeEvaluationofStatisticalAnalysisReportAppendixD:BiographicalInformationofCommitteeandStaffIndex

ReviewoftheScientificApproachesUsedDuringtheFBI'sInvestigationofthe2001AnthraxLetters

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COMMITTEEONREVIEWOFTHESCIENTIFICAPPROACHESUSEDDURINGTHEFBI’SINVESTIGATIONOFTHE2001BACILLUS

ANTHRACISMAILINGS

ALICEP.GAST(Chair),President,LehighUniversity

DAVIDA.RELMAN(ViceChair),ThomasC.andJoanM.MeriganProfessor,StanfordUniversitySchoolofMedicine,andChief,InfectiousDiseaseSection,VeteransAffairsPaloAltoHealthCareSystem,CA

ARTUROCASADEVALL,LeoandJuliaForchheimerProfessorofMicrobiologyandImmunologyandChair,DepartmentofMicrobiologyandImmunology,AlbertEinsteinCollegeofMedicine

NANCYD.CONNELL,ProfessorofMedicine,UniversityofMedicineandDentistryofNewJersey(UMDNJ)-NewJerseyMedicalSchoolandDirector,UMDNJCenterforBioDefense

THOMASV.INGLESBY,ChiefExecutiveOfficerandDeputyDirectoroftheCenterforBiosecurityofUniversityofPittsburghMedicalCenter,AssociateProfessorofMedicineandPublicHealth,UniversityofPittsburghSchoolsofMedicineandPublicHealth

MURRAYV.JOHNSTON,ProfessorofChemistryandBiochemistry,UniversityofDelaware

KARENKAFADAR,JamesH.RudyProfessorofStatisticsandPhysics,IndianaUniversity

RICHARDE.LENSKI,JohnA.HannahDistinguishedProfessorofMicrobialEcology,MichiganStateUniversity

RICHARDM.LOSICK,MariaMoorsCabotProfessorofBiology,HarvardCollegeProfessorandHowardHughesMedicalInstituteProfessor

ALICEC.MIGNEREY,ProfessorofChemistryandBiochemistry,UniversityofMaryland,CollegePark

DAVIDL.POPHAM,ProfessorofMicrobiology,VirginiaPolytechnicInstituteandStateUniversity

JEDS.RAKOFF,UnitedStatesDistrictJudge,SouthernDistrictofNewYork

ROBERTC.SHALER,Director,ForensicScienceProgram,ProfessorofBiochemistryandMolecularBiology,PennsylvaniaStateUniversity

ELIZABETHA.THOMPSON,ProfessorofStatistics,UniversityofWashington

KASTHURIVENKATESWARAN,SeniorResearchScientist,CaliforniaInstituteofTechnologyJetPropulsionLaboratory

DAVIDR.WALT,RobinsonProfessorofChemistryandProfessorofBiomedicalEngineering,TuftsUniversityandHowardHughesMedicalInstituteProfessor

v

Staff

ANNE-MARIEMAZZA,StudyDirector

FRANCESE.SHARPLES,StudyCo-Director(until1/11)

ERICKA MCGOWAN, Program Officer, Board on Chemical Sciences andTechnology(until4/10)

STEVEN KENDALL, Associate Program Officer, Committee on Science,Technology,andLaw

AMANDAP.CLINE,SeniorProgramAssistant,BoardonLifeSciences(until5/10)

KATHIE.HANNA,ConsultantWriter

CAMERONH.FLETCHER,Editor

vi

BOARDONLIFESCIENCES

KEITHR.YAMAMOTO(Chair),ExecutiveViceDean,SchoolofMedicine,andProfessor,DepartmentofCellularandMolecularPharmacology,UniversityofCalifornia,SanFrancisco

BONNIEL.BASSLER,Investigator,HowardHughesMedicalInstitute,andProfessorofMolecularBiology,DepartmentofMolecularBiology,PrincetonUniversity

VICKIL.CHANDLER,ChiefProgramOfficer,Science,GordonandBettyMooreFoundation

SEANEDDY,GroupLeader,HowardHughesMedicalInstituteJaneliaFarmResearchCampus

MARKD.FITZSIMMONS,AssociateDirector,MacArthurFellowsProgram,JohnD.andCatherineT.MacArthurFoundation

DAVIDR.FRANZ,VicePresidentandChiefBiologicalScientist,MidwestResearchInstitute

DONALDE.GANEM,Director,GlobalInfectiousDiseaseResearch,NovartisInstituteofBiomedicalResearch

LOUISJ.GROSS,ProfessorofEcologyandEvolutionaryBiologyandMathematicsandDirector,InstituteforEnvironmentalModeling,DepartmentofEcologyandEvolutionaryBiology,UniversityofTennessee,Knoxville

JOHANDELSMAN,HowardHughesMedicalInstituteProfessor,YaleUniversity

CATOT.LAURENCIN,VicePresidentforHealthAffairsandDean,UniversityofConnecticutHealthCenterSchoolofMedicine

BERNARDLO,ProfessorofMedicineandDirectorofthePrograminMedicalEthics,UniversityofCalifornia,SanFrancisco

ROBERTM.NEREM,InstituteProfessorandParkerH.PetitProfessorEmeritus,InstituteforBioengineeringandBioscience,GeorgiaInstituteofTechnology

CAMILLEPARMESAN,AssociateProfessorofIntegrativeBiology,SectionofIntegrativeBiology,UniversityofTexas

MURIELE.POSTON,DeanofFaculty,SkidmoreCollege

ALISONG.POWER,ProfessorofEcologyandEvolutionaryBiologyandDean,TheGraduateSchool,CornellUniversity

BRUCEW.STILLMAN,President,ColdSpringHarborLaboratory

CYNTHIAWOLBERGER,Professor,DepartmentofBiophysicsandBiophysicalChemistry,JohnsHopkinsUniversitySchoolofMedicine

MARYWOOLLEY,PresidentandCEO,Research!America

vii

Staff

FRANCESE.SHARPLES,DirectorJOL.HUSBANDS,Scholar/SeniorProjectDirectorJAYB.LABOV,SeniorScientist/ProgramDirectorforBiologyEducationKATHERINEW.BOWMAN,SeniorProgramOfficerMARILEEK.SHELTON-DAVENPORT,SeniorProgramOfficerINDIAHOOK-BARNARD,ProgramOfficerANNAFARRAR,FinancialAssociateCARL-GUSTAVANDERSON,ProgramAssociateAMANDAMAZZAWI,SeniorProgramAssistantAYESHAAHMED,ProgramAssistant

viii

COMMITTEEONSCIENCE,TECHNOLOGY,ANDLAW

DAVIDKORN(Co-Chair),ViceProvostforResearch,HarvardUniversity

RICHARDA.MESERVE(Co-Chair),President,CarnegieInstitutionforScience,andSeniorOfCounsel,Covington&BurlingLLP

FREDERICKR.ANDERSON,JR.,Partner,McKenna,Long&AldridgeLLP

ARTHURI.BIENENSTOCK,SpecialAssistanttothePresidentforFederalResearchPolicyandDirector,WallenbergResearchLink,StanfordUniversity

BARBARAE.BIERER,ProfessorofMedicine,HarvardMedicalSchool,andSeniorVicePresident,Research,BrighamandWomen’sHospital

ELIZABETHH.BLACKBURN,MorrisHerzsteinProfessorofBiologyand

Physiology,UniversityofCalifornia,SanFrancisco

JOHNBURRIS,President,BurroughsWellcomeFund

ARTUROCASADEVALL,LeoandJuliaForchheimerProfessorofMicrobiologyandImmunology;Chair,DepartmentofBiologyandImmunology;andProfessorofMedicine,AlbertEinsteinCollegeofMedicine

JOES.CECIL,ProjectDirector,ProgramonScientificandTechnicalEvidence,DivisionofResearch,FederalJudicialCenter

ROCHELLECOOPERDREYFUSS,PaulineNewmanProfessorofLawandDirector,EngelbergCenteronInnovationLawandPolicy,NewYorkUniversitySchoolofLaw

DREWENDY,AssistantProfessor,Bioengineering,StanfordUniversity,andPresident,TheBioBricksFoundation

PAULG.FALKOWSKI,BoardofGovernorsProfessorinGeologicalandMarineScience,DepartmentofEarthandPlanetaryScience,Rutgers,TheStateUniversityofNewJersey

MARCUSFELDMAN,BurnetC.andMildredWohlfordProfessorofBiologicalSciences,StanfordUniversity

ALICEP.GAST,President,LehighUniversity

JASONGRUMET,President,BipartisanPolicyCenter

GARYW.HART,WirthChairinEnvironmentalandCommunityDevelopmentPolicy,UniversityofColorado,Denver

BENJAMINW.HEINEMAN,JR.,SeniorFellow,HarvardLawSchoolandHarvardKennedySchoolofGovernment

D.BROCKHORNBY,Judge,U.S.DistrictCourt,DistrictofMaineALANB.MORRISON,LernerFamilyAssociateDeanforPublicInterestandPublicService,GeorgeWashingtonUniversityLawSchoolPRABHUPINGALI,DeputyDirectorofAgriculturalDevelopment,Global

DevelopmentProgram,BillandMelindaGatesFoundationHARRIETRABB,VicePresidentandGeneralCounsel,RockefellerUniversityBARBARAJACOBSROTHSTEIN,Director,TheFederalJudicialCenter

ix

JONATHANM.SAMET,ProfessorandFloraL.ThorntonChair,DepartmentofPreventiveMedicine,KeckSchoolofMedicineandDirector,InstituteforGlobalHealth,UniversityofSouthernCalifornia

DAVIDS.TATEL,Judge,U.S.CourtofAppealsfortheDistrictofColumbiaCircuit

SOPHIEVANDEBROEK,ChiefTechnologyOfficerandPresident,XeroxInnovationGroup,XeroxCorporation

Staff

ANNE-MARIEMAZZA,DirectorSTEVENKENDALL,AssociateProgramOfficer

x

Preface

In autumn 2001, the tragic deaths, illnesses, andenvironmental contamination caused by the mailing ofBacillus anthracis (B. anthracis) spores in letters sentthrough the U.S. postal system caused tremendous fearand disruption in a nation shaken by the events ofSeptember 11. Efforts led by the Federal Bureau ofInvestigation(FBI)tocharacterizethematerialcontainedin the letters and identify the individual or individualsresponsible for the mailings would involve extensivescientificstudyspanningalmostnineyears.

Itisnotunusualtousesciencetoidentifyandcharacterizeevidenceandtolinkit to a particular individual in a criminal investigation. Indeed, in the 2001B.anthracismailings investigation, physics, chemistry, and biology all played arole.Inthiscase,thefieldofbacterialgenomicswasrapidlyevolvingthroughoutthe investigation. Recognizing the challenges inherent in such a complexscientific investigation, in 2008 the FBI asked the National Research Council(NRC)oftheNationalAcademyofSciences(NAS)toconductanindependentreview of the scientific approaches used during its investigation. In 2009, thecommittee,formedundertheauspicesoftheNationalAcademies’BoardonLifeSciencesandCommitteeonScience,Technology,andLaw,beganthisreview.

Asweundertookthisreview,thecommitteekeptinmindthecontextofthetime,immediately following September 11, 2001, when there were multiple high-profile FBI investigations under way. We recognized that the graveconsequences of these events for public health and national security and theuncertainty about possible additional attacks necessarily influenced the initialdesign and execution of the FBI’s scientific investigation. Throughout itsinvestigation, theFBI embraced and energetically pursued the use of newandemergingsciencethroughanunusualdegreeofinvolvementofoutsidescientists.Inmanyways,thiscaseestablishedandemphasizedthepotentialimportanceof

microbialforensicsintheinvestigationoffutureactsofbioterrorism.

Ascientificstudyismuchmorethanaseriesofwell-executedexperiments.Theplanninganddecisionmakingusedduringastudyareessentialcompo-

xi

nents of the science and can determine its outcome. As we learned from ourreviewofthiscase,itisespeciallyimportantinemergencysituationstohaveaclearly defined approach for undertaking a complex scientific investigationinvolving many experts and collaborators. In any such case, the goal is tointegrate a broad range of experimentalmethods and exploratoryworkwith amorethoroughanddeeperstudyofselectedmethods,alongwithclearguidancefortheinvestigation.Investigatorsmustmaintainahealthydegreeofskepticismand a willingness to challenge their own assumptions. Achieving these goalsrequires considerable thought and planning before a crisis occurs. It is alsoimportant to recognize that when science meets law enforcement there areseveral tensions that need to be balanced: openness and secrecy, collaborationandindependence,anddeliberatenessandexpediency.

Wealsolearnedfromthisinvestigationthatthereisanimmediateandongoingneed from the outset of an investigation to obtain expert advice and haveavailable agroupof advisorswhocanprovide conceptual insight and relevantexpertisetoscientificplans,approaches,andscenarios.

Anunavoidableobservationfromthe2001B.anthracismailingsisthatthebestsubjectmatterexpertsinagivenareaalsomightbeviewedassuspects.Workingwithpotentialsuspectsduringasensitiveinvestigationisachallengethatthelawenforcementcommunitymustcontinuallyaddressthroughitsvettingprocesses.

Throughoutourreview,wefocusedonthescientificaspectsoftheinvestigationand did not evaluate non-science-based investigative material. We haveevaluated the science to the best of our ability, given the materials madeavailable to us.While theremay be additional relevantmaterial to which wewerenotprovidedaccess,webelieve thatour reviewof theavailablematerialhasresultedinmanyusefulfindingsandconclusions.Nonetheless,otheraspectsof, and documents from, the FBI investigation may deserve future study andreview.

Infollowingourcharge,weevaluatedthespecificconclusionsdrawnbytheFBI

based on its scientific analyses. The FBI never provided the committee withthoseconclusionsinwrittenform,althoughFBIconclusionswereofferedinoralpresentations to the committee. We repeatedly sought written statements ofconclusions until the casewas closed by theDepartment of Justice (DOJ) onFebruary19,2010. Inour report,weaddress theconclusionsoffered inverbalreports, as well as the main scientific conclusions as written in the DOJ

AmerithraxInvestigativeSummary.1

In November 2010, after our final report had beensubmitted to the FBI for a security review, the FBIinformed the National Academies that there wereadditionalmaterialsrelevanttothecommittee’sworkthathadnotpreviously

1 United States Department of Justice. AmerithraxInvestigativeSummary.February19,2010.Availableat:www.justice.gov/amerithrax/docs/amx-investigative-summary.pdf.

been sharedwith the committee. TheBureau offered toprovide our committee with these materials and anadditional briefing. After serious discussions with theNationalAcademies’leadership,weagreedtoreceiveandreview thesematerials and reconvene the committee forone finalmeeting in January 2011. The documents andbriefingprovideduswithadditional informationand ledto meaningful changes in this report regarding theorganization of the scientific investigation, samplecollection,andanalyticaltestsundertakenbytheFBIand

its contracting laboratories. This information resulted intheadditionofanewsectioninthereport(3.4.3)andtheaddition of a new finding (3.4) and recommendation(3.1). A benefit of the extension of the project and thedelayinissuanceofthereportisthatimportantadditionalmaterials, now available to the public, provide moreinformationaboutthescientificinvestigation.

LOOKINGTOTHEFUTURE

Whilemuchofoureffortwasfocusedonareviewofthescienceperformedinsupport of the investigation of the 2001 B. anthracis mailings, an equallyimportant aim has been to help ensure that future scientific investigations ofbiological attacks are conducted in themost relevant, rigorous, and thoughtfulmannerpossible.Althoughtheeventsof2001weretragic,theycouldhavebeenmore catastrophic. In the future, among many other requirements, it will beimportant toensuremore timelyresults,moreefficientenvironmentalanalysis,access to globally representative strain collections, and a robust capability forcharacterizing less well studied or less easily cultivated biological agents.Officials also may need to manage expectations among the general public,policymakers, and the scientific community about the conclusions that canrealisticallybeexpectedfromtheuseofmicrobialforensics.

We have been fortunate to work with extremely talented, intelligent, anddedicated individuals in theundertakingof thismultifaceted study.Committeemembers evaluated large numbers of documents under constrainedcircumstances that required exceptional dedication and patience.They listenedintently to speakers, asked probing and insightful questions, and vigorouslydiscussed what was learned, what we could research, and how to word ourfindings.Weareindebtedtothemforall thetimeandenergytheygavetothiseffort.Wearealsomostgratefultothestaff—AmandaCline,CameronFletcher,StevenKendall,ErickaMcGowan,Anne-MarieMazza,andFranSharples—andtotheconsultantwriter,KathiHanna.

AliceP.GastandDavidA.RelmanChairandViceChair

Acknowledgments

Thisreporthasbeenreviewedindraftformbyindividualschosenfortheirdiverseperspectivesandtechnicalexpertise,inaccordancewithproceduresapprovedbytheNationalResearchCouncil’sReportReviewCommittee.Thepurposeofthisindependentreviewistoprovidecandidandcriticalcommentsthatwillassisttheinstitutioninmakingitspublishedreportassoundaspossibleandtoensurethatthereportmeetsinstitutionalstandardsforobjectivity,evidence,andresponsivenesstothestudycharge.Thereviewcommentsanddraftmanuscriptremainconfidentialtoprotecttheintegrityoftheprocess.Wethankthefollowingindividualsfortheirreviewofthisreport:

R.JohnCollier,HarvardMedicalSchoolRitaR.Colwell,UniversityofMaryland

M.BonnerDenton,UniversityofArizonaAshleeM.Earl,BroadInstituteoftheMassachusettsInstituteofTech

nologyandHarvardUniversityPhilipC.Hanna,UniversityofMichiganMedicalSchoolStephenA.Johnston,ArizonaStateUniversityDavidH.Kaye,ArizonaStateUniversityCatoT.Laurencin,UniversityofConnecticutHealthCenter

M.S.Meselson,HarvardUniversityRandallS.Murch,VirginiaPolytechnicInstituteandStateUniversityPaulineNewman,U.S.CourtofAppealsfortheFederalCircuitStanleyA.Plotkin,UniversityofPennsylvania(emeritus)ElizabethRindskopfParker,UniversityofthePacific

R.PaulSchaudies,GenArraytion,Inc.JamesM.Tiedje,MichiganStateUniversityBruceWeir,UniversityofWashington

Althoughthereviewerslistedaboveprovidedmanyconstructivecommentsandsuggestions,theywerenotaskedtoendorsetheconclusionsorrecom

xv

mendations,nordidtheyseethefinaldraftofthereportbeforeitsrelease.Thereview of this report was overseen by Stephen Fienberg, Carnegie MellonUniversity,andFloydBloom,TheScrippsResearchInstitute.AppointedbytheNational Research Council, they were responsible for making certain that anindependent examination of this report was carried out in accordance withinstitutionalproceduresandthatallreviewcommentswerecarefullyconsidered.Responsibilityforthefinalcontentofthisreportrestsentirelywiththeauthoringcommitteeandtheinstitution.

Contents

SUMMARY1

1INTRODUCTION25

1.1Background,25

1.2ChronologyofEventsofFall2001,26

1.3BriefSummaryoftheFBI’sScientificInvestigation,31

1.4SummaryofFBIandDOJScientificConclusions,32

1.5CommitteeProcess,33

1.6IssuesforConsiderationinReadingThisReport,35

1.7OrganizationoftheReport,36

2BIOLOGYANDHISTORYOFBACILLUSANTHRACIS37

2.1Introduction,37

2.2TheBiologyofB.anthracis,37

2.3ClinicalAspectsofAnthrax,38

2.4B.anthracisasaBiologicalWeapon,40

2.5PhylogenyofB.anthracis,41

2.6TheEarlyHistoryoftheAmesStrainofB.anthracis,44

2.7Summary,44

3SCIENTIFICINVESTIGATIONINALAWENFORCEMENTCASEANDDESCRIPTIONANDTIMELINEOFTHEFBISCIENTIFIC

INVESTIGATION47

3.1Introduction,47

3.2ScienceandScientificInvestigationasPartofaLawEnforcementInvestigation,47

3.3TheFederalCoordinatedResponseandAssignmentofLaboratoryWork,55

xvii

3.4CollectionandAnalysisofClinical andEnvironmentalSamples andCrossContamination,60

3.4.1ClinicalandEpidemiologicalSamples,60

3.4.2CrimeSceneEnvironmentalSamples,64

3.4.3SamplesfromanOverseasSiteIdentifiedbyIntelligence,66

3.4.4LetterMaterialandCrossContamination,67

3.5CommitteeFindingsandRecommendations,70

4PHYSICALANDCHEMICALANALYSES75

4.1Introduction,75

4.2SporePreparationandPurification,75

4.3SurrogatePreparationandPurification,78

4.4SizeandGranularityoftheMaterialintheLetters,79

4.5PresenceofSiliconandOtherElementsintheLetterMaterial,80

4.5.1ElementalAnalysis,81

4.5.2SpatiallyResolvedElementalAnalysis,83

4.5.3SiliconintheSporeCoat,84

4.5.4SummaryoftheSiliconAnalysis,87

4.6FeaturesofBacterialGrowthConditionsandProcessingMethods:DetectionofMeglumineandDiatrizoate,87

4.7MediaComponentAnalysis,89

4.8VolatileOrganicCompounds,89

4.9DeterminingWhen theMaterialWas Produced:RadiocarbonDating ofB.anthracisSamples,90

4.10StableIsotopeAnalysis,90

4.10.1B.anthracis,90

4.10.2WaterSamples,92

4.10.3TheEnvelopeMeasurements,92

4.11CommitteeFindings,93

5MICROBIOLOGICALANDGENETICANALYSESOFMATERIALINTHELETTERS97

5.1Introduction,97

5.2IdentificationoftheB.anthracisStrain,97

5.3WastheB.anthracisintheLettersGeneticallyEngineered?,100

5.4B.subtilisContaminationoftheNewYorkSamples,104

5.5IdentificationandCharacterizationofColonyMorphologicalVariantsintheEvidentiaryMaterial,106

5.5.1WhyWastheFBIInterestedinColonyMorphotypes?,106

5.5.2BackgroundInformationonMorphotypes,107

5.5.3 Detection and Characterization of Morphotypes in the Anthrax LettersSamples,109

5.5.4SelectionCriteriaforGeneticVariationsUsedinScreening,113

5.5.5WholeGenomeSequencingofMorphotypeIsolates,114

5.5.6DevelopmentandApplicationofAssaysfortheGenotypes,119GenotypesA1andA3,119GenotypesBandD,119GenotypeE,120


6COMPARISONOFTHEMATERIALINTHELETTERSWITHSAMPLESINTHEFBIREPOSITORY125

6.1Introduction,125

6.2CreationoftheFBIRepository(FBIR),126

6.3UseoftheGeneticAssaystoTestfortheFourGenotypes,130

6.4DerivationofRMR1029Spores,130

6.5AnalysesoftheRepositorySamplesandStatisticalInterpretationoftheEvidence,132

6.5.1TheFBI’sStatisticalAnalysisReport,135

6.5.2CommitteeAssessmentoftheStatisticalAnalysisReport,136

6.6AnalysesBasedonResamplingRMR1029andInterpretationoftheResults,140


BIBLIOGRAPHY153

INDEXOFDOCUMENTSPROVIDEDBYTHEFEDERALBUREAUOFINVESTIGATION161

APPENDIXES

ARadiocarbonDating181BTheForensicsPotentialofStableIsotopeAnalysis183CCommitteeEvaluationofStatisticalAnalysisReport185DBiographicalInformationofCommitteeandStaff193

INDEX205

Tables,Boxes,Figures

TABLES

S-1FBIandDOJConclusionsandCommitteeComments,11

1-1TimelineofKeyEventsintheAnthraxMailingsCase,28

3-1TimelineofScientificEventsintheAnthraxMailingsInvestigation,483-2AnalyticalTechniquesUsedontheEvidentiaryMaterial,58

4-1EstimatedRangesofTotalNumberofSpores,76

4-2EstimatesofMediaVolumeRequiredforSporePreparation,77

4-3MethodsforChemicalAnalysisReferredtoinChapter4,81

4-4SummaryofSiliconMeasurements inEvidentiary andSurrogateSamples,82

5-1PhenotypicCharacteristicsoftheMorphotypes,113

5-2 B. anthracis Isolates Analyzed by the Institute forGenomicResearch(TIGR),115

5-3FurtherGeneticCharacterizationoftheMorphotypeIsolates,116

5-4 Distribution Among the Anthrax Letters of the Genotypes Selected for

RepositoryScreening,118

6-1GeneralResultsoftheScreeningof1,059ViableFBIRSamplesforthePresenceoftheMutationGenotypes,asSummarizedbytheStatisticalConsultanttotheFBI,133

6-2 General Results of the Screening of the 947 Samples that ProvidedDefinitiveResultsforAllFourGenotypes,134

6-3DistributionResults for theFourGenotypeAssays forGenotypesA1,A3,MRI-D,andEinthe947Samples,134

xxi

6-4ObservedandExpected(UnderIndependence)DistributionofPositiveSignaturesofFourGenotypes,1386-5GenotypeAssaysonThreeReplicatesfromTwoSamplesat10DilutionLevels:EntryDenotesNumberofPositiveAssaysonThreeReplicatesatEachDilutionLevel,1396-6ResultsObtainedbyResamplingfromFlaskRMR1029,143

C-1SampleswithPositiveand“Inconclusive”or“Variant”Assays,187C-2Probabilitiesofk4-mutationSamplesinInstitutionF,191

BOXES

S-1ChargetotheCommittee,2

1-1ChargetotheCommittee,27

2-1TheSverdlovskOutbreak,41

3-1BioterrorismInvestigations,54

5-1GenomeSequencing,1015-2ThePolymeraseChainReactionTechnique,1025-3TheTaqManTechnique,106

6-1SubpoenaProtocolforCollectionandSubmissionofAmesStrainSamples,127

FIGURES

2-1WorldwideDistributionandLineagesofB.anthracis,43

3-1TrajectoryandOutcomesofAnthraxMailings,623-2NewYorkPostLetterPowder,633-3LeahyLetterPowder,63

4-1SEMofLeahyandNewYorkPostPowders,854-2StableIsotopeResults

18Oand2H,91

5-1B.anthracisColonyMorphotype“A”,1105-2B.anthracisColonyMorphotype“B”,1115-3B.anthracisColonyMorphotype“E”,112

A-1AtmosphericCO2(NorthernHemisphere),182

Summary

LessthanamonthaftertheSeptember11,2001,attacks,letters containing spores of anthrax bacteria (Bacillusanthracis, or B. anthracis) were sent through the U.S.mail. Between October 4 and November 20, 2001, 22individualsdevelopedanthrax;5ofthecaseswerefatal.

Although it was initiated as a public health investigation, the investigationquickly fell under the purview of the Federal Bureau of Investigation (FBI),when a deliberate act was suspected and letters containingB. anthracis werediscoveredinbothNewYorkandWashington,D.C.,addressedtoTomBrokawof NBCNews, theNew York Post, and U.S. Senators TomDaschle of SouthDakotaandPatrickLeahyofVermont.

Overthecourseofitsinvestigation,theFBIdevoted600,000investigatorworkhourstothecaseandassigned17SpecialAgentstoaTaskForce,alongwith10U.S.Postal Inspectors.Theinvestigationspannedsixcontinents; involvedover10,000witnessinterviews,80searches,26,000emailreviews,andanalysesof4millionmegabytesof computermemory; and resulted in the issuanceof5,750

grandjurysubpoenas.Additionally,29government,university,andcommerciallaboratories assisted in conducting the scientific analyses that were a centralaspectoftheinvestigation(U.S.DepartmentofJustice[USDOJ],2010,p.4).

During its investigation of the anthrax mailings, the FBI worked with otherfederal agencies to coordinate and conduct scientific analyses of the anthraxletter spore powders, environmental samples, clinical samples, and samplescollected from laboratories that might have been the source of the letter-associated spores. The agency relied on external experts, including somewhohadpreviouslydevelopedteststodifferentiateamongstrainsofB.anthracis.

In2008,sevenyearsintotheinvestigation,theFBIaskedtheNationalResearchCouncil (NRC) of the National Academy of Sciences (NAS) to conduct anindependentreviewofthescientificapproachesusedduringtheinvestigationofthe2001B.anthracismailings(seeBoxS-1forcharge).InadditiontoinformingFBIinvestigatorsaboutpossibleleads,muchofthescienceused

1

BOXS-1ChargetotheCommittee

The NRC was asked by the FBI to conduct anindependent review of the scientific approaches usedduring the investigation.The official charge to thecommitteestated:

An ad hoc committee with relevant expertise will evaluate the scientificfoundation for the specific techniques used by the FBI to determine whetherthesetechniquesmetappropriatestandardsforscientificreliabilityandforuseinforensic validation, and whether the FBI reached appropriate scientificconclusionsfromitsuseofthesetechniques.Ininstanceswherenovelscientificmethods were developed for purposes of the FBI investigation itself, thecommittee will pay particular attention to whether these methods wereappropriately validated. The committee will review and assess scientificevidence (studies, results, analyses, reports) considered in connectionwith the2001 Bacillus anthracis mailings. In assessing this body of information, thecommitteewilllimititsinquirytothescientificapproaches,methodologies,and

analytical techniques used during the investigation of the 2001 B. anthracismailings.

Theareasofscientificevidencetobestudiedbythecommitteeinclude,but

maynotbelimitedto:

1. genetic studies that led to the identification of potential sources of B.anthracisrecoveredfromtheletters;

2. analysesoffourgeneticmutationsthatwerefoundinevidenceandthatareunique to a subset of Ames strain cultures collected during theinvestigation;

3. chemicalanddatingstudiesthatexaminedhow,where,andwhenthesporesmay have been grown and what, if any, additional treatments they weresubjectedto;

4. studiesoftherecoveryofsporesandbacterialDNAfromsamplescollectedandtestedduringtheinvestigation;and

5. therolethatcrosscontaminationmighthaveplayedintheevidencepicture.

The committee will necessarily consider the facts and data surrounding theinvestigation of the 2001 Bacillus anthracis mailings, the reliability of theprinciplesandmethodsusedbytheFBI,andwhethertheprinciplesandmethodswere applied appropriately to the facts.The committee will not, however,undertakeanassessmentoftheprobativevalueofthescientificevidenceinanyspecificcomponentoftheinvestigation,prosecution,orcivil litigationandwilloffernoviewontheguiltorinnocenceofanyperson(s)inconnectionwiththe2001B.anthracismailings,oranyotherB.anthracisincidents.

in the investigation formed the basis of a rapidlydeveloping but still nascent scientific field, called“microbial forensics,” involving a series of laboratorytestsusedtodeterminethegeneticidentityofamicrobialagentusedfornefariouspurposes.Thedevelopmentandapplication of microbial forensics would become anessential part of the scientific investigation in thehandsof FBI investigators, who would combine it with

physicochemical analyses to narrow their search for thepossible origin of the anthrax used in the attacks. Keyscientificquestionsfocusedonhow,where,andwhenthematerialmighthavebeenproduced;whetherthematerialin all the evidence collected was identical; whether thematerialhadbeenproducedinsuchamannerastomakeit more easily dispersible; whether it had anydistinguishingphysicalorchemicalpropertiesofvalueincomparison studies; and whether its biologicalcharacteristicscouldprovideleadstoitsorigins.

Thecommitteecarriedoutitsworkmindfuloftheneedtoidentifylessonsthatcould be learned for future investigations in which science might play animportantrole.

UnderthetermsoftheNRCcontractwiththeFBI,thecommitteewasinitiallyprovided with two boxes containing approximately 9,000 pages of materialsregarding the scientific investigations undertaken by the FBI and by externalexpertscontractedbytheFBIduringtheinvestigation.Attheendofthestudyanadditional 641 pages were provided to the committee. Throughout the NRCstudyprocess, theseFBI-providedmaterialswerecoveredbyFOIAExemption7, law enforcement, and were not publicly available.With the release of thisreport,asspecifiedinthecontract,thesedocumentshavebeendepositedinthe

NRCPublicAccessFile.1

In addition to these materials the FBI briefed thecommitteeonseveraloccasions.Someofthesebriefingsweredoneinopensession,whileotherswereconductedin closed sessions covered by FOIA Exemption 7. Thecommitteealsoheardfromanumberofotherexperts.

Inconductingitsreview,thecommitteewasmindfulthat,whileitsfocuswason

thescienceinvolvedinthecase,theFBIdidnotaskthecommitteetoreviewallof the science thatwas used in the investigation. For example, the committeewasnot charged to consideror evaluate anyof the traditional forensic sciencemethods and techniques used in criminal investigations (e.g., hair, fiber,fingerprint,orhandwritinganalysis)(NRC,2009a)nordiditconsideranyofthepsychologicalorbehavioralsciences,suchaslinguistics,usedbytheFBIinitsinvestigation. As such, this report and the committee’s review and evaluationfocused on the application of biological, physical, and chemical sciences toevidentiary materials from the letters, to the collection and analysis ofenvironmentalsamples,totheanalysisoftheflaskdesignatedRMR1029(aflaskcontaining

1 The public can gain access to these materials bycontactingtheNRCPublicAccessRecordsOffice.

anthraxsporesthathadbeenhousedandmaintainedinaU.S. Army Medical Research Institute of InfectiousDiseases [USAMRIID] laboratory at Fort Detrick since1997), and to the collection and analysis of the B.anthracis samples from domestic and internationallaboratoriesandstoredintheFBIRepository(FBIR).

DuringthecourseoftheNRCcommittee’sdeliberations,theDOJannouncedonFebruary19,2010,thatitwasclosingthecasebasedonitsconclusionthatDr.Bruce Ivins, a scientist at USAMRIID, had alone perpetrated the attacks. Dr.Ivins died on July 29, 2008, after taking an overdose of over-the-countermedications.

FBISCIENTIFICCONCLUSIONSANDCOMMITTEEFINDINGS

TheFBIdrewanumberofconclusions fromits scientific investigation,whicharesummarizedinTableS-1attheendoftheSummary.Thecommitteefounditchallenging,however,toidentifyadefinitivesetofscientificconclusionsdrawnbytheFBIinvestigatorsbecausetheydidnotprovidetheminwrittenformandbecause the conclusions provided publicly by DOJ in its briefings and

Investigative Summary2 varied from those provided by FBIofficials in presentations to the committee. For thepurposes of this report, the committee’s analyses arebasedon the scientific conclusionsprovidedby theFBIto the committee on September 24, 2009 (left-mostcolumnofTableS-1) and those issuedpubliclybyDOJonFebruary19,2010,when itclosed thecase (USDOJ,2010) (column second from the left in Table S-1). Thecommitteewasnotinapositiontoofferajudgmentaboutthe importance and strength of the evidence from thescientific investigation relative to the importance andstrength of the evidence from the criminal investigationbecauseitwasnotchargedwith(andlackedtheexpertisefor)reviewingthelatter.

A summary of the committee’s findings with regard to the scientificinvestigationandthescientificconclusionsthatweredrawnfromitbytheFBIisprovidedbelow.Thenumberedstatementsbelowinboldthatarelabeledwithan“S”(e.g.,S.1)summarizesubsetsofthecommittee’sfindingsandareintendedtoorganizethefindingsandhelpguidethereaderthroughthem.

SUMMARYOFCOMMITTEEFINDINGSItisnotpossibletoreachadefinitiveconclusionabouttheoriginsofthe

B.anthracisinthemailingsbasedontheavailablescientificevidencealone.

S.1TheB.anthracisintheletterswastheAmesstrainandwasnotgeneticallyengineered.

2 United States Department of Justice. AmerithraxInvestigativeSummary.February19,2010.Availableat:

www.justice.gov/amerithrax/docs/amx-investigative-summary.pdf.

As background, the Ames strain of B. anthracis wasoriginallyisolatedfromadeadcowinTexasin1981andshipped to USAMRIID in Frederick, Maryland. Overtime it was shared with research and developmentlaboratoriesaroundtheworld.

•ThedominantorganismfoundintheletterswascorrectlyandefficientlyidentifiedastheAmesstrainofB.anthracis.ThescienceperformedonbehalfoftheFBIforthepurposeofBacillusspeciesandB.anthracisstrainidentificationwasappropriate,properlyexecuted,andreflectedthecontemporarystateoftheart.(Finding5.13)

•The initial assessment ofwhether theB.anthracisAmes strain in the lettershad undergone deliberate genetic engineering or modification was timely andappropriate,thoughnecessarilyincomplete.Thegenomesequencesoftheletterisolates that became available later in the investigation strongly supported theFBI’sconclusion that theattackmaterialshadnotbeengeneticallyengineered.(Finding5.2)

S.2 Multiple distinct colony morphological types, or morphotypes, of B.anthracisAmes were present in the letters. Molecular assays of specificgeneticsequencesassociatedwiththesemorphotypesprovidedanapproachtodeterminingrelationshipsamongevidentiarysamples.

As background, when bacteria are grown on agar plates, the descendants ofsingle cells produce colonies. Variation in the appearance, or morphology, ofthesecolonies(i.e.,multiplemorphotypes)canindicatethepresenceofdifferentspeciesorstrainsinthesourcematerial,eachwithadistinctgeneticsignature,orgenotype.

• Multiple colony morphotypes of B. anthracis Ames were present in thematerialineachofthethreelettersthatwereexamined(NewYorkPost,Leahy,andDaschle),andeachof thephenotypicmorphotypeswas found to representoneormoredistinctgenotypes.(Finding5.4)

•SpecificmolecularassaysweredevelopedforsomeoftheB.anthracisAmesgenotypes(thosedesignatedA1,A3,D,andE)foundintheletters.Theseassaysprovided a useful approach for assessing possible relationships among thepopulations of B. anthracis spores in the letters and in samples that weresubsequentlycollectedfortheFBIR(seealsoChapter6).However,morecouldhavebeendonetodeterminetheperformancecharacteristicsoftheseassays.Inaddition, the assays did not measure the relative abundance of the variantmorphotypemutations,whichmighthavebeenvaluableandcouldbeimportantinfutureinvestigations.(Finding5.5)

3 The first number in the findings corresponds to thechapterinwhichtheyarepresented.

•Thedevelopmentandvalidationofthevariantmorphotypemutationassaystookalongtimeandslowedtheinvestigation.Thecommitteerecognizesthatthegenomicscienceusedtoanalyzetheforensicmarkersidentifiedinthecolonymorphotypeswasalarge-scaleendeavorandrequiredtheapplicationofemergingscienceandtechnology.Althoughthecommitteelaudsandsupportstheeffortdedicatedtothedevelopmentofwell-validatedassaysandprocedures,lookingtowardthefuture,theseprocessesneedtobemoreefficient.(Finding5.6)

•AdistinctBacillusspecies,B.subtilis,wasaminorconstituentoftheNewYorkPostandBrokaw(NewYork)letters,andthestrainfoundinthesetwoletterswasprobablythesame.B.subtiliswasnotpresentintheDaschleandLeahy

letters.TheFBIinvestigatedthisconstituentoftheNewYorklettersandconcluded,andthecommitteeconcurs,thattheB.subtiliscontaminantdidnotprovideusefulforensicinformation.Whilethiscontaminantdidnotprovideusefulforensicinformationinthiscase,thecommitteerecognizesthatsuchbiologicalcontaminantscouldprovetobeofforensicvalueinfuturecasesandshouldbeinvestigatedtotheirfullest.(Finding5.3)

S.3TheFBIcreatedarepositoryofAmesstrainB.anthracissamplesandperformedexperimentstodeterminerelationshipsamongthelettermaterialsandtherepositorysamples.ThescientificlinkbetweenthelettermaterialandflasknumberRMR1029isnotasconclusiveasstatedintheDOJInvestigativeSummary.

•TheFBIappropriatelydecidedtoestablisharepositoryofsamplesoftheAmesstrainofB.anthracisthenheldinvariouslaboratoriesaroundtheworld.Therepositorysampleswouldbecomparedwiththematerialfoundintheletterstodeterminewhethertheymightbethesourceofthelettermaterials.However,foravarietyofreasons,therepositorywasnotoptimal.Forexample,theinstructionsprovidedinthesubpoenaissuedtolaboratoriesforpreparingsamples(i.e.,the“subpoenaprotocol”)werenotpreciseenoughtoensurethatthelaboratorieswouldfollowaconsistentprocedureforproducingsamplesthatwouldbemostsuitableforlatercomparisons.Suchproblemswiththerepositoryrequiredadditionalinvestigationandlimitthestrengthoftheconclusionsthatcanbedrawnfromcomparisonsofthesesamplesandthelettermaterial.(Finding6.1)

•TheFBIfacedadifficultchallengeinassemblingandannotatingtherepositoryofB.anthracisAmessamplescollectedforgeneticanalysis.(Finding6.9)

TheresultsofthegeneticanalysesoftherepositorysampleswereconsistentwiththefindingthatthesporesintheattackletterswerederivedfromRMR1029,buttheanalysesdidnotdefinitivelydemonstratesucharelationship.(Finding6.2)•SomeofthemutationsidentifiedinthesporesoftheattacklettersanddetectedinRMR1029mighthavearisenbyparallelevolutionratherthanbyderivationfromRMR1029.ThispossibleexplanationofgeneticsimilaritybetweensporesinthelettersandinRMR1029wasnotrigorouslyexploredduringthecourseoftheinvestigation,furthercomplicatingtheinterpretationoftheapparentassociationbetweenthe

B.anthracisgenotypesdiscoveredintheattacklettersandthosefoundinRMR1029.(Finding6.3)

TheflaskdesignatedRMR1029wasnottheimmediate,mostproximatesourceofthelettermaterial.IfthelettermaterialdidinfactderivefromRMR1029,thenoneormoreseparategrowthsteps,usingseedmaterialfromRMR1029followedbypurification,wouldhavebeennecessary.Furthermore,theevidentiarymaterialintheNewYorklettershadphysicalpropertiesthatweredistinctfromthoseofthematerialintheWashington,D.C.letters.(Finding4.6)

•ThegeneticevidencethatadisputedsamplesubmittedbythesuspectcamefromasourceotherthanRMR1029wasweakerthanstatedintheDepartmentofJusticeAmerithraxInvestigativeSummary.(Finding6.4)

•ThescientificdatageneratedbyandonbehalfoftheFBIprovidedleadsastoapossiblesourceoftheanthraxsporesfoundintheattackletters,butthesedataalonedidnotruleoutothersources.(Finding6.5)

•Biologicalmaterialfromallfourlettersshouldhavebeenexaminedtodeterminewhethertheyeachcontainedallfourgeneticmarkersusedinscreeningtherepositorysamples.(Finding6.7)

S.4Siliconwaspresentintheletterpowdersbuttherewasnoevidenceofintentionaladditionofsilicon-baseddispersants.

Whileanydeliberatemailingofletterscontaininganthraxsporesmightbeconsideredaformofspore“weaponization,”thistermhasbeenmorecommonlyusedtodescribepreparationswithenhancedpropertiesofdispersionandaerosolization.Itiscommonlybelievedthatdeliberateeffortstomakeapowdermoredispersiblethroughtheuseofadditiveswouldsuggestamoresophisticatedlevelofpreparationexpertise.Thus,thepresenceofdispersants,suchasnanoparticulatesilicaorbentonite,wasanimportantfeatureinconsideringwhetherornottheletterscontained“weaponized”anthraxspores.

•AlthoughsignificantamountsofsiliconwerefoundinthepowdersfromtheNewYorkPost,Daschle,andLeahyletters,nosiliconwasdetectedontheoutsidesurfaceofsporeswhereadispersantwouldreside.Instead,significantamountsofsiliconweredetectedwithinthesporecoatofsomesamples.ThebulksiliconcontentintheLeahylettermatchedthesiliconcontentperspore

measuredbydifferenttechniques.FortheNewYorkPostletter,however,therewasasubstantialdifferencebetweentheamountofsiliconmeasuredinbulkandthatmeasuredinindividualspores.Nocompellingexplanationforthisdifferencewasprovidedtothecommittee.(Finding4.3)

• Surrogate preparations of B.anthracis did reproduce physical characteristics(purity, spore concentration, dispersibility) of the letter samples, but did notreproducethelargeamountofsiliconfoundinthecoatsoflettersamplespores.(Finding4.4)

S.5 It isdifficult todrawconclusionsabout theamountof timeneeded topreparethesporematerialortheskillsetrequiredoftheperpetrator.

•Thecommitteefindsnoscientificbasisonwhichtoaccuratelyestimatetheamountoftimeorthespecificskillsetneededtopreparethesporematerialcontainedintheletters.Thetimemightvaryfromaslittleas2to3daystoasmuchasseveralmonths.Givenuncertaintyaboutthemethodsusedforpreparationofthesporematerial,thecommitteecouldreachnosignificantconclusionsregardingtheskillsetoftheperpetrator.(Finding4.1)

S.6Physicochemicalandradiologicalexperimentswereproperlyconductedtoevaluatethesamplesforpotentialsignaturesconnectingthemtoasourcebutprovedtobeoflimitedforensicvalue.

•Thephysicochemicalmethodsusedprimarilybyoutsidecontractors

earlyintheinvestigationwereconductedproperly.(Finding4.2)

•RadiocarbondatingoftheLeahylettermaterialindicatesthatitwas

producedafter1998.(Finding4.5)

S.7 There was inconsistent evidence of B. anthracis Ames DNA inenvironmental samples thatwerecollected fromanoverseas site. (Finding3.4)

At the endof this study, the committeewasprovided limited informationforthefirsttimeabouttheanalysisofenvironmentalsamplesforanthracis Ames from an undisclosed overseas site at which a terroristgroup’santhraxprogramwasallegedly located.Thissitewas investigated

by the FBI and other federal partners as part of the anthrax lettersinvestigation. The information indicates that there was inconsistentevidenceofAmesstrainDNAinsomeofthesesamples,butnoculturableB. anthracis. The committee believes that the complete set of data andconclusions concerning these samples, including all relevant classifieddocuments,deservesamorethoroughscientificreview.

S.8 There are other tools,methods, and approaches available today for ascientificinvestigationlikethisone.

•Investigatorsusedreasonableapproachesintheearlyphaseoftheinvestigationto collect clinical and environmental samples and to apply traditionalmicrobiological methods to their analyses. Yet during subsequent years, theinvestigatorsdidnotfullyexploitmolecularmethodstoidentifyandcharacterizeB.anthracisdirectlyincrimesceneenvironmentalsamples(withoutcultivation).Molecularmethods offer greater sensitivity andbreadth ofmicrobial detectionandmorepreciseidentificationofmicrobialspeciesandstrainsthandoculture-basedmethods.(Finding3.3)

•Pointmutationsshouldhavebeenusedinthescreeningofevidentiary

samples.(Finding6.6)

New scientific tools, methods, and insight relevant to this investigationbecame available during its later years. An important example is high-throughput, “next-generation” DNA sequencing. The application of thesetools, methods, and insight might clarify (strengthen or weaken) theinferenceofanassociationbetweenRMR1029andthesporesintheattackletters.Suchapproacheswillbeimportantforuseinfuturecases.(Finding6.8)Theevidentiarymaterialfromthiscaseisandwillbeimmenselyvaluable,especially in the event of future work on either this case or other casesinvolving biological terrorism or warfare. It is critically important tocontinue to preserve all remaining evidentiary material and samplescollected during the course of this (the anthrax letters investigation) andfuture investigations, including overseas environmental samples, forpossibleadditionalstudies.(Finding6.10)

S.9Organizationalstructureandoversightarecriticalaspectsofascientific

investigation. The FBI generated an organizational structure toaccommodate the complexity of this case and received the advice ofprominentexperts.

•Overthecourseoftheinvestigation,theFBIfoundandengagedhighlyqualifiedexpertsinsomeareas.Itbenefitedfromtheunprecedentedguidanceofahigh-levelgroupofagencydirectorsandleadingscientists.Themembersofthisgrouphadtopsecretnationalsecurityclearances,metregularlyoverseveralyearsinasecurefacility,anddealtwithclassifiedmaterials.TheNRCcommitteeauthoringthisreport,inkeepingwithacommitmenttomakethisreportavailabletothepublic,didnotseethesematerials.(Finding3.1)

•A clear organizational structure and process to oversee the entire scientificinvestigation was not in place in 2001. In 2003, the FBI created a neworganizational unit (the Chemical, Biological, Radiological, and Nuclear[CBRN] Sciences Unit, sometimes referred to as the Chemical BiologicalScience Unit, or CBSU) devoted to the investigation of chemical, biological,radiological, and nuclear attacks. The formation of this new unit with clearerlinesofauthorityiscommendable.(Finding3.2)

•Aswasdoneintheanthraxinvestigation,attheoutsetofanyfutureinvestigationtheresponsibleagencieswillbeaidedbyascientificplananddecisiontreethattakesintoaccountthebreadthofavailablephysicalandchemicalanalyticalmethods.Theplanwillalsoneedtoallowforpossiblemodificationofexistingmethodsandforthedevelopmentandvalidationofnewmethods.(Finding3.5)

LOOKINGTOTHEFUTURE

Whilemuchof the committee’s effortwas focusedon a reviewof the scienceperformedinsupportoftheinvestigationofthe2001B.anthracismailings,anequally important goal has been to help ensure that future scientificinvestigationsofbiologicalattacksareconductedinthemostrelevant,rigorous,andthoughtfulmannerpossible.Althoughtheeventsof2001weretragic, theycould have been more catastrophic. In the future, among many otherrequirements, itwillbe important toensuremore timelyresults,moreefficientenvironmentalanalysis,accesstogloballyrepresentativestraincollections,andarobust capability for characterizing less well studied or less easily cultivatedbiological agents. Officials may also need tomanage expectations among the

general public, policymakers, and the scientific community about theconclusions that can realistically be expected from the use of microbialforensics.

S.10 A review should be conducted of the classified materials that arerelevant to theFBI’s investigationof the2001Bacillus anthracismailings,including all of the data and material pertaining to the overseasenvironmentalsamplecollections.(Recommendation3.1)

Thecommitteedidnotreceivenorreviewclassifiedmaterial.InNovember2010discussionswithFBIandDOJ leadership regarding this report,weweremadeawareofadditionalinformationthatwouldrequirereviewofclassifiedmaterial.Duetothelatenessofthisrevelation,theimportanceplacedonissuingatimelyreport, and the agreement between theNRCand theFBI that allmaterialsweconsideredbepubliclyavailable,thecommitteedidnotundertakethisadditionalreviewofclassifiedmaterial.

S.11 The goals of forensic science and realistic expectations and limitationsregarding its use in the investigation of a biological attack must becommunicatedtothepublicandpolicymakerswithasmuchclarityanddetailaspossiblebefore,during,andaftertheinvestigation.(Recommendation3.2)

TABLES-1FBIandDOJConclusionsandCommitteeComments

Relevantreportfinding/FBIconclusionsDOJconclusionsCommitteecommentsection

“Sporesofsuchhighconcentrationandpurityindicatethattheywerederivedfromhighqualitysporepreparations.Sporesofthisqualityareoftenusedinbiodefenseresearch,includingvaccinedevelopment.Itisimportanttohavehighlyconcentratedsporestochallengemosteffectivelythevaccinebeingtested.Similarly,highlypurifiedsporesarenecessarytopreventobstructionofthemachineryusedinthoseexperiments.Thesefindingsmeantthattheanthraxmailermusthavepossessedsignificanttechnicalskill”(USDOJ,2010,p.14).

Thecommitteefindsnoscientificbasisonwhichtoaccuratelyestimatetheamountoftimeorthespecificskillsetneededtopreparethesporematerialcontainedintheletters.Thetimemightvaryfromaslittleas2to3daystoas

muchasseveralmonths.Givenuncertaintyaboutthemethodsusedforpreparationofthesporematerial,thecommitteecouldreachnosignificantconclusionsregardingtheskillsetoftheperpetrator.

Finding4.1

continued

TABLES-1ContinuedRelevantreportfinding/

FBIconclusions DOJconclusions Committeecomment

section

Nosubstances “Throughoutthecourseof

Althoughsignificant Finding4.3

wereaddedto theinvestigation,repeated

amountsofsilicon

thesporesafter challengeshavebeenraised

werefoundinthe

productionto tothisfindingthatthe

powdersfromtheNew

makethemmore sporeswerenotweaponized.

YorkPost,Daschle,

dispersible(i.e., Thechallengeshavetheir

andLeahyletters,no

therewasno rootinaninitialfindingby

siliconwasdetected

“weaponization” theArmedForcesInstitute

ontheoutsidesurface

ofthespore ofPathology(‘AFIP’)that,

ofsporeswherea

material).Silicon

upongrossexamination,the

dispersantwouldreside.

andoxygenwere

sporesexhibitedasilicon

Instead,significant

presentinside andoxygensignal.However,

amountsofsiliconwere

thesporecoat subsequentanalysisofthe

detectedwithinthe

andnotonthe sporesbySandiaNational

sporecoatsofsome

outside(FBI, Laboratories,usingamore

samples.Thebulk

2009).a sensitivetechnologycalled

siliconcontentinthe

transmissionelectron

Leahylettermatched

microscopy(‘TEM’)–

thesiliconcontentper

whichenabledmaterial

sporemeasuredby

characterizationexpertsto

differenttechniques.

focusitsprobeofthespores

FortheNewYork

tothenanometerscale–

Postletter,however,

determinedthatthesilica

therewasasubstantial

waslocalizedtothespore

differencebetween

coatwithintheexosporium,

theamountofsilicon

anareainsidethespore.

measuredinbulk

Inotherwords,itwas

andthatmeasuredin

incorporatedintothecell

individualspores.No

asanaturalpartofthecell

compellingexplanation

formationprocess”(USDOJ,

forthisdifference

2010,p.14). wasprovidedtothe

Physicalfindings:Spore

committee.

particleshadamassmediandiameterbetween22and38microns,exhibitedanelectrostaticcharge,andweredevoidofaerosolizingenhancers(e.g.,fumedsilica,bentonite,orotherinertmaterial)(USDOJ,2010,p.14).

TABLES-1Continued


Isotopicanalysiswasconductedtodeterminethegeographicalsourceofevidentiarymaterialbasedonwaterandgrownmediasources.ScientistsattheUniversityofUtah,undercontracttotheFBI,concludedthattheresultsfromtheanalysiswereinconsistentwiththesporeshavingbeenproducedatDugwayProvingGroundTheFBIdrewnoconclusionsbaseduponthisanalyticalmethod(FBI,2009).Moregenerally,theresultswereinconclusiveduetothelarge

numberofvariablessuchassourcesofwaterandtheratioofhydrogenandoxygenatomsfoundinculturemedium,whichmadeitdifficulttonarrowdownanexactgeographicsource(FBI,2009).

ItwasnotpossibletoFinding4.5identifythelocationwherethesporeswereprepared.

continued


FBIconclusions

DOJconclusions Committeecomment

section

Testsledtothe “TwoseparateproductionTheflaskdesignated Finding4.6

conclusionthat batchesofanthraxwereRMR1029wasnot

twoseparate usedfortheNewYork

theimmediate,most

production andWashington,D.C.,

proximatesourceofthe

batchesof mailingsbecauseeach

lettermaterial.Ifthe

anthraxwere containeddifferencesin

lettermaterialdidin

usedforthesporeconcentrations,color,

factderivefromRMR-

NewYorkand contaminants,texture,

1029,thenoneormore

Washington, growthmediaremnants,and

separategrowthsteps,

D.C.mailings observeddebris.[But]when

usingseedmaterialfrom

becauseeach coupledwiththegenetic

RMR1029followed

contained analysis,investigatorswere

bypurification,would

differences abletoconcludethatthetwo

havebeennecessary.

inspore distinctbatchesofanthrax

Furthermore,the

concentrations, usedinthe2001attacks

evidentiarymaterialin

color, sharedacommonorigin”

theNewYorkletters

contaminants, (USDOJ,2010,p.15).

hadphysicalproperties

texture,growth

thatweredistinctfrom

mediaremnants,

thoseofthematerialin

andobserved theWashington,D.C.

debris.When letters.coupledwiththegeneticanalysisdiscussedinSectionB,infra,investigatorswereabletoconcludethatthetwodistinctbatchesof

anthraxusedinthe2001attackssharedacommonorigin(FBI,2009).

“Thisstrain,knownas‘Ames,’wasisolatedinTexasin1981,andthenshippedtoUSAMRIID,whereitwasmaintainedthereafter.”(USDOJ,2010,

p.3).

ThedominantorganismFinding5.1foundintheletterswascorrectlyandefficientlyidentifiedastheAmesstrainofB.anthracis.

TABLES-1ContinuedRelevant

FBIconclusions DOJconclusions

Committeecomment

reportfinding/section

Therewasno

“Thesporeparticles...

Theinitialassessment Finding5.2

deliberategenetic

showednosignsofgenetic

ofwhethertheB.

engineeringof

engineering”(USDOJ,2010,

anthracisAmesstrain

theB.anthracis

p.14). inthelettershad

strain(FBI, undergonedeliberate

2009). geneticengineeringormodificationwastimely

andappropriate,thoughnecessarilyincomplete.ThegenomesequencesoftheletterisolatesthatbecameavailablelaterintheinvestigationstronglysupportedtheFBI’sconclusionthattheattackmaterialshadnotbeengeneticallyengineered.

continued


FBIconclusions

DOJconclusions

Committeecomment

section

TheB.subtilis AdistinctBacillus Finding5.3

contamination species,B.subtilis,was

foundinthe aminorconstituent

of

NewYork theNewYorkPostand

samples“did Brokaw(NewYork)

notprovide…a

letters,andthestrain

productive foundinthesetwoavenueinterms

letterswasprobablythe

of…leadsforthe

same.B.subtiliswasnot

investigation” presentintheDaschle

(FBI,2009). andLeahyletters.TheFBI

investigatedthisconstituentof

theNewYorkletters

andconcluded,andthecommitteeconcurs,thattheB.subtiliscontaminantdid

notprovideuseful

forensicinformation.While

thiscontaminantdid

notprovideuseful

forensicinformationinthis

case,thecommittee

recognizesthatsuch

biologicalcontaminants

couldprovetobeof

forensicvalueinfuture

casesandshouldbe

investigatedtotheirfullest.

Therewere Multiplecolony Finding5.4mainlywild morphotypesoftypeB.anthracis

B.anthracisAmes

Amesstrain, werepresentinthebuttherewere materialineachof

significant thethreelettersthat

numbersof wereexamined(New

phenotypic YorkPost,Leahy,and

variantsor Daschle),andeachsubstrains(FBI,

ofthephenotypic

2009). morphotypeswasfound

torepresentoneormoredistinctgenotypes.

TABLES-1Continued

TABLES-1ContinuedRelevant

FBIconclusions DOJconclusionsCommitteecomment

reportfinding/section

Thesephenotypic

“Geneticanalysisof

Specificmolecular Finding5.5

variantscould morphologicalvariants

assaysweredeveloped

bedetectedby identifiedmutationswhich

forsomeofthe

acombination werelaterexploitedto

B.anthracisAmes

ofassaysfor developspecificassays

genotypes(those

fourdifferent toidentifythepresence

designatedA1,A3,

insertion/deletion ofidenticalmutationsin

D,andE)foundin

polymorphisms evidencecollectedduring

theletters.These

(FBI,2009). theinvestigation”(USDOJ,

assaysprovideda

2010,pp.24-25). usefulapproachforassessingpossiblerelationshipsamongthepopulationsofB.anthracissporesinthelettersandinsamples

thatweresubsequentlycollectedfortheFBIRepository(FBIR)(seeChapter6).However,morecouldhavebeendonetodeterminetheperformancecharacteristicsoftheseassays.Inaddition,theassaysdidnotmeasuretherelativeabundanceofthevariantmorphotypemutations,whichmighthavebeenvaluableandcouldbeimportantinfutureinvestigations.

TherewasinconsistentFinding3.4evidenceofB.anthracisAmesDNAinenvironmentalsamplesthatwerecollectedfromanoverseassite.

continued

TABLES-1Continued


“…fifteendomesticlaboratoriesandthreeforeignlaboratorieswereidentifiedasrepositoriesofAmesstrainanthraxatthetimeoftheletterattacks.”(USDOJ,2010,p.17).

“…thecollectionofAmesisolatesfromlaboratoriesbothfromtheUnitedStatesandabroadthatconstitutetheFBIRareacomprehensiverepresentationoftheAmesstrain(USDOJ,2010,p.28).

“Atotalof1,070sampleswereultimatelysubmitted[totheFBIR],whichrepresentsasamplefromeveryAmescultureateverylaboratoryidentifiedbytheFBIashavingAmesstrain”(USDOJ,2010,p.24).

TheFBIrepositorywasdevelopedfromanintensiveefforttoidentifylaboratorieshavingaccesstotheAmesstrain;however,wecannotconcludethatthisapproachidentifiedeverylaboratoryorwasacomprehensiverepresentation.

Section6.2

TABLES-1Continued


“FBIcollaboratedwithvariousexperts…toprovideaclearandthoroughprotocolforthepreparationoftherepositorysubmissions”(USDOJ,2010,p.77).

TheFBIappropriatelydecidedtoestablisharepositoryofsamplesoftheAmesstrainof

B.anthracisthenheldinvariouslaboratoriesaroundtheworld.Therepositorysampleswouldbecomparedwiththematerialfoundintheletterstodeterminewhethertheymightbethesourceofthelettermaterials.However,foravarietyofreasons,therepositorywasnotoptimal.Forexample,theinstructionsprovidedinthesubpoenaissuedtolaboratoriesforpreparingsamples(i.e.,the“subpoenaprotocol”)werenotpreciseenoughtoensurethatthelaboratorieswouldfollowaconsistentprocedureforproducingsamplesthatwouldbemostsuitableforlatercomparisons.Suchproblemswiththerepositoryrequiredadditionalinvestigationandlimitthestrengthoftheconclusionsthatcanbedrawnfromcomparisonsofthesesamplesandthelettermaterial.

Finding6.1

continued

TABLES-1Continued


“In2007,afterseveralyearsofscientificdevelopmensandadvancedgenetictestingcoordinatedbytheFBILaboratory,theTaskForcedeterminedthatthesporesintheletterswerederivedfromasinglespore-batchofAmesstrainanthraxcalled“RMR1029”(USDOJ,2010,p.5.)“Laterintheinvestigation,whenscientificbreakthroughsledinvestigatorstoconcludethatRMR1029wastheparentmaterialtotheanthraxpowderusedinthemailings,..”(USDOJ,2010,p.6).“Afteratime-consumingprocess,thescientificanalysiscoordinatedbytheFBILaboratorydeterminedthatRMR1029,aspore-batchcreatedandmaintainedatUSAMRIIDbyDr.Ivins,wastheparentmaterialfortheanthraxusedinthemailings.”(USDOJ,2010,p.8)...geneticanalysisledtotheconclusionthatRMR1029,locatedatUSAMRIID,wastheparentmaterialmailedsothemailedspores,..:(USDOJ,2010,

p.16“…basedonadvancedgenetictestingcombinedwithrigorousinvestigation,theFBIconcludedthatRMR1029istheparentmaterialoftheevidentiaryanthraxsporepowder,i.e.,theevidentiarymaterialcamefromaderivativegrowthofRMR1029”(USDOJ,2010,p.28).

TheresultsofthegeneticanalysesoftherepositorysampleswereconsistentwiththefindingthatthesporesintheattackletterswerederivedfromRMR1029,buttheanalysesdidnotdefinitivelydemonstratesucharelationship.Thescientificdataalonedonotsupportthestrengthofthegovernment’srepeatedassertionsthatthat“RMR1029wasconclusivelyidentifiedastheparentmaterialtotheanthraxpowderusedinthemailings”(USDOJ,2010,p.20),northerolesuggestedforthescientificdatainarrivingattheirconclusions,“thescientificanalysiscoordinatedbytheFBILaboratorydeterminedthatRMR1029,aspore-batchcreatedandmaintainedatUSAMRIIDbyDr.Ivins,wastheparentmaterialfortheanthraxusedinthemailings”(USDOJ,2010,p.

8).b

Finding6.2

TABLES-1Continued


“IfDr.Ivinspreparedhissubmissiontotherepositoryinaccordancewiththeprotocol,thatsubmissioncouldnotmissallfourofthemorphologicalvariantspresentinRMR1029”(USDOJ,2010,p.79).

ThegeneticevidenceFinding6.4thatadisputedsamplesubmittedbythesuspectcamefromasourceotherthanRMR1029wasweakerthanstatedintheDepartmentofJustice

AmerithraxInvestigativeSummary.

“…theonlycompletegeneticmatchtotheevidencecomesfromRMR1029anditsoffspring”(USDOJ,2010,

p.29).

ThescientificdatageneratedbyandonbehalfoftheFBIprovidedleadsastoapossiblesourceoftheanthraxsporesfoundintheattackletters,butthesedataalonedidnotruleoutothersources.

Finding6.5

continued

TABLES-1Continued


Outofthe1,059viablesamplesfromvariousstockscollectedintheFBIRduringtheinvestigation,8containedall4ofthepolymorphisms;2contained3ofthe4polymorphisms;andafewcontained1or2ofthemutations.(FBI,2009).

TheFBIandcontractscientistsappropriatelyrecognizedthatthemutationsintheletterisolatesprovidedinformationthatmighthelpidentifythesourceoftheB.anthracisusedintheattacks,developedappropriateassaysforfourofthesemutations,andcreatedandscreenedarepositoryofAmesstrainsamples.Basedontheresultsofthatscreening,FBIscientistsappropriatelyconcludedthatthemajorityofrepositorysamplescontainednoneofthefourmutations,although50ofthesamplescontainedoneofthefourmutationsand10sampleshadthreeorallfourmutations(thenumberswithoneormoremutationsarehigherifoneincludessamplesthatwereexcludedintheFBI’sStatisticalAnalysisReport).However,featuresoftherepository,includingunknownprovenance,possiblymultiplesamplesfromthesameflask,thehistoryofsharingandmixingofstockspresentedinvestigativechallenges.

Finding6.1discussion

TABLES-1Continued

aNote that thiswas thefinalconclusionof thescientificinvestigators. An initial finding by the Armed ForcesInstitute of Pathology (AFIP) found, upon grossexamination,thatthesporesexhibitedasiliconsignaland

sometimes exhibited an oxygen signal. Subsequentstudies conducted by Sandia National Laboratories (asdescribedinChapter4ofthisreport)determinedthatthesilicon was localized to the spore coat within theexosporium—thatis,itwasincorporatedintothecellasanatural part of the cell formation process. TheUSAMRIIDscientistwhofirstreviewedtheAFIPresultsand made statements regarding the presence of siliconand possible weaponization retracted those earlierstatements.

bSee for example, “Asnoted above, basedon advancedgenetictestingcombinedwithrigorousinvestigation,theFBI concluded that RMR1029 is the parent material oftheevidentiaryanthraxsporepowder,i.e.,theevidentiarymaterial came from a derivative growth of RMR1029.”(p.28)“Asdescribedindetailabove,over time,geneticanalysisdeterminedthatoneofDr.Ivins’sAmescultures,RMR1029—the purest and most concentrated batch ofAmes spores known to exist—was the parent to theevidentiarymaterialusedintheanthraxmailings.”(p.79)

1

Introduction

1.1BACKGROUND

In fall 2001, shortly after the September 11 attacks in New York City and

Washington,D.C.,U.S. citizens experienced a second set of attacks, this timeinvolving the bacterium Bacillus anthracis (B. anthracis, or more simply,anthrax) placed in at least four and possibly five letters and sent through themail.FromOctober4toNovember20,2001,22casesofanthraxwereidentified—11 inhalational and 11 cutaneous. Five of the inhalational cases were fatal(Jernigan et al., 2002).Twenty infected individuals contracted anthrax asmailhandlers or at worksites where contaminatedmail was processed or received.Twovictimswhodiedfromtheinfectionhadnoknowncontactwithanyoftheworksitesinquestion.Anadditional31peopletestedpositiveforexposuretoB.anthracis spores; approximately 32,000 individuals initiated antibioticprophylaxis (Centers for Disease Control and Prevention [CDC], 2001a;Jerniganetal.,2002).

Overthecourseofitsinvestigation,knownbythecasename“Amerithrax,”theFederalBureauofInvestigation(FBI)devoted600,000investigatorworkhourstothecaseandassigned17SpecialAgentstoaTaskForce,alongwith10U.S.Postal Inspectors. The investigation spanned six continents; involved over10,000witnessinterviews,80searches,26,000emailreviews,andanalysesof4millionmegabytesof computermemory; and resulted in the issuanceof5,750grand jury subpoenas (U.S. Department of Justice [DOJ], 2010, p. 4).Additionally,29government,university,andcommerciallaboratoriesassistedinconductingthescientificanalysesthatwereacentralaspectoftheinvestigation

(U.S.DepartmentofJustice[USDOJ],2010,p.4).

Theinvestigationalsoacceleratedthedevelopmentofanascentscientificfield,calledmicrobial forensics, involvingaseriesof laboratory tests topinpoint thegeneticidentityofamicrobialagentusedfornefariouspurposes.Thisfieldgrewout of the multidisciplinary areas of genomics, microbiology, and forensics,amongothers.Thedevelopmentandapplicationofmicrobial forensicsbecameanessentialpartofthescientificinvestigationinthehandsof

25

FBI investigators, who combined it with physicochemical analyses to narrow

theirsearchforthesourceoftheanthraxusedintheattacks.1

In 2008, seven years into the Amerithrax investigation,

the FBI asked the National Research Council of theNational Academy of Sciences (NAS) to conduct anindependent review of the scientific approaches usedduringtheinvestigationofthe2001B.anthracismailings(seeBox1-1).

DuringthecourseoftheNRCcommittee’sdeliberations,theDOJannouncedonFebruary19,2010,thatitwasclosingthecasebasedonitsconclusionthatDr.BruceIvins,ascientistattheU.S.ArmyMedicalResearchInstituteofInfectiousDiseases(USAMRIID),hadaloneperpetratedtheanthraxattacks.Dr.IvinsdiedonJuly29,2008aftertakinganoverdoseofover-thecountermedications.

Thecommitteecarriedoutitsworkmindfuloftheneedtoidentifylessonsthatcould be learned for future investigations in which science might play animportantrole.

1.2CHRONOLOGYOFEVENTSOFFALL2001

Public health officials in Florida announced on October 4, 2001, that RobertStevens, a photo editor at American Media, Inc. (AMI) in Boca Raton, hadinhalationalanthrax.Thiswas the first reportedcaseof inhalationalanthrax intheUnitedStatesinalmost25years.AfteroneofStevens’scoworkers,ErnestoBlanco, also fell ill and was diagnosed as having contracted anthrax,environmental assessmentsweremade of theAMI facility. These assessmentsrevealed B. anthracis contamination and postexposure prophylactic treatmentwasadministeredtoAMIemployees.Nocontaminatedletterwaseverfound;itisthoughttohavebeendiscardedafterbeingopened(CDC,2001a).AtimelineofthisandsubsequenteventsispresentedinTable1-1.

Lessthantwoweekslater,additionalcasesofapparentanthraxexposurebeganto appear inNewYorkCity. These cases indicated the possible source of theexposureasmostofthoseinfectedhadcomeintocontactwithletterscontainingapowder.TheNewYork lettersaddressed toTomBrokawofNBCNewsandtheNewYorkPost had a Trenton,New Jersey, postmark dated September 18,2001. Sampling of U.S. Postal Service drop boxes in the Trenton area foundanthraxsporesinonlyonemailbox,onNassauStreetinPrinceton(seeChapter3).

A second wave of mailings caused additional cases of anthrax. Two moreanthrax lettersbearing the sameTrentonpostmark,butdatedOctober9,2001,wereaddressedtoDemocraticU.S.SenatorsTomDaschleofSouthDakotaand

1In2008 theNationalBioforensicAnalysisCenterwasestablished in the Department of Homeland Security’sNational Biodefense Analysis and CountermeasureCentertoassistinmicrobialforensicsinvestigations.

BOX1-1ChargetotheCommittee

The NRC was asked by the FBI to conduct anindependent review of the scientific approaches usedduring the investigation.The official charge to thecommitteestated:

An ad hoc committee with relevant expertise will evaluate the scientificfoundation for the specific techniques used by the FBI to determine whetherthesetechniquesmetappropriatestandardsforscientificreliabilityandforuseinforensic validation, and whether the FBI reached appropriate scientificconclusionsfromitsuseofthesetechniques.Ininstanceswherenovelscientificmethods were developed for purposes of the FBI investigation itself, thecommittee will pay particular attention to whether these methods wereappropriately validated. The committee will review and assess scientificevidence (studies, results, analyses, reports) considered in connectionwith the2001 Bacillus anthracis mailings. In assessing this body of information, thecommitteewilllimititsinquirytothescientificapproaches,methodologies,andanalytical techniques used during the investigation of the 2001 B. anthracismailings.

Theareasofscientificevidencetobestudiedbythecommitteeinclude,but

maynotbelimitedto:

1. genetic studies that led to the identification of potential sources of B.

anthracisrecoveredfromtheletters;2. analysesoffourgeneticmutationsthatwerefoundinevidenceandthatare

unique to a subset of Ames strain cultures collected during theinvestigation;

3. chemicalanddatingstudiesthatexaminedhow,where,andwhenthesporesmay have been grown and what, if any, additional treatments they weresubjectedto;

4. studiesoftherecoveryofsporesandbacterialDNAfromsamplescollectedandtestedduringtheinvestigation;and

5. therolethatcrosscontaminationmighthaveplayedintheevidencepicture.

The committee will necessarily consider the facts and data surrounding theinvestigation of the 2001 Bacillus anthracis mailings, the reliability of theprinciplesandmethodsusedbytheFBI,andwhethertheprinciplesandmethodswere applied appropriately to the facts.The committee will not, however,undertakeanassessmentoftheprobativevalueofthescientificevidenceinanyspecificcomponentoftheinvestigation,prosecution,orcivil litigationandwilloffernoviewontheguiltorinnocenceofanyperson(s)inconnectionwiththe2001B.anthracismailings,oranyotherB.anthracisincidents.

TABLE 1-1 Timeline of Key Events in the AnthraxMailingsCase

2001September:LetterscontaininganthraxsporesaremailedtonewsorganizationsinNewYork(ABC,CBS,NBC,andtheNewYorkPost)andFlorida(AmericanMedia,Inc.).Whileonlytwolettersareactuallyrecovered(oneaddressedtotheNewYorkPostandtheothertoTomBrokawatNBC),theexistenceofotherlettersisinferredfromthepatternofinfection(Piggee,2008;Ember,2006).

September18:PostmarkdateonthePostandBrokawanthraxletters.ThepostmarkindicatesthatthelettersweremailedfromTrenton,NewJersey(Cole,2009,p.89).

October:LetterscontaininganthraxsporesaremailedtoU.S.SenatorsThomasA.DaschleandPatrickLeahyinWashington,D.C.TheFBIbeginsaninvestigation—code-namedAmerithrax—intothemailings(Piggee,2008).

October4:RobertStevens,aphotoeditorworkingforAmericanMedia,Inc.,inBocaRaton,Florida,isdiagnosedwithinhalationalanthrax,believedtohavebeencontractedasaresultofcontaminationofhisworkplacebyananthraxmailing.Thediagnosis,initiallymadebyaphysician-microbiologistatthehospitalwhereStevensreceivedcare,wasthenconfirmedattheFloridaStateLaboratoryinJacksonvilleandtheU.S.CentersforDiseaseControlandPrevention(Traeger,2002).

October5:RobertStevensdiesfrominhalationalanthrax.Heisthefirstoffivepersonstodieoftheillness.Intotal,11individualsarebelievedtohavecontractedinhalationalanthraxasaresultofthemailings(Ember,2006;Cole,2009,p.197).

October9:PostmarkdateontheDaschleandLeahyanthraxletters.ThepostmarkalsoindicatesthattheletterswerealsomailedfromTrenton,NewJersey(USDOJ,2010).

October12:TheFBIrecoverstheBrokawletter(USDOJ,2010,p.4).AcaseofcutaneousanthraxisconfirmedinErinO’Connor,anassistanttoTomBrokaw.Sheisthefirstof11personsbelievedtohavecontractedcutaneousanthraxasaresultoftheanthraxmailings(Cole,2009,p.54).

October15:TheDaschleletterisopenedintheSenator’sofficeintheHartSenateOfficeBuilding(Cole,2009,p.89).

October16,17:TheHartSenateOfficeBuildingandotherU.S.SenateandHouseofficebuildingsareclosed(Ember,2006).

October18:TheU.S.PostalService’sTrentonProcessingandDistributionCenterinHamiltonTownship,NewJersey,isclosedforanthraxtesting(Cole,2009,p.92).Onthesameday,inajointannouncementwithPostmasterGeneralJackPotter,FBIDirectorRobertMuelleroffersa$1millionrewardfor“informationleadingtothearrestandconvictionforterroristactsofmailinganthrax”(FoxNews,2001).

October19:TheNewYorkPostletterisdiscoveredandrecovered(USDOJ,

2010,p.4).

October21:ThomasL.Morris,Jr.,apostalworkerattheWashington,D.C.,BrentwoodMailProcessingandDistributionCenter,whichservicedCapitolHill,isthesecondpersontodiefrominhalationalanthraxbelievedtohavebeen

contractedasaresultoftheanthraxmailings.(Cole,2009,p.65).TheBrentwoodMailProcessingandDistributionCenterisclosedthesameday(Cole,2009,p.75).

October22:JosephP.Curseen,Jr.,apostalworkerattheWashington,D.C.,BrentwoodMailProcessingandDistributionCenter,isthethirdpersontodiefrominhalationalanthraxbelievedtohavebeencontractedasaresultoftheanthraxmailings(Cole,2009,

p.65).

TABLE1-1Continued

October31:KathyT.Nguyen,ahospitalworkerattheManhattanEye,Ear,andThroat

Hospital,isthefourthpersontodiefrominhalationalanthraxbelievedtohavebeen

contractedasaresultoftheanthraxmailings(Cole,2009,p.5).

November16:Inajointoperation,theFBI,U.S.EnvironmentalProtectionAgency,andU.S.PostalInspectionServicediscovertheLeahyletterinabagofunopenedmail(Piggee,2008).

November21:OttilieLundgren,anelderlywomaninOxford,Connecticutdiesfrominhalationalanthrax.Sheisthelastpersontodiefrominhalationalanthraxbelievedtohavebeencontractedasaresultofthemailings(Cole,2009,p.108).

December:TheLeahyletterisopenedandexaminedattheU.S.ArmyMedicalResearchInstituteforInfectiousDiseases(USAMRIID)atFortDetrickinFrederick,Maryland(Cole,2009,p.90).

December31:TheDirksenSenateOfficeBuilding,whichisconnectedtotheHartSenateOfficeBuildingbyundergroundcorridors,isreopened(NewYorkTimes,2002).

2002January23:TheHartSenateOfficeBuildingisreopened.Onthesameday,theFBIincreasestherewardforhelpinsolvingthecaseto$2.5million(Gallucci-White,2008,p.8).

June:OfficialssaytheFBIis“scrutinizing20to30scientistswhomighthavehadtheknowledgeandopportunitytosendtheanthraxletters”(Gallucci-White,2008,

p.8).

August6:AttorneyGeneralJohnAshcroftpubliclynamesStevenJ.Hatfill,aformerUSAMRIIDscientistandbiodefenseexpert,asa“personofinterest”intheAmerithraxinvestigation.Hatfillwouldbeclearedin2008(Freed,2010).

2003March:AnthraxdecontaminationbeginsattheAmericanMedia,Inc.,buildinginBocaRaton,Florida,whereRobertStevensworked(BioOne,2005).

June:Searchingforevidencerelatedtotheanthraxmailings,theFBIdrainsapondinFrederick,Maryland.Nothingsuspiciousisfound(Cole,2009,p.195).

August:StevenHatfillsuesAttorneyGeneralJohnAshcroftandothergovernmentofficials,accusingthemofusinghimasascapegoatanddemandingthathisnamebecleared(WashingtonPost,2008,p.11).

December22:TheU.S.PostalService’sBrentwoodMailProcessingandDistributionCenterisreopened(USDOJ,2010,p.3).

2005March14:TheU.S.PostalService’sTrentonProcessingandDistributionCenterinHamiltonTownship,NewJersey,isreopened(USDOJ,2010,p.3).

2007February8:FederalenvironmentalexpertsdeterminethattheformerAmericanMedia,Inc.,buildinginBocaRaton,Florida,hasbeenclearedofanthraxspores(Sarmiento,2007).

2008June:ThefederalgovernmentawardsStevenHatfill$5.82milliontosettlehisviolationofprivacylawsuitagainsttheDepartmentofJustice(DOJ)(Freed,

2010;WashingtonPost,2008).

continued

TABLE1-1Continued

July29:USAMRIIDmicrobiologistBruceE.IvinscommitssuicideastheFBIisabouttofilecriminalchargesagainsthimforhisroleintheanthraxmailings(CBSNews,2008).

August8:DOJofficiallyclearsStevenHatfillofinvolvementintheanthraxmailings(WashingtonPost,2008).

August18:TheFBIholdstwopressbriefings,oneforscientificmediaandoneforgeneralmedia,todescribe“thebodyofpowerfulevidence”thatallowedtheFBItoconcludethatithad“identifiedtheoriginandperpetratorofthe2001Bacillusanthracismailing”(FBI,2008).

September17:FBIDirectorRobertMuellertestifiesbeforetheSenateJudiciaryCommitteeatahearingentitled“OversightoftheFederalBureauofInvestigation.”Atthehearing,MuellerstatesthattheFBIwasseekinganindependentreviewofthescientificevidenceintheanthraxmailingscase.“Becauseoftheimportanceofthesciencetothisparticularcaseandperhapscasesinthefuture,”hesays,“wehaveinitiateddiscussionswiththeNationalAcademyofSciences”to“undertakeareviewofthescientificapproachusedduringtheinvestigation”(Temple-Raston,2008).

2010February19:DOJ,theFBI,andtheU.S.PostalInspectionServiceformallyconcludetheinvestigationintothe2001anthraxattacksandtheDepartmentofJusticeissuesanAmerithraxInvestigativeSummary.Inthesummary,DOJconcludesthat“Evidencedevelopedfrom[the]investigationestablishedthatDr.[Bruce]Ivins,alone,mailedtheanthraxletters”(USDOJ,2010,p.1).

Patrick Leahy of Vermont. The letter addressed toSenator Daschle was opened by a member of the

Senator’s staff on October 15. After discovering theDaschle letter, the House and Senate Office Buildingswere closed for environmental assessment anddecontamination.TheU.S.PostalServicesuspendedmailservicetotheU.S.CapitolandclosedtheHamilton,NewJersey,postalcenterwherethefourrecoveredlettershadbeenprocessed.

Postalofficialssubsequentlydeterminedthat twocontaminatedenvelopeswereprocessed at the U.S. Postal Service Processing and Distribution Center inWashington,D.C. (theBrentwood facility) onOctober 12.Exposure to sporesfromtheunopenedenvelopesat thepostalfacilitieswentundetecteduntilafterthe implicated envelope was opened at the Hart Senate Office Building. OnOctober 21, officials closed the Brentwood facility after a postal worker wasdiagnosedwith ananthrax infection.Severalworkers at thepostal facility thatprocessedtheletterfellillwithinhalationalanthrax,andtwoeventuallydied.

On November 16, 2001 FBI officials, U.S. Postal investigators, and U.S.Environmental Protection Agency hazardous material personnel found anunopened letter addressed to Senator Leahy that appeared to contain anthrax.Theletter,withanOctober9,2001,Trenton,NewJerseypostmark,waslocatedinoneofmorethan230drums—containing642bagsofunopenedmailsenttoCapitolHill—thathadbeensequesteredsincethediscoveryoftheanthraxlettermailed toSenatorDaschle (Beecher,2006).The letterhadaGreendaleSchoolreturn address, block handwriting, and other characteristics similar to theDaschleletter.AmisreadzipcodecausedtheLeahyletter tobemisdirectedtothe State Department mail annex in Sterling, Virginia, where a postal workercontractedinhalationalanthrax.

TheanthraxintheSenateletterswasahighlyrefineddrypowder consisting of about one gram of nearly purespores, as determined in subsequent laboratory analyses(see Chapter 4). The preparation was thus more potent

thanthematerialinthefirst(NewYork)setofmailings.

BythebeginningofDecember2001,itappearedthatthemailingshadended,asno additional letters hadbeendiscovered andno further caseswere identified.ButitwasclearthatmorewasrequiredthanapublichealthresponsebyCDC.TheattackswarrantedamajorlawenforcementinvestigationledbytheFBI,inwhich science would play a leading role. Identifying the source of the lettermaterials could lead to the person or persons responsible for the attacks.Keyquestions focused on the contents of the letters, how, where, and when thematerialsinthelettersmighthavebeenproduced,whetherthematerialinalltheevidence collected was identical, whether the material had been produced insuchamannerastobemoreeasilydispersible,whetherithadanydistinguishingphysicalorchemicalpropertiesofvalueindeterminingthesource,andwhetheritsbiologicalcharacteristicscouldprovideleadstoitsorigins.

1.3BRIEFSUMMARYOFTHEFBI’SSCIENTIFICINVESTIGATION

During its investigation of the anthrax mailings, the FBI worked with otherfederal agencies to coordinate and conduct scientific analyses of the sporepowders recovered from the letters, environmental samples, clinical samples,andsamplescollectedfromlaboratoriesthatmighthavebeenthesourceoftheletter-associated spores.Theagency reliedonexternal experts, including somewho had previously developed tests to differentiate among strains of B.anthracis.

Earlyintheinvestigationthesporesintheletters,aswellasenvironmentalandclinicalisolates,wereidentifiedasthe“Amesstrain”ofanthrax.ThisstrainwasoriginallyisolatedfromadeadcowinTexasin1981andshippedtoUSAMRIIDinFrederick,Maryland.Overtimeitwassharedwithresearchanddevelopmentlaboratories around the world. Thus, the identification of the strain of B.anthracisusedinthemailingswasinsufficienttoidentifyitssource,althoughitnarrowed the possibilities considerably. The evidence had to be examined foradditional unique and distinguishing features that could then be compared tosamples obtained from laboratories holding the Ames strain as a means tonarrowthesearchforthepossiblesourcematerial,andperpetrator(s).

TheFBIsubpoenaedsamplesfromlaboratoriesknowntohaveAmesstrain

B. anthracis and collected them in an FBI Repository (FBIR) that ultimately

included1,070 samples from20 laboratories—17domestic and3 international(inCanada,Sweden,andtheUnitedKingdom).Inaddition,theFBIandpartnerswithin the Intelligence Community collected environmental samples from anundisclosed overseas site at which they had reason to suspect activities by aterroristgroupinproducinganthrax.AlthoughculturesofthesesamplesdidnotproduceB.anthracis,molecularanalysisprovidedinconsistentevidenceforthepresenceofB.anthracisAmesstrainDNAinsomesamples(seesection3.4.3)

ScientistsfromtheDepartmentofDefenseexaminedthesporematerialsintheletters and identified several variants in the samples based on their colony

morphology.2With support from the National Institutes ofHealth, the National Science Foundation, and othergovernment agencies, FBI scientists worked with theInstitute for Genomic Research (TIGR) to identifyseveral genetic mutations associated with the alteredappearance of the cultured variants found in the letters(seeChapter5foranextensivediscussionofthiswork).

FBIinvestigatorscontractedtheassistanceoffourlaboratoriestodevelophighlyspecificmolecular-geneticassaystodetectfourspecificmutationsfoundintheevidence.Thesemutationdetectionassayswereusedintheexaminationof thesamplesintheFBIR,asdescribedinChapters5and6.

The analysis of samples in the FBIR led the FBI to focus attention on aparticular spore-containing flask at USAMRIID known as RMR1029. Theanalysisof the repositorysamplesand thebacteria in this flask isdescribed inChapter6.

In addition, analytical approaches such as scanning and transmission electronmicroscopy,energy-dispersiveX-rayanalysis,carbondatingbyacceleratormassspectrometry, and inductively coupled plasma-optical emission and massspectrometrywereusedtodeterminethechemicalandelementalprofilesofthesporepowders(seeChapter4).Thesestudiesweredonetodeterminewhentheanthraxpreparationmighthavebeenmade,whethertherewerecontaminantsortraceelementsthatwouldprovideacluetotheproductionlocationormaterialsused, and whether there was evidence of an effort to deliberately include

additivestoimprovedispersaloftheanthrax.

1.4SUMMARYOFFBIANDDOJSCIENTIFICCONCLUSIONS

The scientific analyses led theFBIandDOJ todrawanumberof conclusions(seeTableS-1intheSummary).Thecommitteefounditchallenging,however,to identify theFBI’sdefinitiveconclusionsbecause thoseprovidedpubliclybyDOJ in its briefings and investigative summary and those provided by FBIofficialsinoralpresentationstothecommitteevaried.Forthepurposesofthis

2 Morphological variants are observable physical orbiochemical characteristics of an organism. Thesecharacteristics are determined by both genetic makeupandenvironmentalinfluences.

report, the committee’s analyses are based on thescientific conclusions provided by the FBI to thecommittee onSeptember 24, 2009 (left-most columnofTableS-1),thoseissuedpubliclybyDOJonFebruary19,2010, when it closed the case (USDOJ, 2010) (columnsecondfromtheleftinTableS-1),andthoseprovidedbyLouisGrever, EdwardMontooth, andRachel Lieber onJanuary14,2011(FBI/USDOJ,2011).

1.5COMMITTEEPROCESS

Under the terms of theNRCcontractwith theFBI, theFBI initially providedtwoboxescontainingapproximately9,000pagesofmaterialstothecommittee,andtheninDecember2010,theFBIgavethecommitteeanadditional641pagesrelated to the scientific investigations undertaken by the FBI and by variousexternal expertsworking at the behest of theBureau during the course of theanthrax investigation.Throughout theNRCstudyprocess thesematerialswerecovered by FOIA Exemption 7, “law enforcement sensitive,” and were not

publiclyavailable.Uponreleaseofthisreport,asspecifiedinthecontract,these

documentshavebeendepositedintheNRCPublicAccessFile.3

Documents were initially delivered in two batchescontainingreportsofthescientificanalyses(seetheIndexof Documents Provided by the Federal Bureau ofInvestigation for a listing).4 The first batch includedtechnical reviewpanel reports, laboratory analytical testreports and results pertaining to Ames strainidentification,carbondating,stableisotopeanalysis,agarandhemeanalysis,andassaydevelopment,andpublishedpapers. Batch two included materials regarding geneticdiversityandphylogeneticcharacterizationofB.subtilis(another bacterial species); repository screening andmolecular analysis of pathogen strains and isolates andgeneticsof theA1,A3,B,D,andEmutations found inthe evidence; statistical analysis; cross contamination;and chemical and physical properties of the sporepowders. The third batch of documents received inDecember 2010 contained reports of scientific reviewmeetings and some additional information about samplecollection, laboratory notebooks, and reports ofinvestigationsofindividuals.Additionaldocumentswereprovided by the FBI at the committee’s requestthroughout the study; these documents are listed in theIndexofDocumentsProvidedby theFederalBureauofInvestigation under the heading Supplemental

Documents.

No written explanatory materials were provided with these documents thatwould fully inform the committee as towhy the analyseswere done and howthese

3 The public can gain access to these materials bycontactingtheNRCPublicAccessRecordsOffice.

4 In this report, the principal documents received fromthe FBI are referenced according to the followingconvention: “FBI Documents, B*M*D*” where B =Batch,M =Module, D = Document, and * = Number.Thus“FBIDocuments,B1M1D1”wouldrefertothefirstdocument in the first module of the first batch ofmaterialsreceivedfromtheFBI.

documents contributed to the FBI investigations andconclusions.ThematerialregardinganalysesoftheFBIRspecimens was coded, often with different numbers forthesamesampleset.Consequently,thecommitteespentaconsiderable amount of time sorting through andattempting to interpret the available materials before itcould begin to evaluate the science and consider thescientific conclusions. In addition, much of theinformation provided to the committee wascompartmentalizedandsectionsofsomedocumentswereredacted.

WhenthecommitteeposedquestionstotheFBIforclarification,theagencywas

alwaysresponsive;however,responsestoquestionsweresometimesminimalorterse,orweredeflectedas intrudingintothecriminal investigationandbeyondthe purview of the committee despite the committee’s explanation that thequestions were of a scientific nature. Some of these responses may reflecttensionbetweenthescopeofthescientificreviewexpectedbytheFBIandthecommittee’sinterpretationofitscharge.Insummary,theFBIprovidedsomeofthe primary information related to the scientific analyses and was generallyresponsivetocommitteequestions,butearlyonitwasdifficultforthecommitteetoascertaindetailsofwhatwasdoneinthecourseofsomeoftheFBIscientificwork,theidentityofsomeofthesamplesanalyzed,andtherelationshipsamongthesamplesintherepository.

In addition to materials provided directly by the FBI to the committee, FBIofficials also briefed the committee on several occasions. Some of thesebriefings were done in open session, while others were conducted in closedsessionscoveredbyFOIAExemption7.Intheseclosedsessionsthecommitteeheard from a number of DOJ/FBI personnel including: John Fraga, ChristianHassell, Louis Grever, Edward Montooth, and Rachel Leiber. FBI consultantRanajitChakraborty(UniversityofCincinnatiandcurrentlyUniversityofNorthTexas Health Sciences Center) and Daniel Martin (Dugway Proving Ground)alsobriefed the committee in closed session. In addition, the committeeheardfrom a number of other experts:BruceBudowle (formerly FBI;University ofNorthTexasHealthSciencesCenter);RitaColwell(UniversityofMarylandandJohns Hopkins University); Claire Fraser-Liggett (The Institute for GenomicResearch and University of Maryland School of Medicine); Hank Heine(formerlyUSAMRIID);CongressmanRushHolt;PaulKeim(NorthernArizonaUniversity); Joseph Michael (Sandia National Laboratories); Steven Schutzer(UniversityofMedicineandDentistryofNewJersey);JenniferSmith(formerlyFBI;BIOFORConsulting); PatriciaWorsham (USAMRIID); and PeterWeber(LawrenceLivermoreNationalLaboratory).

Inconductingitsreview,thecommitteefocusedonthebiological,physical,andchemical sciences applied to evidentiary materials. The committee was notcharged toconsiderorevaluateanyof the traditional forensicsciencemethodsandtechniquesused(e.g.,hair,fiber,fingerprint,orhandwritinganalysis)(NRC,2009a)nordiditconsideranyofthepsychologicalorbehavioralsciences,suchaslinguistics,usedbytheFBIinitsinvestigation.

The committee met seven times in person in open and closed sessions and

continueditsdeliberationsbyconferencecall.Closedsessionswerereservedforreviewof law enforcementmaterials (FOIAExemption 7) relevant to theFBIinvestigation, committee analyses, deliberations, and report drafting. Publicsessions were convened to gather information from the scientific communityaboutvariousaspectsofthescientificinvestigationorareasofscientificresearchrelevant to the matters at hand. DOJ closed its investigation of the anthraxmailingsafterthecommittee’sfourthmeeting.

InNovember2010,theFBIcontactedtheNationalAcademiesandrequestedtheopportunity to provide the committee with additional materials and anotherbriefing.Thecommitteesubsequentlyreceivedandreviewedthe thirdbatchofmaterials,anadditional641pagesofdocuments,andmetforonefinalbriefingwithFBIandDOJofficialsinmid-January2011.

1.6ISSUESFORCONSIDERATIONINREADINGTHISREPORT

The FBI’s anthrax investigation involved the development and use ofmodernscience inanattempt to solvea crimecommittedwithabiological agent.Theuseofscienceinlegalinvestigationsisnotnew.Scienceiscalledontoanswerquestions, for example, about the safety of drugs, risks from exposure toenvironmentaltoxins,andidentificationofDNAfromarapeormurdervictim.Yet science and the judicial system do not always have an easy relationshipbecauseof differences in culture andoverall objectives.The scientific processtakestime,raisingquestionsandseekinganswers,andchallengingandrevisingaccepted theoriesandnotionsuntilnewhypothesesaregenerated.The judicialsystem,ontheotherhand,aimstosettledisputeswiththeinformationavailableatapointintime.Ittypicallydoesnothavetheopportunitytoconductanotherstudy and wait for complete information. Scientific investigation usually is amore open-ended endeavor than a legal or criminal investigation as scientistsacknowledgeappropriatedegreesofuncertainty—bothsmallandlarge—intheirinvestigations and are inspired to do futurework on the questions of interest,yieldingmorecertaintyandmoreinformation.Incontrast,thejusticesystem,tobe effective, requires decisions to be made rather than deferred, and thusscientificuncertaintyhastobeweighedinlightofallotherevidence.Toleranceforscientificuncertaintymayormaynotbetemperedbythestrengthofother,nonscientificevidence.

As demonstrated in this investigation, the FBI used science in two differentways:1)toidentifyandanalyzeevidenceusingmethodsthatareacceptablefor

presentation in the courtroom; and 2) to identify leads for a criminalinvestigation.Inthelattercase,thescienceperseisnotintendedtobepresentedin the courtroom but it may provide leads to inform and direct the lawenforcement investigation. In either case, the science must be conductedcorrectlyandperformedatahighlevelofscientificstandards.

The committee recognized that forensic science is the application of scientificmethods to matters of interest to the judicial system and must, therefore,consider thenormsofbothscienceand the law(NRC,2009a,Chapter3).Thecommitteealsorecognizedthatsometimespressingnationalinterestorsecurityconcerns, such as those present during this investigation, demand that newlyemergingmethodsbeappliedtotheassessmentofforensicevidenceevenbeforethose methods have been widely adopted or validated by peer review in theforensic and scientific communities. It should be noted that future biologicalattackswillprobablyposegreaterchallengesthandidthisattack:theagentmaybe a member of a species with a more complex and poorly understoodpopulation structure, the agent may be genetically modified in a manner thatfurtherobscuresitsorigin,orasampleoftheattackmaterialmaynotbereadilyavailable (as it was in this case). This last possibility may mean thatenvironmental or clinical samples, with their inherent added challenges, willhavegreaterimportanceinafutureinvestigation.

National security concerns and the pressures of an ongoing criminalinvestigationmayrequirethatthecollectionofsamplesandtheirevaluationbecarried out under circumstances of secrecy that limit the capacity of outsideobservers to assess the validity of the forensic interpretations. Suchcircumstances pose special challenges in which the optimal application andevaluationofscientificmethodsmay insome instances runcounter tosecurityinterests.Thecommittee faced this tensionbetween science and security in itsdeliberations.

In the end, the committee considered the facts and data of the scientificinvestigation, the reliability of the principles and methods used by the FBI,whethertheprinciplesandmethodswereappliedappropriatelytothefacts,andtheconclusionsrelatedtotheseefforts.Thecommitteedoesnot,however,offeraviewontheguiltorinnocenceofanyperson(s)inconnectionwiththe2001B.anthracismailingsoranyotherB.anthracisincidents.

1.7ORGANIZATIONOFTHEREPORT

Based on its review of the materials provided, the committee developed thefindingspresentedinthisreport.ThereportisorganizedtoprovidebackgroundonthescientificcharacteristicsofB.anthracis(Chapter2);describeandreviewthe procedures used in the early stages of the investigation concerning thecollectionofevidenceanditsprocessingandpreservation,aswellasthecreationof a repository of B. anthracis samples collected from around the world forcomparative and investigative purposes (Chapter 3); review and assess thephysicochemicalanalysesoftheanthraxevidence(Chapter4);reviewandassessthebiologicalcharacteristicsofthematerialintheletters(Chapter5);andreviewandassess the analyses and results of theFBI’s comparisonof the evidentiarymaterialagainstthesamplesintheFBIRepository(Chapter6).Thecommittee’sfindings,analysis,andrecommendationscanbefoundinChapters3through6.

2

BiologyandHistoryofBacillusanthracis

2.1INTRODUCTION

Bacillusanthracis(B.anthracis)isaGram-positiverod-shapedbacteriumthatisthe causative agent of the disease anthrax. B. anthracis rods typically havedimensionsofapproximately1µmby4µmandmayoccurinchainsresembling“boxcars”when observed under amicroscope. Theword “anthrax” is derivedfrom the Greek word for “coal” or “black” and was applied to this diseasebecausepatientswiththecutaneousformofthediseaseoftenexhibitblackskinlesions. The disease has been known since antiquity. In the 19th century, thedemonstration byRobertKoch ofB. anthracis as the cause of anthrax was acornerstoneofprovingthegermtheoryofdisease.

This chapter summarizeshistorical, biological, andphylogenetic featuresofB.anthracis, and in particular the Ames strain, as well as clinical features ofanthrax,many of which proved highly relevant as the events surrounding themailingoftheB.anthracisspore-ladenlettersunfoldedinfall2001.

2.2THEBIOLOGYOFB.ANTHRACIS

B.anthracissporesaretheinfectiousagentforallformsofanthrax.Incontrast,B. anthracis vegetative cells are noninfectious in animal models (Mock and

Fouet,2001).Thestructuralcharacteristicsandenvironmentalresistanceof theB.anthracissporearekeytoitsavoidanceofthehostinnateimmuneresponseduring the initial stages of infection (Cybulski et al., 2009;Mock and Fouet,2001). Relative to themajority of bacterial vegetative cells, spores like thoseproducedbyB.anthracis are highly resistant to a variety of commonly lethaltreatments, including dehydration, elevated temperature, UV irradiation,numeroustoxicchemicals,andenzymaticdigestionbyproteasesandlysozymes(Setlow,2006).Inthedormantstate,sporescanretainviabilityfordecadesandlikely for centuries (Nicholson et al., 2000); however, upon arrival in ahospitable environment, the spores can germinate and resume rapid growthwithin

37

hours (Moir, 2006; Setlow, 2003). The stability of spores,with accompanyingeaseofstorageandtransport,isalsoamajorfactorintheirpotentialutilityasabiologicalweapon.

The resilience of B. anthracis spores derives from their unique physical andstructuralcharacteristics.Thesporecytoplasmor“core”isrelativelydehydratedand contains high concentrations of certain low-molecular-weight solutes(Setlow, 2006). These conditions result in complete metabolic dormancy andextremeproteinstability.Thelowcorewatercontentofthesporecorrelateswitha high spore density, a property that is commonly exploited for sporepurification. Centrifugation through density gradients of diatrizoate andmeglumine is a widely used purification method (Tamir and Gilvarg, 1966;NicholsonandSetlow,1990).

Surroundingthesporecoreareamembraneandaspecializedpeptidoglycancellwall,the“cortex.”Thesestructuresplaykeyrolesinlimitingmovementofwaterandsolutes intoandoutof thecore,maintaining thedehydrateddormant state(Setlow,2006).Themembraneandcortexhavenotbeencitedassignificantsitesofmineral associationnor found toplay roles indirect spore interactionswithexternalsurfaces.

Theoutermostlayersofthespore,thecoatandexosporium,aretheprimarysitesofinteractionwiththehostandwithothersurfacesandareimportantfactorsinthe resistance of spores to certain enzymatic and chemical treatments (Driks,2009; Henriques and Moran, 2007; Setlow, 2006). These structures are

composed predominantly of protein and glycoprotein. In some spore-formingbacteria,thesporecoatshavebeenshowntobesitesofassociationofminerals(Johnstoneetal.,1980;Mannetal.,1988;Stewartetal.,1980,1981;Hirotaetal., 2010). These outermost integuments can play major roles in determiningspore adherence to surfaces (Bozue et al., 2007;Brahmbhatt et al., 2007) andmayaffectelectrostaticpropertiesandaggregationwithothersporesorparticles,all of which will affect spore dispersal and infectivity. Efforts at sporeaerosolization and dispersal have been pursued through modification of thespores’surfacestructuresandproperties(Swartz,2001).

The fact thatB. anthracis can exist in the environment as a dormant, highlystablesporemayhaveevolutionarysignificance.WhileadormantenvironmentalstatemayhelpexplainobservationsthatpopulationsofB.anthracishavemuchlessgeneticvariation thanmanyotherbacterialspecies(VanErtetal.,2007a),other factors may also contribute, including the likely recent origin of thisspeciesandthepossibilityoflimitedmeansforhorizontalgenetransfer.

2.3CLINICALASPECTSOFANTHRAX

Anthrax isgenerallyadiseaseofherbivores (e.g.,cattle, sheep,horses),whichacquire the infection by grazing on contaminated soils. Anthrax spores arehighly resistant to environmental insults. These spores allow the bacterium tosurviveforlongperiodsoftimeinsoil,itsnaturalreservoir.Thediseaseoccursworldwide, and there are occasional outbreaks of anthrax in livestock in theUnitedStatesandCanada.

Thecourseandoutcomeofhumananthraxdependondoseandonwhethertheinfectionisacquiredviatheskin,gastrointestinaltract,orinhalation.Mostcasesof human anthrax involve skin lesions (cutaneous anthrax), whereby theinfection is usually acquired as a result of handling infected animal hides orwool, leading tocontaminationof skinabrasionswithB.anthracis. Cutaneousanthrax is the least lethal form of the disease, but still can cause significantmortality of up to 20 percent (Atlas, 2002) if not treated with antimicrobialtherapy. Ingestion of food, such as meat contaminated withB. anthracis, canproducegastrointestinalanthrax,whichisaseriousdiseasewith25to60percentfatality.This formof thedisease isextremely rare indevelopedcountries.Themostfulminantandlethalmanifestationofanthraxisduetoinhalationof

B. anthracis spores, causing a highly fatal disease. Inhalational anthrax is

generally rare and is observed mainly in individuals who work with animalskins.Inthe19thcentury,itwasknownas“woolsorter’sdisease”andisbelievedto havebeen the first documentedoccupational illness (Leffel andPitt, 2006).Inhalational anthrax in individuals not likely to have suffered occupationalexposure,however,canbeasignofabiologicalattackwithB.anthracisspores.

Inhalational anthrax is a rapidly progressive disease with high mortality andmorbidity even when treated with antimicrobial therapy. Anthrax sporesgerminatewhenplacedinbloodorotherhumanoranimaltissuesthatprovideanutrient-rich environment (Inglesby et al., 2002). Upon inhalation, the host’smacrophages(atypeofimmunesystemcell)attackandingestthespores,whichare protected from these host cells by the spore coat. The macrophagesunwittingly transport the spores to lymph nodes in the respiratory system(Liddington,2002),wheretheyarereleased.Thelymphnodesprovidesufficientnutrientstoallowthesporestogerminateandbegintoproliferate.Proliferationinthelymphaticsysteminturnallowsthebacteriaintheir“vegetative”statetospreadintothebloodstreamandbedisseminatedtomultipleorgans.

The genetic determinants of virulence inB.anthracis reside primarily on twolarge plasmids, which are extrachromosomalDNAmolecules. These plasmidsareknownaspXO1andpXO2,andtheycontaingenesthatencodeforanthraxtoxin and a poly-D-glutamate capsule, respectively. Anthrax toxins arecomposed of a combination of three proteins that work together: protectiveantigen(PA),edemafactor(EF),andlethalfactor(LF).WhenPAcombineswithEF and LF, toxicity to host cells and a buildup of fluids (edema) in infectedtissues (e.g., the lungs) are produced. EF is an adenylate cyclase enzyme thatpromotes the accumulation of cyclic adenosine monophosphate (AMP),producing a loss of cellular regulation of water and ion metabolism. LF is ametallopeptidasethatcleavesproteinsofsignaltransductionpathways,resultingin profound effects that range from cell death to interferencewith the cellularfunctionsnecessaryformountingappropriateimmuneresponses.Theeffectsofthe anthrax toxin can kill an infected patient even after the bacteria in thepatient’sbodyhavebeenkilledbyantibiotics(Bouzianas,2009).

InadditiontothePA,EF,andLFtoxincomponents,B.anthracisproducesothercytotoxicenzymes, suchasanthrolysinO, thatcontribute topathogenesis.Thecombination of the protein toxins, which undermine the host’s defenses andinterferewithcellularfunction,andthepolymerizedaminoacidcapsule,whichprotectsthebacteriumfromphagocyticcells,representsaparticularlylethalmix.

Antibodies that neutralize the toxins or promote phagocytosis of encapsulatedcellscanconfersignificantprotection to thehost.Thevaccinecurrently inusefor the prevention of anthrax functions by eliciting neutralizing antibodyresponsestoanthraxtoxin.B.anthracismutantsthatlackeithertoxinproductionorcapsulesareattenuatedforvirulenceandcanbeusedinvaccineformulations(FriedlanderandLittle,2009).

2.4B.ANTHRACISASABIOLOGICALWEAPON

ResearchonB.anthracisasabiologicalweaponbeganmorethan90yearsago,accordingtoInglesbyandcolleagues(2002).Whenappropriatelyprepared,

B.anthracisspores are premier agents for biologicalwarfare and bioterrorismbecause theycanbeproducedinprodigiousquantities inaformthat is readilyaerosolized and inhaled.These factors, plus thehighmortality associatedwithinhalational anthrax, make B. anthracis a serious military and bioterrorismthreat.

Most offensive biological weapons programs were terminated following theratificationorsigningoftheBiologicalWeaponsConventionintheearly1970s(Inglesbyetal.,2002).TheUnitedStates’offensivebiologicalweaponsprogramwas terminated before that by President Nixon in 1969. Nevertheless,international state programs to produce and weaponize B. anthracis haveremainedaconcern.Forexample,in1995IraqacknowledgedhavingananthraxweaponsprogramtotheUnitedNations.

EstimatesoffatalitieslikelytooccurafteramajorattackonanurbanareausingaerosolizedB.anthracisasabioweaponrangeintothemillions(Meselsonetal.,1994; Inglesby et al., 2002). Even an accidental release of aerosolized B.anthracisata formerSovietmilitary facility inSverdlovsk in1979resulted inscoresof infectionsandmanyhumanandanimaldeaths (seeBox2-1) (LeffelandPitt,2006).NoinhalationalanthraxfatalityhadoccurredintheUnitedStatesastheresultofanactofwarorterroruntilthe2001anthraxmailings(Bushetal.,2001).

Theeventsof2001,whenenvelopeswithB.anthracis sporesweredistributedthrough the U.S. mail, showed the potential of this microbe as a biologicalweapon.Theresulting22casesofanthrax,includingfivefatalities,spreadgreatfear and resulted in tremendousdisruption anddislocation asmail distribution

centers,congressionalofficebuildings,andothersitessufferedextensive

BOX2-1TheSverdlovskOutbreak

In1980, reports appeared in the international pressof awidespreadoutbreakofanthraxinthecityofSverdlovsk(now Ekaterinburg) in the Soviet Union (Gwertzman,1980).Sovietmedical,veterinary,and legalpublicationsreported thatanoutbreakhad takenplace inearly1979,involving livestock and humans who ate contaminatedmeat.In1988,threeSovietphysiciansvisitedtheUnitedStates to give formal and informal presentations on theoutbreak (Meselson et al., 1994). In 1990, morediscussion appeared in the Russian press (references inMeselson et al., 1994), leading Soviet President BorisYeltsintocallforfurtherinvestigations.Finally,in1992,Yeltsinwasquotedassaying“thattheKGBadmittedthatourmilitarydevelopmentswerethecause”(referencesinMeselsonetal.,1994).

AgroupofscientistsfromtheUnitedStatescarriedoutanon-siteinvestigationduring twovisits in 1992 and1993; their resultswerepublished inScience in1994 (Meselson et al., 1994; Guillemin, 1999). They concluded that “theoutbreakresultedfromthewindbornespreadofanaerosolofanthraxpathogen,that thesourcewasat themilitarymicrobiologyfacility,andthat theescapeofthepathogenoccurredduringthedayonMonday,April2[1979].”

Theoutbreakhasbeenused tomodelaerosolizedagentreleaseandparameterssuch asplumemigration (Hogan et al., 2007), infectiousdose (Brookmeyer etal., 2001; Wilkening 2006), incubation period (Brookmeyer et al., 2005;Wilkening,2008),andtheimpactofpublichealthintervention(Brookmeyeretal.,2001).A1998studybyPaulKeimandPaul Jackson (Jacksonet al.,1998)

used thepolymerasechain reaction (PCR) technique toanalyze tissue samplesfrom 11 patients from the 1979 Sverdlovsk outbreak. The study found thatsequences representing the entire virulence complement (toxins and capsule)werepresentinallthesamples,andalsofoundthatstrainsfromatleastfourofthe five known strain categories were distributed among the samples, asdeterminedbyvariable regionsequencing (variablenumber tandemrepeats,orVNTRs).Twosubsequentstudieswerepublished:onefoundadditionalevidencefor multiple strains (Price et al., 1999) and the other found no evidence formultiple strains (Okinaka et al., 2008).Whether or not multiple strains wereinvolved in this outbreak and if so the implications of this are not known. Apreviousanalysisof198clinicalsamplesfromnaturallyoccurringanthraxcasesindicatedthepresenceofonlyonestrainineachcase(Jacksonetal.,1997).

contamination(seeChapter3).Over30,000peopleweregivenprophylacticantibiotic treatmentandsixbuildingsrequireddecontamination,allatacostofover$1billion(CDC,2001a).

2.5PHYLOGENYOFB.ANTHRACIS

B.anthracisisamemberofthelargegenusBacillusthatincludesothercommonanddiversespecies,suchasB.cereus,B.subtilis,andB.thuringiensis.

Within the genus, B. anthracis represents a separate lineage that apparentlyevolvedfromtheB.cereusparentspecies.SomestrainsofbothB.cereusandB.thuringiensisareclearlycloselyrelatedtoB.anthracis(Kolstoetal.,2009).Theproductionofendospores that resist chemicals,heat,UV light, anddesiccationwhen the cells experience starvation or other environmental stresses is acommonfeatureofmembersoftheBacillusgenus.

B.anthracishasoftenbeenviewedasahighlymonomorphicspecies,thatis,onethatshowslittlegeneticvariationamongisolates.Whileitisthecasethatmanybacterialspeciesharbormuchmoreextensivegeneticdiversity,newermolecularmethods have allowed researchers to find genetic differences among naturalisolates of B. anthracis, as discussed below. Moreover, all populations ofbacteria are continually generating newmutations.Whilemost newmutationsremainveryrareinapopulation,someofthemmayrisetohighandmeasurableabundances,especiallywhentheyprovideanadvantagetothebacteriainterms

ofincreasingtheirgrowthorsurvivalundercertainconditions.Asdiscussedindetail inChapters5and6, thepresenceofnewmutationsamongthespores intheattacklettersprovidedanimportantleadintheanthraxlettersinvestigation.

ModernscientifictoolsnowaffordanunderstandingatthemolecularlevelofthesimilaritiesanddifferencesamongmembersofthegenusBacillus.Forexample,asnotedabove,thepathogenicityofB.anthracisisrelatedtothepresenceofthepXO1andpXO2plasmids.Thesewereoncebelievedtoseparate

B.anthracisfromtheothermembersofthegenus,Bacillus.Withinthelastfiveyears, however,B. cereus strains containing pXO1 or both pXO1 and pXO2havebeendiscovered, the latter in great apes inAfrica.Thus, the presenceofthese plasmids is no longer believed to be the major factor separating B.anthracisfromothermembersofthegenusBacillus.Rather,aspecificmutationinB.anthracis’global regulatorplcRgene,whichcontrols the transcriptionofvirulence factors inB.cereus andB. thuringiensis,now appears to be the keydifference.AllB.anthracisstrainsinvestigatedcarryamutationthatmakestheplcRgenedysfunctional.ThisfeaturemakesB.anthraciseasilydistinguishablefromitscloserelatives.

Untiltheadventofmodernmolecularapproaches,thegenetichomogeneityofB.anthracis impededefforts toreconstruct theevolutionaryhistoryof thespecies(Keimet al., 1999).Substantial progress has recently beenmade.VanErt andcolleagues(2007a)conductedalargestudyof1,033B.anthracisisolatesusingcanonical SNP (single nucleotide polymorphism) analysis. Based on theirresults,theydividedallB.anthracisisolatesintoA,B,andClineagesthathadbeen previously recognized and further subdivided these into 12 distinctsublineagesorsubgroups(seeFigure2-1).Sublineagedesignationwasbasedonwhole-genomesequencingofsevenisolates that representmajorbranchesonaphylogenetic tree. The five other subgroups, whose genomes were notsequenced,appearatintermediatepositionsalongthesebranches.

VanErtandcolleagues(2007a)pointoutthatdispersalofsporesviacommoditieshasdistributedanthraxworldwidesuchthattherearefociofthediseaseonallcontinentsexceptAntarctica(seeFigure2-1).Themorenumerous“A”lineageisolatesofB.anthracisarethemostwidelydistributed,whiletherearemorerestricteddistributionsof“B”and“C”lineageisolates;forexample,theBlineageisfoundmainlyinSouthAfricaandportionsofEurope.TherearealsodistinctivegenotypesintheWesternHemisphere,withparticularNorthandSouthAmericangenotypesubgroupsthatarerarelyobservedoutsidetheseregions.Basedonmolecularclockestimates,VanErtandcolleagues(2007a)notethattheradiationoftheAlineageseemstohavecoincidedwithperiodsofincreasedanimaldomesticationandexpansionofdomesticanimalpopulations.AlthoughNorthAmericahasasingledominantgenetictype,inmorerecenttimes,tradeinwool,skins,bonemeal,andotherproductsappearstohavecontributedtotheintroductionofanassortmentofrarerB.anthracisgenotypesontheNorthAmericancontinent.Oneofthese,the“Ames”strain,isthestrainfoundinthe2001anthraxmailings.

2.6THEEARLYHISTORYOFTHEAMESSTRAINOFB.ANTHRACIS

TheB.anthracisAmesstrainwasfirstisolatedfromadeadcowinSarita,Texas,in 1981. Texas A&M University shipped this new isolate to the U.S. ArmyMedicalResearchInstituteofInfectiousDiseases(USAMRIID)atFortDetrickin Frederick, Maryland. Because the box used for this shipment bore an oldAmes,Iowa,address,thestraincametobeknownasthe“Ames”strain.Asthisisamisnomer,atleastintermsofthelocationoftheoriginofthefirstsampleofthestrain,Ravelandcolleagues(2009a,b)calledthisspecificisolatefromTexas“B. anthracis Ames Ancestor.” Chapter 5 describes the experiments used todetermine that the Ames strain was the source of the material in the anthraxmailings.

The Ames strain is uncommon in nature. In the large study by Van Ert andcolleagues (2007a), North America was represented by 273 isolates of B.anthracisspanning44genotypesbut theAmesgenotypewasfoundonlyonce,indicatingthatitisrare.AccordingtoKeim(2009),theAmesstrainappearstobe a recent immigrant to North America, with its closest relatives found inChina.InNorthAmerica,thenaturaldistributionoftheAmesstrainappearstobelimitedtoasmallareaofTexas.However,theAmesstrainhasbeenwidelydistributed as a laboratory strain. This fact, coupled with its rarity in nature,“makesitunlikelythatthesourcematerialutilizedinthe2001bioterroristattackwasacquireddirectlyfromnature”(VanErtetal.,2007).

2.7SUMMARY

The bacteriumB. anthracis is the causative agent of the disease anthrax. TheinfectiousagentinallformsofthediseaseisthedormantB.anthracisspore.Thespore’s structural characteristics and properties provide the organism withresistancetoenvironmentalinsults(e.g.,dehydration,elevatedtemperature,UVirradiation) and also enable it to avoid the immune response of an infectedperson. Spores can remain viable in this dormant state for decades or more.Uponarrival inahospitableenvironment(i.e., inahumanoranimalbody)thesporescangerminateandresumerapidgrowthwithinhours,causingillness.

In humans, the course and outcome of the disease depend on dose and onwhethertheinfectionisacquiredviatheskin,gastrointestinaltract,orinhalation.Most human cases involve skin lesions (cutaneous anthrax), whereby theinfection is acquired as a result of handling infected animal hides or wool.Ingestion of food contaminatedwithB. anthracis can produce gastrointestinal

anthrax, a much more serious disease. But the most lethal manifestation ofanthraxiscausedbyinhalationofspores,causingaseverediseasethatisoftenfatal.Inhalationalanthraxisgenerallyrareandismainlyobservedinindividualswho work with animal skins. Inhalational anthrax in individuals not likely tohave suffered occupational exposure, however, can be a sign of a biologicalattackwithB.anthracisspores.

When appropriately prepared, anthrax spores are premier agents for biologicalwarfareandbioterrorismbecausetheycanbeproducedinprodigiousquantitiesin a form that can be aerosolized and inhaled. These factors, plus the highmortality associated with inhalational anthrax, make B. anthracis a seriousmilitaryandbioterrorismthreat.Forthesereasons,theappearanceof

B.anthracis in the2001mailings launchedamajorpublichealthandcriminalinvestigation.

3

ScientificInvestigationinaLawEnforcementCaseandDescriptionandTimelineoftheFBIScientific

Investigation

3.1INTRODUCTION

The discoveries that individuals had contracted anthrax and that letterscontaining B. anthracis had been sent by U.S. mail launched a full-scaleinvestigationbytheU.S.CentersforDiseaseControlandPrevention(CDC),the

U.S.PostalInspectionService(USPIS),andtheFederalBureauofInvestigation(FBI). Numerous investigative techniques were applied throughout theinvestigation,asoutlinedinTable3-1.Thischapterdescribesinbrief theearlystagesof the investigation, specifically thegatheringofevidence, formationofinvestigative teams, and decisions regarding scientific analyses that led to theFBI findingsandconclusions thataresummarizedandevaluatedmore fully in

Chapters 4 through 6.1 It also introduces the concepts ofscience, scientific investigation in law enforcement, and

the different views of uncertainty—and the manner inwhichitisdescribed—inscienceversuslaw.

3.2SCIENCEANDSCIENTIFICINVESTIGATIONASPARTOFALAWENFORCEMENTINVESTIGATION

Inascientificstudy,explanationsforobservablephenomenaaresoughtthroughthe gathering of reliable data and the formulation of testable hypotheses.Scientificobservationsmustbe reproducibleandscientifichypothesesmustberefutable. Science is typically an iterative, collaborative, and open processrequiringtheabilitytoposehypotheses,testthem,andposenewquestionsbasedon the resulting information.At times, a scientific investigation is a divergentprocess,inwhichnewresultsdriveresearchinseveraldirectionsonly

1 Throughout this report, the committee describes itsevaluation of the primary reports and data contained inthematerials provided to the committee by the FBI. Incases where the committee was not able to reviewprimary data, the committee’s assessment of statementsoranalysesofdatabyothersisprovided.

47

TABLE3-1TimelineofScientificEvents in theAnthraxMailingsInvestigation

Agency/institution/individual FBI

Project Final conductingthe document

initiated report work Project number

10/4/01FirstcaseofanthraxreportedN/AtoCDC.The

firstlabconfirmationofaninitialclinicalidentificationofB.anthracisfromavictimoftheletterattacks(Stevens)wasdoneattheFloridaStateLaboratoryinJacksonville.

10/12/01 11/14/01 FBIandlocallaw

Collectionofbiological

N/A

enforcement evidence:4envelopes,17clinicalsamples,106environmentalsamplesalongmailpaths(FL,DC,NJ,NY,CT)

10/17/01 10/19/01 BattelleMemorial

Microbiologicalanalysesof

B2M1D1

Institute(BMI)

lettermaterialidentifies2

B2M13D4

Bacillusspecies:onenonbeta-hemolytic(consistentwithB.anthracis)andonebeta-hemolytic(notfurthercharacterized)

10/01BeecherEnvironmentalsamplingofmailbags

10/01 11/01CDCClinical isolates from strickenN/A patients identified asB.anthracis

10/18/0111/27/01USAMRIIDInitialcharacterizationofletterB1M1D2

material(CFU,EM,visualinspection)

10/01 11/26/01 Battelle Memorial SEM-EDX analysis of letter B2M13D3,InstitutematerialB2M13D8

11/01 ArmedForces SEM-EDXanalysisofletter

AFIP,

Instituteof material 2001Pathology(AFIP)

10/01 05/02 LosAlamos

Materialanalysisforevidence

B1M4

National ofgeneticengineering

Laboratory(LANL)

10/01 9/02 Northern Arizona Identification of USAMRIID B1M3 UniversitysamplesasAmesstrain

TABLE3-1ContinuedAgency/institution/individual FBI


initiated report work Project numberFall 2/28/02 BMI Particlesize

distributionB2M13D11

2001 performedonlettersamples

andsomesurrogatesamples,

butnottheDugwayProving

GroundsurrogatesFall CDC Combined

epidemiological

2001 analysisofallcaserelatedto

theanthraxmailings

11/07/0112/03/01CDCUsingphenotypicsubstrateB2M1D2utilizationand16SrDNAsequencing,BacilluscontaminantidentifiedasB.subtilis

11/09/0111/09/01FBISEManalysisofenvelopesB1M7D16

Fall2001ConsortiumofagenciesN/A(NSF,NIH,DOE,DOJ,FBI,USDA,DOD,andIntelligenceCommunity)formedtoadviseinvestigationandprovidesupportresources

FallBMISiliconincorporationintosporeB2M13D72001coat

Fall2001

Spring2002

TheInstituteforGenomicResearch(TIGR)

CompletionofgenomesequenceforB.anthracisPortonand“Ames2001Floridastrain”(clinicalisolate);publicationinScience,June2002

B1M5D1,B1M5D3

Fall2001

Spring2002

USAMRIID Detectionofphenotypicvariants(“morphotypes”)amongcoloniesderivedfromletterspores

B1M2D12

12/01 12/06 Dugway Proving Reverse engineering of spore B1M13 Ground(DPG)“powders”B1M14

continued

TABLE3-1Continued


Projectinitiated

Finalreport

conductingthework Project

documentnumber

2/02 10/14/02 Lawrence CarbondatingbyAccelerator B1M8

Livermore MassSpectrometryNationalLaboratory(LLNL),NationalOceanSciencesAcceleratorMassSpectrometryFacilityNationalOceanSciencesAcceleratorMassSpectrometryFacility(NOSAMS),WoodsHoleOceanographicInstitute

2/02FBISubpoenasoflaboratoriesforsamplesofB.anthracisAmes

2/02 10/06 SandiaNational

Elementalanalyses(SEM-EDX)

B1M7

Laboratory oflettermaterial,

(SNL) envelopes,andDPGsurrogates(finalreportnotdated)

2/0210/06SNLSiliconanalysesB1M6

2/01/028/13/05UniversityofAgaranalysisB1M10Maryland(UMD)

3/07/022/01/06DPGAnalyticalchemistryanalysisofB1M13sporepowders

3/22/024/14/02FBIVolatileorganiccompoundB1M7D2analysisinevidentiarymaterial

Early 7/6/05 FBI ICP-OES:elemental B1M62002 compositionofletter

material,B1M7

culturemedia,envelopetypes

12/026/1/04TIGRWholegenomesequencingofB1M5MorphsA,B,C,D

8/022/22/04UMDHemeanalysisB1M10

8/028/05EdgewoodChemicalAgarandhemeanalysisB1M10BiologicalCenter(Army)ECBC

8/029/02BMIAgarandhemeanalysisB1M103/035/05UniversityofUtahStableisotopesignaturesB1M9




10/02 2/04 Commonwealth ContracttodevelopMorphA

B2M5

Biotechnologies, assays;onlyA1andA3were

Inc.(CBI) validated3/14/03 10/10/03 Applied 16SrDNA

sequencingofB2M1D4

Biosystems(AB)

BrokawletterB.subtiliscontaminants

7/31/03 8/11/03 Novozymes B. subtilis contaminant B2M1D3 Biotech, Inc.comparedtoB.licheniformis

10/03 5/14/06 TIGR Multiple locus PCR-based B1M5D2 assay for directcomparisonof

B.subtilisstrainstoPostB.subtilis

10/036/4/05TIGRWholegenomesequencingB1M5ofMorphEandB.subtiliscontaminant(PostandLeahy)

10/10/036/30/05ABGenomesequencingofB.subtilis“H2122”(notidentifiedelsewhere)

3/042/06CBIRepository(1104samples)B2M5D8screeningforA1andA3;secondscreening(300samples)7/07-10/07)

6/25/047/05CBIContracttodevelopassaysforB2M6MorphsBandD:bothrejected

7/13/049/14/04USAMRIIDScreeningofselectedsamplesB1M2D13ofFBIRforpresenceofMorphotypes

7/23/04 6/7/05 MidwestResearchInstitute(MRI)

ContracttodevelopassayforMorphsBandD;MorphBassayrejected;MorphDassay

B2M8

accepted11/19/04 4/05 IITResearch

Institute(IITRI)

ContracttodevelopassaysforMorphsBandD;MorphBassayrejected;MorphDassayaccepted

B2M7

12/01/0510/10/07MRIRepositoryscreeningforMorphB2M8D

continued




5/054/24/07IITRIRepositoryscreeningforMorphB2M7D

12/051/07TIGRContracttodevelopassayforB2M9MorphE

10/067/07PacificNorthwestAgarandhemeanalysisB1M11NationalLaboratory(PNNL)

11/0612/07NBFACRepositoryscreeningforB.B2M4D2subtiliscontaminant

2/15/07 12/04/07 CBSU(FBI)

B.subtilisanalysisbyreal-time

B2M4

National PCR:screeningofrepository

Bioforensic andothersamplesAnalysisCenter(NBFAC)

6/25/078/25/07TIGRRepositoryscreeningforB2M9MorphE

8/04/07 11/30/07 NationalCenter

Geneticdiversityand

B2M3

forAgricultural

phylogeneticanalysisofB.

Utilization subtilissamplesResearch(NCAUR)

8/29/078/29/08FBIAnalysisofmeglumineandB1M12diatrizoateinRMR1029,lettermaterial,othersamples

10/05/0710/08/08TIGRFinalizationofB.subtilisB2M2D2genomesequence

1/09/08 6/08/08 TIGR ScreeningofunidentifiedB.

B2M2D3,

subtilisisolatesforpresence

B2M2D4

ofsequencespecifictoPost/

Brokawcontaminant

3/27/08 9/30/08 University of Statistical analysis of FBIR B2M10 Cincinnatiscreeningdata

to converge again when more information informsdecisions about which directions to pursue. Thisdivergence and convergence make a scientificinvestigation different from a law enforcementinvestigation, in which the drive toward convergencedominates to agreaterdegree. In addition, theapproachused to gather, process, and analyze evidence candifferbetween a purely scientific investigation and a law

enforcement investigation.Both typesofapproachesarenecessary in a bioterrorism investigation, which alsorequiresattention topublichealthrisksandsafetyneeds(seeBox3-1).

Animportantfeatureofscienceisthatobservationsaremadeinamannerthatisindependentoftheobserverandontheassumptionthatotherobserverscanandwould make the same observations. Science relies on validated methods forgathering observations and making quantitative measurements systematicallyandreproducibly.Standardsmustbesetforcollectingdataundercontrolledandwell-specified conditions, assessing possible sources of error, establishingcausality (and acknowledging that a relationship is only a correlation whencausalitycannotbeinferredandsupported),andapplyingempiricalfindingstovalidate or refute particular hypotheses. New scientific methods must beassessed for their accuracy and reliability, their limitations, and the range ofcircumstancesunderwhichtheycanbeappropriatelyapplied.

TheQualifiersofCertaintyintheBiologicalSciences

Akeyquestioninthisstudywas“Basedontheavailabledata,howstrongistheapparent association between the letter evidentiary material and a particularsource or sample (e.g., flask RMR1029)?” Some of the committee’s mostimportant findings focus on the strength of a given association and on theconclusions that one should draw from the available scientific data about thenature of the association. Thus, it is important to review briefly the use ofterminologytodescribethestrengthofanassociation.

Quantifyinganassociation,aswellasthedegreeofcertainty(oruncertainty)inthatassociation,involvesstatisticalmethods(seeChapter6).Commonlanguageinvolvesqualifiers,ratherthanquantifiablemeasures,ofthisassociationandthedegreeofconfidenceinit,whichcancauseconfusionamongpractitionersfromdifferentfieldsthatusetheterms.Sincetheinterpretationofthesequalifiersandthewaysinwhichtheyareuseddifferacrossdisciplines(e.g.,statistics,science,law, common language), their use by the committee is clarified here. In thechapters that follow, the committee uses the following four qualifiers ofassociation,listedinorderofincreasingcertainty(decreasinguncertainty):

consistentwithanassociationsuggestanassociationindicateanassociationdemonstrateanassociation

BOX3-1BioterrorismInvestigations

Acrimescenetypicallyisaplacewherethevictimsandperpetratorsmeetintimeandspace.Thetraditionalcrimescene,whichmayhavemultiple locations, is the logicalplace to search for physical evidence leading to theidentityof theperpetrator(s). Identifying,collecting,andpreserving probative evidence combined withinvestigative detective work is the usual approach to asuccessfulprosecution.Solvingthecrimeis theultimategoal of the scene investigation, but there also are otherreasons to investigate the crime scene, including: 1)developing investigative leads for detectives; 2)developing specific information in the formof evidenceor investigativelogic toenableasuccessfulprosecution;3)locating,collecting,andpreservingprobativephysicalevidencethatcanprovideevidenceofinnocenceorguilt;4) developing information and physical evidence thatprovides an accurate reconstruction of the events of thecrime; and 5) linking multiple crimes through theevidence collected across sites (USDOJ, 2000; Fisher,2005).

All crime scene investigations require the integration of multiple forensic

disciplines through the juxtaposition of science and scene investigative skills.Scientific criminal investigations require an amalgamation of capabilitiesincludingsceneexperience,attentiontodetail,askepticalperspective,powersofobservation,andtheapplicationoflogic(Gardner,2005).

Althoughbioterrorismeventsceneshaveelementsincommonwithothercrimescenes, such as the identification, collection, and preservation of the forensicevidence,theycanalsodifferbecauseoftheinherentriskstoinvestigatorsandtothe public. Also, unlike traditional crimes, they may not always involve alocationwheretheparticipantsmeetintimeandspace,asshownbythe2001B.anthracismailingsinwhichthedisseminationofB.anthracisoccurredbymeansof the United States Postal Service (Jernigan et al., 2002). The perpetrator(s)presumablyworkedatadistance,sothecriminal investigationspannedseverallocations,includingthoseoftheenvelopes,postoffices,andstreetpostalboxesthatmighthaveheldcontaminatedenvelopes,and the location(s)where theB.anthracismighthavebeenmanufactured.Eachscenerequiredacomprehensiveand coordinated investigation to find, collect, and preserve the B. anthracisspores.

Future bioterrorism eventsmay differ in the nature of the biological agent ortoxin and in the mode of delivery. In general, bioterrorism incidents can beexpectedtobehandleddifferentlythanthetypicalhomicidesceneinvestigationbecause such events require both traditional scenemanagement skills and thespecial requirements of scenes involving bioagents. Bioterrorism investigatorsmust consider issues such as public safety, operational planning, samplingstrategy, packaging, transport, and storage. The immediate imperative toconsiderpublichealthneedsrequiresfindingandcollectingthebiologicalagentsothatitcanbeidentifiedexpeditiously,aswellasdefiningandcontainingtheenvironmental risk to thosenotyetexposed.The investigationandcontainmentmustbeaccomplishedwhileensuringthesafetyofinvestigatorsandthepublicduring the investigation and while remediating the scene. Sampling strategiesmustcombinethecollectionandpreservationofbioagentswiththecollectionofusualforensicevidence(Budowle,2006).Insomefuturescenarios,deliveryofthe biological agent might occur through natural routes of biologicaltransmission,andthusthe“crimescene”maybelimitedtothesiteatwhichthebiologicalagentwaspreparedordelivered.

The expression “consistent with” is frequently used in

this report and conveys the weakest level of certainty(greatest amount of uncertainty). In general, when theterm “consistent with” is used, it means that anassociationmayormaynotbepresent;theavailabledatacanneitherruleoutnorconfirmanassociation.Theterm“suggests” denotes a greater level of certainty for anassociation than “consistent with,” but even here thenormaluseofthewordinsciencedenotesaweakerlevelof certainty than is implied by the word in everydayparlance. That is, the potential for an association isstronger, and the evidence for the absence of anassociation is weaker, but both are still possible. Incontrast, the terms “indicate” and “demonstrate” denotehigherdegreesofcertaintyandtheseareusuallyreservedforstrongscientificconclusions(i.e., lessuncertainty,orlesslikelihoodofanabsenceofanassociation).Allfourlevels could potentially be quantified with measures of“statistical significance,” but the committee does notassignsuchmeasuresinmost instancesbecausethedataat hand are generally not appropriate for such precisequantificationofthedegreeofuncertainty.

Insummary,thereaderiscautionedtoconsidercarefullytheterminologyinthisreportinlightofthefactthatthequalifiersofcertaintyusedherearethoseusedmost commonly in the scientific literature and that these words can carrydifferentweightincommonlanguageandinthecourtroom.

3.3THEFEDERALCOORDINATEDRESPONSEANDASSIGNMENTOFLABORATORYWORK

Oversight and coordination of a complex scientific study are critical. Largeteams of scientists have been successful at complex studies (e.g., in particlephysicsandgenomicresearch)becausetheyhaveaclearleadershipstructureforthe coordination and planning of their efforts (International Human GenomeSequencingConsortium,2001;Venteretal.2001;NRC,2003).Withpressuresoftimeandexpenseinanystudy,someoneorsomegroupintheresearchteammustmakedecisionsaboutwhichavenuestopursueandwhichtoabandon.

In2001,theFBIhadasciencelaboratoryatQuantico,theHazardousMaterialsResponse Unit, and another team of weapons of mass destruction (WMD)experts,butitdidnothavethecapabilitiestohandleallofthetypesofscientificexperimentsthatwouldberequiredtoexaminetheevidenceintheinvestigationoftheB.anthracismailings.FBIinvestigatorsquicklyrealizedtheneedtoturntooutsidelaboratoriesandexpertsforhelp.TheBureauimmediatelyformedaninternal group that hadmembers from the scientific team, investigative team,and terrorism team. Recognizing the importance of parallel criminal andscientific investigations, the FBI embedded high-level DOJ staff from theinternalteam,whothenadvisedthemthroughouttheinvestigation.

Atthattime,theFBIdidnothavetheorganizationalstructureneededtooverseesuchacomplex,multifaceted,andinvolvedscientificinvestigation.

In 2003, the agency remedied this organizational limitation by forming a newunit focusedon investigations involvingchemical,biological, radiological,andnuclear sciences, called the Chemical, Biological, Radiological, and Nuclear(CBRN)SciencesUnit,ortheChemicalBiologicalScienceUnit(CBSU).

Thecommitteewastoldthattheteamworkingonthescientificinvestigationmetweekly with the law enforcement team for information sharing, strategy, andcoordination.Reportsandnotesfromsomeofthesemeetingsweresharedwiththecommittee late in theprocessof finalizing this report.The reports indicatethecomplexityoftheparalleltracksoftheinvestigationanddocumentprogressof each aspect of the scientific efforts and the decisions to proceed with orabandonparticularlinesoftheinvestigation.

In the early stages of the investigation, the FBI sought the advice of outsideexperts to assist in characterizing the properties of theB. anthracis evidence.Theengagementoftheseexpertswasaidedbythecreationofanadvisorygroupled by the director of the National Science Foundation, the director of the

NationalInstituteofAllergyandInfectiousDiseasesoftheNationalInstitutesofHealth,andfederalofficialsfromnumerousotherscienceagencies.Thisgroupmet regularly in classified sessions with FBI leaders to hear about theinvestigation and to provide advice and the names of potential subjectmatterexpertstheFBIcouldengageforassistance.

In addition, several Technical Review Panels were formed consisting ofscientists from Department of Energy National Laboratories, academiclaboratories, andmembers of theNationalAcademy of Sciences. Panelswereconstitutedtoreviewtheanalyticalplan(thatis,whattestsshouldbedoneandby whom), the progress of the investigation, and the chemistry and biologytechniquesused.We reviewed several reports frommeetingsheld in late2001and thereafter (FBIDocuments,B1M1,B3D1-7).According to thesematerialsandtheDOJreport,“Attheoutsetoftheinvestigation,threepanelscomprisedof33 of the nation’s leading authorities in bioweapons development from theformeroffensivebioweaponsprogram,microbiology,chemistry,andmicroscopywere convened to assist the FBI in developing a comprehensive analyticalframework to evaluate the anthrax powders recovered from the envelopes andthecontaminationfoundintheAMIBuilding”(USDOJ,2010,p.13).

The FBI benefitted from these early informal and regular meetings of seniorleadershipfromotherscienceagencies(FBI,2009;Colwell,2009).TheFBIalsoreceived input from the Department of Defense (DOD), the IntelligenceCommunity, DOJ, CDC, and Armed Forces Institute of Pathology (AFIP)regardingthescientificinvestigation(FBI/USDOJ,2011).

As is shown in Table 3-1, the U.S. Army Medical Research Institute ofInfectious Diseases (USAMRIID) played a central role in the scientificinvestigation.ThefacilityhadprovidedanalyticalservicestotheFBILaboratorysince1998.DuringOctoberandNovember2001,scientistsatUSAMRIIDwereincluded in the team performing on-site testing at the American Media, Inc.(AMI) building in Florida, and they conducted the initial examinations of theletter spore preparations for physical characteristics (using microscopy andelectronmicroscopy) and spore viability (see Chapters 4 and 5). USAMRIIDscientists also conductedmicrobiological analyses and identified the dominantandvariantmorphologicalcolonytypesthatappearedintheevidentiarymaterial(seeChapter5).

The FBI also sought the help of dozens of outside laboratories. In total,

thousandsofsampleswereprocessedandanalyzedby29academic,government,andprivateBSL-3laboratoriesacrossthecountry(Piggee,2008;USDOJ,2010).TheworkcommissionedbytheFBIwashighlycompartmentalized.Mostofthelaboratories conducting analyses were not aware of other analyses under way(Keim, 2009; Michael, 2009; Weber, 2009). Scientists at these laboratoriesresponded rapidlyandprovided thebulkof the scientific studiesonwhich theFBIrelied in its investigation.ThecommitteereadreportspreparedbyoutsidescientistsrespondingtospecificrequestsfromtheFBIandwereceivedreportsof periodic reviews of contracted work by panels of experts (B3D1-7). Thecommittee also reviewed reports of work carried out in parallel at the AFIPalthoughitisnotclearhowcloselyAFIPandtheFBIinvestigativeandscientificteamsworkedtogetherorcoordinatedtheirefforts.

As the scientific investigation proceeded, several laboratories conductedsequential and parallel scientific analyses on the evidentiarymaterial gatheredfrom the letters, environmental samples, and clinical samples (see Table 3-2).These analyses first focused on identifying the nature of the letter andenvironmental materials, their similarities and differences, their biological,chemical, and physical properties, and, eventually, their similarity to othersamplesofAmesstrainB.anthracisinlaboratoriesaroundtheworld.Accordingto the affidavit in support of a search warrant submitted by Postal InspectorThomas

F. Dellafera (Case number O7-524-M-01, October 31, 2007), 16 domesticlaboratoriesand three foreign laboratories (inCanada,Sweden,and theUnitedKingdom)wereidentifiedashavingtheAmesstrainintheirinventoriespriortothe attacks; two additional domestic laboratories were subjected to consentsearches, and one domestic laboratory was subjected to a search warrant. AsubpoenapreparedbytheFBIinearly2002forsamplesubmissionspecifiedthatonly Ames strain samples be submitted; the subpoena protocol for thesesubmissions is described in Chapter 6. In the end, the FBI assembled arepository of over 1,070 Ames strain samples from 20 laboratories, of which1,059were viable.Attributes of the samples in the repositorywere comparedagainstthecharacteristicsoftheevidentiarysamples(asdiscussedinChapters5and6).

According to FBI officials, the focus of these analyses was to provide FBIinvestigators with scientific leads that could be used to assist in its criminalinvestigation(FBI,2009).Chapters4through6providein-depthdescriptionsoftheanalysesconducted,conclusionsreached,andthiscommittee’sfindingsandlessonslearnedforthefuture.

TABLE3-2AnalyticalTechniquesUsedontheEvidentiaryMaterial

FloridaDC,NY,

New

environmentalCT,NJ York

andclinicalclinical PostBrokawDaschleLeahy

Technique samplessamples letterletter letterletter

MLVA(geneticXXXXX

X

analysisofstrain)(B1M3D1)

SequencingofpagAXXXX?genes(B1M4D2)

CompletegenomeXXXsequencingofwild-typeB.anthracis

SequencingandXXXX?PCRanalysistodeterminewhethergeneticengineeringoccurred

PartialgenomesequencingofB.subtilis

XX NoB.subtilis

NoB.subtilis

WholegenomesequencingofB.subtilis

XNotsequenced

NoB.subtilis

NoB.subtilis

Phenotypic X X X Xscreensofvariants (B1M2D14)

(morphotypes)

WholegenomeXXsequencingofmorphotypeA

WholegenomeXXsequencingofmorphotypeB

WholegenomeXsequencingofmorphotypeC

WholegenomeXsequencingofmorphotypeD

Wholegenome

XXsequencingofmorphotypeESEManalysistoXXXassesssizeandshapeofspores

TABLE3-2ContinuedFloridaDC,NY, New

environmentalCT,NJ Yorkandclinical clinical Post Brokaw Daschle Leahy

Technique samples samples letter letter letter letter

AssessmentofbulkX

X

siliconcontent

SEM-EDXbulkXXXanalysisofsilicon

SEM-EDXtoassessXXXsiliconinsporecoat

SEM-EDXbulkXXXelementalanalysis

SEM-EDXforXXspatialresolutionandsensitivity

AssaysforpresenceXXofagar

AssaysforpresenceXXofmegluminediatrizoate

HeadspaceGC-XMSandinfraredspectroscopytodetectVOCs

RadiocarbonanalysisXfordating

StableisotopeXanalysistodeterminegrowthmedium

EnvelopeanalysisXXX

AssaysofgenotypicXXvariants(A,B,C/D,E)

GC-MS = gas chromatography-mass spectroscopy; MLVA = multiple-locusVNTR [variable-number tandem repeat] analysis; PCR = polymerase chainreaction;SEM=scanningelectronmicroscopy;SEM-EDX=scanningelectronmicroscope with energy-dispersive X-ray analysis; VOC = volatile organiccompounds

3.4COLLECTIONANDANALYSISOFCLINICALANDENVIRONMENTALSAMPLESANDCROSSCONTAMINATIONBeginning inOctober 2001, investigators collected biological evidence fromavarietyofsourcesandlocations.Bytheendofthecollectionphase,B.anthracisisolates were gathered from 4 powder-containing envelopes, 17 clinical

specimensobtainedfrominfectedpatients,and106locationsalongthemailpathof the implicated envelopes from Florida, the National Capital Region(Washington, D.C.), New Jersey, New York, and Connecticut, and overseas(Jernigan,2002).

3.4.1ClinicalandEpidemiologicalSamples

AsnotedinChapter2,anthraxisgenerallyararediseasethatmostoftenoccursinpeoplewhohavecontacteitherwithinfectedlivestockorwithcontaminatedanimal skins or other animal products. Inhalational anthrax is particularly rare(Bushet al., 2001), and the fewcases reported in theUnitedStateshavebeenduetooccupationalexposures.Before2001,thelastcaseofinhalationalanthraxin the United States was reported in 1976 (Jernigan et al., 2002). Thus, theoccurrenceofinhalationalanthraxinRobertStevens,anemployeeofAMI,wasnotimmediatelyrecognizedassuch.

LarryBush,aphysicianwith training inmicrobiology,was thefirst tosurmisethattheGram-positivebacilliinthepatient’scerebrospinalfluid(CSF)wereB.anthracis (Cole, 2009). Phil Lee, at the Florida State Laboratory, providedconfirmation based on various tests, including lysis by a B.anthracisspecificvirus.ResearchersinCDClaboratoriesusedstandardmicrobiologicaltechniquestoverifytheputativeagentasB.anthracis(Jerniganetal.,2002).

Bush and colleagues (2001) point out that in the Stevens case, although therewas no typical “occupational exposure” to infected animals or contaminatedanimal products, the victim was apparently infected at work. No spore-containingletterwaseverfound,butcoworkersreportedthatonSeptember19,2001, Stevens had closely examined a suspicious letter containing powder.Anthrax spores were later found on the victim’s computer keyboard, at otherlocations in AMI, in asymptomatic coworkers, in the AMI mailroom, and atregionalandlocalpostalcentersthatservedtheAMIworksite.ThreedaysafterhishospitaladmissiononOctober2,2001,RobertStevensdied,becoming theindexcaseforthis2001bioterrorismevent(Bushetal.,2001).

Inthefollowingweek,asillustratedinTable1-1(Chapter1),casesofcutaneousanthrax were reported in New York City, subsequently linked to exposure toletters containing suspicious powder (Greene et al., 2002). Ultimately, fourenvelopescontainingB.anthracissporeswererecoveredfrommediaoutlets inNewYorkandfromgovernmentofficesinWashington,D.C.,orthecontainment

facilitycreated tostorepotentiallycontaminatedmail.All fourenvelopeswerepostmarkedbytheUnitedStatesPostalService(USPS)TrentonProcessingandDistributionCenterinNewJersey.OnOctober18,acaseofcutaneousanthraxwasconfirmedinapostalworkerattheTrentonCenter,whichwasthenclosed.An investigation by CDC and the New Jersey Department of Health wasinitiated(Greeneetal.,2002).Subsequently,CDCandotheragenciesconductedacombinedepidemiologicalanalysisofallcasesrelatedtotheanthraxmailings(Jerniganetal.,2002).Thisanalysiswasacoordinatedeffortamongmedicalandlaboratory facilities and local, state, and federal public health and lawenforcement agencies. CDC’s Emergency Operations Center supported local,state, and federal public health investigators in Florida, NewYork City, NewJersey,theDistrictofColumbia,andConnecticut.TeamsalsocoordinatedwiththeUSPIS,theDepartmentofDefense,theFBI,andotherfederalorganizations.

Jernigan and colleagues (2002) summarized the early overall epidemiologicfindings. As illustrated in Figure 3-1 from the CDC, a total of 22 cases ofbioterrorism-related anthrax were identified—11 inhalational cases and 11cutaneous cases. Victims were identified in seven states along the east coast:Florida (2 cases),Maryland (3),New Jersey (5),NewYork (8, including oneNewJerseyresidentexposedinNewYork),Connecticut(1),Pennsylvania(1),and Virginia (2). Five of the inhalational anthrax patients died, resulting in afatality rate for inhalational anthrax of 45 percent. Eight of the 11 cases ofinhalational anthrax were confirmed asB. anthracis from analysis of clinicalspecimens.Inmostcases,theexposureslikelyoccurredinthevictim’splaceofwork.Morethanhalfthevictims(12of22)weremailhandlersattheUSPSoringovernment and media industry mailrooms, eight of whom developedinhalationaldiseaseandfourcutaneousdisease.Oftheremaining10victims,6were other media company employees working at locations where mailcontainingpowderwaspresumedtohavebeenreceived(AMI,CBS,NBC,andtheNewYorkPost).A seventh victim, a 7-month-old child had visitedABC’sofficesinNewYork.

Threeothervictims—a61-year-oldManhattanhospital supply roomworker, a51-year-old bookkeeper from New Jersey, and a 94-year-old Connecticutresident—appeared tohavenoworkplaceassociations.Sincepostalprocessingfacilities were widely contaminatedwithB. anthracis, the FBI suggested thatcross-contaminatedmailwasthesourceoftheexposuresforthesethreepatients(Jerniganetal.,2002;Greeneetal.,2002).Jerniganandcolleagues(2002)notedthat “The possibility of B. anthracis exposure from envelopes secondarily

contaminated from implicated postal facilities greatly extended the group ofpotentiallyexposedpersonsinourinvestigation.”

Theepidemiologicalinvestigationsfoundtwodistinctcaseclustersseparatedintime. The first case cluster was related to the two envelopes postmarkedSeptember18,whichwereapparentlymailedinoraroundTrenton,NewJersey,anddelivered inNewYorkCity.Asshown inFigure3-1,11cases inallwereclassifiedaspartoftheclusterrelatedtotheSeptember18mailings.Sevencasesof cutaneous anthrax occurred inmedia company employees inNewYork (5)andpostalworkersinNewJersey(2).Thetwocasesofinhalationalanthrax

U.S. federal government, the image is in the publicdomain.

inFloridawerealsoassignedtothiscluster,basedonthehypothesis that an unrecovered mailing had been sentthere. B. anthracis was isolated from environmentalsamplesatsixpostalfacilitiesalongthepathfollowedbythemailtoAMIaswellasattheAMIbuildingitself.Thedates of onset of illness in the two AMI employees in

Floridawere also consistentwith exposure to envelopesmailed in mid-September. Victims in the cluster thatfollowedtheSeptember18mailingsweremorelikelytohavecutaneousdiseaseandtohavebeenexposedatnewsmediasites(Jerniganetal.,2002).

ThesecondcaseclusterinvolvedthetworecoveredenvelopessenttotheofficesofSenatorsTomDaschleandPatrickLeahy,alsomailed inoraroundTrentonandpostmarkedOctober9,2001.AllfivecasesfromtheD.C.metropolitanareawere part of this cluster and all five of these victims contracted inhalationalanthrax andworked in postal facilities contaminated by theOctober 9 letters.Two additional cases of inhalational anthrax occurred in postal employees inNewJersey.Ingeneral,theOctober9mailingswereassociatedwithmoresevereillness.VictimsinthesecondclusterweremorelikelytohavebeenexposedatmailhandlingfacilitiesalongthepathtotheSenateoffices.

Chapter4examines thedifferences in thephysicalappearanceandconsistencyofthepowdersbetweentheSeptemberandOctobermailingsasreported

bytheFBI(seeFigures3-2and3-3).Inbrief, thepowdersfromtheNewYorkPost and Brokaw letters were multicolored and granular in consistency. Theenvelopesinthesecondwaveofmailingscontainedapowderofuniformcolorand smaller particle size, which would facilitate more efficient airbornetransmissionoftheB.anthracissporesandcouldaccountfortheprevalenceofinhalation cases in the second cluster (Hassell, 2009). In addition, despite thefact that the envelopes in the October mailings were unopened during theirpassagethroughthepostalcenters,theuseofhigh-speedprocessingandsortingmachinesmayhavecontributedtodispersalofthesporesandexposureofpostalworkers. Greene and colleagues (2002) reported that, although the envelopescontaining theB. anthracis were handled in only a small area of the Trentonfacility, environmental sampling found evidence of spores throughout thefacility.

TheB.anthracis isolatescultivatedfromtheclinicalspecimensofpatients, thefour recovered powder-containing envelopes, and over 100 environmentalsamples collected along the suspected path traveled by the contaminatedmailweresubtypedbytheCDCusingmultiple-locusvariable-numbertandemrepeatanalysisandsequencingoftheprotectiveantigengene(pagA).Inaddition,pagAwasamplifiedandsequenceddirectlyfromsomeclinicalspecimens.Allofthe

resultsindicatedthepresenceoftheB.anthracisAmesstrain(Hoffmasteretal.,2002).

Jernigan and colleagues (2002) noted that their epidemiological investigationhad several limitations. Because identification of case patients involvednumerous local, state, and federal officials, data collection methods were notuniform.Thewidespreaduseofpostexposureprophylaxisand thedifficultyofobtaining information about potentially exposed persons prevented accurateestimates of anthrax exposure rates. Some cases may have been overlookedbecause patients might have been administered antimicrobials after beingmistakenlydiagnosedwithothertypesofinfectiousdiseases,withoutphysiciansrecognizing the disease to be anthrax. The lack of prior experience withbioterrorism also forced the investigators to refine methods and redefineinterventionsonacontinuingbasis.

3.4.2CrimeSceneEnvironmentalSamples

TheFBIhazardousmaterials(HAZMAT)team,withtheassistanceofscientistsfromUSAMRIID,CDC,USPIS,EnvironmentalProtectionAgency(EPA),andcontractors,performedenvironmental investigations toassess thepresenceandextent of B. anthracis contamination and to guide decontamination andenvironmentalremediation(Jerniganetal.,2002).Environmentalsampleswerecollected at contaminated worksites and mailboxes by public health, lawenforcement,andothergovernmentandcontractstaff(CDC,2001a;Sanderson,2001, 2002). At the time, the FBI did not have biosafety level 3 (BSL-3)laboratoryfacilitiescapableofhandlingB.anthracis,sosamplesweretestedatlaboratoriesparticipating in the local, state, and federal investigationefforts asdescribedinChapters4through6.

Crimesceneenvironmentalsampleswerecollectedbysurfacesampling,mostlywithRODAC(replicateorganismdetectionandcounting)contactplates.Swabs,wipes,high-efficiencyparticulateair(HEPA)vacuumfiltration,andairsamplingalso were used (Dull et al., 2002; Jernigan et al., 2002; Teshale et al., 2002;Beecher,2006).Basedon thedocumentsprovidedto thecommittee, itappearsthattheFBIandCDCreliedonculture-basedtechniquestodetectB.anthracisintheenvironmentalsamples(Jerniganetal.,2002;Hoffmasteretal.,2002).Inmuch of the work on samples collected from the environment, coloniespropagatedonagarplateswerepresumptivelyidentifiedasB.anthracisbasedon

morphology(Beecher,2006).SelectedcoloniesweredefinitivelyidentifiedasB.anthracisusing standard confirmatory tests. At least one environmental swabsamplefromAMIwassenttoPatriciaWorshamatUSAMRIID(inJune2005)for detection and identification of B. anthracis variant colony morphotypes.Material fromthisswabwasused to inoculatesheepbloodagar.The reportofthis work by Worsham (2009; FBI Documents, B1M2D14) states that B.anthracisvariantmorphotypesA,B,andC/Dwerefound,butnotmorphotypeE,inadditiontothewild-typecolonymorphotype.Furthermore,WorshamstatesthataBacillus strainwas recovered that resembled theB.subtilis found in theNewYorkPost letter. The report states that inOctober 2006, cell suspensionsfrom 34 colonies that exhibited a variantmorphotype, aswell as from theB.subtilis-like isolate, were sent to the National Bioforensic Analysis Center(NBFAC) for DNA extraction, and that the DNA from the variant colonymorphotypesweretobesenttotheInstituteforGenomicResearch(TIGR)forsequencing of the morphotype A, B, C/D, and E genomic regions. However,according to statementsby theFBI to this committee (FBI/USDOJ,2011), theU.S.Attorney’sOfficeadvisedthatthissequencingandfurthercharacterizationofthesecolonymorphotypesfromAMIwouldnotbeundertaken.

TheFBIdidnotusemolecularassaysfordetectingB.anthracisDNAdirectlyinenvironmentalsamples(intheabsenceofacultivatedisolate).MolecularassaystargetingB.anthracis-specificgeneticmarkershadbeendevelopedpriorto2001fordetectingthisorganisminfood(Yamadaetal.,1999)andinenvironmentalsamples(Beyeretal.,1995).Anon-sitepolymerasechainreaction(PCR)-baseddevice was used by the CDC at the Brentwood postal facility in 2001 forpreliminary assessment or adjunct analysis of B. anthracis DNA inenvironmentalsamples,butthisapproachhadnotbeenvalidatedbythematthattime for these types of samples. However, the Biological Aerosol Sentry andInformation System was deployed in 2001 for environmental monitoring andincorporated PCR-based detection methods for a variety of biological agents(Fitchetal.,2003).

The committee recognizes that the FBI may have hesitated to apply a newlyemerging method to the assessment of forensic evidence before it had beenwidely adopted or validated for this purpose; however, under exigentcircumstances, it can be appropriate to proceed without completing fullvalidation.TheFBIstatedtothecommittee(FBI/USDOJ,2011)thatlaboratoriesintheCDCLaboratoryResponseNetworkthatwereequippedforPCRanalysiswere overwhelmed with samples early in the investigation, and that the FBI

obtainedmore timely resultson thepresenceofB.anthracis in environmentalsamplesby relying insteadonRODACplate isolation techniques. Inanycase,by2004,PCRhadbeenvalidatedforvariousbacterialagentsinenvironmentalsamples (Malorny et al., 2004) and could have been performed on theenvironmentalsamplesfrom2001.

Inadditiontotheenvironmentalsampling,nasalswabspecimenswerecollectedfrom potentially exposed individuals to help delineate the area of exposure toaerosolized spores and to determine where persons with inhalational anthraxmighthavebeenexposedbasedonwhere theyworked.According to Jerniganandcolleagues(2002),becausethesensitivityofnasalswabculturesdiminisheswith time following human exposure, attempts were made to obtain cultureswithin sevendaysof exposure.ThepresenceofB.anthracis from nasal swabcultures was determined by Gram stain and colony characteristics as well asthrough confirmatory testing by laboratories participatingwith the local, state,andfederalefforts(Jerniganetal.,2002).

Finally, in the newmaterials provided to the committee it is noted that PCRanalysiswasperformedonhumanremainsfromUnitedflight93on9/11/2001thatwereidentifiedasthoseofthehijackers(B3D1).AnalysiswasperformedatUSAMRIIDandatAFIPforsequencesdiagnosticofB.anthracis.OneassayatUSAMRIIDgavepositiveresults,buttheseresultswerebelievedbytheFBItobe due to laboratory contamination. All other results were negative. As thecommittee learned at the January 2011 meeting, there were no tests done onremainsfromanyoftheotherSeptember11,2001hijackers.

3.4.3SamplesfromanOverseasSiteIdentifiedbyIntelligence

InDecember2010-January2011,theFBIfirstmadeavailabletotheCommittee“AMX Weekly Science Updates” and a newly declassified document thatdescribed the collection and analysis of environmental samples from anundisclosed site outside the continental United States (OCONUS) for thepresence of B. anthracis Ames (FBI/USDOJ, 2011, FBI Documents, WFOReport). Thisworkwas performed as part of the anthrax letters investigation.Fewdetailsweremadeavailabletothecommittee.

At least three sample collection missions were conducted by the FBI and/orpartners from the intelligence community at an overseas site because ofinformation about efforts by Al Qaeda to develop an “anthrax program”

(FBI/USDOJ, 2011). InMay2004, theFBI and partners from the intelligencecommunityvisitedanoverseaslocationatwhichtheyhadbeentoldananthraxprogramhadbeenoperating,andbroughtbackswabandswipesamples to theUnitedStates.NoneofthesamplesgrewB.anthracisafterincubationinculturemedia.However,threeswabsampleswerereportedaspositiveforB.anthracisandfor

B. anthracis Ames-specific sequences by PCR, including swabbings from theoutsideofanunopenedmedicinedropperpackage,asink,andasinkdrainhose.Repeat testingof these threepositivesamplesaspartofagroupof15blindedsamples, including soil samples,water blanks and non-AmesBacillusspecies,againyieldedpositiveresultsfortwoofthethreesamesamples(andfornoneofthe other samples). However, not all replicates of the DNA extracts from thepositivesamplesgavepositiveresults.Apparently,anearliercollectionmissiontothissite,priortoMay2004,byothersintheintelligencecommunityhadalsoyielded sampleswithpositivePCR results forB.anthracis DNA and negativeculture results. As a result of these findings, a third collection mission wasconducted in November 2004 and this time large portions of the site werereturned intact to the United States, including the entire sink, drain, andassociated plumbing that had been the source of the positive March 2004samples.Theseitemswereextensivelysampled,andagaintestedforbothviableB.anthracisandforB.anthracisDNA.This time,according to theJune2008declassified document, all the tests were negative (FBI Documents, WFOReport).

The committee was provided only fragmentary information about and limitedprimary data from this work and received them very late in our study. Weconsider these data to be inconclusive regarding the possible presence of B.anthracisAmesatthisundisclosedoverseassite.Severalscientificandtechnicalissuesshouldbeexploredinmoredetail,suchastheperformancecharacteristicsoftheassays,whetherornottheassayswerevalidatedforusewiththesesampletypes,thedegreetowhichsamplesorsamplelocationsgaverepeatedlypositiveresults, interpretationof inconsistentpositive results,whetherornot theAmesgeneticmutations in theanthrax lettersweredetected in anyof theseoverseassamples,andthenaturaldistributionofB.anthracisstraintypesinthisoverseasgeographicregion.

3.4.4LetterMaterialandCrossContamination

Material was collected directly from the Daschle, Leahy,New York Post, andBrokawletters,butinvaryingquantities.Asindicatedabove,noletterwasfoundat the AMI building in Florida, thus only environmental and clinical sampleswere available for analysis.The decision by theU.S.Attorney’sOffice not topursuemolecular analysison theAMIcrimescene samples (FBI,2011) limitsthe ability to definitively connect this attack to the material in the recoveredlettersfromNewYorkandWashington,D.C.

TheletteraddressedtoTomBrokawatNBCwasfoundafterErinO’Connor,anassistant to Brokaw, developed cutaneous anthrax after opening a lettercontaining a white powder (Cole, 2009). As mentioned before, the FBIlaboratory was not equipped to handle B. anthracis, so all of the New Yorksamples were sent to the New York Department of Health in Albany orelsewhere. The New York City Health Department officials who tested theBrokaw letter accidentally lost most of the sample and contaminated thelaboratory,renderingthespaceunusableforacriticalperiodoftimeaftertheB.anthraciswasdiscoveredandbeforethefullscopeoftheproblemwasknown.Asaresult,littlematerialfromtheBrokawletterremainedavailableforfurtheranalyses.AbiopsyobtainedfromablackescharlesionthatformedonO’Connorandwas sent toCDC testedpositiveby immunohistochemical staining for thecellwallantigenofB.anthracis.

TheNewYorkPostletterhadbeenthrownintoabinforhatemailandwasfoundafteremployeeJohannaHudenbecameillwithcutaneousanthrax.Theletterhadbeenunopenedandtheenclosedmaterialwasavailableforfutureanalyses.

On October 15, when staff in Senator Daschle’s office opened an envelopecontainingawhitepowder,policequarantinedtheofficeandsurroundingrooms,shut down the Capitol’s mail system, and suspended public tours. Lawenforcementofficials inprotectivebiohazardsuits tookoverSenatorDaschle’soffice.Unlikeprevious samples,whichwere sent to theCDCforanalysis, theFBI sent the Daschle letter to USAMRIID because of its biocontainmentfacilities(FBI,2009).Sincemuchofthematerialwasdispersedwhentheletterwasopened,limitedmaterialwasavailableforfutureanalyses.

On November 16, 2001, FBI and EPA HAZMAT personnel found the letteraddressed to Senator Leahy in one of 280 barrels of unopenedmail collectedfromCapitolHillafter thediscoveryof theanthrax-contaminated letter sent toSenatorDaschle(FBI,2008a).ThesearchthatresultedinretrievaloftheLeahy

letter was conducted by teams of HAZMAT workers from the FBI and EPACriminal Investigative Division (FBI, 2008a). They developed a samplingprotocolintendedtoeliminatetheneedforHAZMATteamstosiftthrougheachpieceofmailin642trashbagstofindcontaminatedmail(FBI,2008a).

Themailwassampledandsortedinacontainmentfacilityconstructedinalargewarehouseandmaintainedwithnegativeairpressure.TheintakeandexhaustairwaspassedthroughHEPAfilters,whichtrapessentiallyallparticlesthesizeofanthraxspores.Airsamplerswereusedtomonitortheairinsideandoutsidethecontainmentareaforthepresenceandquantityofairbornespores.Investigativeworkersweremonitoredforexposurebysamplingtheirclothing.Ingeneral,theonlyinvestigatorswhowerecontaminatedwerethosewhohandleda“hot”bagcontainingtheletteraddressedtoLeahyladenwithspores(Beecher,2006).

Eachbagwasshakeninanattempttodistributeanysporesthatcouldbepresent.A swabwas then inserted into a smallhole in eachbagandwipedaround theinside.Aftertheswabwaswithdrawn,theholewassealedwithducttapeandtheswabwasused to inoculateaPetridish,whichwas sent to theNavalMedicalResearchCenter(NMRC)foranalysis(FBI,2010b).

Trace contamination was detected in 62 bags, likely due to a high level ofshedding from cross-contaminated mail (Beecher, 2006). Five bags producedmore than 100 bacterial colonies from a swab and were considered “hot.”Innovative air sampling was used to maximize the recovery of the targetorganism (Beecher, 2010). One bag, the one containing the Leahy letter, wasorders of magnitude more contaminated than the others. This bag producedbetween19,000and23,000spores(or760to920colony-formingunitsperliterofairsampled)(Beecher,2006).

Beecher (2006)examined someof the issues related tocrosscontaminationofmail,mailbags,andthelocalenvironmentbytheLeahyletter.Hedocumentedextensivecontaminationofpersonnelespeciallyduringphysicalhandlingofthespore-laden letter. In the conduct of this work, the investigators followedthoughtful and appropriate practices and procedures for the purpose ofminimizingartifactualcrosscontamination.Ofnote,theyidentifiedacorrelationbetween the degree of letter contamination and the previous handling of theletter by sorting machines in the Trenton and Washington, D.C., postalprocessinganddistributioncenters.Theinsightsfromthisworkprovedusefulindeveloping a more detailed understanding of the route taken by the anthrax

letters from the point of deposit through theU.S. Postal System to the site ofdelivery. Although suggestive of a mechanism and scenario by which OttilieLundgren,aresidentofOxford,Connecticut,mighthavedevelopedinhalationalanthrax (cross contamination of mail en route to her home), the committeelacked sufficient information (e.g., other possible exposures, unusualsusceptibility to low numbers of anthrax spores) with which to assess theplausibilityandlikelihoodofthismechanismandthisparticularscenario.

On September 1, 2001, Defence Research Establishment Suffield (DRES) inCanada released the results of a study (FBI Documents, B2M11D1) that hadbeen designed to measure and better understand the dispersion of spores thatmight occur after the opening of an envelope containingB. anthracis spores.Thisstudyinvolvedaseriesofexperimentsinwhichenvelopescontainingeither

0.1or1.0gramofB.globigii sporesat aconcentrationof~1×1011cfu/g(asasurrogateforB.anthracisspores)wereopenedinaDRES aerosol test chamber that was configured torepresent amail roomor office.The chambermeasured18×10×10 ft (i.e.,withavolumeof1,800cu ft) andhadarecirculatingairhandlingsystemoperatingat1,050cu ft/min.Thepresenceof spores atvarious sites in thechamber was assessed using culture-based approaches,notmoleculardetectionmethods.Theresultsshowedthattheactofhandlingoropening theseenvelopeswas“farmore effective than initially suspected” in causingdispersion of spores in the chamber. Particles ofrespirable size were released quickly and spreadthroughout thechamber,suchthatafter theopeningofa0.1gsporeenvelope,10minutesofexposuretotheairinthechamberwouldhaveprovidedadose480-foldgreaterthantheamountneededtokillahumanwith50percentprobability. The investigators noted that envelopeswere

more likely to cause cross contamination of the localenvironment,includingtheenvelopehandler,iftheopencorners of the envelopewere not deliberately sealed bythepreparer.The investigatorsatDRESdidnotseal thecornersoftheenvelopestheyusedintheseexperiments.However,thecornersoftheenvelopesmailedthroughtheU.S.PostalSystemwithB.anthracissporesinSeptemberandOctober2001apparentlyweresealed.Thisstudywasvaluableinrevealingthepotentialspeed,magnitude,andspatial distribution of environmental contamination bysporessubsequenttothehandlingoropeningofaspore-ladenenvelope.

3.5COMMITTEEFINDINGSANDRECOMMENDATIONS

The events of autumn2001 unfolded rapidly,withCDC’s initial public healthresponsequicklyextendedtoamajorcriminalinvestigationunderthecontrolofthe FBI. A multipronged investigative strategy emerged for the scientificinvestigation, with one set of activities focused on understanding thecharacteristics of the material in the letters and another on developing andconductingacomparativeanalysisoftheseevidentiarymaterialsagainstsamplescollectedfromthescientificcommunity.Thescienceandtechnologythatformedthebasisfortheseanalysesevolvedrapidlyandhadamajorimpactonthefieldofmicrobial forensics.Although the public health crisis largely subsided afterthe last victim died in November 2001, the scientific investigation continueduntil2008andthecriminalinvestigationcontinueduntilthecasewasclosedinFebruary2010(seeTable3-1).

Finding 3.1: Over the course of the investigation, the FBI found andengaged highly qualified experts in some areas. It benefited from theunprecedented guidance of a high-level group of agency directors andleading scientists. The members of this group had top secret national

securityclearances,metregularlyoverseveralyearsinasecurefacility,anddealtwithclassifiedmaterials.TheNRCcommitteeauthoringthisreport,inkeepingwithacommitmenttomakethisreportavailabletothepublic,didnotseethesematerials.

Inacomplexinvestigationduringaperiodofextremenationalurgencysuchasthis one, it is imperative to recruit and make wise use of the best and mostrelevantexpertise.TheBureauregularlybriefeditshigh-leveladvisorygroupina secure setting where classified material was reviewed. They also relied onsubject matter experts and expertise from other government agencies. Thecommittee recognizes, inagreementwith theFBI (FBI/USDOJ, Jan2011) thatthe unique skills and expertise needed to conduct microbial forensicexaminationsmight,insomecasessuchasthisone,requirerelianceonthesameset of scientific personnel, who also might be considered potential suspects.Thus it is important to have in place an external oversight structure with thecapacitytorecognizewhererelevantexpertiseandconflictsmightresideandtoprovide guidance and coordination for the overall scientific investigation. Inaddition,thesmallnumberoflaboratorieswithexpertiseinagivenpathogenortechniquerequiresthatacertainlevelofindependentoversightbemaintained.

Finding 3.2: A clear organizational structure and process to oversee theentire scientific investigation was not in place in 2001. In 2003, the FBIcreated a new organizational unit (theChemical, Biological,Radiological,andNuclear[CBRN]SciencesUnit,sometimesreferredtoastheChemicalBiologicalScienceUnit,orCBSU)devotedtotheinvestigationofchemical,biological,radiological,andnuclearattacks.Theformationofthisnewunitwithclearerlinesofauthorityiscommendable.

Weekly meetings occurred between the science team and those leading thecriminalinvestigation.Theresultsofthesemeetingsandthewaythescientific,criminaland legalaspectsof the investigation interplayedwillbebeneficial inpreparingforfutureattacks(B3D1).Inaddition,thedevelopmentofanew

U.S.Governmentmicrobialforensicsinfrastructureandresearchstrategywillbeimportantstepstowardenhancingfuturecapabilitiesforattributionintheeventofabiologicalattack(Pesenti,2010).

Finding3.3:Investigatorsusedreasonableapproachesintheearlyphaseoftheinvestigationtocollectclinicalandenvironmentalsamplesandtoapply

traditionalmicrobiologicalmethodstotheiranalyses.Yetduringsubsequentyears, the investigatorsdidnot fullyexploitmolecularmethods to identifyandcharacterizeB.anthracisdirectlyincrimesceneenvironmentalsamples(without cultivation). Molecular methods offer greater sensitivity andbreadthofmicrobialdetectionandmorepreciseidentificationofmicrobialspeciesandstrainsthandoculture-basedmethods.

Thecommitteerecognizesthatthecircumstancesof2001createdanabundanceof samples and associated work for the LRN (laboratory response network)resulting inadecision touseRODACplates foranalysisof theenvironmentalsamples. Thus, the FBI did not confirm the presence of the letters-associatedgenetic mutations in the environmental samples. In the nine years since thattime,dramaticadvancesinhighthroughputsequencingtechnologyhavegreatlyimproved the ability to detect and characterize rare strains and species in

complexenvironmentalsamples2(forfurther,relateddiscussion,

2 Multiple studies in each of a number of diverseenvironmental settings have demonstrated the feasibilityand reliability of resolving strain-specific fine genomicstructure (at the level of SNPs and local structuralrearrangements) in highly complex biological samplesusing next-generation sequencing technology andmetagenomic approaches. Examples include strainresolutionandgene

see Finding 6.8 in Chapter 6). In thinking of futureincidents,itisimperative,basedonlessonslearnedfromthisinvestigation,toanticipatethetypesofsituationsandcircumstances that might affect evidence collection,preservation, and documentation, and to employprotocols and procedures that ensure the best possible

outcomes.

Finding3.4:TherewasinconsistentevidenceofB.anthracisAmesDNAinenvironmentalsamplesthatwerecollectedfromanoverseassite.

Attheendofthisstudy,theNRCcommitteewasprovidedlimitedinformationfor the first timeabout the analysisof environmental samples forB.anthracisAmes from an undisclosed overseas site at which a terrorist group’s anthraxprogramwasallegedlylocated.ThissitewasinvestigatedbytheFBIandotherfederal partners as part of the anthrax letters investigation. The informationindicates that therewas inconsistent evidenceofAmes strainDNA in someofthese samples,butnoculturableB.anthracis.The committeebelieves that thecomplete set of data and conclusions concerning these samples, including allrelevantclassifieddocuments,deservesamorethoroughscientificreview.

Finding3.5:Aswasdoneintheanthraxinvestigation,attheoutsetofanyfuture investigation the responsible agencies will be aided by a scientificplan and decision tree that takes into account the breadth of availablephysicalandchemicalanalyticalmethods.Theplanwillalsoneedtoallowforpossiblemodificationofexistingmethodsandforthedevelopmentandvalidationofnewmethods(seeChapter4,Section12).

Thescientificinvestigationofanyfuturebiologicalattackwouldgreatlybenefitfromrobust independentoversight andongoing review.Toaccomplish this, thegovernment shouldmaintain a standing body of scientific expertswith propersecurity clearances who are fully briefed on matters of importance forpreparedness and response to a biological attack. When an investigation islaunched,membersofthisgroupcouldhelpguidethescientificinvestigation.

Inpreparingforfutureinvestigations,allrelevantU.S.governmentagenciesanddepartmentswillneedtoworktogethertoensurethatindependent,

family structure in ocean surface water (see multiplepublicationsfromtheSorcererIIGlobalOceanSamplingExpedition), evidence for rare strain ecotypes in acidmine drainage (see multiple publications from theBanfield group at UC Berkeley), and gene family

sequence microheterogeneity in prophage genomes thathave been reconstructed from shotgun sequencing ofvirus-likeparticlesinhumanfeces(e.g.,seesupplementaldatafromNature466:334,2010).Thislargeandgrowingbody of work strongly suggests that the application ofthese same techniques to some of the environmentalsamples from the anthrax letters case might provideadditional clarity about B. anthracis genome sequencevariants and the relationships among the strains in thesamplesandtheletters.

high-quality, external science advice is available fromindividualswithexpertiseincriticalscientificareaslikelyto be relevant given anticipated scenarios for scientificinvestigations. By identifying a core set of externalexperts and convening this group ahead of time,productive working relationships could be establishedbetween this group and members of the government’sbioforensics community. When a new investigation islaunched,thiscoresetofexternalexpertscouldassistinrecruitingotherswithmorespecificexpertiserelevanttothe investigation under way. For example, the FBIincludedsomeexpertscientistsearlyintheanthraxlettersinvestigation,butitdoesnotseemtohavesoughtformalexpertise in statistics until the investigation was nearlycompleted. Because many inferences depend on thedesignandanalysisofdatasets thatmaybecomplex,as

inthiscase,foranysimilarinvestigationinthefuture,itwill be important that the FBI consult with expertstatisticians throughout the processes of experimentaldesignandplanning,samplecollection,sampleanalysis,and data interpretation. The committee recognizes thatmuch has been done by the government over the pastseveral years to build and enhance this importantinfrastructureandsetofresources,althoughthescopeofthis study did not include assessment of the currentinfrastructure.

When an investigation is launched, the panel of external experts couldrecommend and review strategies, protocols, and procedures; help with thedevelopmentofnewmethodsand scientific approaches;provideadviceon theselection of contract scientists and additional outside experts; assist in datainterpretation; andhelpgenerate alternativehypotheses.Membersof thepanelshouldnotbedirectlyinvolvedinconductingthescientificinvestigationitself.Itwillalsobeimportantthatthepanelhaveanongoingrolethroughoutthecourseoftheinvestigationandbebriefedonallofthesciencethatiscontemplatedandpursued. Consistent documentation of recommendations and input, and thesubsequentresponses,willbeofgreatvalue.

Inthefuturerelevantagenciesshouldreviewandperiodicallyupdateappropriateprotocols and experimental designs to use best strategies for preservingevidence,exploitingsamplesforscientificinformation,andmeetingsubsequentlegal challenges. Protocols should ensure that clinical and environmentalevidence is properly collected, preserved, documented, and analyzed tomaximize the utility of the samples collected. Furthermore, state-ofthe-artmolecularmethods,suchasnext-generationnucleicacidsequencingtechniques,offergreatpotential forcharacterizingclinicalandenvironmental samples (seeFinding6.8inChapter6).

Recommendation 3.1: A review should be conducted of the classifiedmaterials that are relevant to the FBI’s investigation of the 2001Bacillusanthracismailings, includingallof thedataandmaterialpertainingtothe

overseasenvironmentalsamplecollections.

Thecommitteedidnotreceivenorreviewclassifiedmaterial.InNovember2010discussionswithFBIandDOJ leadership regarding this report,weweremadeawareofadditionalinformationthatwouldrequirereviewofclassifiedmaterial.Duetothelatenessofthisrevelationandtheimportanceweplacedonissuingatimelyreport,andtheagreementbetweentheNRCandtheFBIthatallmaterialwe considered be publicly available, the committee did not undertake thisadditionalreviewofclassifiedmaterial.

Recommendation 3.2: The goals of forensic science and realisticexpectations and limitations regarding its use in the investigation of abiological attack must be communicated to the public and policymakerswith as much clarity and detail as possible before, during, and after theinvestigation.

Communicating with the public and policymakers is extremely important inordertoensurethataccurateinformationisavailableandtominimizeunrealisticexpectations.Specialattentionwillneedtobepaidtocommunicatingscientificinformationtothesegroupsinanaccurateandcrediblemanner,especiallyiftheinformationwillplayacriticalroleintheinvestigation.

When presenting to the public the findings of an investigation that involvescientific evidence, especially one as important as the anthrax lettersinvestigation,officialswillneedtomakeeveryefforttohavescientistsverifytheaccuracy of the scientific information they report. The inaccurate reporting offactsortheoverstatementofscientificevidenceisadisservicetothepublic.Intheanthraxlettersinvestigation,therewererepeatedclaimsthatalloftheattackletters contained all of the genotypic variants (seeChapter 6 andFinding 6.7)thatimplicatedflaskRMR1029asthesourceoftheanthraxspores,wheninfactnotallof the letterswerechecked for thesevariants.Ofevengreaterconcern,because it suggested possible deception by the suspect, the strength of theevidencewasoverstatedthatadisputedsamplesubmittedbythesuspecthadnotcomefromthepropersource(seeChapter6andFinding6.4).Similarmistakescanbeavoidedinthefuturebyinvolvingtherelevantscientistsinfactcheckingofthereportsbeforetheyarereleased.

4

PhysicalandChemicalAnalyses

4.1INTRODUCTION

Early in its investigationof the2001Bacillusanthracis (B.anthracis) attacks,the FBI hired several laboratories and conducted some of its own analyses toascertain physical and chemical characteristics of evidentiary materials, assummarized in Table 3-1 (Chapter 3). These analyses of the letter powdersfocusedonthesizeandgranularityoftheparticulates,elementalcontent,ageofthe spores, and identification of chemical signatures that might provide cluesrelatedtothesourceorproductionprocessesused.

The physical and chemical analytical methods used by the FBI and outsidecontractorswereconductedproperlyandmostwerewellestablishedatthetimeandvalidatedforuseinlawenforcementinvestigations.Insomeinstances,newmethods were developed to accomplish the desired analytical measurements.Other methods, such as mass spectrometric analysis and various microscopytechniques,arewellacceptedbutwereappliedinnewwaysandfornewtargetanalytes in this investigation (e.g., heme, agar, and additive analysis, isotopicanalysis of letter evidence, silicon determination in spores). This chapterdescribesandevaluatesthechemicalandphysicalanalysesperformed.

4.2SPOREPREPARATIONANDPURIFICATION

One lineof inquiry in theFBI investigationconcerned theexpertise, time,andtechnology needed to produce the material used in the attacks. That is, whatskills, tools, or procedures would be needed to cultivate, purify, and dry thespores,andhowlongwouldtheentireprocesstake?Whileaspectsofthisissuepertaintothetraditionalcriminalcomponentof theinvestigation,otheraspectspertain to the science component. The committee restricted its review andanalysistotheselatteraspects.Committeeeffortswerecomplicatedbythefactthat theFBIdidnotpublishorprovide thecommitteewithspecificordetailedconclusionsonitstheoriesregardingthemethodsusedforcultivat

75

ing,purifying,ordryingthesporesfoundintheletters.Indiscussionswiththe

FBI at the January 2011 meeting, the FBI stated that some of its consultingexperts referred to the letter preparations as beingof “vaccine quality”,whichnarrowed the list of potential suspects. Nonetheless, the Bureau investigatedindividualswithoutregardtotheirspecificskillsets.TheFBIfurtherstatedthatthetimeforpreparationandequipmentusedinpreparationofthelettermaterialswasdifficulttoascertainbecauseofnumerousvariables.However,FBIofficialsindicatedthatinferencesaboutrequiredskillsortimeforsporepreparationwereneverthesolecriterionforeliminatingsuspects(FBI/USDOJ,2011).

With regard tocultivationof thespores in liquidversussolid (i.e.,agar-based)medium, assays for the presence of residual agar in the letter material wereinconclusive, as described below.TheDOJAmerithrax Investigative Summary(USDOJ,2010)describesthepotentialuseofeitherafermentororanincubatorwith shaken flasks and liquid media. That document also suggests that aminimumof500mlofliquidculturewouldberequiredtoproducethesporesinthelettersbutthenstates“wecannotsaywithcertaintyhowmuchmaterialwasusedintheletters.”

Giventheinformationavailableonthenumberofsporesbelievedtohavebeenplaced in the letters and knowledge of spore yield from various types ofcultivationmethods,arangeofrequiredculturevolumescanbeestimated.Fourspore-containing letters were recovered and evidence indicates that oneadditional letterwassent toAmericanMedia,Inc.(AMI)(Cole,2009).Ahighestimateforthetotalnumberofsporessentthroughthemailwouldincludefive

letters, each containing 1 gram of spore-containing powder with 2 × 1012

sporespergram,foratotalof1.0×1013spores.Alowestimate for the totalnumberof spores sent through themailwould include five letterswith 0.8 gram of spore-containing powder per letter. Two of the letters (LeahyandDaschle)mighthavecontained2×1012sporespergramwhiletheothers(NewYorkPost,Brokaw,andAMI)might have contained 2 × 1011 spores per gram (FBIDocumentsB1M2D1,B1M2D3,B1M2D6),foratotalof

3.7×1012spores(seeTable4-1).

TABLE4-1EstimatedRangesofTotalNumberofSpores

Numberofletters,gramsporesperletter,Totalsporescontainedinalllettersandspores/gram

Lowestimate=3.7×10122letterswith0.8gramsporesperletterand2×1012sporespergram3letterswith0.8gramsporesperletterand2×1011sporespergram

High estimate = 1.0 × 1013 5 letters with 1 gram spores perletterand2×1012sporespergram

ApublishedyieldofB.anthracisAmessporesgrownonsolidmedium is8×109 sporesperPetri 150-mmplate(Carreraetal.,2007).Productionoftherequirednumberofsporesonplates,with theconservativeassumptionofno spore losses during purification, would thereforerequire 463 to 1,250 plates. Expert testimony to thiscommittee indicated that 15 liters of liquid culture in afermentor could yield 2 × 1013 B. anthracis spores(Heine, 2010). Cultivation in shake flasks or lossesduringsporepurificationcouldcertainlyreducethisyieldseveralfold. RMR1029, was a well-characterized large-scalesporepreparationhousedattheU.S.ArmyMedicalResearchInstitute for InfectiousDiseases (USAMRIID).

Theinitial1-literpurifiedsporepreparationinRMR1029wasderivedfromapproximately160litersofcultureandcontainedanestimated3×1013spores.Thus,cultivationin the range of 2.8 to 53 liters of liquidmediumwouldhavebeenrequiredtoproducethesporesrequiredfortheletters(seeTable4-2).

Sporepurificationistypicallyaccomplishedbyrepeatedcentrifugation,disposalofthesupernatant-containingcellulardebris,andresuspensionofthesporepelletinfreshliquid.Highsporepurityisgenerallyachievedbycentrifugationofthesporesthroughagradientofahigh-densitycompound.Themostcommonlyusedhigh-density compound, meglumine diatrizoate, was not detected in the lettermaterial(seeSection4.6below),suggestingthatthisprocedurewasnotusedorthatthesporeswereextensivelywashedaftersuchaprocedure.Purificationbyany method would involve some liquid washing steps and would require arelatively large-capacity centrifuge. Such instruments are commonly found inmicrobiology laboratories. The spores in the Leahy and Daschle letters wereaccompaniedby lesscontaminatingdebris, and thuswereofhigherpurityandconcentration,thanthoseintheNewYorkPostletter(FBIDocuments,B1M2D2,B1M2D6).

Thereareseveralmethodsfordryingsporesuspensionstoproducepowderslikethose found in the letters: these include chemical desiccation, air drying, andfreeze drying (lyophilization), any of which could require several hours toseveraldays.Dryingofsurrogatesporepreparationsusingvarious

TABLE4-2EstimatesofMediaVolumeRequiredforSporePreparation

Totalsporesneededforallletters

Sporespreparedonplatesat8×109spores/plate

Sporespreparedinliquid

Lowestimate=3.7×1012 463plates 2.8litersina

fermentor,based

onHeine’s(2010)estimateof2×1013sporesfrom15liters

Highestimate=1.0×10131250plates53litersinafermentor,based on RMR1029 having 3 × 1013 spores from 160liters

methods produced particle size distributions similar tothose found in letter samples (described below), butvolatileorganiccompoundsthatmighthavebeenusedfordrying were not detected at significant levels in lettersamples (see Section 4.8 below). The FBI thereforeofferednoconclusionsconcerningthemethodusedtodrytheattackspores.

Asaresultofthedifferentpossibleproductionschemesthatmighthaveyieldedproduct with the observed characteristics of the evidentiary materials, thecommitteefindsthatthetimerequiredforthisworkcouldbeaslittleas2or3days to as much as several months. The differences are based on differentestimatesof the timerequiredforpropagation,purification,anddrying,amongothervariables,aswellasthestateofthestartingmaterial.Inparticular,itisnotknownwhethersomeoftheinitialstepsmighthaveoccurredwellinadvanceoftheletterattacks.Thecommitteecannotresolvethesedistinctionsbecauseithadno information identifying a production method or the steps involved inproduction.

The FBI did not present a definite theory on how and when propagation,purification,anddryingtookplace,noronwhatspecificskillswouldberequiredtoperformthesetasks.Nonetheless,inferencesmadebytheFBIconcerningthe

time, skill, and equipment required for spore preparation were said to besignificant considerations in its narrowing of the list of potential suspects(USDOJ, 2010, pp. 29-33, 36-38), but were never the sole criteria foreliminating suspects (FBI/USDOJ, 2011). Without further specification withrespecttosporepreparationsvariables,thecommitteefindsnoscientificbasisonwhichtoaccuratelyestimatetheamountofthetimeorspecificskillsetneededtopreparethesporematerial.

4.3SURROGATEPREPARATIONANDPURIFICATION

In pursuit of greater clarity on the issues discussed above, the FBI askedscientistsatDugwayProvingGrounds(DPG)tocreatesurrogatesamplesinanattempt to mimic the physical properties of the letter samples. Chemicalpropertiesof the surrogateswerealsocompared to thoseof the letter samples.Surrogates designated in theWeeklyUpdates as “Buran” and “Abshire”werecharacterized with respect to physical and chemical properties, but thepreparation procedures for these sampleswere not provided to the committee.Thecommitteedidnotlearnofthescientificrationalebehindthechoiceofthevariouspreparationsstudied,otherthananapparentefforttostudyawiderangeofpreparationconditions.

Surrogates were grown by both plate and fermentation methods (using B.anthracis from the Leahy letter as the starter source) (FBI Documents,B1M13D3). Plate methods included various combinations of growth media(sheepbloodagar,newsporulationmedium),washing(DPG,Patrickmethods),drying(oven/air,lyophilizer,acetone,speedvac),andmilling(ballmill,mortarand pestle, sieve)—36preparations in all. Fermentationmethods included twotypes of antifoam agents, one containing silicon in the form ofpolydimethylsiloxane(AntifoamC)andonesilicon-free(Antifoam204)(DPG,2006). A separate set of B. anthracis Sterne preparations was analyzed atLawrence Livermore National Laboratory (LLNL) beginning in 2006 for thespecific purpose of studying silicon uptake into the spore coat. In situationswhereitwasinconvenientorunsafetoworkdirectlywithB.anthracis,surrogatesamplesofotherBacillusspecieswere prepared.The results of these analysesarediscussedbelow.

4.4SIZEANDGRANULARITYOFTHEMATERIALINTHELETTERS

In fall 2001 at the request of USAMRIID, the Armed Forces Institute ofPathology(AFIP)performedscanningelectronmicroscopy(SEM)todeterminethe size and shape of particulates in the letter material. SEM is a commonmicroscopic imaging technique used extensively across scientific disciplines.ResultsofthisanalysisdemonstratedthatthesizeandshapeofsporesfoundintheletterswereconsistentwithB.anthracis (FBIDocuments,B1M2D4;AFIP,2001).The imagesshowed individual sporesaswellasclustersof spores,andother solid (crystalline)material suchas calciumcarbonate (Kuhlman,2001b).While themorphologymayhavebeen affectedby sample preparationprior toanalysis (autoclave), the imageswereconsistentwithmeasurementsofparticlesize.

In fall 2001,BattelleMemorial Institute (BMI) evaluated size distributions ofaerosolized particles from untreated letter material and surrogate samples todeterminewhetherrespirable-sizeparticleswerepresentandwhethertheamountofsuchparticleswouldhaverequiredspecializedprotocolsforpreparation(e.g.,

dispersants).1Well-establishedcommercialinstrumentswereused to aerosolize the samples (AeroDisperser®, TSI,Inc.) and measure their particle size distributions(Aerosizer®,TSI, Inc.).TheAeroDisperserusesahigh-velocitygastoliftanddispersesolidparticles(powders)off a plate into the airflow.TheAerosizermeasures theaerodynamic diameters of individual particles byaccelerating the aerosol in a sonic airflow anddetermining the velocity by time of flight between twolaserbeams.TheAerosizerwascalibratedwithNationalInstitute of Standards and Technology traceable particlesize standards between 5 and 20 micrometers (µm) indiameter.

TheDaschle andLeahy letter samples had bimodal particle size distributions,with one mode around 1.5 µm in diameter, corresponding to the size of an

individualB.anthracisspore,andanothermodegreaterthan20µmindiameter,corresponding to the size of clusters of large numbers of spores and othermaterial

1 To remove the implication that autoclaving of lettersampleswasstandardpractice, theword“untreated”hasbeeninsertedintothissentence.Theinsertionrepresentsa modification to the text that appeared in theprepublicationeditionofthisreport.

(FBI Documents, B2M13D11). In the Daschle sample,0.05percentof the totalvolume (mass)ofparticleswasfoundinthesmallerdiametermode.IntheLeahysample,1 percent of the total mass was found in the smallerdiametermode.

Several Bacillus subtilis var. niger culture preparations made using onlycentrifugationforconcentrationand lyophilizationfordryingalsohadbimodalsize distributions, with the smaller (1.5 µm diameter) mode constitutingapproximately1percentofthetotalaerosolizedmass(Kuhlman,2001a,c).Thesimilarity between the letter and these size distributions showed that powderswith dispersion characteristics similar to those of the letter material could bemadewithouttheadditionofadispersant.

Particle size distributions for theDugway surrogate sampleswere reported asmean particle diameter, which unfortunately is a less informative indicator ofparticlesizewhenthedistributionisbimodal.Nonetheless,manyoftheDugwaypreparations gave mean particle diameters in the same range as the lettersamples,2to4µm,consistentwiththenotionthatdispersantswerenotrequiredtoproducepowderswiththeseparticlesizedistributions(DPG,2006).

A recent report in the scientific literature describes production ofB. anthracissporesinamannertoenhanceformationofparticlesofabout1.5µmindiameter(Baron et al., 2008). The Baron preparation involved a proprietary dryingprocedure, ball milling, and the addition of 20 percent amorphous silica

fluidizingagent.The sizedistribution showeda sporemodearound1.5µm indiameter and a smallermode around 0.5 µm in diameter. It is not possible tocomparetheparticlesizedistributionsofthelettersampleswiththeBaronworksincethelatterstudyincludedanimpactortoremoveparticleslargerthanabout5µm.

4.5PRESENCEOFSILICONANDOTHERELEMENTSINTHELETTERMATERIAL

While any deliberate mailing of letters containing anthrax spores might beconsidereda formofsporeweaponization, this termhasbeenmorecommonlyused to describe preparations with enhanced properties of dispersion andaerosolization.Itiscommonlybelievedthatdeliberateeffortstomakeapowdermoredispersiblethroughtheuseofadditiveswouldsuggestamoresophisticatedlevel of preparation expertise. Thus the presence of dispersants, such asnanoparticulate silica or bentonite, was an important feature in consideringwhether or not the letters contained “weaponized” anthrax spores. The FBIcommissioned several studies to determinewhether siliconwas present in the

lettersand,ifso,toascertainthenatureofthesilicon.2

2 The elemental analysis methods discussed in thischapterdeterminetheamountofsiliconinthesamplebutprovidenodirectinformationaboutthechemicalformofthis element other than what can be inferred fromsimultaneousdetectionofotherelements.Therefore, theterm “silicon” is used in reference to the results ofelementalanalysis.

4.5.1ElementalAnalysis

Elementalanalysiscanbeperformed inseveralways,providingvarying levelsof sensitivity and spatial resolution. The FBIworkedwith laboratories havingseveral capabilities and these techniques are summarized in Table 4-3. Oneapproach is to measure the presence of heavy elements while performingelectronmicroscopy.Asstatedpreviously,AFIPperformedSEMinfall2001to

characterize the morphology of the letter samples. A complementary tool inmany scanning electronmicroscopes, energy-dispersiveX-ray analysis (SEM-EDX),providesalow-resolutionanalysisofthespatialdistributionofelementsin the samples. SEM-EDX is a well-established analytical technique thatmonitorstheX-rayemissionfromthesmallregionofsamplethatisirradiatedbyelectrons. The wavelengths of X-ray emission identify the elements present,whilethecorrespondingintensitiespermitquantificationofeachelementintheirradiatedregion.Theseinitialmeasurementsshowedhighlevelsofsiliconinthelettermaterial(USAMRIID,2001).

In early 2002 the FBI performed elemental analysis on letter and surrogatesamplesusing inductivelycoupledplasma-optical emission spectroscopy (ICP-OES)(FBI,2009).ICP-OESisawell-establishedchemicalanalysistech-

TABLE4-3MethodsforChemicalAnalysisReferredtoinChapter4

MethodPrimaryuseintheinvestigationInductively coupled plasma-optical Bulk elemental composition of a sampleemissionspectrometry(ICP-OES)dissolvedinaqueoussolution

ScanningelectronmicroscopywithenergySize-resolvedelementalcompositionofadispersiveX-rayanalysis(SEM-EDX)solidsample,withsizeresolutionofabout1µmorless

Nanometersecondaryionmass

Sizeresolvedelementalandmolecular

spectrometry(nano-SIMS)

compositionofasolidsample,withsize

resolutionofabout1µmorless

Acceleratormass spectrometry (AMS)Measurement of the ratio of14C to12Cinasample

Isotope ratiomassspectrometry (IRMS)Measurementof the ratiosof2H to

1Hand18Oto16Oinasample

Gaschromatography-massspectrometry

Separationandidentificationofvolatile

(GC-MS) organiccompounds(i.e.,compoundsthatcanbevaporizedwithoutdecomposition)

Liquid chromatography-mass spectrometry Separation and identification ofpolar,(LC-MS)nonvolatileorganiccompounds

Aerodynamic particle sizer (Aerosizer®) Size distribution of anaerosol,bytimeofflightinsonicflow

AerodisperserSizedistributionofanaerosolparticlebylift

niquethatprovidesaccurateandprecisemeasuresofbulkelementalcomposition(weightpercentagesof individualelements in the sample),butgivesno informationaboutthe spatial distribution of the elements in the sample.ICP-OESanalysisshowedquitehighweightpercentagesof silicon (Table 4-4): 10 percent in theNewYorkPostmaterialand1.4percentand1.8percentfortwoseparatesamples of the Leahy material. Control samples of B.subtilis with and without the addition of a dispersant,werealsoanalyzed,andthesamplecontainingdispersantshowedahighermeasured silicon content thanmatchedsampleswithoutdispersant.

The large amount of silicon in the letter materials fueled speculation thatdispersantssuchassilicondioxideorbentonite(analuminumsilicateclay)may

have been present, possibly indicating intent to “weaponize” the material byenhancingitsabilitytodisperse.Acontrolsampleofbentonitewasanalyzedaspart of the original AFIP work for USAMRIID (FBI Documents, B1M2D4).Bentonitewas ruled out rather quickly as an additive to thematerial from theNewYorkPost,Leahy,andDaschlelettersowingtothelackofacommensurateamount of aluminum and other elements in the SEM-EDX images. Silicondioxide remainedapossibilityandwas investigatedusingSEM-EDX toprobethespatialdistributionofelementsinthesamples.

TABLE 4-4Summary of SiliconMeasurements in Evidentiary and SurrogateSamples

Bulk silicon content SEM-EDXSEM-EDX% sporesSampleaICP-OESAnalysiswithSiincoat

Leahy1.4-1.8%1-2%perspore97/124=76%

Daschle1-2%perspore73/111=66%

NewYorkPost10%1-2%perspore91/141=65%

RMR10290/98=0%0/115=0%0/191=0%

RMR-1030(shake 6/94=6%flaskpreparation) 0/118=0%

7/113=6%Dugwaysurrogates

0.2-5%(10agar 42/163=26%

preparations) 17/161=11%50/172=29%

(fermentationusingLeighton-Doimedia)

0%(2agarpreparations)

NOTES:ICP-OES=inductivelycoupledplasma-opticalemissionspectrometry;SEM-EDX=energydispersiveX-rayanalysis;SI=silicon.

aSimilar analysis of Brokaw letter material was notperformedduetothesmallamountofsampleavailable.

Bulk elemental analysis by ICP-OES of surrogatepreparations from Dugway, without the addition of adispersant, was reported for 10 of the 36 preparations(FBIDocuments,B1M7).Allof thesurrogatesanalyzedwere found to contain silicon at a level of 0.2 weightpercentormore,andfourcontainedsiliconinthe2-to5-percentrange.TheseresultsindicatethatitispossibletopreparesporeshavingasiliconcontentintherangeoftheLeahy letter sample without adding a dispersant (FBIDocuments,B1M7).

Trace levels of several elements including aluminum, sodium, magnesium,potassium, calcium, chlorine, iron, manganese, zinc, and tin were detected inletter and surrogate samplesaswell as inpreparation reagents.While someofthese elements are ubiquitous impurities in the environment, others are lesscommonandsometimesareusedinchemicalprofilinginforensicinvestigations.TheAmerithraxScienceUpdatedocuments(B3D1)describemultipleeffortstolink stainless steel fragments contained in letter material with productionmethodsorother sourcesof stainless steelparticles (e.g.,drugs,growthmediacomponents, pens). As of 2005 Michael and Kotula of Sandia NationalLaboratories believed that the tin and iron present in the powders may haveprovidedausefulchemicalsignature;however,thecommitteewasnevershownany evidence to indicate that this possibility was pursued further (FBIDocuments,B1M1D7)orthatthesediscussionsledtoanyconclusionsaboutthesourceofmaterialorproductionmethods.

4.5.2SpatiallyResolvedElementalAnalysis

SandiaNationalLaboratories (SNL)offered instrumentationwithbetter spatialresolution and detection sensitivity than the instruments at AFIP and FBI.

Beginning inFebruary2002, theFBI soughtSEM-EDXanalysesof letter andsurrogatesamplesusinginstrumentsandalgorithmsdevelopedbystaffscientistsat SNL that elucidate correlations among the spatial distributions of differentelements.These enhanced capabilities greatly aided themeasurements and theconclusionsthatcouldbedrawnfromthem.

SEM-EDXcanbeperformedinlow-orhigh-spatialresolutionmodedependingon how tightly the electron beam is focused. Low-resolution operation canprovideanestimateofthebulkelementalcompositionofasample,althoughitisa less reliable indicator of bulk composition than ICP-OES. Low-resolutionSEM-EDX analysis of both the New York Post and Leahy samples gaveestimatesof1to2percentforthebulksiliconcontent.TheconsistencybetweenICP-OES and SEM-EDX measurements (Table 4-4) of the Leahy material,which appeared to be a refined and homogeneous powder, argues against theexistence of a method-dependent artifact that would lead to an incorrectmeasurementbyonemethodortheother.Ontheotherhand,theICP-OESandSEM-EDXmeasurementsoftheNewYorkPostmaterialdifferedbyanorderofmagnitude. Further complicating the comparison, the 2001 AFIP SEM-EDXimagesoftheNewYorkPostmaterial(obtainedbytheFBIduringthecourseofthisstudy)showedregionsenrichedwithsiliconbutnotoxygen,suggestingthepresence of a reduced form of silicon (FBI Documents, AFIP, 2001). TheseregionswereapparentlymissingornotreportedintheSNLSEM-EDXimages.When asked at the January 2011meeting, the FBI stated that the presence ofreducedsiliconwas“justanobservation”andthatdifferencesbetweenthetwomethods (AFIP and SNL) could account for the discrepancy as one of themethodsreliedonsectionedsporeswhiletheotherdidnot.

4.5.3SiliconintheSporeCoat

High-resolution SEM-EDX of the Leahy and New York Post samples gavesimilarspatialdistributions—siliconwasconcentratedwithinthesporecoat,butnonewasdetectedintheexosporiumwhereadispersantwouldreside(Figure4-1).While the SEM-EDX images appear convincing, two questions should beconsidered: (1) Is silicon in the exosporium detectable in the experiment, ormight it be lost during sample preparation? (2)Howwas silicon incorporatedintothesporecoatanddoesitspresenceinthesampleshaveforensicvalue?

TheFBIapproachedthefirstquestionbypreparingasampleofB.subtilisspikedwith 20 percent Syloid 244, a commercial nanoparticulate silica product, to

simulateaflow-enhancedsample.Whenthissamplewasmountedandanalyzedin the same manner as the letter samples, silicon dioxide nanoparticles wereclearlyobservedonthesporesurface.Althoughthisexperimentindicatesthatadispersantwould have been detected if it were present in the sample, amoredefinitive experiment would have been to spike an actual letter sample withdispersantatlevelscomparabletothebulksiliconcontent.Thelatterexperimentwasnotperformed.

Initially, theFBIprovidedevidentiary samples toSNL thatwerealready fixedandstained.Itisunclearwhetherornotthispreparationwouldhaveresultedinseparation of the dispersant from the spores prior to analysis. The FBIsubsequently provided letter samples to SNL that had been treated only bygamma irradiation, which should not have resulted in loss of dispersant. Theirradiatedsamplesalsoshowedsilicononlyinthesporecoat.

Insight into the second question can be gained from a study showing thatB.cereus spores accumulate silicon in the coat (Stewart et al., 1980). The FBIobtainedarchivedsamplesfromthatstudyandhadSNLanalyzethembySEM-EDX. The images showed silicon in the spore coat, indicating that siliconincorporationwasnotuniquetothelettersamples.SNLsubsequentlycomparedthe characteristics of the spore coat in the letter samples to a surrogatefermentationsamplepreparedatDugwayusingLeighton-Doimedia(Table4-4).MorethanhalfofthesporesanalyzedfromboththeLeahyand

LeahyLetterMaterial

NewYorkPostLetterMaterial

NewYorkPostletterscontainedsiliconinthecoat.Incontrast,onlyaboutone-fourthofthesporesintheDugwaysamplecontainedsiliconinthecoat.SNLanalysisofmaterialtakendirectlyfromRMR-10303(preparedfromshakenflasksatUSAMRIID)showedonlyafewsporeswithsiliconinthecoat,andanalysisofmaterialfromRMR1029showednosporeswithsiliconinthe

3RMR-1030wasthelabelofanotherflaskofaB.anthracisAmesstrainsporepreparationproducedbyandhousedatUSAMRIID.RMR-1030didnotresembleRMR1029orthelettersporesinitsphysiochemicalorgeneticproperties(FBIlettertothecommittee,December7,2009).Thefourgeneticsignatures(A1,A3,D,andE[seeChapters5and6])werenotdetectedinRMR-1030.

coat. Two of the 35 Dugway plate preparations were

evaluated for silicon by ICP-OES, and one showed 5percentandtheother0.7percentsilicon.Neitherofthesesamplesshowedanysporescontainingsiliconinthecoat.

Beginning in 2005, LLNL conducted a detailed study of silicon incorporationintothecoatsofB.anthracisspores.Chemicalanalysiswasperformedbynanotime-of-flightsecondaryionmassspectrometry(nano-SIMS),amethodinwhichaprimarybeamofionsisfocusedontoasmallregionofthesample.Theimpactoftheprimaryionsonthesamplecausesmaterialfromthesamplesurfacetobeejectedassecondaryions,whicharethencharacterizedbymassspectrometry.Achemical image is producedby scanning the primarybeamacross the sample.Theparticularstrengthofnano-SIMSwithrespecttothisstudywasitsabilitytodetecttracelevelsofsiliconinthesporecoatatrelativelyhighspatialresolution.Measurements were performed on a total of 57 spore samples that includedexisting samples provided by the Department of Homeland Security andcollaborating laboratories (Weber, 2009; FBI Documents, B1M1D7) and B.anthracis Sterne surrogates produced by LLNL researchers under a variety ofconditions. The LLNL researchers were not given the opportunity to performnano-SIMS on FBI evidentiarymaterial. The goals of the Sterne experimentsweretomanipulatesiliconlevelsinthesporesandtoquantitativelycomparetheamounts incorporated with previous work and predictions based on silicatesolubilityandprecipitation.

Siliconwas found inalmostallof thesporesexamined.Thehigh incidenceofsilicon detection in these experiments is most likely due to the enhancedsensitivity of nano-SIMS (impurity detection at ppm level in submicrometerregions)overSEM-EDX(Weber,2009).Theamountofsiliconincorporatedinthe coat varied by over two orders ofmagnitude.While silicon incorporationtendedtoincreasewiththesilicateconcentrationinthegrowthmedium,amuchstronger correlation was found with the amount of iron in solution. Theseobservationsandtheinsighttheygiveintothemechanismofsiliconuptakearethe subject of a peer-reviewed publication by the LLNL team (Weber, 2009).However,theamountofsiliconincorporatedintheseexperimentsdidnotmatchthelettersamples.Mostpreparationshadasiliconcontentbelow

0.1 percent per spore, with 0.3 percent being the highest amount detected—much lower than the amounts reported by SEM-EDX analysis of the lettersamples,whichwereontheorderof1to2percentperspore.

Recently,Hirotaandcolleagues(2010)publishedastudyofsiliconincorporationinB.cereussporecoatsinwhichtheyfoundsiliconinsporesgrowninaculturecontaining silicate, but none in spores grown in the absence of silicate andsubsequently exposed to it. These results suggest that silicon becomesincorporated into the mother cell and then accumulates in the spore duringmaturation.Theresearchersalsofoundthattheamountincorporatedwasstrainspecific—YH64tookup15timesmorethanitsnearestrelativeNBRC15305—andthatsiliconincorporationenhancesacidresistance.

4.5.4SummaryoftheSiliconAnalysis

ThesubstantialeffortdevotedtothecharacterizationofsiliconinBacillussporecoats resulted in new fundamental insight into microbial processes and thedevelopmentofneworenhancedanalyticalmeasurementtechnology.(Table4-4presents a summary of the analytical results.) Elemental analysis of the lettersamplesshowedthat(1)thesiliconcontentwashigh,(2)mostofthesiliconwasincorporated in the spore coat, (3) the majority of spores in the samplescontained silicon in the coat, and (4) no siliconwasdetected in the formof adispersantintheexosporium.

ThebulksiliconcontentintheLeahylettercouldbecompletelyexplainedbytheamount of silicon incorporated in the spores during growth. (Not enoughmaterial was available to make this comparison for the Daschle letter.) Incontrast, the New York Post letter had significant bulk silicon content, farexceedingthatcontainedinthespores.

No studies have considered the effect of the chemical form of silicon (e.g.,silicate impurity versus polydimethylsiloxane antifoam agent) on uptake. Theinabilityoflaboratoryexperimentstoreproducethesiliconcharacteristicsofthelettersamplesisnotsurprisinggiventhecomplexityoftheuptakemechanism.

AfewsporesanalyzedfromRMR-1030containedsiliconinthecoat,butnoneofthesporesanalyzedfromRMR1029containedsiliconinthecoat.Therefore,thelettersamplescouldnothavebeentakendirectlyfromtheflasks—aseparategrowthpreparationwouldhavebeenrequired.

ThematerialintheDaschleandLeahyletterswasreportedtohave“ahighlevelofpurity”and tohaveelectrostaticproperties thatcaused it todisperse readilyuponopeningof the letters.Theseproperties shouldbe regardedasqualitative

observationssince theywerenotbasedonquantitativephysicalmeasurements.The committee received testimony (Martin, 2010) stating that some Dugwaypreparations, particularly those utilizing lyophilization but no dispersant, gaveproducts with similar appearance and electrostatic dispersibility as the lettersamples, suggesting that thesepropertieswerenot necessarily connected to anintentional effort to increase dispersibility through addition of a dispersant.Exogenous silicon and bentonite, which enhance the dispersibility of sporepreparations,werenotfoundintheLeahyandDaschleletters.

4.6FEATURESOFBACTERIALGROWTHCONDITIONSANDPROCESSINGMETHODS:DETECTIONOF

MEGLUMINEANDDIATRIZOATEBacterialendosporessuchasthoseproducedbyB.anthracisachievetheirlong-termdormancyandmostoftheirresistancepropertiesviarelativedehydrationofthesporecoreorcytoplasm.Thisdehydrationresultsinthesporeshavingahighdensity relative to the other components that remain following cultivation. Acommonsporepurificationmethodtakesadvantageofthisdifferenceindensity(TamirandGilvarg,1966)bycentrifugation throughasolutionof intermediatedensity.Theculturedebrisremainsontop,whilethehigh-densitysporesformapelletatthebottomofthetubeandcanberesuspendedandwashed.Generally,severalroundsofcentrifugationandresuspensioninfreshwateraresufficienttoremovemost of the chemicals used to produce thedensity gradient.Themostcommonly used density-gradient compound is a mixture of diatrizoate andmeglumine (sold under the trade name RenoCal). The presence of diatrizoateand/ormeglumineinevidencesamplescouldprovideleadstotheirsource.

In fall 2006, scientists at the FBI developed assays for the detection ofdiatrizoate and meglumine using liquid chromatography mass spectrometry(MS)withelectrosprayionizationmethods.Usingpurifiedstandardcompounds,FBIscientistsdevelopedfour-tieredassaysfor thedetectionofeachcompound(FBIDocuments,B1M12D4).

Iohexolandmetrizamide,chemicalssimilartodiatrizoateandalsousedforsporepurification,weresubjectedtotheMSanalysis.Thesechemicalsdidnotproducea false diatrizoate signal. The ions produced from these compoundswere notdescribed,and it is thereforenotclearwhether theassayswouldhavedetectedthemhadtheybeenusedtopreparetheletterspores.

Detection of diatrizoate and meglumine in purified spore samples was firstdemonstrated with control samples of B. cereus spores (FBI Documents,B1M12). The spores were purified using the standard method for sporepurification at USAMRIID, involving centrifugation through two RenoCal-76gradients, followedby twowaterwashes.Theywere thengamma-irradiated inthe same manner as the attack letter samples. The spores were dried andextracted with pure water and then removed by centrifugation, and thesupernatant was assayed for the presence of diatrizoate andmeglumine. BothcompoundswereclearlydetectableinthecontrolB.cereussporespurifiedusingRenoCal-76.

Samples takendirectly fromRMR1029 testedpositive formeglumine inassaytiers1-4andfordiatrizoateinassaytiers3and4.SamplesfromtheLeahyandNewYorkPost letters testednegativeforbothmeglumineanddiatrizoate inalltiersofbothassays.

Thisanalysiswassufficienttoansweronequestion:WerethelettersporestakendirectlyfromRMR1029andusedwithoutfurtherpurification?Thepresenceofdiatrizoate andmeglumine in the RMR1029 samples and their absence in thelettersamplesisconsistentwiththeideathatthelettersporeswerenotderiveddirectly from RMR1029. These data, in conjunction with evidence discussedelsewhereinthisreport(primarilyconcerningsiliconinthesporecoat),ledtheFBItoconcludethatthelettersporeswereproducedbyafurthercultivationofasample from RMR1029. The resulting spores were either not purified usingdiatrizoate and meglumine or they were washed extensively followingpurification,loweringthediatrizoateandmegluminetolevelsbelowtheassays’limitsofdetection(0.01µg/mlfordiatrizoateand0.001µg/mlformeglumine)(FBIDocuments,B1M12D4).

4.7MEDIACOMPONENTANALYSIS

Spores can be grown either in suspension or on agar. The FBI contractedscientistsattheUniversityofMaryland,BattelleMemorialInstitute,andPacificNorthwest National Laboratory to develop new methods for determiningwhetherthecompositionandsourceofthegrowthmediumforsporescouldbeascertained.Analyses centered on detection of trace amounts of agar or bloodagar,aspecializedformofagar.(Forarecentreviewofmethods,seeWahletal.,2010.)

ResultsofeffortstodetectagarintheLeahyandNewYorkPostsamplesthroughthepresenceofproductsresultingfromcleavageofagarcoupledtoavarietyofmass spectrometry techniqueswere inconclusive (Fenselau,2005;Wunscheletal.,2008;Wahletal.,2010).

Blood agar is sometimes employed as a rich medium for growth of certainmicroorganisms.Nutrientsinbloodprovideasuperiorgrowthmediumforsomemicrobes. When nutrients become depleted, the microbes go into a spore-formingstage.Heme ispresent inbloodcellsand is released into themediumduringsporeformation,dueprimarilytohemolysis.Traceamountsofhemeinasporesamplewouldindicatethatbloodagarwasusedforsporegrowth.Matrix-assisted laser desorption/ionization time-of-flightmass spectrometry (MALDI-TOF-MS) measurements of heme (Whiteaker et al., 2004) showed clear andspecificMSsignalsinBacillussporesamplesgrownonbloodagarbutnosignalon those prepared otherwise. The protocolwas able to identify heme in someFBIsamplesfromUSAMRIIDwiththeexceptionofanirradiatedB.anthracisSterne grown on blood agar. This result may indicate that spore irradiationcompromisesthereliabilityofresultsfromthismethod.

In a validation study (FBI Documents, B1M10), the agar and blood agaranalyses were highly sample dependent and too sensitive to experimentalconditions for the FBI to draw any conclusions from these studies.While thestudiesdescribedheresuggestthat,undersomeconditions,bothagarandbloodagarcanbedetectedattracelevelsinspores,theinformationgleanedfromthesestudies was not helpful in leading to sources for the spores used in the letterattacks.Thus the studyof agar andbloodagar in theevidentiarymaterialwasinconclusiveandnotpursuedfurtherbytheFBI.

4.8VOLATILEORGANICCOMPOUNDS

Volatile organic compounds (VOCs) are organic compounds that have a highvaporpressureandlowwatersolubility.Theyareproductsofmicrobialactivity,and they are also sometimesused in thepreparationof spores.They areoftenemittedasgasesfromcertainsolidsorliquids,includingcommonproductssuchaspaint,cleaningsupplies,pesticides,andofficeequipment.TheFBIconductedanalysestodetectVOCsinlettersamplesthatmayindicatemethodologyusedtopreparethespores(FBIDocuments,B1M7D2).

TheFBIusedheadspacegaschromatography-massspectrometry(GC-MS)and

infraredspectroscopy todetectVOCs.HeadspaceGC-MSwasperformedonlyon the Leahy letter sample. Trace amounts of ethanol, acetone, and t-butanolwere identified. Distributions of VOCs were slightly different for Leahy andsurrogatecultures,buttheFBIplacedlittlesignificanceontheresultsowingtothemanypotentialsourcesofthesecompounds.

4.9DETERMININGWHENTHEMATERIALWASPRODUCED:RADIOCARBONDATINGOFB.ANTHRACISSAMPLES

Blinded samples for radiocarbon analysis (14C) were treated withstandard protocols and sent to the LLNL Center forAccelerator Mass Spectrometry (CAMS) and to theNational Ocean Sciences AMS Facility (NOSAMS) atWoodsHoleOceanographicInstitutionduringwinterandspring2002.

Theradiocarboncompositionsof thesampleswerereportedas∆14Cin‰(partsperthousand)relativetothestandarddateof1950.Positive values are the result of either the rising (pre-1963 but post-1950) or the falling portion of the 14Ccalibration curve (see Appendix A). Negative valuesindicate samples prepared before 1950. Seven sampleswere analyzed by NOSAMS and three sets of sampleswereanalyzedbyCAMS(Set#1,20samples;Set#2,1sample;andSet#3,5samples).Assigningactualcalendardates to specific samples is complicatedby thepossibleincorporation of different amounts of fossil fuel carbon(with essentially no 14C content) in the sample(potentiallyfromorganicsolventsusedinthepreparation

ofthesampleorfossilfuelburning).

CAMSassigneddatesassumingapristineenvironmentremovedfromsourcesoffossil fuel carbon while NOSAMS added assumptions of varying amounts offossil carbon. The analyses indicated that the Leahy sample was producedbetween1998and2001.Radiocarbonanalysiswasnotperformedon theotherlettersamples.

4.10STABLEISOTOPEANALYSIS

4.10.1B.anthracis

Overthecourseoftheanthraxmailingsinvestigation,theFBIsubmittedaseriesof samples to the Stable Isotope Ratio Facility for Environmental Research(SIRFER)at theUniversityofUtah for analysisof the stable isotope ratiosof

hydrogen (2H/1H),carbon (13C/12C), nitrogen (15N/14N),andoxygen (18O/16O)using itsFinneganMAT isotoperatio mass spectrometer. These included a variety ofsamplesfromvariouslocationsinFortDetrickconsistingofsporeculturesonagarmedia.Thesamples in thissethave δ18O values ranging from 11.6‰ to 14.9‰ andδ2Hvaluesbetween−95‰and−76‰.Theδ13Cvaluesranged from −23.9‰ to −15.2‰ and should reflect theisotopic composition of the growth medium (Kreuzer-MartinandJarman,2007).Amongthesamplesofgrowthmedia, therearetwodistinctgroups:sampleswithδ13Cfrom−19.1‰ to−15.9‰and sampleswithvalues from−25.6‰to−22.5‰.ThetrianglesinFigure4-1representthe relationship between the δ18O and δ2H for these

samples.

The Leahy letter evidentiary sample was received at SIRFER in late January2004 and the analysis was completed in February 2004. The stable isotope

results are δ2H = −83±0.3‰, δ13C = −24.5±0.3, δ15N =+8.7±0.01,andδ18O=+18.2±0.4.Therelationbetweenδ2Handδ18OvaluesisshownasthediamondinFigure4-2.Itisapparentthatthehighδ18Ovalueisinconsistentwiththerestofthesporesamplesanalyzed.Inanattempttounderstandtherela-

FIGURE4-2Stableisotoperesults18Oversus2H.StableisotopicdatawereobtainedfromanumberofsamplesanalyzedbytheStableIsotopeRatioFacilityforEnvironmentalResearch(SIRFER).ThetrianglesarefromAmessporesamplesobtainedfromvariouslocationsinFortDetrick.ThesampleslabeledS(open

circles)andN(filledcircles),receivedinNovember2003,havedifferentδ13Csignaturesof~23.5and~18.6,respectively,indicatingdifferentgrowthmedia.ThesquareisfromasampleofRMR1029andthediamondisfromtheLeahyevidentiarysample.SOURCE:CourtesyofAliceMignerey.

tiveisotopicvalues,EhleringerandKreuzer-MartinusedtheisotopiccompositionsofknownsporepreparationsofB.subtilistoconstructamodelthatrelatedtheδ2Hinthesample to that of themediumandwater used (Kreuzer-Martin et al., 2005). Using the δ2H of water fromDugwayasaninput(δ2H=−121‰)andcomparingtheresultstheyobtainedforδ2Hofthemediumtothatofallknownmedia,theyconcludedthatitwashighlyunlikelythatDugwaywaterwasusedtopreparethesample(FBIDocuments,B1M9D5).

The extensive work on the influence of the growth medium on the isotopicsignature of the resultant spores suggests that the Leahy sample was not

producedinaliquidmedium.Thereshouldbearelationshipbetweentheδ2Hand δ18O in the water used in the spore preparation(meteoric water line) (Kendall and Coplen, 2001); thisconstrainsthepossiblevaluesofδ18Ousedintheinitialspore preparation. Using data gathered for B. subtilis(Kreuzer-Martin et al., 2003) and other strains of B.

anthracis (A0256 and Sterne), Ehleringer and Kreuzer-Martin concluded that data for the Leahy sample areinconsistent with the sample having been grown in aliquidmediumwithpurifiedtapwater.Theypositthatitwas likely to have been grown on agar medium withsignificant enrichment in the δ18O signature due toevaporation(FBIDocuments,B1M9D14).Stableistotopeanalysiswasnotperformedontheotherlettersamples.

Additionalresearchontheeffectsofgrowthmediaandcultureconditionsontheresultant spore isotopic composition could develop this approach into a usefulforensictoolthatmightprovideleadsinfutureinvestigations.

4.10.2WaterSamples

TheFBIcollectedsamplesoftapwaterat20locationsin18citiesandsentthem

to SIRFER for δ2H and δ18O analysis. The correlationbetween the δ2H and δ18O values is very close to themeteoricwaterline(MWL)(KendallandCoplen,2001).As expected, the samples fromDugway inUtah are thelightest (most negative)with δ2H= 120‰ and δ18O=−15‰. Those from Miami are very close to zero (thevalueofmeanoceanwater).ThreesamplesweretakenintheFrederickarea,oneatUSAMRIID,onefromawell,and one from the city municipal supply. All gaveconsistent values around−45‰ for δ2H and−7.5‰ forδ18O. These results proved useful in providing starting

waterisotopicratiosasinputfortheinterpretationofthespore sample isotopic ratios presented in Section 4.10.1(FBIDocuments,B1M9D1).

4.10.3TheEnvelopeMeasurements

SIRFERconductedstableisotopeanalysisforδ2H,δ13C,andδ18Oonfour sets of envelopes, each with three samples: oneuntreated, one subjected to the decontaminationprocedureof irradiation,andone treatedwithninhydrin.Thewhole envelopematerialwasmeasured, aswas thecelluloseextractedfromtheenvelopematerial.Therewasno statistical difference in any of the untreated samplesfor δ13C, which had an average value of −23.7‰.Comparison with the irradiated samples showed nomeasurable change, but the ninhydrin treatmentdecreasedtheδ13Cbyabout0.66‰.Therewasa smalldifferenceintheδ18Obetweenthesamplesdesignatedas“O”andthoseas“N”,withthe“O”samples29.3±0.3‰compared to 30.4±0.1‰ for the “N” samples. The FBIdrew no conclusions based on these results (FBIDocuments,B1M9D9).

4.11COMMITTEEFINDINGS

Finding 4.1: The committee finds no scientific basis on which to accuratelyestimatetheamountoftimeorthespecificskillsetneededtopreparethesporematerialcontainedintheletters.Thetimemightvaryfromaslittleas2to3days

to as much as several months. Given uncertainty about themethods used forpreparation of the spore material, the committee could reach no significantconclusionsregardingtheskillsetoftheperpetrator.

The DOJ Amerithrax Investigative Summary indicates that, because of theextraordinaryhighsporepowderconcentrationsandtheexceptionalpurityofthematerial in the Washington, D.C., letters, “the anthrax mailer must havepossessed significant technical skill” (USDOJ, 2010, p. 14). The FBI did notpresentadefinitetheoryonhowandwhenpropagation,purification,anddryingtookplace,noronwhatspecificskillswouldberequiredtoperformthesetasks.Nonetheless, inferences made by the FBI concerning the time, skill, andequipment required for spore preparation were said to be significantconsiderationsintheirnarrowingofthelistofpotentialsuspects(USDOJ,2010,pp.29-33,36-38).IndiscussionswiththeFBIattheJanuary2011meeting,theFBI informed thecommittee thatsomeof itsconsultingexperts referred to theletter preparations as being of “vaccine quality”, which narrowed the list ofpotential suspects but that they investigated any and all individuals withoutregardtotheirspecificskillsets.TheFBIfurtherstatedthatthereweretoomanyvariablestobeabletoquantifythetimeorspecifytheequipmentusedtopreparethe letter materials. However, FBI officials indicated that inferences aboutrequired skills or time for spore preparation were never the sole criterion foreliminatingsuspects(FBI/USDOJ,2011).

There are distinct professional judgments of the time that this work wouldrequire,with some estimates as lowas 2 or 3 days andothers in the rangeofmany months. The distinctions are based on different judgments of the timerequiredforpropagation,purification,anddrying,amongothervariables,aswellasthestateofthestartingmaterialavailabletotheindividual(s)inquestion.Inparticular,itisnotknownwhethersomeoftheinitialstepsmighthaveoccurredwell in advance of the letter attacks. The committee cannot resolve thesedistinctions because a particular production method or the steps involved inproductionwerenotidentified.

Finding 4.2: The physicochemical methods used primarily by outsidecontractorsearlyintheinvestigationwereconductedproperly.

Electron microscopy, aerosol particle size analysis, and bulk and spatiallyresolvedelemental compositionswere appropriate first steps for characterizingthe samples. Follow-up experiments to determine the spatial distribution of

silicon in letter material and the mechanism of silicon uptake also wereappropriate, as were efforts to search for possible elemental and molecularsignatures.Whilerecognizingthecircumstancesofthetime,andtheurgencyofthesestudies, thecommitteenotesthatthephysicalscienceinvestigationswerenotpursuedtotheextentthattheycouldresolveimportantdiscrepancies.

The FBI plan for the chemical and physical characterization of samples (FBIDocuments,B1M1D5)containsaflowdiagramdefiningtheapproachandrefersto the key scientific methods to be used. The committee, however, was notshownany recordofhow theFBImadedecisionsaboutwhichexperiments topursueorwhichapproachestoabandon.Thecommitteealsodidnotseerecordsofdetaileddiscussionsaboutwhichsamplesweretobestudiedwitheachofthecharacterization techniques (see Table 3-2 in Chapter 3 for information thecommitteewasabletogatherfromallofthesubmittedmaterials).

Finding 4.3: Although significant amounts of silicon were found in thepowdersfromtheNewYorkPost,Daschle,andLeahyletters,nosiliconwasdetectedontheoutsidesurfaceofsporeswhereadispersantwouldreside.Instead,significantamountsofsiliconweredetectedwithinthesporecoatsofsomesamples.Thebulksiliconcontent in theLeahy lettermatchedthesilicon content per spore measured by different techniques. For theNewYork Post letter, however, there was a substantial difference between theamountofsiliconmeasuredinbulkandthatmeasuredinindividualspores.No compelling explanation for this difference was provided to thecommittee.

ICP-OESanalysis indicated a silicon content of the bulkNewYorkPost lettermaterial of 10 percent by mass, while SEM-EDX performed by SNLdemonstrated silicon in individual spore coats at a level corresponding to 1percentbymassperspore.AttheJanuary2011meeting,theFBIattributedthisdifference to a limited amount of sample available (only one replicate wasperformed for ICP-OES analysis) and the heterogeneous character of theNewYorkPost letter.An explanation based on the heterogeneous character impliesthatthespecificsamplesanalyzedwerenotrepresentativeofthelettermaterial.In such a case, additional samples should have been analyzed to determinerepresentativeness.Ifsuchdataexist, theywerenotprovidedtothecommittee.Lacking this information, one cannot rule out the intentional addition of asilicon-based substance to the New York Post letter, in a failed attempt toenhance dispersion. The committee notes that powders with dispersion

characteristics similar to the letter material could be produced without theadditionofadispersant.

Early in the investigation,AFIPperformedSEM-EDXanalysisof aNew YorkPost lettersampleandfoundregions in thesamplehavinghighsiliconcontentbut no oxygen, suggesting the presence of silicon-rich material that was notrelated to nanoparticulate silica. While this observation could have led to anexplanation for the difference between the bulk and individual sporemeasurements, follow-up experiments apparently were not performed. ThecommitteenotesthatthisinformationwasnotmadeavailabletoitortotheFBIuntilspring2010.

Finding4.4:SurrogatepreparationsofB.anthracisdidreproducephysicalcharacteristics (purity, spore concentration, dispersibility) of the lettersamples, but did not reproduce the large amount of silicon found in thecoatsoflettersamplespores.

SurrogatepreparationsbyDPG,usingB.anthracisfromtheLeahyletterasthestarter source (FBI Documents, B1M13D3), reproduced the general physicalcharacteristics of the letter samples (purity, spore concentration, dispersibility)butnot thesiliconchemicalcharacteristics.Surrogatepreparationsshowedthatsampleshavingbulksiliconcontentup to5percentcouldbepreparedwithoutintentionaladditionofsilicondispersant.However,noneof theDPGsurrogatepreparationsanalyzedforsiliconinthesporecoatweresimilartotheNewYorkPost,Daschle, andLeahy letter sampleswith respect to either the amount perspore of silicon incorporated in the coat or the fraction of spores observed tocontain silicon in the coat. Furthermore, the committee sought, but could notobtain,adetailedexplanationofthethoughtprocessthatwentintoselectionoftheDPGmethods or their relationship to theBuran andAbshire preparations.Thecommitteeacknowledgesthatthereweremanymorepossiblescenariosforspore preparation than could have been feasibly explored with availableresources and in a reasonableperiodof time.However, itwasnot clear to thecommittee how the subset of surrogate preparationmethods was selected andwhether these choices were based on an understanding informed by theinvestigationoronotherassumptionsabout theapproach taken toproduce theevidentiarymaterials.

Finding4.5:RadiocarbondatingoftheLeahylettermaterialindicatesthatitwasproducedafter1998.

Thesporesintheletterwerenottakendirectlyfromastockpileproducedmanyyearsago.Oneormorerecentgrowthstepswouldhavebeenrequired,althoughit is not possible to pinpoint the time frame for that growth. Comparing thehydrogenisotoperatiosfromwaterfromDPGtoamodelforallknownmedia,Kreuzer-Martinandcolleaguesconcludedthatitwashighlyunlikelythatwaterfrom DPG was used to prepare the sample; however, it was not possible toidentifythelocationwherethesporeswereprepared.

Finding4.6:The flaskdesignatedRMR1029wasnot the immediate,mostproximate source of the letter material. If the letter material did in factderivefromRMR1029,thenoneormoreseparategrowthsteps,usingseedmaterial from RMR1029 followed by purification, would have beennecessary. Furthermore, the evidentiarymaterial in the NewYork lettershadphysicalpropertiesthatweredistinctfromthoseofthematerialintheWashington,

D.C.letters.

SEM-EDXmeasurementsshowednosiliconinthecoatsofsporestakendirectlyfromRMR1029, whereas themajority of spores analyzed from theNew YorkPost,Daschle,andLeahylettermaterialscontainedsiliconinthecoat.Basedonrecentstudiesofthemechanismofsiliconincorporation,siliconcouldhavebeenincorporatedinthecoatsofthelettersporesonlyifsporesfromRMR1029weresubjected to one or more subsequent growth steps. Another observationconsistentwithaseparategrowthstepwasthedetectionofB.subtilisintheNewYorkPostandBrokawlettermaterialbutnotinRMR1029(discussedinChapter5). The detection of meglumine and diatrizoate in RMR1029 but not in theLeahyandNewYorkPostsamplesalsoisconsistentwiththisfinding;however,itisnotconclusivebecauseitmighthavebeenpossibletorinsetheseimpuritiesawaywithoutrequiringlatergrowth.Someofthesefindings,aswellasothers,indicate that theNewYork lettermaterialswere prepared separately from thematerialsintheWashington,D.C.,letters.ThepresenceofB.subtilisintheNewYorkbutnottheWashingtonlettermaterialsandthedifferentphysicalpropertiesof the materials indicate that the two sets of letter materials were preparedseparately.

5

MicrobiologicalandGeneticAnalysesofMaterialintheLetters

5.1INTRODUCTION

AsdiscussedinChapter2,thebacteriumBacillusanthracis(B.anthracis)isthecausative agent of the disease anthrax. Anthrax generally affects grazingmammals (e.g., cattle, sheep, horses). Anthrax in humans, particularlyinhalational anthrax, is rare andoccursmainly in individualswithoccupationsinvolvingthehandlingofhides,hair,orbonefrominfectedanimals.Inhalationalanthrax can also be a sign of a biological attack with B. anthracis spores,especially in individuals without likely exposure to infected animals or theirproducts.Thiswasthecasein2001,whenanumberofcasesofbothcutaneousandinhalationalanthraxwerediagnosedamongmediaandpostalemployeesandothers after the delivery of letters containing suspicious powders in severalplaces.

In October 2001, clinical reporting of human anthrax cases spurred a broadepidemiological investigation to identify the source of the illnesses aswell asany other infected parties (see Chapter 3 for a timeline and a more detaileddiscussionof this epidemiological investigation). IdentificationofB. anthracisasthecauseofthe22casesofillnessandfivedeathsin2001wasdeterminedbyclinicallaboratorymeans(CDC,2001a,b,c).Becausetheseillnessesanddeathsappeared to have resulted from a bioterrorist attack, immediate efforts wereundertaken to identify a common source for theoutbreak, includingmoleculargeneticanalysisofthecausativeagent.

5.2IDENTIFICATIONOFTHEB.ANTHRACISSTRAIN

The first step in the search for a sourceof theanthrax-causingpowders in the2001mailings was to identify which “strain” (or strains) ofB. anthraciswasusedintheattack.DisparatecasesofhumananthraxinConnecticut,NewYork,Florida,andWashington,D.C.,andcontaminatedenvironmentallocationsinthelastthreeofthesesiteswerealllinkedwhenasingleB.anthracis

97

strain, the Ames strain, was identified in all of these cases and associatedlocations(Keimetal.,2008).AsnotedinChapter2,B.anthracis isoneofthemost genetically homogeneous microorganisms known (Keim et al., 2000).Nonetheless, even in the most homogeneous species there are usually somedifferences in genome sequences among populations. These sequencedifferences,althoughfewinnumber,aresufficienttocharacterizesubgroups,or“strains.”Strainsaremembersof thesamespecies,but theirdifferencesreflectthe divergence of sublineages as they evolved over time (Keim et al., 2000).Among B. anthracis populations, a variety of strains had already beenrecognized even before sequencing technology enabled detailedcharacterizationsofgenomedifferencesamongstrains.

WorkperformedbyPaulKeimandotherswellbeforethe2001anthraxattackshadresultedinthedevelopmentofseveralmolecularmethodstodetectgeneticdifferencesamongBacillusspeciesaswellasamongisolatesofB.anthracis.Inthe mid-1990s, work by Hendersen and colleagues (1995) and Anderson andcolleagues (1996) led to the identification of a 12-nucleotide variable numbertandem repeat (VNTR) sequence (called vrrA for “variable repeat regionA”)thatprovided the firstmolecularmarker thatdistinguishedamongB. anthracisstrains.Thebasisof thismarkerwasshowntobedifferencesinthenumberofrepeated sections of this genetic sequence, and five different variations weredetected.Subsequently,VNTRanalysisatmultiplegeneticloci(MultipleLocusVNTRAnalysisorMLVA)enabledthecharacterizationof426

B.anthracisisolateswith89distinctgenotypes(Keimetal.,2000).

Another approach, amplified fragment length polymorphism (AFLP) analysis,hasbeenparticularlyusefulforexaminingdifferencesbetweenB.anthracisandcloserelatives,suchasB.cereusandB.thuringiensis(Keimetal.,1999,2008;Hoffmasteretal.,2002;Keimetal.,2008).TheAFLPtechniquehadbeenusedtoidentifyabout30variableregionsandprovidedanabilitytoprofileportionsofthegenomeofalargenumberofdiverseB.anthracisstrains.Inaddition,thepagAgene,whichencodestheprotectiveantigen(PA)protein(oneofthethreeanthraxtoxinproteinsdiscussedinChapter2)alsohadbeensequenced(Priceetal.,1999).BecauseoftheimportanceofpagA inthedevelopmentofimmunityto anthrax, this genewasof interest indeterminingwhether aparticular strainmighthavebeengeneticallyaltered,or“engineered,”forincreasedeffectivenessasaweapon(Hoffmasteretal.,2002).

Thesenewmolecularapproaches,combinedwiththecreationofacollectionofstrainsfrommanyoftheworld’sgeographicregions,greatlyenhancedscientificcapabilitiesforidentifyinggeneticvariationsamonganthraxstrainsatthattime.Using these methods, Keim and colleagues (1999, 2000) had established apicture of the evolutionary lineages ofB. anthracis.Thesemethods for rapid,reliablemolecularsubtypingwerealsocriticalindeterminingtheidentityoftheclinical and environmental isolates in the 2001 anthrax attacks. Although acomplete genome sequence provides the most effective genetic signature foridentifying an organism, at the time of the attack mailings no B. anthracisgenomeshadyetbeencompletelysequencedandpublished(Keimetal.,2008).

UsingMLVAateightlociidentifiedbyKeimandhiscolleagues,scientistsfromtheCentersforDiseaseControlandPrevention’s(CDC’s)LaboratoryResponseNetworksubtyped135B.anthracisisolates(samples)collectedfromtheattackvictims, letter powders, and environmental samples, and determined that allthese isolates were likely to have been derived from a common source(Hoffmasteretal.,2002).Inaddition,CDCscientistssequencedthepagAgenesfromasubsetoftheseisolatesandconcludedthatnoneoftheisolatesappearedtohavebeenengineered (Hoffmasteretal.,2002). (Theuseofadditional testsperformed under the aegis of the FBI to assess the possibility of geneticengineeringisdiscussedbelow.)Allattack-associatedisolateswereidentifiedasMLVAgenotype62andPAgenotypeI.Genotype62isthegenotypeoftheAmesstrain commonly used worldwide for laboratory research for vaccinedevelopment. The PA I genotype was also identical to the Ames strain PAgenotype.TheseresultsledCDCtoconcludethattheB.anthracisstrainusedintheattackswasindistinguishablefromtheAmesstrain(Hoffmasteretal.,2002).

AsearlyasOctober2001,samplesfromthespore-ladenenvelopesandclinicalisolates (including one fromRobert Stevens, the deceased Florida patient andindex case) were also sent to Paul Keim’s laboratory at Northern ArizonaUniversity.TheKeimlaboratoryhadalreadyestablishedtheB.anthracisMLVAsequencedatabasethatcontainedinformationonmorethan1,000samplesfromaround theworld.Thisdatabaseproveduseful for identifying theB.anthracisstrainintheforensicsamples.

Beginning in January2002,Keimalsobeganconductinggenetic testingat therequest of the FBI on isolates ofB. anthracis provided by the United StatesArmyMedicalResearch Institute for InfectiousDiseases (USAMRIID),whichhadreceivedevidentiarysamplesfromtheFBI.Thefirst18evidentiarysamples

(designated Batch E0001) received by the Keim laboratory were handledaccordingtoFBIchainofcustodyrequirementsandwereinitiallynotidentified.AninitialMLVA-8analysisfoundthatallbuttwoofthesamples(“Connecticutsamples”) provided by USAMRIID were identical to and consistent with theAmes strain genotype (Keim, 2002a).An expanded analysis of 15MLVA loci(Keim,2002b)yieldedsimilarresults,withallbut3forensicsamplesmatchingtheAmesstrain.OnesampledifferedfromtheAmesstraininthatithadlostthepXO2plasmid(seeChapter2),butwasotherwiseidenticaltotheothers.Keimnotedthatplasmidsarecommonlylostduringcultureandthatthislossmayhaveoccurred prior to shipment of the sample to his laboratory, butwas not to beinterpretedasnecessarilyindicatingthattheoriginalforensicsamplewaspXO2negative. The two Connecticut samples proved to be distinct from the otherevidence,butwereidenticaltoeachother.Theirgenotypesmatched10isolatesfrom China in the Keim laboratory database that did not have a genotype orstrain designation at that time, but most closely resembled reference strainGenotype61.Atthetime,KeimnotedthatthesesamplesclearlydidnotmatchtheAmes strain. These samples proved to be from a separate case (Malakoff,2002)andnot fromtheelderlyConnecticutpatientwhodiedfrominhalationalanthrax.TheFBIwasinitiallyinterestedinthesesamplesuntilitwasdeterminedthat they were not related to the anthrax letter investigation. The Keimlaboratory continued to receive and strain type the B. anthracis samplessubmittedtotheFBIRepository(seeChapter6)overthenextsixyears.

5.3WASTHEB.ANTHRACISINTHELETTERSGENETICALLYENGINEERED?

AnimportantinvestigativeissuewaswhethertheB.anthracisstrainusedinthemailingshadbeengeneticallyengineered.Forexample, theFBIwas interestedinwhetherantibiotic resistancegenesorvirulence factorshadbeen introducedintotheattackstrain’sgenomefromotherstrainsorspecies,andwhethertherewereanyothermutationsinthesample,engineeredorotherwise,thatmighthelpinvestigatorsdetermineitssource.

To address some of these questions, Paul Jackson and colleagues at the LosAlamos National Laboratory (LANL) analyzed samples from the attackenvelopesfromOctober2001throughmid-2002.Thematerials tested includedsamples of the progenitor Ames strain isolated from the dead cow in 1981(Ames “Ancestor”); an isolate from the deceased Florida patient, RobertStevens; isolates from the Brokaw, New York Post, and Daschle letters; and

severalstocksfromUSAMRIID,includingasamplefromflaskRMR1029.Thesamplesweretestedforpossible indicationsofgeneticengineeringusingDNAsequencing (see Box 5-1) and polymerase chain reaction (PCR) amplification(seeBox5-2)tolookfor(1)thepresenceofgenesencodingresistanceagainsttheantibioticsciprofloxacin,tetracycline,erythromycin,bleomycin,kanamycin,andchloramphenicol;(2)modificationsoftheprotectiveantigen,edemafactor,and lethal factorproteingenes; and (3) inserted sequencesderivedeither fromcloning vectors (plasmids) known from the literature to have been used toengineerB.anthracisorfromtheinsertionofthecereolysingenesofB.cereus,reported(Pomerantsevetal.,1997)tohaveconferreduponthestrainanabilityto evade protective immunity induced by some anthrax vaccines (FBIDocuments,B1M4D2).TheLANLscientistsreportedthat“noneoftheisolatesassayedshowedanyindicationofgeneticmanipulationbasedonthepresenceofmarkersnormallyassociatedwithgeneticmanipulationofB.anthracis.”

It should be noted that the LANL investigators did not look for other lessobvious alterations that also might have indicated that the organisms in theevidentiary samples had been genetically engineered. Indeed, theyacknowledged that a well-financed laboratory could exploit or develop othercloning vectors and other methods for genetic manipulation without leavingclearmolecular

BOX5-1GenomeSequencing

Untilthe1970s,DNAwasthemostdifficultmoleculeinbiologytoanalyzebecauseofitsenormouslengthandits“monotonous”chemicalstructure.TheDNAmoleculeisa stringofchemicalbuildingblocks—thenucleotidesor“bases” adenine (A), thymine (T), cytosine (C), andguanine (G). DNA sequencing is the process ofdeterminingtheexactorderofthesebuildingblocksinapieceofDNA.Forexample,“ATCGGCTAA”ispartofaDNA “sequence.” Today, DNA sequencing has becomeindispensable for basic research and for numerous

applications, such as disease diagnostics, biotechnology,systematics, and forensic biology.The earliest DNAsequencing methods were developed in the 1970s andwere laborious and very slow. But theHumanGenomeProject,whichbeganin1990andwaslargelycompletedin 2003, greatly stimulated the development of newsequencing technologies. These are largely automatedandareordersofmagnitudefasterthanearlierefforts.Forexample, in2001 itcould takeaboutayear tosequencethe genome of a bacterium like B. anthracis, whereastodaysuchaprocessrequiresonlyafewhours.

Genome sequencing generally requires that the genome being studied first bebrokenintosmallerpieces.Thisprocess isusuallycarriedoutbyenzymesthat“cut” theDNAintoshort fragments. Inanalternateapproachcalled“shotgun”sequencing, the DNA of interest is mechanically broken into randomoverlapping fragments. Each fragment is sequenced numerous times, and thegenome is reassembled using computational methods to order the fragmentsbased on the regions of overlap. If there is sufficient overlap, the genomesequence can be considered complete or “closed.” In bacteria, which usuallyhavecircularchromosomes, thismeans that theentirecircleof thesequence isknown.Thistechniqueworksparticularlywellforsmallgenomes,suchasthoseof bacterial species that donot have extensive regions of repetitive nucleotidesequences. For the much larger genomes of animals and plants, the DNA ofinterest may also be broken into pieces and then cloned by inserting thefragments intobacteria,whichmakecopiesof theDNAwhen theydivideandreproduce. The cloned fragments are then mapped against the genome beingsequenced.Themappingreducesthelikelihoodthatregionscontainingrepetitivesequences (much more common in eukaryotic genomes) will be assembledincorrectly.

TheenormousincreasesinspeedandefficiencyofDNAsequencinghaveledtoa revolution in scientists’ ability to identify mutations quickly and precisely.Recently, the term“deep sequencing”was coined to describe this approachof

simultaneoussequencingofmassivenumbersofshortfragmentsderivedfromamixtureofgenomes,suchasmightexistinanevolvingpopulationderivedfromasinglecellthathas,overtime,accumulatedgeneticvariants.Thesemillionsofshort sequences are then ordered by computer programs, enabling theidentification of single nucleotide polymorphisms (SNPs) and other geneticvariants.

BOX5-2ThePolymeraseChainReactionTechnique

The PCR technique provides a rapid means to amplify(increase the number of copies of) DNA segments ofinterest.KnowledgeoftheDNAsequencetobeamplifiedisused todesign twospecific,but fairlyshort,syntheticDNA strands, or oligonucleotides, that arecomplementarytothesequenceofDNAtobeamplified.These oligonucleotides serve as “primers” for DNAsynthesisanddetermine thesegmentofDNAamplified.InPCR,theoriginaldouble-strandedDNAisfirstheatedto separate the strands. The separated strands are thencooled in the presence of an excess of the twoprimers,which hybridize with the complementary sequences inthe strands of DNA being studied. The mixture is thenincubatedwiththenucleotidesthataretherawmaterialsfor DNA synthesis and an enzyme called “DNApolymerase.”ThisenzymesynthesizesnewDNAstartingfrom theprimers andcopying theDNAstrand towhichtheprimerisbound.TheresultofthefirstcycleofPCRproduces two newdouble-strandedDNAmolecules thateach contains one strand of the original DNA and one

strand from the primer. This cycle of denaturation,hybridization,andsynthesisisthenrepeatedmanytimes,increasingthenumberofcopiesoftheDNAsequenceofinterest.Eachcyclegeneratesfresh templatesfor furtheramplification, and only the sequences bracketed by theprimers are amplified, while regions that lack primingsitesarenot.Afteranumberofthesecycles,asubstantialproportion of the reaction mixture corresponds to theamplifiedDNA.

SOURCE: Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., and P.Walter. (2002).Molecular Biology of the Cell, 4th ed. New York: GarlandScience.

signatures,oratleastthesignaturesthattheinvestigatorssought. However, the subsequent completion of thegenomicsequencesoftheAmesAncestorandlateroftheletter isolates (see Section 5.5.5 below) stronglysupportedthefindingsoftheLANLgroup(seealsoBox5-1 on genome sequencing). As noted by Read andcolleagues(2002), identificationofgenesthathavebeenalteredorinserteddeliberativelyinpotentialbioweaponsagentsisfacilitatedbycompletegenomesequencing.Nofurthertestingrelatedtotheissueofgeneticengineeringof the attack powders was performed after mid-2002asidefromthegenomesequencing.

Priortothe2001letterattacks,theInstituteforGenomicResearch(TIGR)hadbegun sequencing the genome of the “Porton Down” isolate of B. anthracis(Readetal.,2002).Thegenomesofmanybacteriahavemultipleparts,typically

includingasinglelargecircularchromosomeandoneormoresmallerplasmids.In particular, most B. anthracis strains carry two plasmids, called pXO1 andpXO2, thatencodeproteinsrequiredforvirulencebut thatarenotessential forthe bacteria to grow under laboratory conditions. However, the Porton isolatehad been rendered avirulent by “curing” (eliminating) both plasmids from theAmes strain (Read et al., 2003), making it a less than optimal choice for areference genome. Shortly after the letter attacks, the National ScienceFoundation provided critical funding that allowed TIGR to sequence to draftquality the isolate fromthespinal fluidof thedeceasedFloridapatient,RobertStevens(Ames“Florida”).Here,“draftquality”referstoagenomesequenceforwhich a small fraction of the nucleotide bases remains uncertain, there areremaining small gaps in the coverage of the complete genome, or both. ThisworkwasdescribedinapaperbyReadandcolleaguesin2002,andincludedacomparisonoftheFloridaandPortonisolatesandanexaminationofagroupofisolatesof theAmesstrainobtained fromvarious laboratoriesprior to2001aswellasan isolate fromaTexasgoatobtained in1997.The latterwas theonlyotherisolateoftheAmesstrainknowntohavebeencollectedinthefieldasidefromthe1981deadcow“Ancestor”isolate.Inthisinitialsetofcomparisons,11sequence differenceswere found between the chromosomes of the Porton andFloridaisolates(Readetal.,2002);however,theseldifferenceswerealsofoundinalltheotherAmesisolatestested.Fromthesedata,andtheunderstandingthatthePortonDownstrainwasderivedfromearlierisolatesatUSAMRIID(Readetal.,2002), itwas inferred that themutations in thePortonstrainoccurredafterthe 1982 transfer of the strain to Porton Down. No sequence differencesappeared to distinguish the isolate of the Florida victim from many of theisolatesoftheAmesstrainpresentinvariouslaboratoriesbeforetheattack.

In spring 2003TIGR also initiated the sequencing of theAmesAncestor andcompletedthisworkinOctober2003(Ravel,2010).UnliketheFloridaisolate,which was sequenced only to draft quality, the Ames Ancestor sequence was“closed,” that is, assembled into one contiguous sequence. Annotation andanalysis of theAmesAncestor sequence continued untilmid-2004 and it wasreleased to GenBank on June 1, 2004. (The paper announcing the AmesAncestor sequence was not, however, published until 2009; see Ravel et al.,2009). The Ames Ancestor sequence served as the high-quality referencegenomeneededfor thecomparativegenomicswork thatTIGRlaterperformedoncolonymorphotypesidentifiedfromtheattacklettermaterials(seebelow).

Theapparentabsenceofchromosomaldifferencesbetweentheattackstrainand

Ames strains had important implications, both positive and negative, for theinvestigation.Onthepositiveside,thefindingsstronglysupportedtheinferencethat the attack strain had come—directly or indirectly—from a laboratory thatpossessedtheAmesstrain.Alsoonthepositiveside,thefindingssupportedtheconclusionthattheattackstrainhadnotbeenengineeredtomakeitresistanttotreatment or more virulent. On the negative side, the absence of distinctivesequences in theattackstrain seemed tomean that itwouldnotbepossible tousegeneticmarkerstotracetheattackmaterial tooneparticularsourceamongthe various institutions (and laboratorieswithin the institutions) that possessedtheAmesstrain.

Nonetheless,animportantclueastothesourceofthestrainintheletterscamefrom microbiologists at USAMRIID who discovered that the attack materialcontained at low tomoderate frequency several subtypes ofB. anthracis thatproduced morphologically distinct colonies. These colony morphologicalvariants (or “morphotypes”) retained their distinctive appearance when singlecellsfromthecolonieswereregrownintonewcolonies.Thispersistencemeantthat the morphotypes were genetically distinct from the standard (wild-type)Ames strain in the samples; that is, they apparently contained a mutation ormutations that caused them to produce their distinctive-looking colonies. Thatseveralmorphotypescouldbedistinguishedindicatedthatdifferentmorphotypescontained different mutations. The morphotypes appeared to be spontaneousmutants that arose during the preparation of the batches of spores that wereeventuallyusedintheattacks.

As described and discussed later in this chapter and in Chapter 6, TIGR alsosequenced the genomes of several of these morphotypes to identify themutationsthatdistinguishedthemfromthewild-typeAmesstrainandfromeachanother(FBIDocuments,B1M5D1-2).Thesemutationsplayedacentralroleinthe investigation (USAMRIID, 2005; FBI Documents, B1M2D12; Worsham,2009),helpingtheFBItotraceattackmaterialstoapossiblesource.

TheinvestigationsummariesprovidedtothecommitteeinDecember2010refertothepresenceofapE03vectorreferredtoasan‘Israelicloningvector’amongcertainrepositoryisolates(B3D1).OntheJanuary11,2011meetingtheFBIwasaskedtoclarifywhatwasknownaboutthevector.ThecommitteewastoldthatthisvectorwasaderivativeofthecommonlyusedcloningvectorpBR322thatwasfoundinsomeisolates,andthatithadnoforensicvaluetotheinvestigation(FBI/USDOJ,2011).

5.4B.SUBTILISCONTAMINATIONOFTHENEWYORKSAMPLES

Afindingthatwasinitiallyofhighforensicinterestwasthediscovery,basedoncultivationtechniques,thatthepowderfromtheletterssentonSeptember18totwoNewYorkCity addresses (theNew York Post and TomBrokaw atNBC)containedamixtureofB.anthracisand,atafrequencyofabout1to5percent,anon-B.anthracisbacterium.Thecontaminatingbacterialspecieswas identifiedbytheCDCasB.subtilisonthebasisof16SrRNAgenesequencingandlaterwholegenomesequencing(CDC,2001a;FBIDocuments,B2M1D2).B.subtilisis a ubiquitous bacterial species that is readily isolated from environmentalsamples from around the world. The identification of the contaminant as B.subtiliswasatfirstconsideredofpotentialimportancebecausecertainstrainsofB.subtilisarewidelyusedinacademicandindustriallaboratories.Hence,ifthecontaminant had proved to be a particular laboratory strain, it might haveprovidedacluetotheoriginoftheNewYorkCitypowders.

Wholegenomesequencinganalysiscarriedoutandreportedin2004byTIGRofan isolate referred toasGB22from theNewYorkPost letter showedhigh (98percent) similarity, but not identity, to the published sequence of the standardlaboratorystrainB.subtilis168(Kunstetal.,1997). In2006,TIGRdeveloped95PCRassaysfor23B.subtilislociintheevidentiarysamplethatdifferedfromthe reference (B. subtilis 168) genome. The amplified DNA regions werecomparedusinggelelectrophoresis.(DNAsequencingoftheseamplifiedregionswouldhavebeenamoredefinitiveapproach.)TheB.subtilis isolates fromtheNewYorkPost andBrokaw letterswere identical to each other at all 23 loci,indicatingthattheywerethesamestrain.(ThewholegenomesequenceoftheB.subtilis from theBrokaw letterwas not determined, however, so their identitywasnotdefinitivelydemonstrated.)

Becausethe95PCRassayswouldhavebeencumbersometoperformonlargercollectionsofsamples,theFBILaboratorynextidentifiedfourgeneticmarkersintheGB22letterstrain,threeofwhich(designatedID65,ID91,andID107byTIGR)were rare in a surveyof72B.subtilis strains isolated fromaround theworld. These strains were obtained from the NRRL (formerly the NorthernRegionalResearchLaboratory)collectionoftheU.S.DepartmentofAgricultureandtheAmericanTypeCultureCollection,andtheyweremeanttorepresentageographically and genetically diverse collection (FBIDocuments, B2M4D2).ThethreeraremarkersdistinguishedtheGB22strainfromtheotherstrains.Thefourthmarker (sboA)wascommontoall theB.subtilisstrainsexamined.This

combinationofmarkerswasdesignedfirsttodeterminewhetheranyB.subtiliswaspresentinadditionalsamplesbasedonthepresenceofthesboAmarkerand,second, to determine whether such samples contained a strain that might besimilartoorthesameastheGB22strainfromtheNewYorkletters.

TheFBILaboratorydevelopedTaqMan(seeBox5-3)real-timePCR(RT-PCR)assays for the four markers, and these assays were provided to the NavalMedicalResearchCenter(NMRC)andtheNationalBioforensicAnalysisCenter(NBFAC) at the Department of Homeland Security’s National BiodefenseAnalysisandCountermeasuresCenter,wheretheassayswerevalidatedbyblindtesting. NMRC andNBFAC used the assays to evaluate over 300 evidentiarysamples. Only two B. subtilis strains from these samples were found thatmatched GB22 at all four loci. But when TIGR followed up by furthercharacterizingthesetwosamplesusingthecompletesetof95PCRassays,theyprovedtobegeneticallydifferentfromGB22(FBI,2009).NBFAClater(2007)alsotestedallAmesstrainsamplesintheFBIRepository(seeChapter6)forthepresence of B. subtilis contamination (see NBFAC analytical result reports,November2006-December2007,FBIDocuments,B2M4D3-15).Although322out of 1057 repository samples tested positive for the sboA nucleic acidsequence,furthertestingshowedthatnoneofthese322sampleswaspositivefortherareID65markerinGB22(NBFAC,2007;FBIDocuments,

BOX5-3TheTaqManTechnique

TheTaqMan®techniqueishighlysensitive(Easterdayetal.,2005a)andreliableasadiagnostictool(Easterdayetal., 2005b), and allows the detection of geneticdifferences between samples even at the level of SNPs(VanErt et al., 2007b). Itusesanoligonucleotideprobethat anneals to both the wild-type and mutant targetDNA. The probe is labeled with both a fluorescent tagandafluorescencequencherandbindstightlytotheexactcomplementary sequence in the target DNA. PCR isinitiated using primers that anneal nearby.One of these

primersisdesignedsuchthatitannealsonlytoatemplatecontainingthespecificallele tobedetected.Twoprimersetsaregenerallyused,one that isspecificfor thewild-type allele and a second that is specific for the mutantallele. If the primer anneals and Taq polymerasesynthesizes a new strand along the template, then theboundfluorescentTaqManprobewillbedigestedbytheexonuclease activity of the advancing polymerase,therebyreleasingthefluorophoreandproducingasignalthat indicates thepresenceof theparticularallele. If theprimer does not anneal and Taq polymerase does notsynthesize DNA, then the oligonucleotide probe willremainboundandintact,andthefluorescenttagwillnotemitdetectablefluorescenceduetothecloseproximityofthequencher.

B2M4D13). (If any samples had been positive for thepresenceoftheID65marker,analysesfortheID91andID 107 markers would have also been performed.) Inshort,many repository sampleswere contaminatedwithB.subtilis,althoughapparentlynotbythesamestrainasintheNewYorkPostletter.Ultimately,theFBIconcludedthat the testing for B. subtilis did not provide usefulinformationleadingtothesourceoftheNewYorklettermaterials—GB22isapparentlyanenvironmentalstrainofunknownoriginthatcouldnotbetracedtoanyparticularsource.

5.5IDENTIFICATIONANDCHARACTERIZATIONOFCOLONYMORPHOLOGICALVARIANTSINTHEEVIDENTIARYMATERIAL

5.5.1WhyWastheFBIInterestedinColonyMorphotypes?

Anymicrobialgeneticistcanattesttothefactthatclosescrutinyrevealsunusualindividualvariantsinapopulationofmicrobes.Suchvariantsoftencarrygeneticalterationsthatproducenoticeablephenotypicchangesinphysiology,behavior,or morphology. When the variants are observed at the level of the physicalappearance of colonies as they grow on agar plates, those variants are oftenreferredtoas“morphotypes.”Astheselectivepressuresforrapidgrowthunderlaboratory conditionsmaydiffer from those experiencedbyorganisms in theirnatural environment, genetic variants (mutants) may arise that replicate fasterand, over time, replace the “wild-type” strain during repeated cycles oflaboratoryculture.Thisprocessrepresents,ineffect,rapidevolutionleadingtoapopulationbecomingbetteradaptedtoitsparticularlaboratoryconditions.

AnalysesofthesporesamplesfromtheattacklettersfromasearlyasNovember2001(FBIDocuments,B1M2D7)revealedthepresenceofcolonymorphotypeswhose stability suggested that they resulted from the presence of geneticallydistinct subpopulations. The specific set of genetic alterations in a populationmightprovideausefulprofileforthatpopulationandifitcanbedemonstratedtobepresentintwodifferentsamplepopulations,mightsuggestthatthetwowerederived fromacommonsource.Withsufficientknowledgeabout the identitiesof such genetic variations and the frequencies with which they arise in thepopulation under specific culture conditions, the statistical significance of thesimilarities between the two populations might, in principle, be calculated.Under some circumstances, the chance of two independent populationscontaining the same genetic variant subpopulations might be so small that itcouldbeconcludedwithhighconfidence that theywerederived froma singlesource. With this goal, the FBI pursued detailed characterization of thephenotypicandgeneticvariationamongtheevidencesamples.

5.5.2BackgroundInformationonMorphotypes

Given the rapid generation times (many generations per day) and largepopulations (often billions of cells) typically observed in laboratory bacterialcultures,itishighlylikelythatgeneticvariantswillariseinculturedpopulations.Ifageneticvariantinapopulationisabletoinitiategrowthmorequickly,grow

more rapidly, or sustain growth longer as conditions become less favorable, ittends to increase its frequency in the population. This process can lead tocultures with multiple genetically distinct subpopulations. While this basicprocess of selection drives evolution in nature, it can present problems forgenetic studies in the laboratory, as the characteristics of an organism maychangeoverthetimeframeofascientificstudy(ElenaandLenski,2003).

Microbiologists usually seek to avoid this phenomenon using two primarymethods.First,stockculturesarestoredundernongrowthconditionsineitheradriedorfrozenstate,sincemostmutationsdonotariseintheabsenceofgrowth.Freshsamplesofthatnonvaryingstockarethenusedtoinitiateeachnewsetofanalyses. Second, cultures retrieved from the stock are streaked on growthmedium in such a way that individual colonies are obtained and each colonycontainsapopulationderivedfromasinglecell(i.e.,aclonalpopulation).Somegeneticvariantsthatmakeupaminorproportionofthestockpopulationmayberecognizablebasedontheiratypicalcolonymorphology,andthosevariantscanbeavoidedinthegenerationofculturesforfurtheruses.

Itiswellknowninlaboratoriesthatstudybacterialsporeformation(sporulation)thatlow-frequencysporulating(oligosporogenous)variantsappearinabacterialpopulationatareadilyobservablefrequency(Veliceretal.,1998;Micheletal.,1968). Furthermore, laboratory selective pressures can result in these variantsbecomingdominantmembersofthepopulationandsupplantingthesporulation-proficient parent strain. Two phenomena may increase the abundance ofasporogenous (nonsporulating) or oligosporogenous (sporulating with reducedfrequency) variants above that of other types of variants.When a populationencounters adecline innutrients, sporulation-proficient cells ceasegrowthandenter the dormant spore state. Sporulation-deficient variants may continuegrowth to somedegreeat theexpenseof theirneighbors, increasing theirownrelativeabundanceinthepopulation.Ifthisagedpopulationofcellsandsporesis eventually transferred to fresh medium, the nonsporulated cells may alsoresumegrowthmorerapidlythanthespores,againincreasingtheirabundanceintheresultingpopulation.Thesemaybeselectivefactorsleadingtothefrequentobservationofoligosporogenousvariantsinlaboratorycultures.

StudiesofB.anthracis phenotypic variants,manyofwhichwere shown to beoligosporogenous,werepreviouslycarriedoutbyPatriciaWorshamandMicheleSowers at USAMRIID (Worsham and Sowers, 1999) as part of an effort toidentifyusefulstrainsforvaccineresearch.Workingwithmultiplestrains(e.g.,

Ames, Sterne, Vollum, and others) of B. anthracis, Worsham and Sowersidentified numerous phenotypic variants (Worsham and Sowers, 1999). Theirinitialscreenforphenotypicvariationwasgrowthonmediumcontainingadyecalled Congo red. The dye accumulated by wild-type B. anthracis coloniesresultsinthecoloniesassumingasalmon-pinkcolor.Poorlysporulatingcoloniesdo not accumulate the dye, resulting in awhite appearance that can be easilyidentifiedbyeye.Furtherstudyof thewhitecolonymorphotypesrevealed thatthey were sporulation deficient. While there is no clear physiologicalexplanation for the coincidence of the absence of Congo red binding andsporulationdeficiencies,nosuchexplanationisrequiredforthepracticaluseofthis morphotype screening method. One of these phenotypic variants wascarefully characterized as carrying a mutation in spo0A, a primary regulatorygeneforentryintosporulation(Hoch,2000).Indeed,mutantsofspo0Aandothergenes governing entry into sporulation were historically recognized inexperiments in which cultures of cells sporulating in rich medium weremaintainedforprolongedperiodsoftime(Micheletal.,1968).

Reinforcing the view that laboratory conditions frequently enrich for mutantsdefective insporulation,arecentpublicationbySastallaandcolleagues(2010)reports that repeated passage of B. anthracis on rich medium results in theaccumulationofsporulationmutants,whichwererecognizedbytheirdistinctivecolonyphenotype.Manyof themutantscontainedmutations in thesporulationregulatorygene spo0A, as had been observed by PatriciaWorsham (Worshamand Sowers, 1999). These included point mutations as well as deletions andinsertions. Sastalla and colleagues further showed that the spo0A mutantsexhibited a shorter lag time than did the wild type when inoculated on richmedium.Thus,onefactorcontributingtotheenrichmentofsporulation-deficientmutantsuponrepeatedpassageisthatatleastsomesporulationmutantsresumerapid(exponentialphase)growthmorequicklythandoesthewildtype.

5.5.3DetectionandCharacterizationofMorphotypesintheAnthraxLetterSamples

Part of the analysis of the anthrax letter evidence samples involved thepreparation of relatively low-density liquid suspensions of spores recoveredfromthelettersandthespreadingofthesesuspensionsonthesurfaceofasolidgrowthmedium.Thisprocedureallowedeachviablespore togerminate,grow,anddivide in isolation, producing a clonal populationofmillionsof cells—allderived from a single cell—in the form of a colony. This is a standard

microbiologicalpracticeusedfortheenumerationofviablesporesinasample.Themethod also allows the visual detection of variants in the population thatmay carry genetically heritable traits responsible for altered phenotypicproperties, although the sensitivity and discriminatory capacity of thismethodare low. Relatively early in the investigation (fall 2001), Terry Abshire, amicrobiologist at USAMRIID, observed the presence of multiple phenotypicvariants (morphotypes) among the colonies generated from the anthrax lettersporesamples.TheanalysisofthesevariantseventuallybecameamajorfeatureintheFBI’sefforttoidentifythemostlikelysourceofthespecificcultureusedto produce the letter samples. To the advantage of the FBI, PatriciaWorshamwasoneoftheUSAMRIIDscientiststhatassistedwiththeanalysisoftheletterspore samples. Due to her extensive experience with B. anthracis and herspecificexperiencestudyingB.anthracisphenotypicvariants,Worshamandhercolleagueswerereadilyabletoapplymethodsforthestudyandcharacterizationofphenotypicvariantsinthelettersamples.

InearlyanalysisoftheLeahylettersample,sporeswereplatedonsheepbloodagar and incubated for three days. On these plates, Abshire observed thepresence of morphotypes with more defined colony edges and a yellow tintrelativetothemajorityofthecolonies.Basedonthisobservation,Worshamwasasked to develop a protocol to characterize morphotypes in the multipleevidentiary samples. The goal of this analysis was the identification ofphenotypic and eventually genetic signatures of the letter samples that mightcontributetodeterminingthesourceofthelettermaterial.

Spore samples from the Daschle, Leahy, and New York Post letters weresuspendedinwateratalowdensity,suchthattheycouldbespreadtogiverisetobetween 10 and 50 colonies per plate, allowing the observation of individualcolony phenotypes. Thousands of colonies from each sample were visuallyinspected.Theinitialplatingwasonsheepbloodagarplates toallowthemostefficientcolonyrecovery.Theplateswereincubatedateither26°Cor37°Cwithroomair,orat37°Cwithanatmospherecontaining20percentCO2.Theseconditions were employed to allow the detection ofvariants that might be apparent only under particulargrowth conditions. While the characteristics of colonymorphotypes aremost obvious following incubation for

48 hours or more after nutrient exhaustion andsporulation, these starvation conditionswere avoided inorder to diminish the likelihood of the selection of newvariants that were not present in the initial evidentiarysamples. Plates were inspected after 18 to 22 hours ofincubation, which was sufficient to allow thedevelopmentofsizablecolonies.Mostclassesofcolonymorphotypes were identified predominantly by thepresenceof a yellow tint (although the committee notesthatthistintisnotapparentinFigures5-1,5-2,and5-3,whileoneclassexhibitedasmaller,moreopaquecolonyappearance.

FIGURE5-1B.anthracisColonyMorphotype“A.”PhotographofcoloniesformedbygrowthofB.anthraciscellsonbloodagar.Thecolonyonthetopdisplaysthemorphologydesignated“TypeA.”Thecolonyonthebottomdisplaysthetypicalwild-typemorphology.

SOURCE:USAMRIID.ThisimageisaworkoftheUnitedStatesArmyMedicalResearchInstituteforInfectiousDiseases,takenormadeduringthecourseofanemployee’sofficialduties.AsaworkoftheU.S.federalgovernment,theimageisinthepublicdomain.

FIGURE5-2B.anthracisColonyMorphotype“B.”PhotographofcoloniesformedbygrowthofB.anthraciscellsonbloodagar.Thecolonyonthetopdisplaysthemorphologydesignated“TypeB.”Thecolonyonthebottomdisplaysthetypicalwild-typemorphology.SOURCE:USAMRIID.ThisimageisaworkoftheUnitedStatesArmyMedicalResearchInstituteforInfectiousDiseases,takenormadeduringthecourseofanemployee’sofficialduties.AsaworkoftheU.S.federalgovernment,theimageisinthepublicdomain.

Upontheidentificationofeachcandidatevariantcolony,thecolonywasscrapedfromthecultureplate.Toproduceanarchivalstockofthevariant,50percentoftherecoveredmaterialwasstoredat−70°C.Thisisastandardmicrobiologicalpracticeforthemaintenanceofstrainsunderconditionsthatpreventthefurtheraccumulationofgeneticvariation.Theremainderofthe

recoveredmaterialwasagainstreakedontoafreshsheepbloodagarplateandincubatedat37°Cfor18to22hoursinordertoproducenumerouscoloniesforeachcandidatemorphotype.Thisnewgrowthwasinspectedandcomparedtoanonvariantisolatetodeterminewhetherthephenotypeofeachcandidatemorphotypewasstableovermultiplegenerations,indicatingthepresenceofanunderlyinggeneticchange(mutation),andwhethertherecoveredmaterialproducedauniformcolonyappearance,indicatingapure(clonal)cultureofthemorphotype.Colonieswerecollectedfromthissecondplatetoproduceanadditionalstockat−70°Cthatcouldbeusedformultipleanalyseswithoutsacrificeofthearchivalstock.

FIGURE5-3B.anthracisColonyMorphotype“E.”

PhotographofcoloniesformedbygrowthofB.anthraciscellsonbloodagar.Thecolonyonthetopdisplaysthemorphologydesignated“TypeE.”Thecolonyonthebottomdisplaysthetypicalwild-typemorphology.SOURCE:USAMRIID.ThisimageisaworkoftheUnitedStatesArmyMedicalResearchInstituteforInfectiousDiseases,takenormadeduringthecourseofanemployee’sofficialduties.AsaworkoftheU.S.federalgovernment,theimageisinthepublicdomain.

Eachmorphotypicvariantrecoveredwassubjectedtonumerousphenotypicteststhathadbeenusedinearlierstudies(WorshamandSowers,1999)todifferentiatevariantsfromthewild-typestrain.Ineachcase,candidatevariantswereobservedsidebyside,onthesameplateofmedium,withcoloniesofthewild-typestrainaswellasisolatesofseveralpreviouslyidentifiedmorphotypeclasses.VariantswereexaminedforCongoredbinding,degreeofcolonyspreading,degreeofcolonypigmentation,and“bull’s-eye”appearanceofthecolony(whichmightbeduetovariationbetweenconcentricringsincolonythickness,pigmentation,orotherphysiologicaltraits).Sporulationwasscoredbymicroscopicobservationofmaterialrecoveredfromcoloniesontwodifferentmedia:sheepbloodagar,whichisarichmediumnotconducivetoefficientsporulation,andLeighton-Doimedium,aminimalmediumthatis

normallyusedforsporeproduction.Hemolysis,therupturingofredbloodcellsandalterationoftheredhemecolor,canbedetectedaftergrowthonsheepbloodagarfor48hoursat37°Cfollowedbyrefrigerationfortwoweeks,duringwhichthehemolyticreactionbecomesmorevisiblypronounced.

TABLE5-1PhenotypicCharacteristicsoftheMorphotypes

Colonysize

Colonyspread Pigmentation Hemolysis

MorphotypeA Large Yes Bull’seye ++

MorphotypeB Large Yes Altered ++

MorphotypeC/D Large Yes Bull’seye +

MorphotypeE Small No Opaque −

Following extensive testing, four predominantmorphotype classes were identified in the Leahy lettermaterial, although there were also other less abundanttypes.ThefourwerenamedA,B,C/D(twoinitialsimilarclasses,C andD,were eventuallymerged), andE (alsocalled opaque) (see Table 5-1). All of themorphotypeswere oligosporogenous, producing significantly fewerspores than the wild type on all media tested.Morphotypes A, B, and C/D exhibited large zones ofhemolysisthatwerenotproducedbythewild-typestrain.ThedefiningcharacteristicofmorphotypeAwasalarge

spreadingcolonywithabull’s-eyecenter.MorphotypeBwas similar toA in colony sizebut didnot produce thebull’s eye and produced slightly different pigmentationon some media. Morphotype C/D was also similar inappearance to A, but produced a smaller zone ofhemolysis and was somewhat more proficient at sporeproduction. Morphotype E was unique in producingcolonies that were smaller than those of the wild type,had more regular edges, and were more opaque.Morphotype E also exhibited temperature-sensitivesporulation,producingsignificantlymoresporesat26°Cthanat37°C,yet still fewer than thewild typeat eithertemperature.ScreeningofsamplesfromtheDaschleandNew York Post letters revealed morphotype classes thatwere phenotypically similar to those from the Leahyletter. (Due to a scarcity of material from the Brokawletter, no screening of samples for colony morphotypeswasperformedonthatmaterial[Hassell,2010].)

5.5.4SelectionCriteriaforGeneticVariationsUsedinScreening

Classesofmorphotypespresentinthelettersampleshadbeencharacterizedbythe scientists at USAMRIID as early as January 2002, and the FBI began toconsidermethodsforscreeningtheFBIRepository(FBIR)(seeChapter6)ofB.anthracisisolatestoidentifyasourceculture.PhenotypicscreeningoftheFBIRsamples was deemed an impractical method for several reasons. First,phenotypic screens of the types described above are relatively slow, laborintensive,andhighlydependentonthetrainedeyesoftheinvestigatortoidentifyvariant colonies. Second, similar phenotypic variations can be associatedwithdifferent genetic alterations located in either the same or widely separatedgeneticloci.Thepresenceofsimilarcolonymorphotypesintwosampleswould

not provide direct genetic evidence to link the two sample populations.Third,phenotypic screens are insensitive and do not reliably detect rare variants.Identificationofthespecificmutationsassociatedwitheachphenotypicvariationwasrequiredfor thedevelopmentofdefinitiveassaystodetect thepresenceofshared mutations in multiple strains within the repository. Such DNA-basedassays are rapid, sensitive, compatible with high-throughput methods, anddefinitivetothelevelofnucleotidesequence.

Scientists selected representativemorphotype isolates aswell as control wild-type isolates from each of three letter samples for detailed genetic analysis.Severalcriteriawereusedforthisselection.First,thescientistsneededtobeabletodistinguishthevariantsfromthewild-typecoloniesonplates.Second, theseparticularmorphotypesmusthavebeenpresentatahighenoughfrequencyforthe scientists to identify them repeatedly. The third essential criterionwas theapparentpresenceofthemorphotypeineachofthethreelettersamples(Leahy,Daschle,NewYorkPost)thatweresubjectedtothisanalysis.Thefinalselectionofmorphotypesfocusedonfourvariants:A,B,C/D,and

E.Therewereothermorphotypesfoundinthelettermaterials,buttheywerenotused for further forensic testing. Worsham and colleagues at USAMRIIDquantified the percentages of variants by randomly picking about 370 isolatedcoloniesfromplatesmadeusingdilutionsfromtheLeahyletter.Thesecolonieswere79percentwild-typemorphology,6.7percentC/Dmorphotype,1.1percentBmorphotype,1.3percentAmorphotype,and4.9percentEmorphotype(othermorphotypes accounted for the remaining fraction) (FBI Documents,B1M2D12).Itisimportanttonotethattwoidentical-lookingmorphotypesneednot,andoftendidnot,havethesamegenotype.Indeed,asdiscussedinthenextsection, two independent isolatesexhibiting similarcolonymorphotypesmighthavemutationsindifferentgenesorevendifferentmutationsinthesamegene.Also, some colonies identified asmorphotypic variantsmay not have had anymutation, as the distinction between genetic and nongenetic variation is notalwaysclear.Thus,itwascrucialtoidentifydifferencesinnucleotidesequenceasanunambiguoussignatureofdifferentmutantsubpopulations.

5.5.5WholeGenomeSequencingofMorphotypeIsolates

To determine whether the genetic alterations associated with each colonymorphotypemightbesuitableforuseasforensicmarkers,thegenomesequencesofmultiplemorphotypeisolatesweredetermined(Table5-2).GenomicDNA

TABLE 5-2 B. anthracis Isolates Analyzed by the Institute forGenomicResearch(TIGR)

TIGRID

FBIID Origin Morphotype Sequencingstatus

GBA AmesPorton PortonDown Wildtype 12X–

closed

GB6 AmesAncestor Texas/USAMRIIDWildtype 12X–

closedGB8 LL10/E3 Leahyletter A 8X–closedGB9 LL9/E2 Leahyletter B 8XGB10 LL1/E1 Leahyletter Wildtype 8X

GB11 PL10/E6 NewYorkPostletter A 8X

GB12 PL9/E7 NewYorkPostletter B 8X–closed

GB13 PL1/E9 NewYorkPostletter Wildtype 8X–closed

GB15 DL10/E4 Daschleletter A NSGB16 DL9/E5 Daschleletter B NSGB17 DL1/E8 Daschleletter Wildtype NSGB18 LL6/E10 Leahyletter C 12XGB19 LL7/E11 Leahyletter D NSGB23 LL18 Leahyletter E 12XGB24 LL19 Leahyletter E NS

NS=notsequencedSOURCE:FBIDocuments,B1M5D1-2.

extracted from the colony morphotypes identified byUSAMRIIDwasprovidedbyPaulKeimtoTIGR,whereit was prepared for genome sequence analysis. Plasmidlibraries were produced from the genomic DNAs andshotgun sequencing was carried out to produce

approximately8X(insomecases12X)averagecoverageof the genome. However, most of these genomesequences were not closed (i.e., assembled into onecontiguous sequence). In some cases (e.g., the samplesfrom theDaschle letter), no sequencingwas performed.In these cases the TIGR scientists used PCR to testwhether the same genetic differences in the sequencedsampleswerealsopresentintheunsequencedones(FBIDocuments, B1M5D1). Again, as previously noted,efforts were not undertaken to identify these or othermorphotypesintheBrokawletter.

Control wild-type isolates from each letter were found to possess no geneticdifferences from theAmesAncestor strain (FBIDocuments,B1M5D1-2).Thegenome sequences of each of the chosen morphotype isolates did, however,exhibitdifferencesfromthewild-typeisolatesinparticulargeneticregionsoftheB. anthracis chromosome or, in the case of the E morphotype, in the pXO1plasmid. Some samples of morphotype A contained a single nucleotidepolymorphism(SNP)whileotherscarriedaduplication.SNPswerealsofoundformorphotypesBandC,whileDcontainedachromosomaldeletionandEhadadeletionin thepXO1plasmid.Followingtheidentificationof thesesequencedifferences, unsequenced morphotype isolates were further tested for thepresence of the same sequence differences using PCR amplification andsequencing of the amplified DNA. The results are summarized in Table 5-3.Thesedatarevealedthatmultipleisolatesofsomeofthemorphotypes(e.g.,B)wereassociatedwithasinglegeneticchangewhileothers(e.g.,isolatesoftheAmorphotype) exhibited several different sequence variations in the samechromosomalregion.

ThegenotypesoftheAmorphotypeisolatesfromthreeletters(Leahy,NewYorkPost,andDaschle)wereoftwogeneralkinds.ThefirstwasaSNP

TABLE5-3FurtherGeneticCharacterizationoftheMorphotypeIsolatesGenotype

Morphotypeclass

Affectedlocus

Typeofmutation

examinedingreaterdetail

Lettersource

Assaymethoddeveloped

A Onecopy Insertions A1,2024

bp Leahya qPCRof16S indifferent DaschlebrRNAgene

sitesNewYorkPostb

overlappinga16SrRNA

A2,2608bp

NewYorkPosta

Notused

geneA3,823bp

LeahybDaschlea

qPCR

NewYorkPostb

B spo0Fpromoter

SNPinintergenicregion

B LeahyaNewYorkPosta

Notused

C Sensorhiskinase

SNPproducingstopcodon

C Leahya Notused

DSensorhis258bpDLeahyaTaqman+kinasedeletionPCR

E Putative 9or21bp

E,9bp Leahya Taqman+

response deletion,ordeletion Daschlec PCR

regulator SNP NewYorkPostc

(plasmid)

NOTE:bp=basepair;qPCR=quantitativepolymerasechainreaction;SNP=singlenucleotide

polymorphism.

a Source of original morphotype isolate with thisgenotype.

b Sample subsequently found to contain DNA carryingthisgenotype.

cSourceofsubsequentmorphotypeisolatedemonstratedto have this genotype. SOURCE: FBI Documents,B1M5D1-2.

in a gene encoding aK+uptake protein.This SNPwasfoundonlyintheNewYorkPostlettermaterial,butnotinthe Porton Down, Ames Ancestor, or Leahy genomes,and no further forensic use wasmade of thismutation.The second kind of A morphotype genotype involvedlarge insertions in one of the eleven copies of the B.anthracis 16S rRNA gene. A 2024 bp (base pair)insertion (later termed theA1 genotype) and an 823 bpinsertion(A3genotype)werebothfoundinthematerialsfromtheLeahy,Daschle,andNewYorkPost letters,andboth were subsequently chosen for the development of

two separate assays to be used to screen the FBIRepository of Ames strain samples (see Chapter 6). A2608 bp insertion (A2 genotype)was found only in theNewYorkPostletterandanassaywasdevelopedforthedetection of this genotype by CommonwealthBiotechnologies, Inc., (CBI) in Richmond, Virginia. Invalidation testing, the performance of theA2 assaywassurpassedbythatoftheA1andA3assays.Consequently,the FBI decided not to use the A2 assay for evidenceanalyses. Eventually, many more variants ranging from822 to 2608 bp were found among other samplesprovidedtoTIGR(FBIDocuments,B1M5D4).

BmorphotypeisolatesfromtheLeahyandNewYorkPostwerefullysequenced(Table 5-2),while themorphotype isolate from theDaschle letterwas studiedusingPCR.Thesequencing revealed that theLeahyandNewYorkPost letterscontainedanidenticalSNPinthenoncodingregionbetweenthespo0Fgeneandanadjacentgene,andthisSNPwasnotpresentintheAmesAncestororAmesPortonreferencesamples.PCRamplificationandresequencingoftheampliconconfirmed the presence of the same SNP in the same region of the DaschlemorphotypeB isolate. The SNPwas the replacement of a thymine (T)with acytosine (C).The twoopen-reading frames are divergently transcribed, so thisintergenic region likely contains the promoters for these genes, one of which(spo0F)playsakeyroleingoverningentryintosporulation.Thismutationmayexplain why the morphotype is sporulation deficient. The gene expressionpatternsofthesemutantswerenotexaminedbecausethesekindsofexperimentswereconsideredoutsidethescopeoftheinvestigation.Theconsistentpresenceof the B SNP provided a second potential genotypic signature for comparingFBIRsamples to the lettermaterials,but itwasnotusedby theFBI to screenrepositorysamplesbecausemultipleeffortsbycontractorstodevelopassaysforthisSNPfailed (seebelow),norwereanyotherSNPsrepresentingsinglebasepairmutationsusedtothatend(seeChapter6).

ThemorphotypeC andD isolates shared a very similar phenotype. For theC

morphotype, only material from the GB18 sample from the Leahy letter wassequenced(Table5-2).Again,theTIGRteam’sreasoningwasthatothersamplesdidnotneedtobesequencedbecauseanypolymorphismsfoundinGB18couldbe tested by PCR later. The GB18 C morphotype analysis found one SNPcorresponding to anucleotide change fromG toA, creatinga stopcodon in ahistidinesensorkinase(“hiskinase”)gene,amemberofafamilyofproteinsthatregulategeneexpression.WhentheDmorphotypesampleGB19fromtheLeahyletter was subsequently examined for the presence of the C SNP in the samegene sequence, a 258 bp deletion was found instead. This genetic deletionresulted,inturn,inadeletionof86aminoacidsinthesamehiskinaseprotein.TheSNPfoundinGB18islocatedinthechromosomalregionthatisdeletedinGB19.ThusthesimilarityintheCandDphenotypescouldbeexplainedsinceboth the C SNP and the D deletion likely produced a nonfunctioning proteinfromthesamehiskinasegene,whichplaysaroleinsporulation.TheNewYorkPostandDaschleletterswerenottestedfortheCandDmorphotypes.

ThemorphotypeEisolatefromtheLeahyletter(GB23)wassequencedandhadnochromosomalmutations.Instead,thisstrainhada21bpdeletioninthepXO1plasmid.Thedeletionwaslocatedinageneencodingaputativegeneexpressionregulator. A PCR/resequencing assaywas used to test for the presence of thesamedeletion in theGB24Leahy letter sample.This samplecontaineda9bpdeletioninthesamegeneticlocus.ThePCR/resequencinganalysiswasrunonaseriesofotherblindsamples relevant to the investigationaccording toTIGR’s2005report(FBIDocuments,B1M5D4).Someoftheseadditionalstrainscarriedthe9or21bpdeletions,butotherscontainedaSNPrepresentingasinglepointmutation(CGT→TGT)inthesamelocusthatappearedtocreatedefectsinthecorresponding proteins severe enough to interfere with normal function,althoughitwasbeyondthescopeofTIGR’sworktotestthis.

Table5-4providesasummaryof thedistributionamongthecase lettersof themorphgenotypesthatwereultimatelyusedforscreeningoftheFBIR.

TIGRcompletedthisstageofthestudybyanalyzingasetofsamples(listedinTable7,FBIDocuments,B1M5D4)thatincludedadditionalevidentiarysamplesas well as noncase strains from the Keim laboratory’s scientific collections.TIGRtestedthesesamplesusingtheassaysdevelopedforthevariousgenotypes.These additional analyses showed that only the colony morphotype samplesthemselvescontainedthespecificpolymorphismsidentifiedbytheTIGRteam,whichwasinterpretedtomeanthatthesegenotypesrepresented

TABLE5-4DistributionAmongtheAnthraxLettersoftheGenotypesSelectedforFBIRScreening

LetterGenotypeA1GenotypeA3GenotypeDGenotypeELeahy++++

Daschle++NT+

NewYorkPost++NT+

BrokawNTNTNTNT

NT=nottestedSOURCE:Hassell(2010).

uniquemarkerssuitableforfurtherforensicuse.TheA1,A3, D, and E genotypes were employed for thedevelopmentofvalidatedassaysthatwereusedtoscreensamplesoftheAmesstraincollectedbytheFBIfromalldomesticandforeignsourcesthatitwasabletoidentify.ThedetailsofthisscreeningareprovidedinChapter6.

5.5.6DevelopmentandApplicationofAssaysfortheGenotypes

GenotypesA1andA3

CBIwasthecontractorselectedbytheFBItodevelopthegeneticassaysfortheA1andA3morphotypesandtesttheFBIRepositorysamples(Chapter6).CBIbegan work for the FBI in mid-2002 (FBI Documents, B2M5D2). The Amorphotypes thatwere analyzedmost thoroughlywere found to contain largeinsertionsoverlappinga16SrRNAgene(FBIDocuments,B1M5D1).AlthoughtheinsertionwasofadifferentsizeineachofthethreeAmorphotypes,allthreehadinsertionsinthesamelocus.TheA1genotypehada2024bpinsertionandwas originally identified in an isolate from the Leahy letter. During assaydevelopment by CBI this allele was also detected in DNA derived from bulk

Daschle and New York Post letter spores (Hassell, 2010). The A3 genotypecontainedan823bpinsertionthatwasoriginallyidentifiedinanisolatefromtheDaschleletter,butduringassaydevelopmentbyCBIthisallelewasalsodetectedin DNA derived from bulk Leahy and New York Post letter spores (Hassell,2010).TheA2genotypehada2608bpinsertionandwasoriginallyidentifiedinan isolate from the New York Post letter. No acceptable assay for A2 wasdeveloped, and it is not known whether the A2 allele was present in sporematerialfromtheotherletters.TheassaysdevelopedbyCBIusedtheTaqMananalyticaltechnique(Didenko,2001),whichisanadaptationofPCR(seeBoxes5-2and5-3).

CBIcompleteditsvalidationstudiesinFebruary2004.Limitsofdetectionwereestimatedat0.005percentfortheA1genotypeassayand0.001percentfortheA3 assay in a background of 20 nanograms (ng) of Ames Ancestor DNA.Appropriate reaction controls were also developed. Sequencing of ampliconsservedasafinalconfirmatorystep.TheA1andA3genotypeassayswerechosenbytheFBIforuseinanalyzingtheFBIRsamples(seeChapter6)asproblems(e.g., high number of false positives) with the validation of the assay forgenotypeA2ultimately caused this assay tobe abandoned. It shouldbenotedthatassaydevelopmentandvalidationtookalmosttwoyears.

GenotypesBandD

Three companies—CBI, IITResearch Institute (IITRI), andMidwestResearchInstitute(MRI)—werehiredtodevelopassaysfortheBandDgenotypes.Noneof thecontractorswassuccessful indevelopinga reliableBassay. Inaddition,theFBIexpressedapreferencenottouseassaysdirectedatSNPs(FBI,2009).Consequently, the FBIR samples were not screened for the B genotype. TheIITRIandMRIassays for theDgenotypewerebothacceptedby theFBIandusedtoscreentheFBIRsamples.TheDgenotypehada258bpdeletionandwasoriginallyidentifiedinanisolatefromtheLeahyletter.Itwasneverdeterminedwhetherthisallelewaspresentinthesporepopulationsintheotherevidentiarylettersamples.Assaydevelopmentandvalidationineachcasetookalmostoneyear.

IITRI assay (FBI Documents, B2M7): A technical proposal for assaydevelopment for the D genotype was submitted by IITRI in July 2004 andvalidation of this assay was completed in April 2005. Using TaqMan/PCR

(Boxes5-2and5-3) thisassaydetectedthegenotypeDwhenitwaspresentatlevelsaslowas

0.01 percent relative to the Ames Ancestor background. The repositoryscreenings using the IITRI assay for theD genotype began inMay 2005 andwerecompletedinearly2007.

MRIassay(FBIDocuments,B2M8):MRIsubmitteditstechnicalproposalfordevelopment of the D deletion assay to the FBI in July 2004 and assaydevelopmentwascompletedinJune2005.ItusedRT-PCRandhadadetectionlimitof

0.01percentintheAmesAncestorbackground.Threeapproacheswereusedtoincreasesensitivity:closelyspacedprimers, shortannealing time (15seconds),and confirmation of reaction amplicon with melt curve and fragment sizeanalysis.ThissecondDassayalsowentforwardforscreeningtherepositoryandother samples. Screening began in December 2005 and was completed inOctober2007.

GenotypeE

TheEmorphotypewasidentifiedfromtheLeahy,Daschle,andNewYorkPostletters (FBI Documents, B1M5D4). Although there were apparently severaldifferent mutations that produced the “opaque” phenotype, all appeared toinvolvethesamegeneonthepXO1plasmid.OneisolatefromtheLeahylettermaterialcarrieda21bpdeletioninthisgene,andanotherisolatealsofromtheLeahyletterhada9bpdeletioninthesameregionofthatgene.Bothofthesedeletions were also found in E isolates from the Daschle andNew York PostlettersusingPCRandsequencingofthislocus(Hassell,2010).OtherEisolatescontainedasinglebpsubstitutioninthesamegene.

The 9 bp deletion was chosen for the development of an assay for use inscreening theFBIR samples.The assaywasdeveloped in2005usingTaqMantechnology,validated,andappliedtotherepositorybyTIGRundercontractfromtheFBI in2007(FBIDocuments,B2M9).Preparationsofpurifiedmutantandwild-typeDNAweremixedinamountscoveringa1,000,000-foldrangeofratiosof mutant-to-wild-type DNA, and the assay was shown to reliably detect themutantgenomewhenpresentat0.01percentofthetotalDNAinthesample.Inaddition,theassaydidnotproducefalsepositivesforthepresenceofthemutant

alleleusingvaryingamountsofwild-typeDNA.ThisassaywasapprovedbytheFBIfortestingrepositorysamples,whichwasperformedfromJunetoAugustof2007.


Finding 5.1: The dominant organism found in the letters was correctly andefficientlyidentifiedastheAmesstrainofB.anthracis.ThescienceperformedonbehalfoftheFBIforthepurposeofBacillusspeciesandB.anthracis strainidentification was appropriate, properly executed, and reflected thecontemporarystateoftheart.

Finding5.2:The initialassessmentofwhether theB.anthracisAmesstrain inthe letters had undergone deliberate genetic engineering or modification wastimely and appropriate, thoughnecessarily incomplete.Thegenome sequencesof the letter isolates that became available later in the investigation stronglysupportedtheFBI’sconclusionthattheattackmaterialshadnotbeengeneticallyengineered.

Inthefirstfewmonthsfollowingtheattacks,isolatesfromthelettersandothersources were examined only for the presence of some obvious and expectedsigns of genetic engineering. This examinationwas not exhaustive andwouldhavemissedlessobviousorlesswellrecognizedsignaturesofdeliberategeneticalteration.Had the casenot involved theAmes strain ofB.anthracis,with itsrelativelybriefhistoryandhighdegreeofcharacterization,thislimitationcouldhavebeenaseriousone.

Finding5.3:AdistinctBacillusspecies,B.subtilis,wasaminorconstituentoftheNewYorkPostandBrokaw(NewYork)letters,andthestrainfoundinthese two letterswasprobably thesame.B.subtiliswasnotpresent in theDaschleandLeahyletters.TheFBIinvestigatedthisconstituentoftheNewYorklettersandconcluded,andthecommitteeconcurs,thattheB.subtiliscontaminant did not provide useful forensic information. While thiscontaminant did not provide useful forensic information in this case, thecommitteerecognizesthatsuchbiologicalcontaminantscouldprovetobeofforensicvalueinfuturecasesandshouldbeinvestigatedtotheirfullest.

Although theB. subtilis isolates in the two New York letters appeared to beclosely related, the B. subtilis isolate in the Brokaw letter was not fully

sequenced, and therefore the presumed identity of the two isolates was notdefinitively demonstrated. Although B. subtilis was found in several hundredrepositorysamples,thestrainsinthesesamplesdidnotmatchtheisolatesfoundin the New York letters. Biological contaminants could prove to be of greatforensicvalueandshouldbeinvestigatedtotheirfullestinfuturecases.

Finding 5.4: Multiple colony morphotypes of B. anthracis Ames werepresentinthematerialineachofthethreelettersthatwereexamined(NewYork Post, Leahy, and Daschle), and each of the phenotypicmorphotypeswasfoundtorepresentoneormoredistinctgenotypes.

This important discovery greatly facilitated the subsequent laboratoryinvestigationandisatestamenttothecriticalimportanceofattentive,thoughtfulscientistswhowere prepared to explore unexpected results in the setting of aforensicinvestigation.

Finding 5.5: Specificmolecular assays were developed for some of theB.anthracisAmesgenotypes(thosedesignatedA1,A3,D,andE)foundintheletters. These assays provided a useful approach for assessing possiblerelationshipsamongthepopulationsofB.anthracissporesinthelettersandinsamplesthatweresubsequentlycollectedfortheFBIRepository(seealsoChapter 6). However, more could have been done to determine theperformancecharacteristicsoftheseassays.Inaddition,theassaysdidnotmeasure the relative abundance of the variant morphotype mutations,which might have been valuable and could be important in futureinvestigations.

InthecourseofdevelopingtheassaysthatwereusedtoscreentheFBIRsamplesfor the four genotypes, procedures were employed to examine both thespecificityandsensitivityof theassays, includinganalysesofdefinedmixturesof genotypes at known proportions. However, the repository included bothhomogeneous and heterogeneous samples, in unknown proportions, and theextent of genetic diversity in the heterogeneous samples was also unknown.More could have been done to determine the performance characteristics,includingreproducibilityof results,under theactualconditionsassociatedwiththerepositorysamples.

Inaddition,theseassayswerenotusedtoquantifytherelativeabundanceofthegenotypes in theFBIRsamplesand theevidentiarymaterials.Measurementof

relative abundance of genotypes might have helped clarify the relationshipbetweentheevidentiarysporesamplesandwhethertheywerederivedfromthesameordifferentcultivationevents.

Finding 5.6: The development and validation of the variant morphotypemutation assays took a long time and slowed the investigation. Thecommitteerecognizesthatthegenomicscienceusedtoanalyzetheforensicmarkers identified in the colonymorphotypeswas a large-scale endeavorandrequiredtheapplicationofemergingscienceandtechnology.Althoughthecommitteelaudsandsupportstheeffortdedicatedtothedevelopmentofwell-validated assays and procedures, looking toward the future, theseprocessesneedtobemoreefficient.

Future casesmaynot allow for a time frameas lengthy as that of the anthraxlettersinvestigation.Assaydevelopmentandvalidationtookalmosttwoyearsinsome cases, for reasons that are not clear to the committee. The committeerecognizes that theexperiencegained in thecase, aswell as fasterandgreatlyimproved technologies, could help speed future investigations. These factorsalone,however,maynotbesufficientforallcontingencies.Inparticular,futurecases could involve less well documented or less easily grown species andstrains, and precious investigation time could be lost because of the need toestablishbasicinformationabouttherelevantorganism’sbiologyandpopulationgenetics. In addition, original attack material (in this case, the powder in theanthraxletters)maynotbeavailableinallbioterrorismscenarios.Also,insomefuturecasesofbioterrorism theattacksmaycontinueuntil theperpetrators areidentifiedandapprehended.

6

Comparisonof theMaterial in theLetterswithSamplesintheFBIRepository

6.1INTRODUCTION

The previous chapters have established the followingmain conclusions: First,thephysicalevidenceassociatedwiththeanthraxsporesintheattacklettersdidnotrevealthesourceofthematerialsintheletters.Second,geneticanalysesof

thesporesestablishedthattheanthraxbelongedtoaparticularsubtypeknownastheAmesstrain.Third,whilenogeneticdifferenceswereobservedbetweenthepredominantBacillusanthracisgenotype in the lettersand thecanonicalAmesstrain,FBIcontractorsdiscoveredseveralsubpopulationsofB.anthraciscellsinthelettersthateachproduceddistinctcolonymorphologiesaftergrowthonagarplates, and some of these subpopulations possessed specific geneticmutationsthatcouldbeidentifiedbyspecificsequence-basedassaysdevelopedbytheFBIanditscontractors.

In thischapter,wedescribetheestablishmentof theFBIRepository(FBIR)ofAmes strain samples, created by the FBI to look for the B. anthracissubpopulations found in the letterswith the hopeof being able to identify thesourceoftheB.anthracis sporesused in theattacks.ThechapteralsooutlinesthehistoryofRMR1029, the spore-containing flaskat theU.S.ArmyMedicalResearchInstituteforInfectiousDiseases(USAMRIID)thatbecameafocusofthe FBI investigation, at least in part because it contained all of thesubpopulationgenotypesthatwereassayed.

The results obtained from screening the repository samples are described,includingtheevidencethatappearedtoimplicateRMR1029asthesourceofthespores in the attack letters. Particular attention is given to the limitations ofapplyingformalstatisticalmethodstotheseresultsandoftryingtoquantifythestrengthoftherelationshipbetweenthesporesintheattacklettersandthoseinRMR1029.Thecommitteethendiscussesevidencebearingonadisputedsamplesubmittedbythesuspectinthiscaseaswellasfollow-upexperimentsperformedby theFBI todeterminewhether that samplehadcome from its stated source,RMR1029(seeAmerithraxInvestigativeSummary,

125

USDOJ,2010,pp.75-79,foradescriptionofthecircumstancessurroundingthedisputed sample). The chapter ends with the committee’s major findingsregardingthegeneticevidencerelevanttothesourceoftheB.anthracis sporesusedintheletterattacks.

6.2CREATIONOFTHEFBIREPOSITORY(FBIR)

At the time of the anthrax mailings, the federal government had notsystematically collected information on which laboratories possessed anthrax.

Althoughthe“SelectAgents”programwascreatedin1996bytheAntiterrorismand Effective Death Penalty Act (Public Law 104-132), this statute governedprimarily the transfer of biological agents between research laboratories. TheAct directed the Secretary of Health and Human Services (HHS) to issueregulations governing the transport of biological agents with the potential toposeseverethreatstopublichealthandsafetythroughtheiruseinbioterrorism,the so-called “select agents” (NRC, 2009). The authority to regulate selectagentswasdelegatedby theHHSSecretary to theCentersforDiseaseControland Prevention (CDC). To ensure that these agents were transferred onlybetween responsible parties, CDC required that any laboratories that mighttransfer select agents be registered and that transfers be reported to CDC andconducted under a permitting system. As long as the select agents were nottransferred,specific informationabout thefacilities thatpossessed theseagentsdidnothavetobereported(NRC,2009b).

Thedeterminationthat theAmesstrainofB.anthraciswasused in theattacksledtoaprocessbywhichtheFBIsearchedforandacquiredsamplesofknownand accessible derivative stocks of that strain for comparison. As noted inChapter 2, the Ames strain had been widely distributed among laboratoriesaroundtheworldforresearchandvaccinetrials,sotheFBIfirsthadtoidentifyall laboratories that maintained stocks of the strain. Next, the Bureau had toobtain samples of these Ames strain derivatives, which would constitute theFBIR and be screened for the presence of themutant genotypes found in theletters(seeChapter5).Tothisend,theFBIpreparedandissuedasubpoenathatincluded a protocol for the collection and submission ofAmes strain samples(Box 6-1). This subpoena was sent in February 2002 to 16 laboratories orfacilitiesintheUnitedStatesthathadbeenidentifiedaspossessingstocksoftheAmes strain. (It was subsequently determined that one of these domestic

laboratories did not possess the Ames strain1.) In addition to thesubpoenas,

1 According to the FBI (discussion with committee,December11,2009),16U.S.laboratorieswereoriginallyidentified as candidates for having the Ames strain.According to the DOJ case closing summary, however,

only 15 domestic laboratories were confirmed asrepositories of the Ames strain (USDOJ, 2010, p. 17).CDC provided a listing of all laboratories registered towork with B. anthracis, and FBI investigators createdtheirownlistbasedonCDCselectagenttransferrecordsdocumentingeverytransferofanthraxbetween1997and2001aswellasanthraxinventoryrecords

BOX6-1SubpoenaProtocolforCollectionandSubmissionofAmesStrainSamples

Purpose: Provide guidance in the preparation andshipmentofBacillusanthracisAmesstraintotheUnitedStates Army Medical Research Institute of InfectiousDiseases, Fort Detrick, Maryland. As per FBI request,submit two culture agar slants of each distinct B.anthracis Ames strain stock in your possession, whichdiffersinsourceorinotherparametersprescribedbytherequestingAgency.

Materials:

TrypticSoyAgar(TSA)Slants(Remelcatalog#08932,orequivalent)SterileinoculatingloopsAdhesiveLabels,permanent,waterproofSTP-100InfectiousSubstanceShippingContainer(Saf-T-Pac),orequivalent

Procedure:

1. Collect each B. anthracis Ames strain stock as per your institutionalinventoryandpersonalknowledge.

2. Prepareaminimumof twoTSAslant tubesperstockbyprelabelingwithpermanent waterproof labels. Include the following information on the

label: “B. anthracis Ames strain.” Other designators used by yourlaboratory,date,andyourlabname.Additionalinformationforeachstockshallbeprovidedseparately.

3. Arepresentativesampleofeachstockshallbeusedfor inoculationof theTSAslants. If thestockisanagarculture,donotuseasinglecolony,butrather use an inoculum taken across multiple colonies. Thawed frozenstocksorother liquidsuspensionsshallbewellmixedprior to transferofinoculumtotheTSA.

4. InoculateeachTSAslantsinazigzagmanneroverthesurfaceoftheagar.5. Incubateslantsat35-37Cfor12-18hourstoconfirmculturegrowth.6. Individuallywraptheslantsinpackingmaterialsapprovedforthetransport

ofinfectiousselectagentsinaccordancewithregulationsfortheshipmentofsuchmaterials.

Source: FBIDocuments, Preparing and ShippingTSASlants forB. AnthracisAmes.

consent searches were conducted at USAMRIID inMaryland and at Dugway Proving Ground (DPG) inUtah, and a search warrant was executed at a privatecompanyinOhio(BattelleMemorialInstitute)in2004toensure that samples were obtained from every stockderived from the Ames strain in those three facilities.Voluntarysubmissionswerealsorequestedfromthethreeforeign

thatwere subpoenaed from over 100Biosafety Level 3(BSL-3)laboratoriesintheUnitedStates.“Theserecordswere supplemented by information culled from FBIinterviewsofscientistsworkingateachoftheselabs,andthrough FBI reviews of relevant scientific publicationsmentioningtheAmesstrain”(USDOJ,2010,p.17).

laboratories (in the United Kingdom, Canada, andSweden)knowntopossesstheAmesstrain,twoofwhichprovidedsamples.

TheFBIdescribedtheirapproachtoassessingAmesstrainsinternationallyinthefollowingway:Complementingthisinitiativewasaseparateintensiveeffort toassesswhetheraforeigngovernmentoraterroristorganizationmayhavegainedaccess to theAmesstrainandperpetrated theattacks.Anexhaustive initiative,onethatcontinueduntiltheclosingofthecase,addressedthisconcern.Aswiththedomesticinvestigation,itincludedreviewsofpublishedresearchandtransferrecords. In addition, it involved an assessment of available intelligence onforeigngovernmentandforeignactorcapability,meetingswithforeignexperts,witness interviews, and the collection of Ames strain isolates from certaingovernments.(FBI/USDOJ,2011).

LaboratoriesinpossessionofstocksderivedfromtheB.anthracisAmesstrainweredirectedtoobtainsamplesfromeachofthestocksinboththeirinstitutionaland personal inventories for submission to the FBIR.The subpoena asked forlittleinformationabouttheprovenanceofthesamplesbeingsubmitted,withnospecific requirement that such information be provided. For this reason,information about the derivationof the various stocks from the originalAmesstrainwasnotstandardizedandvariedgreatlyinitsamountandspecificity.

Ingeneral,stocksofB.anthracisarestoredindifferentwaysdependingonthephysiological state of the bacteria (spores or vegetative cells) as well as theprocedures used in a particular laboratory. For example, somemicrobiologicalstocks are isolated from single colonies, cultured for only a few additionalgenerations, and then stored in freezers; these stocks are expected to begeneticallyquitehomogeneous.Otherstocksexistasconfluentlawnsofcellsonslants or asmultiple colonieson agarplates; such stockshave thepotential toaccumulategeneticdiversityiftheyarekeptinthesestatesforlongperiods.Stillother stocks, including the one designated RMR1029, aremixtures of severalindependent large-scale culture preparations; such mixtures may also harborgenetic diversity. The sampling protocol in the subpoena directed that a“representativesample”ofeachstockbeprovided.Itspecifieddifferentmethodsforobtainingthesample,dependingonhowthestockwaskeptinthelaboratory.For agar-based stocks, the submitters were told to take an inoculum frommultiplecolonies(“donotuseasinglecolony”,seeBox6-1),althoughaprecise

numberwas not specified.Samples taken from thawed frozen stocks or liquidsuspensionswererequiredtobe“wellmixed”priortoinoculatingtheslantthatwas to be delivered to the FBIR. The submitters were apparently not asked,however,toprovideinformationaboutwhichmethodstheyhadusedtomaketheinocula for submission nor in what form the stocks were stored. In addition,there was no effort to standardize the number of spores or cells that weresubmitted. These omissions limit the committee’s knowledge (and that of theinvestigators)aboutthequalityofthesamplessubmittedand,inparticular,howwelleachsubmissionmettherequirementofbeinga“representativesample.”

Inall,20laboratories(15domestic,twoforeign,plusUSAMRIID,Dugway,andBattelle)submittedtotheFBIRatotalof1,070(USDOJ,2010,p.24)samplesofstocks derived from the Ames strain of Bacillus anthracis. Of these, 1,059sampleswere screened and results reported for thepresenceor absenceof thefourmutantgenotypes—A1,A3,D,andE(FBIDocuments,B2M10D2)—usingtheassaysdescribedinChapter5.TheFBItoldthecommitteethattheother11sampleswerenotviable,failedtogrowtheAmesstrainofB.anthracis,orfailedtogrowB.anthracisat all.The results and interpretationof this screeningaredescribedbelow.

Itisimportant,however,torecognizenotonlyseveralinherentlimitationsoftheFBIR collection that make it difficult to assess the evidence in any formalstatistical sense, but also the effects of the decision to require growth of thesamples before testing for the fourmutant genotypes. In addition to issues ofrepresentativeness,thereareissueswiththeindependenceofthesamples.

First,statisticalanalysestypicallyassumethatsamplesaretakenatrandomfromadefinedpopulation.TheFBIaimedtocreateacomprehensiverepositorythatencompassedtheentirepopulationofstocksderivedfromtheAmesstrain,ratherthan a representative fraction thereof. Given uncertainties in the extent of theentirepopulationofAmesstocksworldwide(highlightedbyconcernabout thepossibility of clandestine stocks held by terrorist organizations—see section3.4.3), the lack of specificity in the subpoena protocol, the uncertainties incompliancewiththesubpoenaprotocol,theincompleteinformationontransfersof Ames-derived stocks between laboratories, and the possibility that somestockswere produced but later destroyed, the repositorywas unlikely to have

been comprehensive.2 DOJ states in its Summary, “Thecollection ofAmes isolates from laboratories both from

theUnitedStatesandabroadthatconstitutetheFBIRarea comprehensive representation of the Ames strain”(USDOJ, 2010, p. 28). Section 6.5 provides furtherdiscussionofthisissue,alongwiththeimplicationsoftheviolation of this assumption of “representativeness” forthestatisticalinferences

Second,therewerecomplexandvaryingdegreesofgeneticrelationshipsamongthestocks,reflectingtheircommondescentfromtheoriginalAmesisolateandthe history of transfers, single-colony isolations, and mixtures of materialswithinandbetween institutions.Asaconsequence, somesetsof relatedstockswere likely to be represented by many samples and others by few samples,renderingitimpossibletoassesstherelevantfrequenciesofgenotypesacrossthepopulationofinterest.

Third, statistical analyses are critically dependent on replication to providemeasuresofthesensitivityandspecificityofassays,andsuchreplicationshould

2TheissueofoverseassamplesdiscussioninChapter3,section 3.4.3 raises additional questions about thecomprehensivenessoftheFBIR:butasstatedpreviously,theseissueswerebeyondthescopeofthiscommittee.

bepartofaprotocolateverybiologicalandtechnologicallevel at which it is feasible. Although scientists wererequired to submit duplicate samples to the repository,only one sample was analyzed for the genotypes ofinterest while the other was retained in an archive.Subpoenarecipientsshouldhavebeenrequiredtosubmitthree or more samples of each stock, so that replicategenotypic assays could have been performed. Such

replication would have allowed a more rigorousassessmentoftheresultsofthegenotypicassays.

Finally, even for replicate samples from the same stock, other sources ofpotentialvariation—suchas in thedensityofcellsor thespatialdistributionofgenotypes in the original stock—might have introduced statistically importantdependence across the several assays performed on the samples. Thus, theresults obtained from the several genotypic assays cannot be assumed to beindependent.

6.3USEOFTHEGENETICASSAYSTOTESTFORTHEFOURGENOTYPES

Either during or after the development of the genetic assays, a decision wasmadethattheyshouldbeperformedonsamplesofB.anthracisAmesonlyafterthe cells in the sample were cultivated once again (in addition to the priorcultivationstepbytherecipientsofthesubpoenaprotocolbeforesubmittingthesamples).AccordingtotheFBI,thisfurthercultivationstepwasdoneinparttostandardizethenatureofthesubmittedsamplespriortothetesting(FBI,2009).In addition, itwouldhave expanded the amount of sample available forDNApreparation and subsequent testing by the different contractor laboratoriesinvolvedingeneticassaydevelopment(FBI,2009).However,thesensitivityofeachofthemolecularassayswouldnothavenecessarilyrequiredthisadditionalcultivation step. Furthermore, the committee notes at least two problemswiththis additional cultivation step. First, the samples that were submitted to therepositorybutthenfailedtogrowinculturecouldnotbetested;however, theycould have been tested if the genetic assays had been directly applied to thesamples.Second,eachcultivationsteppotentiallycreatedadditionalbiasinthesample, because not all cell genotypes grow at the same rate under the sameconditions (see also Section 5.5.2). Some variant cell typesmight growmoreslowlyandfallinabundancerelativetotheothercelltypes,suchthattheyarenolonger detectable, while others may grow more quickly and become moredominant.Thecommitteewasunabletoassesstheextenttowhichthebenefitsofsamplecultivationoutweighedthesepotentialproblems.

6.4DERIVATIONOFRMR1029SPORES

Thepresenceof genetic variants in the anthrax letter samples (as described in

Chapter5)and in thesporepopulation inRMR1029and insamplesknown tohave come fromRMR1029 (as described below) led theFBI to conclude that“RMR1029 is the parent material of the evidentiary anthrax spore powder”(USDOJ, 2010, p. 28). Based on their investigation, FBI officials stated thatRMR1029wasproducedin1997as“aconglomerationof13productionrunsofspores by Dugway, for USAMRIID, and an additional 22 production runs ofspore preparations at USAMRIID that were all pooled in this mixture” (FBI,2008c, p. 55). The resulting 164 liters of spore productionwere concentrateddowntoaboutaliter.Originally,thesporepreparationwasdividedintotwoone-literErlenmeyerflasks,oneofwhichwaseventuallydepleted,sothatonlyoneflaskremainedforsubsequentrepeatedsampling,as isdiscussedfurtherbelow(FBI,2008c,pp.58-59).

This spore preparationwasmaintained atUSAMRIID,where records indicate

that it contained approximately 3 × 1010 spores per ml(USAMRIID, 1997). Between 1998 and 2003, aliquotswerewithdrawnfromRMR1029foruseinanimalstudiesduring the development of anthrax vaccines andtherapeutics.TheamountofRMR1029consumed in thecourse of these studies is documented in terms oftransfersoutoftheflask(B3D14,B3D16),butgiventheconcentrated nature of the preparation, a very smallvolumewould have provided amplematerial for use asinocula toproduceadditional sporepreparationssuchasthosethatmighthavebeenusedintheletters.

Theproductionhistoryof theRMR1029 sporepopulationprovides aprobableexplanation for the presence of subpopulations of genetic variants, includingvariants thatcouldgiverise tovisuallydistinctivecolonies(morphotypes).Forexample, colonies of genetic variants that are defective in spore formationtypically have surface textures and coloration that distinguish them from thewild type (see Chapter 5). While it is standard practice in microbiology toprepare new cultures by starting from single-colony purified stocks, thisprocedure was deemed impractical (Martin, 2010) for the preparation of the

large volumes of spores that were combined to produce the RMR1029 stock.Thissporesuspensionwasintendedforuseasareferencestock,orpopulation,that could be drawn upon for many studies, thereby allowing comparison ofresultsacrossstudiesperformedatdifferent times.The largenumberofsporesrequired for an extended set of studies could not, however, be prepared in asingle batch (especially given the biosafety level required for work with adangerouspathogen).BecausethefermentorsusedforgrowinglargebatchesofsporeswerenotavailableatUSAMRIID,thebulkofthematerialinRMR1029wasgeneratedatDugway.

It is likely that some or all of the genetic variants (including especially thosediscoveredon thebasisofatypicalcolonymorphologies)present inRMR1029were present in the material provided by Dugway, for the following reasons.First, the DPGmaterial was prepared using inocula that had not been startedfromasinglecolonybutinsteadcamefromstocksthathadbeenobtainedfromUSAMRIID.Bulkmaterialfromthestockwasthenusedtoinoculateblood-agarplates.

AphotographshowntothecommitteeofadenselawnofB.anthracisgrownonsuchaplateatDugwayrevealsthepresenceofmanypapillae,smalloutgrowthsofbacteriaindicativeofmutantsthatareovergrowingtheirneighborsinthelawnor have some other distinctive feature (Martin, 2010). The scientist whorepeatedly prepared these materials for the multiple production runs told thecommitteethatthepresenceofnumerouspapillaewasthetypicaloutcome.Thecommitteebelievesthatthepresenceofthesepapillaecanbetakenasevidencethat the agar slants already contained mutants, that growth of the bacterialpopulation on the blood agar plates selected formutants, or both. BecauseB.anthraciscellssporulateonbloodagaroncetheyreachhighdensity,thepapillaecouldhavebeenoutgrowthsof sporulation-defectivemutants thatcontinued togrow for several generations after other nonmutant cells stopped growing andformedspores.

Second, according to one DPG scientist, the biological material (lawns,includingpapillae)scrapedfromtheseplateswasthenuseddirectlytoinoculatethefermentorsatDugway(Martin,2010).Thismaterialwascollectedfromtheplatesafterthelawnpopulationhadlargelyconvertedtospores.Becausesporesmust germinate before growth can resume, unsporulated cells (including fromsporulation-defectivemutantsintheinoculum)wouldlikelyhavehadagrowthadvantageinthefermentor.BecausematerialpreparedatDugwaycomprisedthe

bulk of thematerial that was pooled in the RMR1029 flask, and because theinoculausedtopreparethesporeshadvisibleevidenceofmutantsthatmayhavebeendefectiveinsporeformation,thecommitteesuggeststhatatleastsomeofthe morphotypes identified in RMR1029 originated from Dugway. Variousbiologicalfactorswouldhaveaffectedtheresultingpresenceandabundanceofthe genetic variants, including their growth rates, germination rates, andsporulationefficienciesunderthespecificcultivationconditionsusedaswellastherateatwhicheachvariantarisesbymutation.

6.5ANALYSESOFTHEREPOSITORYSAMPLESANDSTATISTICALINTERPRETATIONOFTHEEVIDENCE

The assays developed by the various contractors (CommonwealthBiotechnologies, Inc. [CBI], Midwest Research Institute [MRI], IIT ResearchInstitute[IITRI],andtheInstituteforGenomicResearch[TIGR])anddescribedinChapter5wereusedtoanalyzethesamplessubmittedtotheFBIRunderthesubpoenaprotocolorgatheredthroughthesearchesperformedbytheFBI.Thepurposeoftheseassayswastosearchforthepresenceofthegeneticmutations—A1,A3,D,andE—amongtheFBIRsamplestodeterminewhetheranyofthesampleshadageneticprofile thatmatchedthatof theevidentiarymaterialandcouldbeapossiblesourceofthematerialusedintheletters.

ItisimportanttoreiterateherethattheexistenceinlaboratoriesoftheAmesstrainofB.anthracisdatesbackonlyto1981andthatallsamplesoftheAmesstrainatlaboratoriesoutsideUSAMRIIDhadbeenderivedeitherdirectlyorindirectlysometimebetween1981and2001fromUSAMRIID’sstocksoftheorganism.ThisfactisofsubstantialsignificanceandlimitsaquantitativestatisticalanalysisoftheresultsoftheFBIRassays.

ThecommitteereviewedthereportprovidedtotheFBIbyacontractorwhoperformedstatisticalanalysisoftheassayresultsforthe1,059viableFBIRsamples.Theresultsoftheassaysweresummarizedbythecontractorinatable(reproducedhereasTable6-1)intheStatisticalAnalysisReport.

TheFBIdidnotseekformalstatisticalexpertiseuntilithadcompletedthegenotypeassaysoftherepositorysamples.Thus,theutilityofthestatisticalanalysiswaslimitedinpartbythefactthattheexperimentaldesignwascreatedwithoutinputfromstatisticians.TheFBIcontractoranalyzedthedatafor

A1A3MRI-DIITRI-DE“Neg-u”——21——

Positive3521645823

Negative9659469409631,008Inconclusive20323424—Variant829———Pending———1—NoBacillus

DNA1717——15Nogrowth1414—1313

TOTAL1,0591,0591,0591,0591,059

NOTES:MRI-DandIITRI-DrefertothetwoindependentassaysfortheDmutationgenotypedevelopedandperformedbytheMidwestResearchInstitute(MRI)andIITResearchInstitute(IITRI),respectively.

—indicatesthatnosampleswerereportedinthatcategory.“Neg-u”=Themeaningofthisdesignationisnotcleartothecommittee.“Neg-u”designationappearsintheStatisticalAnalysisReportbutitisnotdefinedanddoesnotappearinanyofthefinalreportsonDassayssubmittedtotheFBIfromMIRandIITRI.SubsequentanalysiscombinedthesesamplesfromMRIwithMRI’s940“negative”results.Variant=samplesimilartobutnotidenticaltothegenotype.Pending=sampleanalysisincompletewhenStatisticalAnalysisReportsubmittedtoFBI.SubsequentreportfromMRIindicatesthatMRIclassifiedthesampleas“inconclusive.”Inconclusive(IITRI)=inconsistentresultsonreplicates.Inconclusive(MRI)=inconsistentresultsonreplicatesornogrowthornoBacillusDNA.ThusIITRIseparatedthethreecategorieswhile

MRIclassifiedallthreecategoriesas“inconclusive.”(ThefinalreportfromMRI[FBIDocuments,B2M8D7]divides35“inconclusive”samplesintonine“inconclusiveanalysis”(i.e.,“failuretoproduceaclearreproduciblepositiveindicationforthepresenceoftheMorphDdeletion”),19insufficientDNA,and7nogrowth.SOURCE:FBIDocuments,B2M10D2.

947ofthesamplesthatprovideddefinitiveresultsforallfour genotypes. For genotype D, the IITRI data weredropped because they did not provide results in allcategories, so analysis of theD genotypewas based onthe MRI data only. An additional 112 samples wereomitted from further investigation because the resultswith these samples were recorded as “inconclusive,”“variant,”“nogrowth,”or“noDNA”foroneormoreofthe four genotypes. Tables 6-2 and 6-3 summarize thedatafromtheStatisticalAnalysisReportintwoways,ineach case using only the 947 samples that produceddefinitive results. Table 6-2 shows the frequencies ofpositiveandnegativeresultsforeachofthefourgeneticassays, and Table 6-3 shows the frequency of samplesthat showed a given profile of positive and negativeresultsacrossthesetofassays.

TABLE6-2GeneralResultsoftheScreeningofthe947SamplesthatProvidedDefinitiveResultsforAllFourGenotypes

A1A3MRI-DEPositive27165116

Negative920931896931

TOTAL947947947947

SOURCE:FBIDocuments,B2M10D2.

TABLE6-3DistributionResultsfortheFourAssaysforGenotypesA1,A3,MRI-D,andEinthe947Samples

A1A3MRI-DECountpositivepositivepositivepositive8

positivepositivepositive—2

positivepositive——2

positive—positive—4

—positive—positive3

——positivepositive

2

positive———11

—positive——1

——positive—35

———positive3

————876

TOTAL947

NOTE:—=negativeresultSOURCE:FBIDocuments,B2M10D2.

6.5.1TheFBI’sStatisticalAnalysisReportThe Statistical Analysis Report prepared for the FBI (FBI Documents,B2M10D2)containsfiveconclusionsthataresummarizedbelow.

(a)Frequencyof4-positivesamples

Thefractionofthe947sampleswithpositiveresultsforall4genotypeswas8/947=0.00845.Thisfractionwasofferedasanestimatefortheprobabilityof

theoccurrenceofa4-positive(++++)sample.(“Retainingonlypositiveandnegativeresultsofthefourassays,inthe947repositorysamples,eightshowedsimultaneouspositiveresults++++forallfourassays(i.e.,0.84percent),withexact95percentconfidenceintervalof0.0037to0.0166(i.e.,from1in270to1in60)”(FBIDocuments,B2M10D2).(b)

Dependenceamongassays

The interdependence among the assay outcomes (co-occurrence of genotypes)was examined using various statistical tests and it was concluded that theoccurrenceofthefourgenotypesdidnotappeartobeindependent.

Briefly,aspresentedinthereport,iftheoutcomeswereindependent,andnotingthat27ofthe947samplesshowedA1,16ofthe947samplesshowedA3,51ofthe947samplesshowedMRI-D,and16ofthe947samplesshowedE,thenonewouldexpectthefractionofsamplestestingpositiveforallfourgenotypestobe0.4383permillion,orlessthanoneinamillion.Sincetherewere947samples,onewouldhaveexpectedtosee947timesthisfraction,or0.0004sampleswithall fourmutations.That is, if theassayoutcomes reallywere independent,one

wouldnotexpecttohaveseenanysampleswithallfourmutations.3Infact,however, the FBIR contained eight samples that werepositive for all four mutations, a number considerablylarger than the expected 0.0004 samples based on theassumption of independence. Similarly, the report notedthat the fraction of samples that would be expected tohave only one of any of the four genotypes can becalculated as 0.1072; hence one would expect to see(0.1072×947)=101.5ofthe947sampleswithonlyonepositive assay. The FBI observed 50 samples with onepositiveassay.

(c)Relationshipsamongsamples

Given the existence of historical relationships among the 947 repository

samples, theStatisticalAnalysisReport described twomethods to assess theserelationships.The firstwas a “network analysis” applied to the distribution ofcombinationsofassayresultsfor thefourgenotypesamongtheFBIRsamples.Table6-3providesthenumberofeachofthese11combinationsamongthe947

3 Under the assumption that the assay results areindependent,theprobabilityofjointoutcomesequalstheproduct of the probabilities of each separate outcome.The observed frequencies are estimates of theprobabilities.Multiplying the four observed frequenciestogether yields 0.0285 × 0.0169 × 0.05385 × 0.0169 =0.4383 × 10−6 for 4 positive genotypes, and 0.9715 ×0.9831 × 0.94615 × 0.9831 = 0.8884 for 4 negativegenotypes.

FBIR samples that were considered in the StatisticalAnalysis Report. The network analysis provides arepresentation of the relationships among the genotypicconfigurations, informed by their frequencies and themutationaldifferencesamongthem.IntheReport,itwasconcludedfromthisrepresentationthattheeightsamplesthat were positive for all four genotypes were mostclosely associated with the two samples that werepositiveforthreegenotypes(A1,A3,D)andwiththesixsamplespositivefor twoof these threegenotypes.Fromtheanalysis, itappearedthat the16samplespositiveforEwerelesscloselyrelated.

The second method in the Statistical Analysis Report to assess relationships

among samples relied on a spreadsheet of dates (rows) and labs (columns)indicating the datewhen a sample “from” one institutionwas transferred “to”anotherfacility.(Thespreadsheetindicatedthattransfersoccurredbutitdidnotindicatedthespecificrelationshipsamongsamples.)TheFBItoldthecommitteethat the spreadsheet was generated from laboratory reports and included allrecorded transfers ofAmes samples between 1981 and 2001. The spreadsheetshowed known direct or indirect relationships between Laboratory F(USAMRIID)andmost(butnotall)oftheotherninelaboratoriesorinstitutionsthatsubmittedsamplesthatwerepositiveforoneormoreofthefourgenotypes.Basedonthesetwomethods,theStatisticalAnalysisReportconcludedthattherewereassociationsbetweentheeightsamplestestingpositiveforfourgenotypesandtheothersamplesthatwerepositiveforoneormoreofthegenotypes.

(d)Sensitivityandspecificity

ThestatementofworkthatprecededthesubmissionoftheStatisticalAnalysisReportindicatedplanstoprovideestimatesofsensitivitiesandspecificitiesoftheassaysforthefourgenotypes.Thecommitteewasabletofindonlyalimitedanalysisofthissortinthefinalreport.(e)

Significanceof“LaboratoryF”asthesourceofsevenoftheeightsamplesfoundtobepositiveinallfourassays

TheStatistical Analysis Report concluded that the chance of finding eight 4-positivesamplesshouldbeverylow(i.e.,lessthan0.0166,orlessthan1in60).Thereportalsonotesthatsevenoftheeight4-positivesamplescamefromonlyone institution (“Laboratory F”), that the eighth sample came from anotherinstitution, and that “its occurrence in [the other laboratory] is explained by arecentsampletransfer”(FBIDocuments,B2M10D2).

6.5.2CommitteeAssessmentofStatisticalAnalysisReport

Representativeness,Randomness,Independence

Many of the methods used in the Statistical Analysis Report rely on theassumption that the 947 FBIR samples were a representative and randomcollection, independently sampled from some well-defined population of B.anthracis samples. In fact, there is no meaningful population beyond therepository itself, and the repository could not be a random collection of

independent samples due to the relationships and sharing among laboratories.Further,theFBIRsometimescontainedsamplesfromthesamesource.TheFBIconfirmed (1/14/2011 meeting) one such instance: FBIR samples 006-002(Statistical Analysis Report, p.28) and 067-001 (StatisticalAnalysis Report, p. 57) were duplicates made from thesame submission (“the disputed sample”).Although thelack of independence among the genotype assays wasproperlyacknowledgedintheStatisticalAnalysisReport,statistical calculationswereperformed that reliedon theassumption of independence among the 1,059 samples(e.g., chi-squared tests on p. 10, Statistical AnalysisReport).

Committee concerns about each part of the Statistical Analysis Report aredescribedbelow:

(a)Frequencyof4-positivesamples

Becausetheeight4-positivesamplesandthe947overallsampleswerenotindependentsamples,theproportion(8/947)asanestimatefortheprobabilityoftheoccurrenceofa4-positive(++++)sampleisnotmeaningfulnorisa

calculated“95%confidenceinterval”forthisprobability.4(b)

Dependenceamongassays

Table 6-4 from the Statistical Analysis Report shows all possible ways ofobtainingsampleswithnopositiveassays,and1-,2-,3-,orall4-positiveassays.TheStatistical Analysis Report includes a formal statistical test to assess thesignificance of the differences between these “observed frequencies” and the“expected frequencies.” However, the inference from this test is not validbecause,asnotedabove,the947sampleswerenotindependent.

Thecommitteeidentifiedtwopotentialsourcesofdependenceamongrepositorysamples in the results of the four genotype assays. The most obvious is the

genealogicalstructureofdescentfromtheoriginalAmesancestorinlightofthehistoryof transfer ofB.anthracis samples and stocks among laboratories (seediscussion below). Another possible source of dependence among the assayresultsstemsfromvariationamongsamplesintheprocessofpreparation(undertheguidanceof the subpoenaprotocol) and in theamountofDNA, leading tosample-specificvariationinprobabilitiesofdetectionofthegenotypes.

4Theconfidenceintervaliscomputedfromthebinomialdistribution (Snedecor and Cochran, 1989). Thiscalculation is valid only if the 947 samples areindependent.As an aside, even if itwere appropriate tocalculatea95percentconfidenceintervalwiththesedata,the calculation is misinterpreted in the StatisticalAnalysisReport.Thecalculatedinterval(0.0037,0.0166)is actually a 95 percent confidence interval for theprobability thata sample from the targetpopulation isa4-positive sample, not for the occurrence of eight 4-positivesamplesinthetargetpopulation,asstatedintheStatisticalAnalysisReport (FBIDocuments,B2M10D2;citedaboveinSection6.1(a)).

SOURCE:FBIDocuments,B2M10D2.

(c)

RelationshipsamongsamplesGiventhecountsofthedifferentgenotypicconfigurationsinTable6-3,theresultofthenetworkanalysisisinevitableandaddsnonewinsights.Thisfrequency-dependentanalysisisdominatedbythe876samplesshowingnoneofthefourgenotypes,whiletheappearanceofamoredistantrelationshipofEpositivesamplesresultsbothfromtheirlowfrequencyandtheabsenceofEintheonly2samplespositiveforthreegenotypes.Note,however,thatonly16ofthe23Epositivesampleswereincludedinthisanalysis,andthatseveralofthe7otherEpositivesamplesexcludedfromthestatisticalanalyseswerealsopositiveforothergenotypes(AppendixC,TableC-1).Further,thenetworkanalysisisdesignedtodisplayrelationshipsamongnaturallyevolvingorganisms.NoconclusionscanbedrawnfromitsapplicationtotheFBIRsamples,withoutknowingtheircomplexhistoryoftransferandmixingrelationships.(d)

Sensitivityandspecificity

As noted above, the statement of work that preceded the submission of theStatisticalAnalysisReport includeda request forestimationsofsensitivityandspecificityof assays; however, the committee foundonly a limited analysis ofthissortinthefinalreport.Whileassaysensitivityandspecificitywereassessedbytheassaydevelopmentcontractorsinanidealizedandartificialsettingduringthedevelopmentoftheassays,amoremeaningfulassessmentinthecontextofthetestingofactualrepositorysampleswouldrequirereplicationoftestsusingFBIRsamples,orthecreationofmultipletypesofsimulatedstocksamples.

Toassess thesensitivityof theassays,dilutionexperimentswereconductedontwosamples,RMR1029and“SPS.266Tube#5,”usingthreereplicatesateachof10dilutionlevels.Unfortunately,theanalysisrevealssubstantialvariationintheassay results. InRMR1029, thegenotypeswereusuallynotdetectedbeyondadilutionof1:10.ForSPS.266Tube#5,oneof the three replicateswaspositiveforE out to a dilution of 1:100,000, yet two replicatesTABLE 6.5GenotypeAssays on Three Replicates from Two Samples at 10 Dilution Levels: EntryDenotesNumberofPositiveAssaysonThreeReplicatesatEachDilutionLevel

Sample1:RMR1029

Dilutionlevel

10.110.210.310.410.510.610.710.810.910.10

A13—————————

A331—1——————

D:IITRI3—————————

D:MRI3—————————

E11————————

Sample2:SPS.266Tube#5

Dilutionlevel

10.110.210.310.410.510.610.710.810.910.10

A131————————

A3333211—1——

D:IITRI321———————

D:MRI321———————

E11111—————

NOTE: — denotes either negative or no growth SOURCE: FBI Documents,B2M10D2.

were negative even at just a ten-fold dilution and onereplicate for A3 was positive even at a 1:100,000,000dilution, whereas most were negative at 1:100,000 to1:10,000,000 dilutions. Table 6-5 provides the resultsfrom these dilution experiments summarized in theStatisticalAnalysisReport.

The lackofagreementbetweenMRIand IITRIassays for theDgenotypehasalreadybeennoted(Table6-1),andfurther illustrates thedifferingsensitivitiesandspecificitiesoftheassays.Theresultsofthetwoassaysagreedfor945ofthe1,059 samples, for a concordance rate of 92.1 percent, but disagreed for 30

samples.5 While concordance is informative, the 30samples with discordant results provide a valuableopportunity to assess the validity of the assay and gainfurther insight into these samples; this opportunity wasnotfullyexploited.

5 The decision to set aside the IITRI results seemssomewhatarbitrary.ThereportstatesthatIITRIobtained“nogrowth”with 13 samples butMRIobtained growthonall samples.However, theFinalReport fromMRI-D(FBIDocuments,B2M8D17),dated21July2006,notes“Nogrowth”on7samples.ThetotalnumberofsamplesyieldingnoinformationwassimilarforbothMRI-D

(35)andIITRI-D(37).

In its conclusions, the FBI paid particular attention tosamples carrying three or four of the genotypes.However, the FBI did not address the issue of falsenegative results. In connection with this issue ofsensitivity of the assays, amajor concern regarding theStatisticalAnalysisReportistherestrictionofitsanalysesto the 947 samples that contained no inconclusive orvariant results. Additionally, no errors and uncertaintiesof detection, nor variation in sample preparation, are

taken into account for the analysis. Four of the 112disregarded samples scored positive for the remainingthreegenotypeassays(seeAppendixC,TableC-1).

ThelackofreplicationintheassaysoftheFBIRsamplesmakesitimpossibletoquantify the strength of any finding relating to the presence or absence ofgenotypesintherepositorysamplessincesomeabsencesmaybefalsenegatives.Because samples were not retested and because the dilution experimentsdemonstrate the potential for different results on the same sample, one cannotquantify the strength of any finding related to the absence or presence ofgenotypesintherepositorysamples:thus,sometestresultsof“negative”couldwell be false negatives (“present but unable to detect”). Consequently, thefinding of all four genotypes in both RMR1029 and seven samples from onelaboratory clearly suggests a relationship betweenRMR1029 and three of thefourattackmaterials,butitisimpossibletocalculateanymeasureof“statisticalstrength”forthisassociation.

(e)SignificanceofLaboratoryFas the sourceof sevenof theeight4-positivesamples

AssuggestedintheStatisticalAnalysisReport(p.2),onfirstinspectionitmayseem noteworthy that seven of the eight samples scoring positive for all fourgenotypescamefrom“LaboratoryF.”However,LaboratoryFsubmittedalmosttwo-thirds(63percent)of the947samples.(Most laboratoriessubmittedfewerthan15samples;sevensubmitted18to74samples,andLaboratoryFsubmitted598samples;seeTableC-2inAppendixC.)Consequently,LaboratoryFcouldhavesubmittedmostorevenalloftheeight4-positive(++++)samplesmerelybychance.Moreprecisely,theprobabilitythatseven,orevenalleight,ofthe4-positivesampleswouldendupamongtheLaboratoryF598submissions,merely

bychance,is0.14223(about1in7).6

6.6 ANALYSES BASED ON RESAMPLING OFRMR1029 AND INTERPRETATION OF THERESULTSIn late February 2002, Bruce Ivins and a technicianwhoworkedwith him at

USAMRIIDpreparedduplicatesamplesoffourAmes-derivedstocksfrom

6 This probability calculation arises from thehypergeometricdistributionfortheprobabilitythat7or8ofthe8samplesariseinasubsetof598samplesfromthetotal947samples;seeAppendixC,TableC-2.

his laboratory for submission to the FBIR. The secondcopyofeachsamplewassenttoPaulKeim’slaboratoryat Northern Arizona University. According to theDepartmentofJustice(USDOJ,2010,p.78),onorbeforeMarch28,2002,theUSAMRIIDstaffwhohadcollectedthat laboratory’sAmes strain samples for submission totheFBI“advisedDr. Ivinsandhis laboratory technicianthattheirsubmissionswerenotpreparedaccordingtotheFBIRprotocol.”TheDOJreportcontinues:“Specifically,Dr.Ivinsandhislabtechnicianusedhomemadeslantsasopposed to the commercially available Remel slantsspecifiedby theprotocol, so the four slantspreparedonFebruary 27, 2002 were rejected by the FBIR, and Dr.Ivins was told to resubmit his culture samples on theappropriate slants” (p. 78). The FBI disposed of itsrejected samples but, importantly, the duplicate copiessent to Keim’s laboratory were retained, including onethathadcomefromflaskRMR1029andthatwouldlaterbe shown to contain all four genotypes. In April 2002,Ivins submitted four newly prepared samples, again induplicate,and this time thesampleswereaccepted.One

copyofeachsampleenteredtheFBIRandtheduplicateswereagainsenttoNorthernArizonaUniversity.

Inthecourseoftheinvestigation,certaindoubtswereraisedaboutwhetherIvinshadsubmittedsamplesofallrelevantAmesstocksinhispossessiontotheFBIR.InApril2004, theFBI securedRMR1029andotherB.anthracissamples thathadbeenunderIvins’scontrol,andtheseweretransferredelsewhereforvarioustests.Fromthesetests,theFBIconcludedthat:“Geneticanalysisdeterminedthat...RMR1029—thepurestandmostconcentratedbatchofAmessporesknowntoexist—wastheparenttotheevidentiarymaterialusedintheanthraxmailings”(USDOJ,2010,p.79).Thecommitteeaddressedthisconclusion(seediscussionabove)andalsofocusedonsecondaryanalysesperformedinordertoreconcilecertaindiscrepancies in themultiple samples thatwere submittedby IvinsandpurportedtocomefromflaskRMR1029.

FBIinvestigatorshadobservedthatthesecondRMR1029samplesubmittedbyIvins in April 2002 did not score positive for any of the four genotypesdiscovered in theattack lettersand inRMR1029aswellas insamplesderivedfromRMR1029thatweresubmittedbyotherscientists.Basedonthisapparentdiscrepancy,inlate2006theFBIobtainedfromNorthernArizonaUniversitytheduplicate of the first RMR1029 sample that Ivins had submitted in February2002,whichwas thenput into theFBIRandanalyzed.This earlierRMR1029samplescoredpositiveforallfourofthegenotypesthatwereassayed(A1,A3,D,andE),whereasthelatersamplehadscoredpositivefornoneofthem.

TheFBI sought todetermine thecauseof thisdiscrepancybetween theearlierandlatersubmissionsbyIvins thatbothweresupposedtohavecomefromthesameRMR1029 flask.One possibility (the null hypothesis)was that repeatedsamples from RMR1029 following the FBI protocol might produce variableresults.Thispossibilitycouldreflect,forexample,insensitivityofthemoleculartestsatthegenotypicfrequenciespresentinthatflask.Toaddressthatissue,andontherecommendationofexternalscienceadvisors(the“RedTeam”;USDOJ,2010, p. 79), the FBI directed that an experiment be performed in which theRMR1029 flask was sampled in an identical manner 30 times following thesubpoena instructions.These replicate sampleswere processed at theNationalBioforensicAnalysisCenterandtheresultingmaterialanalyzedbyscientistsatCBI,MRI,IITRI,andtheUniversityofMarylandforthepresenceofthesamefourgenotypes.According to theDOJsummaryof thecase (USDOJ,2010,p.

79), the resultswere as follows: “Occasionally, only three of the four geneticmutations were detected, and at no time were less than three detected. ItfollowedthatifDr.Ivinspreparedhissubmissiontotherepositoryinaccordancewiththeprotocol,thatsubmissioncouldnotmissallfourofthemorphological

variantspresentinRMR1029.”7

Table6-6presentstheresultsofthese30analyses.Thesedata demonstrate substantial variability among the 30replicates.Sixteenof the30 samples scoredpositive forall fourmutations and another eight scored positive forthreeofthefourmutations;however,fivesamplesscoredpositiveforonlytwomutations,andonesample(Sample20) scored positive for just one of the four mutations.(Inconclusive results, as well as cases where the IITRIandMRI tests for theDgenotypegavedifferent results,wereconsidered“negative”outcomesbythecommittee.)None of the 30 samples scored negative for all fourmutations.

Given these assay results of the 30 replicates, what is the probability thatanalysis on an additional sample, taken from RMR1029 in the samemanner,wouldyieldnegativeresultsforallfourgenotypesbychancealone?Sincenoneofthe30sampleswasnegativeforallfourassays,theprobabilityofobtainingasampleoffournegatives,bychancealone,cannotbeveryhighand,accordingto

thebinomial distribution8it isunlikelytobehigherthan0.095(9.5percent).

At the January 2011 meeting with the FBI, the committee was told that thesecondcopyofthisdisputedsubmissionwasalsoanalyzedandtestednegativefor all four geneticmarkers.That is, the second copy of Ivins’ disputedApril2002submission,keptatNAUalongwiththesecondcopiesofallsubmissions,was moved into the FBIR, given a new identification number, retested, and

found to be negative for all four genetic markers. If the assays on these twosampleswereasindependentofoneanotherastheassaysperformedonthe30samplesinTable6-6,thentheprobabilitythatbothsetsofassayswouldyield

7 On October 15, 2010, The DOJ issued an erratumstating: “Based on a ‘Red Team’ recommendation,experiments were prepared at the direction of the FBILabtoaddresstheFBIRsubmissionprocesswithregardto RMR1029. RMR1029 was sampled 30 times inaccordance with the subpoena instructions. In a fewinstances, fewer than three markers were detected.However, in none of the 30 attempts were no markersdetected. It followed that if Dr. Ivins prepared hissubmission to the repository in accordance with theprotocol, that submission could notmiss all four of themorphologicalvariantspresentinRMR1029.”

8 That is, if the probability of a sample having fournegatives were greater than 0.095, the probability ofobtaining no such samples among the 30would be lessthan(1−0.095)30=5%.

TABLE6-6ResultsObtainedbyResamplingfromFlaskRMR1029

A1A3DIITRIDMRIESample1+++++

Sample2+++++

Sample3+++++

Sample4+++++

Sample5+++++

Sample6+++++

Sample7++Negative++

Sample8+++++

Sample9+++++

Sample10Inconclusive+InconclusiveInconclusive+

Sample11+++++

Sample12Inconclusive+InconclusiveNegative+

Sample13Inconclusive+NegativeInconclusive+

Sample14Negative++++


Sample16+++++

Sample17+++++


Sample19+++++

Sample20NegativeInconclusiveNegativeNegative+

Sample21+++++


Sample23+++++

Sample24Negative+NegativeNegative+

Sample25Inconclusive++++

Sample26+++++


Sample28+++++


Sample30Negative+InconclusiveNegative+

NOTES:+=positive;IITRI=IITResearchInstitute;MRI=MidwestResearchInstituteSOURCE:FBIDocuments,B2M10D2.

negativeresultsforallfourgenotypeswouldbe(0.095×0.095)=0.009025(or

0.9percent).Whileitisstillpossiblethattwosuchresultscouldhaveoccurredbychancealone,thischanceisverysmall(lessthanonepercent).


During the course of the committee’s review of the scientific evidence, theDepartmentofJusticeofficiallyclosedtheinvestigation.Thecommitteeisnotinapositiontoofferajudgmentabouttheimportanceandstrengthofthescientificinvestigationrelativetotheimportanceandstrengthofthecriminalinvestigativecomponentofthiscasebecauseitwasnotchargedwith(andlackedtheexpertisefor)reviewingthelatter.Ourmajorfindingisthat:

ItisnotpossibletoreachadefinitiveconclusionabouttheoriginsoftheB.anthracisinthemailingsbasedontheavailablescientificevidencealone.

Finding6.1:TheFBIappropriatelydecidedtoestablisharepositoryofsamplesoftheAmesstrainofB.anthracis thenheld invarious laboratoriesaround theworld.Therepositorysampleswouldbecomparedwiththematerialfoundintheletters to determine whether they might be the source of the letter materials.

However,foravarietyofreasons,therepositorywasnotoptimal.Forexample,the instructions provided in the subpoena issued to laboratories for preparingsamples (i.e., the“subpoenaprotocol”)werenotpreciseenough toensure thatthelaboratorieswouldfollowaconsistentprocedureforproducingsamplesthatwouldbemostsuitableforlatercomparisons.Suchproblemswiththerepositoryrequired additional investigation and limit the strength of the conclusions thatcanbedrawnfromcomparisonsofthesesamplesandthelettermaterial.

The FBI and contract scientists appropriately recognized that themutations intheletterisolatesprovidedinformationthatmighthelpidentifythesourceoftheB.anthracisusedintheattacks,developedappropriateassaysforfourof thesemutations,andcreatedandscreenedarepositoryofAmesstrainsamples.Basedon theresultsof thatscreening,FBIscientistsappropriatelyconcluded that themajorityofrepositorysamplescontainednoneofthefourmutations,although50ofthesamplescontainedoneofthefourmutationsand10sampleshadthreeorall fourmutations (the numberswith one ormoremutations are higher if oneincludes samples that were excluded in the FBI’s statistical report). However,featuresoftherepositoryincludingunknownsampleprovenance,andthehistoryofsharingandmixingofstocks,presentedinvestigativechallenges.

The first challenge with the repository was the lack of independence amongsamplesandan incompleteunderstandingof theprovenanceofsamplesdue tothe known history of sharing. Sharing of samples between laboratories is animportantpartofscientificresearchandiscriticaltotestingreproducibilityandfurthering scientific analysis. Prior to the attacks of 2001, several institutionssharedsamplesof theAmesstrain todifferentextents, resulting invariation inthenumbersandkindsofsamplestheysubmittedtotheFBIR.Also,thissharingextended not only to substrains but also tomixtures of several substrains thatwere grown in separate batches and then pooled. While such sharing andtransfers are important in scientific research, they make it more difficult toidentify a unique source of the mutations found in the attack materials. Inrecognition of this important issue, FBI scientists and investigators sought todetermine the history of shipments among institutions and the genealogicalrelationships among samples in the repository, but they never obtained acompleterecord.

Another challenge with the repository was that, since the importance of themutant genotypes was not fully understood when the subpoena protocol waswritten, thedocumentwasvague(e.g.,“useaninoculumtakenacrossmultiple

colonies”), andwasnotwritten inaway thatwouldmaximize thechance thatvariantgenotypesinamixedstockpopulationwouldbesubmitted.Thus,ifthefour assayed genotypes had been present in a laboratory culture at lowfrequency, it is not clear whether they would have found their way into thesampleof theculturesubmitted to the repository, sinceas fewas twocolonieswould have satisfied the instructions provided in the subpoena protocol.Aftertheimportanceofthemutantgenotypesbecameknown,therewasnorequestforadditional samples using a revised protocol that might have improved thesampling.

A final challengewas that the repository collection processwas based on theintegrity of the individuals asked to provide samples. If the motive for therepositorywastoidentifythesourceofthelettermaterial,standardsofcustodyof evidence would dictate that agents of the FBI should have obtained thesamples. In most instances, holders of the material were asked to providesamplesandsend them in.Thesendercouldhavebeen the instigatorandmaynothavecompliedwithinstructions,astheFBIallegeswithrespecttoDr.Ivins.

Finding 6.2: The results of the genetic analyses of the repository sampleswere consistentwith the finding that the spores in the attack letterswerederived fromRMR1029,but theanalysesdidnotdefinitivelydemonstratesucharelationship.

The scientific data alone do not support the strength of the government’srepeated assertions that “RMR1029 was conclusively identified as the parentmaterialtotheanthraxpowderusedinthemailings”(USDOJ,2010,p.20),norstatementsabouttheroleofthescientificdatainarrivingattheirconclusions,asin “the scientific analysis coordinated by the FBI Laboratory determined thatRMR1029, a spore-batch created andmaintained atUSAMRIID byDr. Ivins,wastheparentmaterialfortheanthraxusedinthemailings”(USDOJ,2010,

p.8).

Thecommitteeagreesthat thegeneticevidenceisconsistentwithandsupportsanassociationbetweentheRMR1029flaskandtheB.anthracisusedinthethreeattacklettersthatweretested;however, thereareseveralimportantcaveats.Asdiscussed above, the nature of the repository collection, including theincompletelydocumentedhistoryof sharingandmixingofAmes strain stocksand the ambiguity in the subpoena protocol,makes it difficult to quantify the

strengthoftheevidencelinkingRMR1029totheletters,becauseofthecomplexand ill-defined nature of the reference population. Thematerials from at leastthreeof thefourattacklettersharboredamixtureofseveralvariantgenotypes.By contrast, the repository may include samples recently derived from singlecolonies, as is customary for microbiological research, but it is unlikely thatthesesamplesbyvirtueofthisrecenthistorywouldhavemorethanoneofthevariant types. Moreover, owing to the practice of sharing samples amonglaboratories,manysampleswereessentiallyduplicatesofoneanother.Itisalsopossible that the sample repository was incomplete because the globaldistributionofAmesstockswasnotknownorbecausesomestocksmighthavebeendestroyedprior to thesubpoena.These limitationsmade it impossible forthe committee to generate any meaningful estimate of the probability of acoincidentalmatchbetweentheB.anthracisgenotypesdiscoveredintheattacklettersandthoselaterfoundbyscreeningsamplesfromtheRMR1029flask.

Finding 6.3: Some of themutations identified in the spores of the attackletters and detected inRMR1029might have arisen by parallel evolutionrather than by derivation from RMR1029. This possible explanation ofgenetic similarity between spores in the letters and inRMR1029was notrigorously explored during the course of the investigation, furthercomplicating the interpretationof theapparentassociationbetween theB.anthracis genotypes discovered in the attack letters and those found inRMR1029.

Another challenge with determining the cause of the apparent associationbetweensomeoftheB.anthracisgenotypesintheattacklettersandthosefoundin RMR1029 stems from the possibility that the same mutations might havearisenrepeatedlyinotherAmesstrainpopulations.Colonyvariantswithsimilaroreven identicalmutationsmightarise repeatedly inotherpopulationsfor tworeasons.First,wedonotknow thenumberand rateofpossiblemutations thatcould produce similar phenotypes.Research byWorsham and colleagues (see,forexample,WorshamandSowers,1999)identifiednumerousoligosporogenousvariantswithphenotypessimilartothosedescribedintheletters.Inresponsetoquestionsraisedbythecommittee,theFBIindicatedthatamongthe296AmessubmissionstotheFBIRbyWorsham,onlytheMorphDgenotypewasdetected,anditwasinthreesamples.TheA1,A3andEmutationswerenotdetectedinanyoftheWorshamsamplessubmittedtotheFBIR(FBI,2010a).Second,undertheconditionsusedtogrowB.anthracisforthelarge-scaleproductionofspores,there may well have been inadvertent selection that favored oligosporogenic

mutants,whichwould cause the frequency of thesemutants to be higher thanexpected for mutations that conferred no advantage to the cells, therebyincreasingthelikelihoodofparallelevolutioninreplicatesporeproductions.TherecentpublishedworkbySastellaandcolleagues(2010)highlightsthepossiblerole of repeated passage of B. anthracis in the enrichment of sporulation-deficientmutants.

Finding6.4:ThegeneticevidencethatadisputedsamplesubmittedbythesuspectcamefromasourceotherthanRMR1029wasweakerthanstatedintheDepartmentofJustice,AmerithraxInvestigativeSummary.

The committee reexamined the data that the FBI obtained following thediscoverythatoneofthesamplessubmittedbyBruceIvins,whichwassupposedto have been taken from RMR1029, did not test positive for any of the fourassayed mutations (A1, A3, D, and E) in either of two copies analyzed. Asdiscussed in Section 6.6, an experiment was performed in which 30 replicatesamplesweretakenfromRMR1029accordingtotheFBIsubpoenaprotocolandtestedforthefourmutations.Basedontheseresults,thecommitteefoundthatitis, in fact, possible that the disputed sample came from RMR1029, and theprobabilityof thisoutcome—that anactual sample fromRMR1029would testnegativeforallfourgenotypesintwosetsofassays—mightbeontheorderof1percent. Hence, while the evidence is strongly suggestive that the disputedsamplewasnot takenfromRMR1029, it is lesscertainthanis indicatedintheoriginalversionofthecase-closingsummaryissuedbytheDOJ,whichassertedthat all 30 additional samples scored positive for at least three of the fourgenotypes, and concluded that “It followed that if Dr. Ivins prepared hissubmission to the repository in accordancewith the protocol, that submissioncould not miss all four of the morphological variants present in RMR1029”

(USDOJ,2010,p.79).9

Finding6.5:The scientificdatageneratedbyandonbehalf of the FBI provided leads as to a possiblesource of the anthrax spores found in the attackletters, but these data alone did not rule out othersources.

Thecommitteewasnotchargedwithreviewing,norwasitgivenaccessto,thefindingsfromthecriminalinvestigationcomponentofthiscase.Thecommitteetherefore could not assess the potential value of additional scientificinvestigationwithrespecttobetterestablishingthesourceoftheB.anthracis

9Seefootnote7.

attack spores.Additional experimentsmightbeofvaluewere there a need to strengthen the scientific aspects ofthe case (in particular, if the case depended on thefindings of the scientific investigation component asopposedtothecriminalevidence).

Finding 6.6: Point mutations should have been used in the screening ofevidentiarysamples.

TheFBIchosetostudyonlyasubsetofthemutationalvariantsofB.anthracisfound in the attack letters. In particular, certain point mutations were notinvestigated, apparently because FBI scientists regarded them as less stable,more difficult to screen for their presence in the repository, or both. In thecommittee’s view, key evidentiary samples in the repository (along withappropriatecontrols)shouldhavebeenscreenedforallofthemutationsfoundinalloftheletters.Thestabilityofpointmutationsshouldnothavebeenaconcern,asmostpointmutationshaveextremelylowratesofreversionandmostof themethods and data used to track the spread of infections rest on this stability.Moreover, the forward rate of deletion mutations and both the forward andreverse ratesof insertionmutations (the typesofmutations screened for in theA1,A3,D, andE genotypic assays; seeChapter 5 andTable 5.2) are usuallyhigherthanthecorrespondingratesforpointmutations.Thus,thepossibilitythatidenticalmutationsmayariseindependently(inparallel)isgreaterforinsertionsand deletions than for point mutations.With regard to the concern about thegreater difficulty of screening for point mutations, it should be feasible tosequence directly the relevant genes in a large number of samples, includingthose that are genotypic mixtures such as RMR1029 and the letter samples,usinghigh-throughput“next-generationsequencing”methods(seeFinding6.8).

Finding 6.7: Biological material from all four letters should have beenexamined to determine whether they each contained all four geneticmarkersusedinscreeningtherepositorysamples.

TheFBIobservedmorphologicalvariants in theB.anthracis isolatedfromtheattacklettersandusedthosevariantstoidentifymutationsthatwerethenusedasgenetic markers during the systematic screening of the repository samples.However, theFBIdidnotsystematicallyexaminethematerialsfromtheattackletters to determine whether all of these markers were present in each lettersample (possibly owing to concerns about the limited amount of biologicalmaterialfromsomeoftheletters).AndalthoughgenotypesA1,A3,andEwereidentified in all three letters that were examined (New York Post, Leahy,Daschle), the D genotype was found only in the New York Post letter. Thematerial in theBrokaw letterwasnotexamined for thepresenceofanyof thegenotypes,sotheFBIcouldonlyinferfromothernongeneticevidencethatthebiologicalpropertiesofthematerialsinthisletterwerethesameasorsimilartothematerialsintheNewYorkPost,Leahy,andDaschleletters.

Inthecommittee’sview,itwouldhavebeenusefultodeterminewhetherallofthe genotypes were present in all of the letters and, if so, at what relativeabundances. This issue is important because similarmorphotypes can and didarise from different mutations, and multiple mutations producing similarphenotypeswere present in some of the letters. Thus, the presence of similarmorphotypes in different letters does not mean that the same genotypes werepresent in all the letter samples. Given the conspicuous differences in thephysicalpropertiesof someof the letter samples, it is evenmore important toestablish their genetic similarities and, if relevant, consider the possibleimplicationsofanygeneticdifferencesthatmighthavebeenfound.

Finding 6.8: New scientific tools, methods, and insight relevant to thisinvestigationbecameavailableduringitslateryears.Animportantexampleishigh-throughput “next-generation”DNA sequencing.Theapplicationofthese tools,methods, and insightmight clarify (strengthenorweaken) theinferenceofanassociationbetweenRMR1029andthesporesintheattackletters.Suchapproacheswillbeimportantforuseinfuturecases.

When the committee began its deliberations the FBI’s anthrax lettersinvestigation was still open, although the FBI had publicly declared itsconfidence in having identified the sole person responsible for the mailings.

During the committee’s review of the scientific evidence, the DOJ officiallyclosedtheinvestigation.Thecommitteeisnotinapositiontoofferajudgmentabout the importance and strength of the scientific investigation evidencerelative to the importance and strength of the criminal investigation evidence,because it was not charged with (and lacked the expertise for) reviewing thelatter.

Since2001,importanttechnologicaladvanceshaveoccurredthatwouldallowamore thorough and systematic analysis of genetic similarities and differencesbetweenkeyevidentiarysamples, repositorysamples,andappropriatecontrols.Whentheinvestigationbegan,bacterialgenomesweretypicallysequencedatanaveragecoverageoffewerthan10readspernucleotide(see,forexample,Readetal.,2002).Given theerror rates inherent insequencing technologiesand thecostsofwhole-genomesequencingatthattime,itwasfeasibletosequenceonlya few well-chosen clones as was done first to compare an isolate from theFloridavictimwiththePortonDownAmesstrain(Readetal.,2002),andthensearchformutationsinseveralmorphotypes(asdescribedinChapter5).

Itwasalsonoteasyin theearly2000s tosequenceandinterpret thedatafrommixturesofbacterialgenomes.However, thatchangedduringthecourseoftheinvestigation and it is now possible to sequence bacterial genomes, evenincludingminority components of heterogeneous populations, tomuch greaterdepths of coverage much more quickly, allowing discovery of geneticpolymorphisms (mutationalvariants) inbacterialpopulationswithout requiringphenotypic discrimination (Barrick and Lenski, 2009; Holt et al., 2009).Appropriatestatisticalmethodsmust thenbeused todistinguishvariations thatreflect errors in sequencing from mutations actually present in the mixture.Importantly, any application of next-generation sequencing technologies tosamples and evidentiarymaterial from this case would have required that themethodsbevalidatedforusewithsamplesandmaterialofthistypeandfortheintendedpurposesandquestionsathand.

With such technologies and methods, one would likely have discoveredadditionalpolymorphismsinRMR1029andthelettersamples.Totheextentthatanynewpolymorphismswerefoundtobeconcordantbetweentheseevidentiarymaterials(withinstatisticallimits),thatwouldstrengthenthegeneticassociationbetweenthebacteriafromthelettersandthosefromthesuspectflask.Itisalsoconceivable,however,thatsuchadditionalanalysesmighthaverevealedfurthermutations in the samples from the attack letters that were not present in

RMR1029, thereby weakening the link between the evidentiary material andRMR1029. Thus, while it was not feasible at the start of the investigation,investigators should have subsequently examined the value from a forensicperspectiveof “deep sequencing”of key samples.Given the limitationsof theexisting repository, it is uncertain whether this further scientific investigationwould have identified an altogether different source of theB.anthracisattackspores,butitcouldhaveprovidedadditionalinformationontheprocessusedtogeneratethematerialintheattackletters.

More generally and looking forward, the committee anticipates that deep-sequencing methods (including, as appropriate, metagenomic analyses ofenvironmentalsamplesofdiversemicrobialcommunities)willbean importantforensic tool in future investigations of any similar event involvingmicrobialpathogens.

It should be noted that future biological attacks may pose even greaterchallengesthandidthisattack.Forexample,thebiologicalagentmaybelongtoaspecieswithamorecomplexandlesswellunderstoodpopulationstructure,itmay be genetically modified in a manner that obscures its origin, or a directsampleof theattackmaterialmaynotbeavailable.This lastpossibilitymeansthat environmental and clinical samples,with their additional challenges,mayhavegreaterimportanceinafutureinvestigation.

Finding 6.9: The FBI faced a difficult challenge in assembling andannotatingtherepositoryofB.anthracisAmessamplescollectedforgeneticanalysis.

Muchofthechallengeinassemblingandannotatingtherepositorywasinherenttothetypesofmaterialsinvolved,whichincludedstocksthathadbeenderived,sampled(orcombined),andthenstoredfordifferentperiodsinseveralwaysandfor diverse scientific purposes in many laboratories. The FBI collectedsubstantial information, or metadata, about many or all of these samplesincludingdetailsofhistoricalderivation,modeofsamplingandstorage,andsoforth.Aswasappropriate,thegeneticscreeningofthesamplesinthecollectionwasperformedina“blind”fashionsothatthisinformationwouldnotinfluencethe test results. If a future bioterrorism event requires the establishment of asamplerepository,attentionshouldbepaid to thestructureof thedatabaseandtheinclusionof(orwaystolinkto)anyandallrelevantmetadata.

Finding 6.10: The evidentiary material from this case is, and will be,immensely valuable, especially in the event of future work on either thiscaseorothercasesinvolvingbiologicalterrorismorwarfare.Itiscriticallyimportant to continue to preserve all remaining evidentiarymaterial andsamplescollectedduringthecourseofthis(theanthraxlettersinvestigation)andfutureinvestigations,includingtheoverseasenvironmentalsamples,forpossibleadditionalstudies.

Recent and future advances in scientificmethods and insightmay provide themeans to extract additionalvaluable information fromcase-associatedmaterialand samples. In addition, in the event of a future biological attack, thesematerials and samples may prove useful for comparative analyses. Therefore,despite the closure of this case, all remaining case-associated materials andsamplesshouldberetainedandpreservedforpossiblefurtherstudies.

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IndexofDocumentsProvidedbytheFederalBureauof

Investigation1

BATCH1

Module1:TechnicalReviewPanelMeetings

TechnicalReviewPanels

1. November5,2001WFOAnthraxExpertPanelReview(pp.2-27)2. December7,2001ExternalTechnicalReviewPanel(pp.28-39)3. December12,2001ExternalReviewofAnalyticalPlan(pp.40-46)4. June11-12,2002ProgresstoDateReview(pp.47-65)5. August8,2005ChemistryReviewPanel(pp.66-195)

Module2:USAMRIID

Initial examination of the letter spore preparations for physical characteristics(microscopyandelectronmicroscopy)andsporeviabilitystudies.

1. 18Oct2001SPS02.57(Daschle)CFUReport(p.3)2. 21Oct2001EMReportofDasch1eLetter(pp.4-19)

1 The Committee on the Review of the ScientificApproachesUsedDuring the FBI’s Investigation of the2001Bacillus anthracisMailings initially received FBImaterials related to the scienceof theAnthraxMailingsInvestigationintwobatches.Athirdbatchofdocumentswas provided later. In addition to the documents in thethree principal batches of materials, the FBI alsoprovidedseveralsupplementaldocuments in response tocommittee requests. Unless otherwise noted, this indexreflects the order in which the materials were received

from theFBI, themanner inwhich thedocumentswereorganizedbytheFBI,andthelanguageusedbytheFBIwhen describing the documents. Page numbers inparentheses after document titles are the page numbersassigned to thedocumentby theFBIor, in caseswherethe FBI did not assign page numbers, the number ofpagesintherespectivedocument.

161

1. 24Oct2001SPS02.88.1(NYPost)CFUReport(pp.20-21)2. 25Oct2001EMDaschle“Si”Report(pp.22-25)3. 25Oct2001SimonsLetterreNYPost(pp.26-28)4. 28Oct2001EM/CFUReportofNYPost(p.29)5. 27Nov2001ReportonIsolatesfromDaschleandNYPostLetters

(p.30)

SpecialPathogensSampleTestLaboratoryAnalyticalTestReports:Results ofAnalysisofLetterMaterial

1. SPS.02.88NYPostPowder10/22/2001(pp.32-33)2. SPS.02.44BrokawEnvelope11/4/2001(pp.34-35)3. SPS.02.57DaschleLettersandPowders11/9/2001(pp.36-45)4. SPS.02.266LeahyPowder(pp.46-53)

Microbiological examinations and identification of phenotypic variants(Morphotypes) which appeared different than the predominant “Ames”phenotype

1. Report#1AnalysisofEvidentiaryMaterial(pp.55-68)2. Report#2AnalysisofRepositorySamples(pp.69-79)3. Report#3AnalysisofEnvironmentalSamples-AMI(pp.80-90)4. Report#4ExaminationofRepositorySporePreparations:Screeningforthe

HemolyticB.subtilisContaminant(pp.91-95)5. Report#5AnalysisofRepositorySamples(pp.96-99)

Isolation of Morphological Variants from FBI Repository Samples FBIR 049004(Leahy)PowerPointPhotos

17. Isolation of Morphological Variants from FBIRepository Samples FBIR 049 004 (Leahy) PowerPointPhotos(pp.101-109)

Module3:AmesStrainIdentification

Reports

1. Forensic Analysis of Putative Anthrax Samples, Batch E0001 v02.01.02(MLVA-8)(pp.3-10)

2. MLVA-15MolecularTyping(BatchE0001)4/16/02(pp.11-14)3. Laboratory Reports NAU-0001 (May 30, 2002) to NAU-0038 (May 5,

2008)(pp.15-167)

GuidelinesandStandardOperatingProcedures

1. GuidelinesandStandardOperatingProceduresforForensicAnalysisV09.2303(pp.168-204)

2. GuidelinesandStandardOperatingProceduresforForensicAnalysis:Real-TimePCRspeciesSpecific,CanonicalandStrainSpecificSNPGenotypingofBacillusanthracisandFrancisellatularensisV11.03.04(pp.206-231)

3. Real-TimePCRspeciesSpecific,CanonicalandStrainSpecificSNPGenotypingofBacillusanthracisV04.11.07(pp.232-272)

4. QualityAssuranceStandardsforForensicDNAAnalysis(pp.273-280)

Literature

8.“Multiple-LocusVariable-NumberTandemRepeatAnalysisRevealsGeneticRelationshipswithinBacillusanthracis”(Keimetal.,2000,182:2928-2936)(pp.282-291)

ProgressReports

9.NAUDNA-BasedStrainTypingofAnthraxSamples(pp.293-649)2

Module4:AnalysisforEvidenceofGeneticEngineering

Reports

1. 19October2001LANLReceiptletter(pp.3-4)2. LaboratoryReports(pp.5-55)

Presentation

3.AnalysisoftheAmerithraxB.AnthracisAmesIsolatesforEvidenceofGeneticEngineering(pp.57-79)

Module5:GenomicSequencing

Publications

1. “GlobalGeneticPopulationStructureofBacillusanthracis,”M.VanErtetal.,(2007)PLoSONE2(5);e461.doil-.1371/jpornal.pone.0000461(pp.2-11)

2. “TheCompleteGenomeSequenceofBacillusanthracisAmes‘Ancestor,’”J.Raveletal,J.Bacteriology,Jan2009,Vol191,No.1,

2TheFBIdidnotassignanametothisdocument.Thedescriptorwasassignedbythecommittee.

p.445-446(pp.12-13)

Reports

1. GenomicAnalysisofBacillusanthracisIsolatesRelevanttotheAmerithraxInvestigation,June1,2004(MorphA,B,C,D,Wildtype)(pp.17-75)

2. GenomicAnalysisofBacillusanthracisIsolatesRelevanttotheAmerithraxInvestigation,June4,2005(MorphE(Opaque,Post/LeahyB.s.)(pp.76-159)

3. MultipleLocusPCR-basedAssayfortheDirectComparisonofunknownB.subtilisisolatestoB.Subtilis,NewYorkPost,May15,2006(pp.160-

220)

TIGRProgressReports

6.TIGRProgressReports(pp.221-458)

Module6:SandiaNationalLaboratory(SNL)

Reports

1.SandiaNationalLaboratoryFinalReport(pp.3-42)

HistoricalLiterature

1. “DistributionofCalciumandOtherElementsinCryosectionedBacilluscereusTSpores,determinedbyHigh-ResolutionScanningElectronProbeX-RayMicroanalysis,”M.Stewart,A.P.Somlyo,A.V.Somlyo,H.Shuman,J.A.Lindsay,W.G.Murrell,J.Bacteriology,July1980,Vol143,No.1,pp.481-491(pp.44-54)

2. “ScanningElectronProbeX-RayMicroanalysisofElementalDistributioninFreeze-DriedCryosectionsofBacilluscoagulansSpores.”M.Stewart,A.P.Somlyo,A.V.Somlyo,H.Shuman,J.A.Lindsay,W.G.Murrell,J.Bacteriology,Aug.1981,Vol147,No.2,pp.670-674(pp.55-59)

3. “AutomatedAnalysisofSEMX-RaySpectralImages:APowerfulNewMicroanalysisTool,”P.G.Kotula,M.R.Kennan,J.R.Michael,Microsc.Microanal.,9,1-17,2003(pp.60-76)

4. SiliconSummaryBibliographyofSelectPublications(pp.77-81)

PowerPointFilesofIndividualSampleResults

6.PowerPointFilesofIndividualSampleResults(pp.82-532)

Module7:ChemistryUnit,FBILaboratory

FBILaboratoryReports

1.020322006April15,2002

2.020605001June18,20025-6)

3.020110004August26,2002

4.020605001October16,200211-12)

5.040624018June28,200413-14)

6.030519001October15,200315-22)

7.031008001October30,2003

8.031124029December11,200326-28)

9.030819015June29,2004

10.050321004April18,2005

11.050118006April18,2005

12.050408005May18,2005

13.050408005July6,2005

ElementalAnalysisSummaryQ13SPS02.88(Post)(pp.3-4)Q12WhitePowder(Leahy)(pp.

Q12,Bc14579,Bs/Ba+/(pp.7-10)Q13SPS02.88Elemental(pp.

LeahyPowderElemental(pp.

CultureMediaElemental(pp.

Burans12Elemental(pp.23-25)10DPGgrowthsElemental(pp.

CommercialMedia(pp.29-34)MediaSaltsandSpores(pp.35-37)19DPGStubsSEM(pp.38-54)2DPGStubsSEM(pp.55-67)2DPGStubsSEM(pp.68-91)

14.1ElementalAnalysisSummaryTable(pp.93-94)

15. Comparison of results from 2 different instruments (by 2 different FBIexaminers,2yearsapartin2differentLabs(HQVSQuantico)

EnvelopesandParticleTransport

16. FBI Laboratory Electron Microscopy of Envelope surfaces and particletransportthruenvelopes(pp.96-111)

Module8:Carbon-14(14C)DatingCenter for Accelerator Mass Spectrometry (CAMS), Lawrence LivermoreNationalLaboratory

1.CAMSbackgroundinformation(pp.3-14)

2.Proposal(pp.15-16)

3.QuantitatingRadiocarbonConcentrationsinIsolatedSamplesofBiologicalOrigin:StandardOperatingProceduresforFBIMeasurementsofNatural14C(pp.17-23)

4.LLNLReport10/14/02(pp.24-29)

5.LLNLReport1/16/04Addendum(pp.30-33)

6.LLNLDataTable3/30/04DPGSamples(pp.34-39)

7.ScientificReferences(pp.40-50)

NationalOceanSciencesAcceleratorMass Spectrometry Facility (NOSAMS),WoodsHoleOceanographicInstitute

8.NOSAMSbackgroundinformation(pp.52-55)

9.GeneralStatementofC-14Procedures(pp.56-57)

10.AmerithraxSampleHandlingProcedures(pp.58-72)

11.FinalReport(pp.73-83)

12.ScientificReferences(pp.84-112)

Module9:StableIsotopes

StableIsotopeReports

1.IsotopicCharacterizationofWaterSamples3/14/03(pp.3-10)

2.IsotopicCharacterizationofAnthraxSamples7/30/03(pp.11-19)

3.IsotopicCharacterizationofWaterSamples10/15/03(pp.20-25)

4.IsotopicCharacterizationofSporeSamples1/5/04(pp.26-31)

5.Stable IsotopeCharacterizationofAnthraxSampleSPS02.2662/22/04(pp.32-44)

6.IsotopicCharacterizationofMicrobialSporeSamples5/20/04(pp.45-53)

7.AReportontheStableIsotopeRatiosofEnvelopeSamples9/17/04(pp.54-58)

8.AReport on the Stable IsotopeRatios of Treated andUntreated Envelopes11/7/04(pp.59-64)

9.AReport on the Stable IsotopeRatios of Treated andUntreated Envelopes12/22/04(pp.65-77)

10.IsotopicCharacterizationofRMR10295/6/05(pp.78-80)

11.ATabulationofStableIsotopesofTapWaterSamplesAnalyzedfortheFBI3/23/05(pp.81-247)

ProposalsandSOWS

12.ProposalsandSOWS(pp.248-269)Kreuzer-MartinPublications

13.Kreuzer-MartinPublications(pp.271-310)

StableIsotopeRatiosandtheForensicAnalysisofMicroorganismsPowerPoint

14. Stable Isotope Ratios and the Forensic Analysis of MicroorganismsPowerPoint(pp.312-342)

Module10:AgarandHemeAnalysis

AgarAssayUniversityofMaryland

1.UnsolicitedProposalSOW(pp.3-8)

2.ProgressReports(pp.9-55)

3.SummaryofChromatograms(pp.56-72)

4.FinalAgarReport(pp.73-94)

5.ReductiveHydrolysisProcedureSteps(pp.95-96)

6.ReductiveHydrolysisLiteratureReferences(pp.97-118)

7.ASMSAbstractPoster(pp.119-120)

AgarAssayValidation-BMI

8.Task4SpotReport27Aug2002(pp.122-126)

9.Task4InterimReport17Sept2002(pp.127-136)

HemeAssay(UMD)

10.SOW(pp.138-149)

11.ProgressReports(pp.150-182)

12.AnalysisofHemebyMALDIProcedure(pp.183-190)

13.FilterSterilizationValidation(pp.191-192)

14.HemeFinalReport(pp.193-216)

15.HemeA.C.publication(pp.217-222)

HemeAssayValidation–ECBC

16.InterimReport(ECBC)(pp.224-238)

17.AnalyticalTestReport(ECBC)(10Aug2005)(pp.239-246)

AgarOverview

18.AgarOverview(pp.248-251)

Module11:PacificNorthwestNationalLaboratory

Reports

1. ResidualAgarDeterminationinBacterialCulturesbyESI/MS,DHSFinalReport(pp.3-99)

2. DetectionofAgar,byAnalysisofSugarMarkers,Wunscheletal.,J.MicrobiolMethods,74,(2008),57-63(pp.101-107)

3. ResidualAgarDeterminationinBacterialCulturesbyESI/MS,K.L.Wahletal.submittedforpublication(pp.108-141)

Presentation

4.FBISamplesDataSummaryPowerPoint(pp.143-197)

Module12:FBILaboratoryRenocalAssay

Reports

1.FBILabReport070829018(pp.2-3)StandardOperatingProcedures

1. MeglumineDiatrizoateAnalysisbyLC/MS/ESI(pp.5-26)2. PerformanceMonitoringProtocol(QA/QC)fortheFinniganLTQLC/MS

(ESI)Instrument(pp.27-45)

Reference

4.DetectionofTraceAmountsofMeglumineandDiatizoatefromBacillusSporeSamplesUsingLiquidChromatography/MassSpectrometry;submittedtoAnal.Chem.(pp.47-74)

Module13:DPGProductionMethods

TestPlan

1.TestPlan(pp.3-24)ProjectOverviewPowerPoint

2.ProjectOverviewPowerPoint(pp.26-60)

INDEXOFDOCUMENTSPROVIDEDBYTHEFBI

FinalReportDated2/1/06

3.FinalReportDated2/1/06(pp.62-118)SEMPhotosofProduction

4.SEMPhotosofProduction(pp.120-255)

Module14:PreparationofB.anthracisAmesonCommercialandPreparedMedia

Report

1.PreparationofB.anthracisAmesSporesonCommerciallyPreparedMediaandMediaPreparedatUSAMRIID(pp.3-6)

BATCH2Module1:B.SubtilisContaminant

Battelle

1.ReportofisolationofcontaminantfromBrokawletterin“SummaryofMicrobiologicalAnalysis19Oct2001”(pp.3-5)

CentersforDiseaseControl

2.ReportofidentificationofcontaminantsubmittedbyBMIasB.subtilisincludingmorphology,hemolysis,gramstaining,antibioticresistanceand16srRNAsequencematch(pp.7-19)

Novazymes

3.IdentificationoftheNYPostcontaminantasB.licheniformis(p.21)AppliedBiosystems

4.IdentificationoftheB.subtilisisolatedfromtheBrokawandNYPostlettersby16sribosomalRNAgenesequenceanalysis(pp.23-32)TIGR

GenomicAnalysisofBacillusanthracisIsolatesRelevanttotheAmerithrax

Investigation,June4,2005(MorphE(Opaque),Post/LeahyB.s.)(seeBatch1,Module5,Document3)

MultipleLocusPCR-basedAssayfortheDirectComparisonofUnknownB.subtilisIsolatestoB.subtilis,NewYorkPost,May15,2006(seeBatch1,Module5,Document4)

Module2:WholeGenomeAssemblyofB.subtilisIsolate

TechnicalProposalsandSOW

1. MicrobialGeneticServicesinSupportoftheAmerithraxInvestigation(FBISOW)(pp.3-42)

2. WholeGenomeAssembly,ClosureAnnotationofBacillussubtilisGB22(TO1)(pp.43-57)

3. MultipleLocusPCR-BasedAssayoftheDirectComparisonofOneUnknownB.subtilisIsolatetoB.subtilisNewYorkPost(TO3)(pp.58-74)

4. TrainingforUseofAffymetrixComparativeGenomicHybridizationArrays(TO4)(pp.75-91)

5. DevelopanAnnotationFileSpecifictotheFBIB.SubtilisComparativeGenomicHybridizationArrays(NewTO5)(pp.92-101)

ProgressReports

1. WholeGenomeAssembly,ClosureandAnnotationofBacillussubtilisGB2212/07-4/08(TO1)(pp.103-106)

2. MultipleLocusPCR-basedAssayoftheDirectComparisonofOneUnknownB.subtilisIsolatetoB.subtilisNewYorkPost1/08-4/08(TO3)(pp.107-114)

3. MultiplelocusPCR-BasedAssayoftheDirectComparisonofOneUnknownB.subtilisIsolatetoB.subtilisNewYorkPost5/08(TO3)(pp.115-122)

4. AffymetrixGB22TilingGeneChipSOP(pp.124-136)

StandardOperatingProceduresFinalReports

1. Whole Genome Assembly, Closure and Annotation of Bacillus subtilisGB22(TO1)(pp.138-142)

2. Multiple Locus PCR-Based Assay of the Direct Comparison of OneUnknownB.subtilisIsolate toB. subtilisNewYorkPost (TO3) (pp. 143-

166)3. GenomeMTVManual(TO4)(pp.167-185)

Module 3: Genetic Diversity and Phylogenetic Characterization of B.subtilis

StatementofWork(SOW)

1.GeneticDiversityandPhylogeneticCharacterizationofB.subtilis(pp.3-14)

ProgressReports

2. 8 Monthly and 1 Quarterly Report 12/06-11/07 (pp. 16-79) StandardOperatingProcedures

3.Allele-SpecificOligonucleotide(ASO)TypingAssayforBacillussubtilis(pp.81-82)

FinalReports

4.GeneticDiversity and PhylogeneticCharacterization ofB. subtilis11/30/07(pp.84-124)

Module4:B.subtilisScreening

StandardOperatingProcedures

1.BacillussubtilisAnalysisbySingleplexReal-TimePCRStandardOperatingProcedure(pp.3-10)

SynopsisofAssayDevelopment

2.SynopsisofAssayDevelopment(pp.11-55)

StandardOperatingProcedures

3.AnalyticalPlan,AcceptanceofWork(AOW)40(pp.57-104)

AnalyticalPlans

1. AnalyticalPlan,AcceptanceofWork(AOW)46(pp.106-115)2. AnalyticalPlan,AcceptanceofWork(AOW)47(pp.116-183)3. AnalyticalPlan,AcceptanceofWork(AOW)57(pp.184-187)4. AnalyticalPlan,AcceptanceofWork(AOW)58(pp.188-193)5. AnalyticalPlan,AcceptanceofWork(AOW)62(pp.194-239)6. AnalyticalPlan,AcceptanceofWork(AOW)68(pp.240-244)

AssayValidation10.SerialDilution/LOD

NBFAC.061117.001(pp.246-253)

11.AssayValidation NBFAC.061019.002(pp.254-259)

LaboratoryReports

12.LocationSearches NBFAC.061113.001(pp.261-279)13.RepositoryScreening

NBFAC.070215.0001,

NBFAC.070314.0001toNBFAC0.70314.003(pp.280-359)

14.EnvironmentalSamples

NBFAC.070723.0001(pp.360-380)

15.LocationSearches NBFAC.070727.0001,NBFAC.071102.0001,NBFAC.080828.0001(pp.381-440)

Module5:MolecularAnalysisofPathogenStrainsandIsolatesandGeneticMutationsA1andA3

TechnicalProposalandFBISOWs:GeneticDiscriminationofBacillusanthracisIsolatesUsingMolecularBiologicalTechniques

1.TechnicalProposalandFBISOWs:GeneticDiscriminationofBacillusanthracisIsolatesUsingMolecularBiologicalTechniques(pp.3-65)

AssayDevelopmentMorphA1-A3

2.ProgressReports:May2002-Jan2004(pp.67-499)AssayDevelopmentMorphA1-A3

1. FinalReport(pp.501-603)2. ValidationStudyMorphA-1Protocol(pp.604-712)3. ValidationStudyMorphA-2Protocol(pp.713-820)4. ValidationStudyMorphA-3Protocol(pp.821-932)

AssayDevelopmentMorphA1-A3

7.MorphAOverviewPowerPoint(pp.934-957)

RepositoryScreeningMorphA1andA3

8.ProgressandFinalReports,March2004-Oct2007(pp.959-1295)

Module6:GeneticMutationsBandD(CBI)

AssayDevelopment:TechnicalProposals

1. DNAAssaysforMinorGeneticVariantsofBacillusanthracis(pp.3-76)2. Task1TechnicalProposalMorphBSNP(pp.77-94)3. Task2TechnicalProposalMorphDDeletion(pp.95-114)

AssayDevelopment:ProgressReports

1. MorphB(pp.116-160)2. MorphD(pp.161-222)

AssayDevelopment:StandardOperatingProcedures

1. MorphBProtocol(pp.224-268)2. MorphDProtocol(pp.269-310)3. Appendix1-10(pp.311-867)

Module7:GeneticMutationsBandD(IITRI)


1. TechnicalProposal(pp.3-51)2. Task1DNAAssayDevelopment–MorphBSNP(pp.52-97)

3. Task2DNAAssayDevelopment–MorphDDeletion(pp.98-137)4. CapabilitiesBrief(pp.138-176)


1. DNA Assays for Minor Genetic Variants Meeting Minutes 2/23/05 (pp.178-179)

2. Task1MorphBSNPProgressReports(pp.180-215)3. Task2MorphDProgressReports(pp.216-251)

AssayDevelopment:Validation

1. ValidityTestReportsBSNP(pp.253-408)2. ValidityTestReportDDeletion(pp.409-661)

AssayDevelopment:StandardOperatingProcedures

1. StandardOperatingProcedureReal-TimePCRAssay for theDetectionoftheMorphBSNP(pp.662-686)

2. StandardOperatingProcedureReal-TimePCRAssay for theDetectionoftheMorphDDeletion(pp.687-710)

RepositoryScreeningMorphD:ProgressReports

1. MonthlyStatusReports(pp.711-800and803-955)2. FBIKick-OffMeeting(pp.956-996)3. B.anthracisMorphDDNAScreeningProgressReviews(pp.997-1016)

RepositoryScreeningMorphD:FinalReports

1. TechnicalRepositoryScreeningFinalReports(pp.1018-1134)2. OptionPeriodI,II,IIIFinalReports(pp.1135-1212)

Module8:GeneticMutationsBandD(MRI)


1. Volume1TechnicalProposal(pp.3-57)2. Task1DNAAssayDevelopment–MorphBSNP(pp.58-80)3. Task2DNAAssayDevelopment–MorphDDeletion(pp.81-102)


1. Task1MorphBSNPProgressReports(pp.104-170)2. Task2MorphDDeletionProgressReports(pp.171-242)

AssayDevelopment:Validation

6.MorphBSNPAssayValidityTestReport(pp.244-260)

7. Morph D Deletion Assay Validity Test Report (pp. 261-272) AssayDevelopment:StandardOperatingProcedures

8.ExtractionofDNAfromBacillusanthracisCultures(pp.274-281)

9.PCR-BasedAssayfortheDetectionofMorphBSNP(pp.282-315)

10.PCR-BasedAssayfortheDetectionofMorphDDeletion(pp.316-350)

AssayDevelopment:FinalReports

11.Task1MorphBFinalAdministrativeReport(pp.352-358)

12.Task2MorphDFinalAdministrativeReport(pp.359-366)

13.DNAAssayDevelopmentMorphBSNP(pp.367-380)

14. DNA Assay Development Morph D Deletion (pp. 381-396) RepositoryScreeningMorphD:TechnicalProposals

15.Volume1TechnicalProposal(pp.398-435)RepositoryScreeningMorphD:ProgressReports

16.DNAScreeningofAmesStrainAnthraxSamplesforMorphD(pp.437-628)

RepositoryScreeningMorphD:FinalReports

17.TechnicalRepositoryScreeningReport(pp.630-670)

18.TechnicalRepositoryScreeningReportAddendum1(pp.671-675)

19.TechnicalRepositoryScreeningReportAddendum2(pp.676-680)

Module9:GeneticMutationE(TIGR)


1.LL19DetectionAssay:ValidationStudyAnalysisPlan(pp.3-7)

2.LL19DetectionAssay:AnalysisPlanfor1060BlindSamples(pp.8-13)


1. June2004thruSept/Dec2005(seeBatch1,Module5)AssayDevelopment:StandardOperatingProcedure

2. OpaqueAssayDevelopmentSOP(pp.16-58)AssayDevelopment:Validation

3. B.anthracisLL19DetectionAssay:ValidationStudy(pp.60-123)AssayDevelopment:FinalReport

6.GenomicAnalysisofBacillusanthracisIsolatesRelevanttotheAmerithraxInvestigation,June4,2005(MorphE(Opaque,Post/LeahyB.s.)(seeBatch1,Module5)

RepositoryScreeningMorphE:FinalReport

7.AnalysisofaRepositoryofBacillusanthracisforthePresenceoftheLL19OpaqueDeletionGenotype(pp.126-323)

Module10:StatisticalAnalysis

TechnicalProposalandSOW

1.DeterminationoftheSignificanceoftheMarkersDiscoveredintheEvidentiaryMaterialofAmerithraxInvestigation(pp.3-21)

FinalReport

2.StatisticalReportofAmerithraxData(Sept30,2008)(pp.23-135)

Module11:CrossContamination

Reports

1.“RiskAssessmentofAnthraxThreatLetters,”DefenseR&DCanada,TechnicalReportDRES-TR-2001-048,September2001(pp.3-36)

2.“ForensicApplicationofMicrobiologicalCultureAnalysistoIdentifyMailIntentionallyContaminatedwithBacillusanthracisSpores,”D.J.Beecher,AppliedEnvironmentalBiology,Aug2006,p.5304-5310(pp.37-43)

Module12:DeclassifiedReports

TechnicalReviewPanelMeetings(NAS-1)

1.November14,2001TechnicalReviewPanelMeeting(pp.3-13)

AgarandHemeAnalysis(NAS-1):AgarAssayValidation-BMI

2.StatementofWorkB-Task-04ChemicalProcessTroubleshooting(pp.14-20)

3.SPOTReportonB-Task-04TechnicalProgressSummary(pp.21-27)

ChemicalandPhysicalCharacteristics(NAS-2)(seeBatch2,Module13)

Module13:ChemicalandPhysicalProperties

Reports

1. Determination of Concentration of Culturable Bacteria in Sample02.57.03(Daschle)Oct17-18,2001(pp.3-7)

2.PreliminarySPOTReportonParticleSizeAnalysisOct18,2001(pp.8-22)

3.SEMImagesSampleA(Daschle)Oct19,2001(pp.23-28)

4.SummaryofMicrobiologicalAnalysesOct19,2001(pp.29-31)

5.PreliminarySPOTReportonSampleAnalysesOct22,2001(pp.32-57)

6.PreparationStepsandAssociatedEquipmentOct25,2001(pp.58-61)

7.AnalysisofSiliconandSilicainPowderSamplesNovember21,2001(pp.62-74)

8.Analysis of Silicon andSilica in Powder Samples SEM/EDSAnalysisNov26,2001(pp.75-88)

9.TheAnalysisofSurrogateDryPowderBacillusSporeProductDecember28,2001(pp.89-98)

10.AnalysisofSPS02.266.02C(Leahy)Feb12,2002(pp.99-144)

11.SummaryofSampleAnalysis(SPS02.266.02C)28Feb02(pp.145-178)

Module14:ASMBioDefenseMeetingPresentations

CBI,Mr.ThomasA.Reynolds

1.TheScienceBehindtheAmerithraxInvestigation:MorphA1andA3Assays(pp.3-11)

MRI

2.MorphotypeDAssayDevelopmentandValidation(pp.13-33)

NAU,Dr.PaulKeim

3.TheAmesStrain:Frequency,Distribution,andForensicAnalysis(pp.35-51)

SNL,Dr.JosephR.Michael

4.ElementalMicroanalysisofBacillusanthracisSporesfromtheAmerithraxCase(pp.53-71)

TIGR,Dr.JacquesRavel

5.TheGenomicsBehindtheAmerithraxInvestigation(pp.73-106)

BATCH33

1.AmerithraxScienceUpdate–04-26/2002–11/25/2005(271pages)

2. ECdated2/8/2005–TechnicalReviewPanelMeetingAgenda,Anthrax

ReviewPanel,AmerithraxPanelSummary11/14/2001;12pagePowerPoint(17pages)

3. ECdated12/21/2001–MeetingofAnalyticalChemistryExpert(279A-WF-222936-LABserial37and1A579)(7pages)

4. ECdated2/11/2002–ProposedLabAnalysisandR&DStrategyAnalyticalFlowChart(279A-WF-22936-LABserial65)(5pages)

5. ECdates11/14/2005–CaseAgentMeeting(attachedWFOForensic&Investigationupdatemeeting11/05/2001)(279A-WF22936-LABserial1308and1A6553)(30pages)

6. ECdated11/14/2005–ScientificReviewPanelMeetingJune11-222002(279A-WF-22936-LABserial1310and1A6554)(22pages)

3TheFBIdidnotassignpagenumberstothisbatchofmaterials.

1. ECdated12/21/2001–ExternalExpertReviewofAnalyticalPlan(279A-WF-22936-LABserial25and1A533)(7pages)

2. U.S.ArmyMedicalResearchInstituteofInfectiousDiseases,ReferenceMaterialReceiptRecord(1page)

3. 279A-WF-222936-BATTELLESerial#9144. 279A-WF-222936-SC118Serial#3(8pages)5. 279A-WF-222936-USAMRIID/BEISerial#19(7pages)6. 279A-WF22936-USAMRIIDSerial#1418(2pages)7. Dr.IvinsUSAMRIIDLaboratoryNotebook#4010(30pages)8. IncomingShipmentRecordsforthe8PositiveFBIRepositorySamples

(originisFBIRDatabase)(19pages)15A.UnitedStatesDistrictCourtSearchWarrantApplicationandAffidavitCase#07-524-M-01(33pages)15B.UnitedStatesDistrictCourtSearchWarrantApplicationandAffidavitCase#07-525-M-01(27pages)15C.UnitedStatesDistrictCourtSearchWarrantApplicationandAffidavitCase#07-526-M-01(28pages)15D.UnitedStatesDistrictCourtSearchWarrantApplicationandAffidavitCase#07-527-M-01(28pages)15E.UnitedStatesDistrictCourtSearchWarrantApplicationandAffidavitCase#07-528-M-01(28pages)

15F. United States District Court Search Warrant Application and AffidavitCase#07-529-M-01(39pages)

16.FBIRepositoryShipmentRecords5(32pages)

SUPPLEMENTALDOCUMENTS6

1.AFIP Materials related to USAMRIID SpecimensOctober2001(41pages)

2. PreparingandShippingTSASlantsforB.AnthracisAmes(1page)3. FBIWFO[WashingtonFieldOffice]ReportonSamplesfromanOverseas

SiteIdentifiedbyIntelligence7(18pages)

4WhilethisdocumentwaslistedintheBatch3tableofcontentsprovidedtothecommitteeby

theFBI,thisdocumentwasnotprovidedtothecommitteebecauseofitssecurity

classification.5TheFBIdidnotassignanametothisdocument.Thedescriptorwasassignedbythecommittee.6TheFBIdidnotassignpagenumberstosupplementaldocumentsprovidedtothecommittee.7TheFBIdidnotassignanametothisdocument.Thedescriptorwasassignedbythecommittee.

AppendixA

RadiocarbonDating

The technique of radiocarbon dating was pioneered byLibbyintheearly1950s.Thistechniqueisbasedonthe

halflifeof14Cofapproximately5,700yearsandthefactthat the radioisotope is made through cosmic rayinteractionsintheatmospherebythereactionofenergeticneutronson14Ntoproduce14Candaproton.The14Cmixes in the atmosphere andbecomes incorporated intoCO2.Alllivingthingshaveratiosof14C/12Cthatreflectthe value in the atmosphere at the time and place inwhich they are living. Once a plant or animal dies, itstops incorporating modern CO2 and the 14C slowlydecays away.The standard is to reference everything totheratioof14C/12Cintheatmospherein1950.Theunitsareinthechangerelativetothisvalueas∆14Cinunitsor‰(partsperthousand.)Allpositiveratiosarefromafter1950 and all negative ratios correspond to before 1950,due to the loss of 14Cbecause of its radioactive decay.The complication is in determining what the 14C/12Cwas at the time the animal or plant was living. Thehistorical ratio is influencedby thecosmic ray flux inagivengeographical region(it is strongernear theearth’spolesandsensitivetotheearth’smagneticfield)andthesolar activity that is a dominant source of the earth’scosmic rays. Tree rings and corals have been used ascalibrationstorelatethe∆14Ctoactualcalendarages.

Modernhumanshavemodifiedthepurelycosmogenic14Ccontentoftheatmosphere in two ways. Fossil fuel burning hasintroduced into theatmosphereold (dead)carbon that isso old that it has essentially no14C left.Therefore, the“modern”14C/12Cratiowasfallinguntilthemid-1950s,when atmospheric nuclear testing created what is nowcalled the “bomb spike.” (see Figure A-1) The testingraisedthe14Ccontentoftheatmospheretoalmosttwicethe pre-bomb value in the Northern hemisphere at itspeak around 1965. With the cessation of atmospherictesting, the exchange of the atmosphere with the oceanhas gradually reduced the levels to values of∆14C lessthan 100‰, down from the peak of >800 ‰. Thisbehaviorisclearlyseeninthefigurebelow,whichshowsamodern∆14CcurveusingdatafromLevinandKromer(2004).This

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FIGUREA-1AtmosphericCO2(NorthernHemisphere).

Changeinvaluesof14CforatmosphericCO2since1959.Therisefromvaluesnearzeroresultsfromatmospherictestingofnuclearweapons.Thedeclinefrompeakvaluesreachedinlate1963resultsfromtheexchangeofCO2betweentheatmosphereandocean.As

aresultofthisexchange,levelsof14Cintheoceanhaveslowlyrisen.Thegapinthecurvebetween1973and1976isduetoalackofatmosphericdatafor1974and1975.SOURCE:CourtesyofAliceMignerey.

rapidriseandfallenablesthedatingofmodern(youngerthan1950)samplestowithinafewyearsinsomecases.

Radiocarbondatinghasadvancedtremendouslywiththeadventofthetechnique

ofacceleratormassspectrometry(AMS)toidentifyindividual14Catoms.Thisisadirectcountingmethodanddoesnotrelyon

detectingtheradiationthatisemittedwhenthe14Catomsdecay.Thisimprovementhasledtothecapabilitytoradiocarbondatesamplesoflessthan1mginmass.Thisnewtechniqueenabledtheanthraxsamplestobeanalyzed.

AppendixB

TheForensicsPotentialofStableIsotopeAnalysis

Typically,elementsinnaturebehavethesamechemicallyand biologically, independent of their isotopic identity.However, there are differences in a numberof physical,chemical, and biological processes that produce smallvariations in the ratios ofminor isotopes of elements totheirmajorcomponents.Thesedifferencesaresmall,butwith good analytical instrumentation they can bedetected.Oneexampleistheprocessofevaporationandcondensation. Water that has been evaporated from alarge source, such as the ocean, tends to be deficient intheheavier isotopesofhydrogenandoxygen,relative tothe original source. In the reverse process ofcondensation the heavier isotopes will condense morereadily, yielding rain that is enriched in the heavierisotopes. This continual cycle of evaporation andcondensation makes the ratio of 2H/1H and 18O/16Olowerforwatersourcesthatarefartherremovedfromtheoceans(themajorultimatewatersource).Theresultsare

also a function of temperature (summer versus winterprecipitation)andaltitude.Anextensivesamplingofriverwaters in theUnited Stateswas performed to provide abaseline for further studies (Kendall andCoplen, 2001).River water can come from a number of sources—groundwateraswellassurfacerunoff—butevenwiththiscomplexity there are some clear geographical trends inthe resultant maps of the deviation of the 2H and 18Ocompositionofthewaterwhencomparedtomeanoceanwater, expressed as δ2H and δ18O in ‰ (parts perthousand). Negative values mean that the water isdepleted in the heavier isotopes relative to oceanwater.Since this process affects both the2H/1Hand18O/16Oratios,aplotofδ2Hversusδ18Ogivesaroughlystraightlinecalledthemeteoricwaterline(MWL).Samplesthatfallbelowthislineareusuallyfromaridregionswithlowhumidities. While different regions have different locallinearrelationships,theoveralltrendcanbeusedtoinferanaveragevalueofδ18Ofromagivenδ2Hvalue.

Theisotopesofcarbonareinfluencedbythebiologicalprocessesthatareusedinthesynthesisoftheorganiccompoundsbyanorganism.Thereareverydifferent

ratios of 13C/12C for the C3 and C4 photosyntheticpathways. A C3 plant will typically exhibit a δ13Caround−25‰,whenreferencedagainsta

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standard limestone, while a C4 plant will give δ13C values around−9‰.A δ13C of about −16‰might represent either amixtureof the two sourcesor a plant that hasgrown inwater (algae or a hydroponically grownplant).Animalswillacquire theδ13Csignatureof their foodsources. Inthe case of bacteria cultured in the laboratory, thesignatureof thegrowthmediumwillbereflected in thatof the spores produced. Tests of this expectation forliquidgrowthmediumbearthisout(Kreuzer-MartinandJarman,2007).

Inthecaseofδ2Handδ18O,culturedmicroorganismsrecordtheisotopicsignatureofthewaterintheculturemediumas well the nutrients in the medium. Since the anthraxattacksof2001therehavebeenextensivestudiestestingthese relationships using the nonpathogenic Bacillussubtilis. On average about 70 percent of the oxygenatoms in spores produced come from the water, whileonlyabout30percentofthehydrogenatomscomefromthe water (Kreuzer-Martin et al., 2003, 2005) Resultsshowa strongpositive correlationbetween the δ2Handδ18Ocontentoftheculturewaterandresultantsporesforliquid cultures (Kreuzer-Martin and Jarman, 2007;Kreuzer-Martinetal.,2003).Samplesgrownonanagar

mediumarealsosubjectedtoisotopicfractionationfromevaporationofwaterfromthemedium.Itwasfoundthatthe influence of evaporation was small for δ2H, butsignificant for δ18O (Kreuzer-Martin et al., 2005).Exchange of water vapor with the ambient atmospherecould alsobe important if the agarwasprepared inonelocation and used in another, or anytime there is adifferent isotopicsignatureof thewaterusedandthatofthesurroundingwatervapor,as in thecaseof studiesatLawrence Livermore National Laboratory, where thesource of the tap water is largely the SierraMountainsandtheambientatmospherecanhaveasignificantmarineinput(Kreuzer-Martinetal.,2005)

Theforensicvalueofisotopicmeasurementsonbacteriaculturesdependsontheindividualcircumstances.Ithasthepotentialtoruleoutcertaincombinationsofwater and growth media as well as to provide a distinguishing marker fordiscriminationbetweenproductionbatchesofspores.

AppendixC

CommitteeEvaluationof

StatisticalAnalysisReport

TheStatistical Analysis Report (B2M10)was submittedin response to a contract with the FBI, to analyze theresultsoftheassaysonthe1,070FBIRepository(FBIR)samplesanddeterminewhether the resultsof theassayscould be related to those obtained on the evidentiary

material. If such a relationship could be identified, asecondaryissuewasthedevelopmentofameasureofits“statistical strength.” Two of the attack letters assayedpositiveforthefourmutationsA1,A3,D,andE.Resultson 1,059 of the 1,070 samples were tabulated in theStatisticalAnalysisReport.Eightsamplestestedpositiveforallfourmutations;sevenoftheseeightsamplescamefrom one institution (USAMRIID) and the remainingsample came from a different institution (BattelleMemorial Institute [BMI]). A table of documentedtransfers of samples from one institution to anothershowed a transfer of sample material from the firstinstitution(USAMRIID)tothesecondinstitution(BMI).This Appendix discusses the validity of the inferencesand calculations in the Statistical Analysis ReportsubmittedtotheFBI.

AsnotedinChapter6,thestatisticalanalysesusedinthereport(e.g.,95percentconfidence interval for the proportion of samples with four mutations, chi-squaredtestsofindependence)requiretwokeyassumptionstobevalid:

1. Representativeness:The1,059samplesareassumedtobearepresentativeand random collection of samples from somewell-defined population ofsamples.

2. Independence:The1,059 samples are assumed tobe independentofoneanother(i.e.,havenoconnectionwitheachother,beyondthattheyallcomefromthesamepopulation).

TheStatistical Analysis Report acknowledges that neither assumption can bevalidated from these data. The committee agrees with this assessment. As aconsequence,manyofthestatisticalmethodsappliedtothesedatacannotbe

185

validated. The consequences of the violation of these assumptions and theirimpactsarelistedbelow.

1.FBIRisnotarepresentativeandrandomcollectionofsamples fromawell-definedpopulationofB.anthracissamples.

The1,059samplesdonotappeartosatisfyassumption1.Theywereobtainedinresponse toa request from theFBI.No information isavailableon samples inthepopulationthatwerenotsubmitted.Infact,the“targetpopulation’’seemsnottohavebeendefined.ItcouldbethepopulationofalluniquepreparationsofB.anthracis Ames in the United States, or in the world, or from selectedinstitutions.The absenceof a definitionof “well-definedpopulation”makes itdifficult to assess representativeness of the collection. The elimination ofsamplesthathad“inconclusive”resultsonassaysalsoappearstobenonrandom,assomeinstitutionshadmanymore“inconclusive”assaysthanothers.

2.The1,059samplesintheFBIRarenotindependent.

The FBI submitted to the committee a table of known transfers of samplesbetweeninstitutions.Hence,thesecondassumptionisviolated.Thus,theresultsofthechi-squaredtestsforindependenceofthemutationsthatarecalculatedinthereportarenotmeaningful.Further,theconfidenceintervalfortheproportion8/947isnotappropriate.Thecorrectdenominatorforthisproportionislikelynot947.Amoreaccuratenumeratoranddenominatormightrefertothenumberofknown independent preparations rather than the number of samples, but suchinformationmaynotbepossibletoobtain.

3.Violation of assumptions renders invalid the inferences from the statisticalanalyses.

BecausetheFBIRisnotarepresentativeandrandomcollectionofindependentsamples, theresultson theassaysfromtherepositorymaybebiased.Virtuallyallstatisticalproceduresassumethattheunitsonwhichmeasurementsaremadecomprise a random, representative collection from the target population. (Theeffectsofbiasedsamplingon inferenceshavebeenwelldocumented;see,e.g.,Freedman et al., 2007). Without an appropriate model that characterizes thenonrepresentative-ness and the degree of dependence among the samples, it is

notpossibletocalculateameaningfulmeasureof“statisticalsignificance”intheresults.

4.Resultson112samplesbeyondthe947samples

TheStatisticalAnalysisReporteliminatedfrommostofitstablestheresultsoftheassayson112samplesthatshowed“inconclusive”forA1,A3,MRI-D,orE.Twenty-one of these 112 sample that were eliminated from the statisticalanalysisassayedpositivefor1,2,or3mutations.TableC-1liststhesesamples.(Five samples—05-022, 49-014, 53-014, 53-068, 54-008—are listed twicebecausetheywerereportedas“inconclusive”or“variant”ontwoassays.)

TABLEC-1SampleswithPositiveand“Inconclusive”or“Variant”Assays

FBIRNumberA1A3MRI-DIITRI-DE+Mutations039-010inc+---A3

044-034var--+-IITRI-D

049-014incvar++-MRI-D,IITRI-D

053-004var+--+A3,E

053-010var++++A3,MRI-D,IITRI-D,E

053-014varinc--+E

053-068incinc+--MRI-D

054-008inc++inc+A3,MRI-D,E

061-030inc-++-MRI-D,IITRI-D

066-015incinc++-MRI-D,IITRI-D

005-022+var-inc+A1,E

017-006-var++-MRI-D,IITRI-D

049-014incvar++-MRI-D,IITRI-D

049-018-var++-MRI-D,ITRI-D

053-014varinc--+E

053-068incinc+--MRI-D

054-066+inc+++A1,MRI-D,IITRI-D,E

054-068-var+-+MRI-D,E

005-020--+inc-MRI-D


043-016--+inc-MRI-D

044-020-+-inc+A3,E

052-026+++inc-A1,A3,MRI-D


054-022--+inc-MRI-D

057-036--+inc-MRI-D

inc=inconclusiveIITRI=IllinoisInstituteforTechnologyResearchInstituteMRI=MidwestResearchInstitutevar=variant

Inadditiontothetwo3-positivesamples(+++)amongthe947samples,thefoursamplesbelowalsotestedpositivefor3mutations(orderedbyFBIRnumber):

052-026+++inc-A1,A3,MRI-D

053-010var++++A3,MRI-D,IITRI-D,E


054-066+Inc+++A1,MRI-D,IITRI-D,E

Thefollowingfoursamplesrevealedpositiveassaysfor2of the 4mutations, in addition to the 11 samples notedamongthe947samples(orderedbyFBIRnumber):


044-020-+-inc+A3,E

053-004var+--+A3,E

054-068-var+-+MRI-D,E

DILUTIONEXPERIMENTS

Dilution experimentswere conducted to assess the sensitivity of the assays tovariousconcentrations.ThirtysampleswerepreparedfromRMR1029atdilution10.0. As with the other samples, some of the assays were “inconclusive.”GenotypeEtestedpositiveinall30samples;all4mutationstestedpositivefor16 samples. But in the remaining 14 samples, assays for one or more of thegenotypeswerenegative.Infact,onesampletestednegativeforA1,A3,andD;itwas positive for onlyE.Five sampleswere positive for twomutations only(A3andE),andeightsampleswerepositiveforonlythreeofthefourmutations(7 for A3, D, E; 1 for A1, A3, E). Thus, 6 of the 30 replicate samples (20percent)testedpositiveforonly1or2ofthemutations.Giventhat50ofthe947FBIR samples showed only 1 positive, and 11 of the 947 showed only 2positives, this variation indicates that someof the samplesmayhaveharboredmutations that went undetected. Absent any repeat testing of these samples,however,itisdifficulttoknowhowsuchfalsenegativesmighthaveaffectedtheinferences.

Additional experiments were conducted on RMR1029 and another sample,“SPS.266Tube#5,”at10dilutions levels (10.1, . . .,10.10).Theresultsof thethreereplicatesateachdilutionlevel,foreachofthefivegenotypes,forsamples

from both RMR1029 and SPS.266 Tube#5 were reported in Chapter 6.Variability in the resultson replicates,even from thesamesampleat thesamedilution level, demonstrates the value, and need for, replicate testing. Forexample, the results on the three replicates from RMR1029 at dilution 10.1,orderedasA1,A3,MRI-D,IITRI-D,E,were:(-++++),(-+++-),(++++-).Clearly,dilutionaffectstheassayresult:thegreaterthedilution,themorelikelytheassay isnegative.Moreover, it isperhapsunexpected thatgreaterdilutionssometimesgivepositiveresultswhennotallreplicatesatlesserdilutionsdidso.

CONCORDANCEOFTESTSFROMIITRI-DANDMRI-D

The FBI retained both the Illinois Institute for Technology Research Institute(IITRI)andMidwestResearchInstitute(MRI)toconducttheDassays.Becausetheassayson the1,059 samples canbeconsidered tobe independentbetweenIITRI and MRI, the Statistical Analysis Report (Table 3, p. 7, as presentedbelow)tabulatestheresultsoftheDassaysfromthetwofacilities:

IITRI-DMRI-DInconclusiveNegativeNogrowthPendingPositiveTotalInconclusive022120034Negative179091112940Negative-u12000021Positive612004664TOTAL24963131581,059

TheStatisticalAnalysisReportcombined the “negative-u”resultswiththe“negative”results,andeliminatedthe12 samples that showed “no-growth” by IITRI-D and“inconclusive” byMRI-D aswell as the one “pending”sample,toyieldthefollowingtable:

IITRI-DMRI-DInconclusiveNegativePositiveTotal

Inconclusive022022

Negative1892912959

Positive6124664

TOTAL24963581,045

Eliminating the14“no-growth”and“pending” samples,the concordance rate is 975/1045 = 0.933, with a 95percent confidence interval (0.916, 0.947). Thus, theagreement between the facilities is unlikely to be lowerthan91.6percentandlikelydoesnotexceed94.7percent.Ofgreaterinterest,however,arethe12samplesthatwerepositive by IITRI-D but negative for MRI-D, the 12samples that were negative by IITRI-D but positive byMRI-D,andthesixsamplesthatwerepositivebyMRI-Dbut inconclusive by IITRI-D. While concordance isinformative, these 30 samples with discordant resultsmight provide increased information about the samplesandtheassayprocess.Ontheotherhand,wealsoknowfromtherepeatedassaysofthedilutionseriesthatsomediscordance also arises owing to variation even whenusingthesameassayprocedure.

Inanycase,becausegenotypeDistheonlyoneofthefourgenotypesthatwassubjected to independent testing by a second organization one cannot saywhethertheresultsontheothergenotypesmighthavebeendifferentiftheyalso

hadbeensubjectedtoindependenttesting.

“SIGNIFICANCE”OFSEVEN(++++)SAMPLESFROMINSTITUTIONF

TheStatisticalAnalysisReportnotesinitsconclusions:

“Insummation,thoughtherandomchanceofoccurrenceof the sample type (++++) is 8 out of 947 (i.e., 0.84%)withexact95%confidence intervalof0.0037 to0.0166(I.e.,from1in270to1in60),thissampletypehasbeenfound in only two institutions thus far sampled(USAMRIID and BMI), and its occurrence in BMI isexplained by a recent sample transfer from USAM toBMI, since there is no documented record of sampletransfersintheotherdirection.’’(p.2)

As noted in Chapter 6, 598 of the 947 samples (63percent) came from Institution F. (Twelve of theinstitutions submitted 6 or fewer samples; 4 institutionssubmitted15-31samples,and4institutionssubmitted49-74 samples.) Therefore, one would not be surprised tofindmore“mutation-positive”samplesfromInstitutionFthan,say,fromInstitutionB(whichcontributedonlyonesample). One might naturally ask: How unusual is theoccurrence of seven “4-mutation” samples—or even alleight—from Institution F? Given that Institution Fcontributedalmost2/3ofthe947samples,howmanyofthe4-positive(++++)sampleswouldInstitutionFreceiveif the4-mutationsamplesweredistributedcompletelyat

random?

Theanswertothisquestionisgivenbytheprobabilitiesofobserving0or1or2or...or8oftheeight(++++)samplesfromInstitutionF,giventhatInstitutionFsubmitted598ofthe947samplesthatyieldeddefinitiveresultsontheA1,A3,MRI-D,andEassays.Theseprobabilities(fromthehypergeometricprobabilitydistribution)(Johnsonetal.,2005)areshowninTableC-2.

TABLEC-2Probabilitiesofk4-MutationSamplesinInstitutionF

k=012345678Probability0.00030.00450.02760.09550.20580.28260.24150.11740.0248

This table shows that thechanceof InstitutionFhavingendedupwithsevenoreightoftheeight(++++)samplesis (0.1174+ 0.0248) = 0.1422, or about 1/7. Therefore,while theobserveddata showing that sevenof theeight(++++) samples appeared in Institution F is notcompletely typical, it also could hardly be consideredextreme.

AppendixD

BiographicalInformationofCommitteeandStaff

AliceP.Gast(Chair)(NAE)becameLehighUniversity’s13thpresidentonAugust1,2006.PreviouslyshewastheRobert T. Haslam Professor of Chemical Engineering,Vice President for Research, and Associate Provost atMassachusettsInstituteofTechnology.PriortomovingtoMIT in 2001, she spent 16 years as a professor of

chemical engineering at Stanford University and at theStanford Synchrotron Radiation Laboratory. In herresearchshestudiessurfaceandinterfacialphenomena,inparticular the behavior of complex fluids. Some of herareas of research include colloidal aggregation andordering,proteinlipidinteractions,andenzymereactionsatsurfaces.In1997Dr.GastcoauthoredthesixtheditionofPhysicalChemistryofSurfaceswithArthurAdamson.Professor Gast received her B.S. in ChemicalEngineering from theUniversityofSouthernCalifornia.After earning her Ph.D. in chemical engineering fromPrincetonUniversity,GastspentapostdoctoralyearonaNATOfellowshipattheÉcoleSupérieuredePhysiqueetdeChimieIndustriellesinParis.Shereturnedthereforasabbatical as a Guggenheim Fellow. She was a 1999Alexander von Humboldt Fellow at the TechnicalUniversity in Garching, Germany. She received theNational Academy of Sciences Award for Initiative inResearch, and the Colburn Award of the AmericanInstitute ofChemical Engineers. Shewas elected to theNational Academy of Engineering in 2001 and to theAmerican Academy of Arts and Sciences in 2002.Professor Gast has served on numerous advisorycommittees and boards, including the Board of theAmerican Association for the Advancement of Science(AAAS) and the National Space Biomedical ResearchInstitute Board of Directors. She is a member of the

American Chemical Society, the American Institute ofChemicalEngineers,andtheAmericanPhysicalSociety.

DavidA.Relman(ViceChair)istheThomasC.andJoanM.MeriganProfessorin the Departments of Medicine and of Microbiology and Immunology atStanfordUniversity,andChiefofInfectiousDiseasesattheVAPaloAltoHealthCareSysteminPaloAlto,California.HereceivedanS.B.(Biology)fromMIT

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(1977) and M.D. (magna cum laude) from Harvard Medical School (1982),completed his clinical training in internal medicine and infectious diseases atMassachusettsGeneralHospital,servedasapostdoctoralfellowinmicrobiologyatStanfordUniversity,andjoinedthefacultyatStanfordin1994.Dr.Relman’scurrentresearchfocusisthehumanindigenousmicrobiota(microbiome),andinparticular the nature and mechanisms of variation in patterns of microbialdiversity in the human body as a function of time (microbial succession) andspace(biogeographywithinthehostlandscape)andinresponsetoperturbation,e.g.,antibiotics(communityrobustnessandresilience).Oneofthegoalsofthisworkistodefinetheroleofthehumanmicrobiomeinhealthanddisease.Thisresearch integrates theory and methods from ecology, population biology,environmentalmicrobiology, genomics, and clinicalmedicine.During the pastfewdecades,hisresearchdirectionshavealsoincludedpathogendiscoveryandthe development of new strategies for identifying previously unrecognizedmicrobial agents of disease. Thiswork helped to spearhead the application ofmolecular methods to the diagnosis of infectious diseases in the 1990s. Hisresearch has emphasized the use of genomic approaches for exploring host-microbe relationships.Past scientificachievements include thedescriptionofanovelapproachforidentifyingpreviouslyunknownpathogens,theidentificationof a number of new human microbial pathogens, including the agent ofWhipple’s disease, and some of the most extensive and revealing analyses todateofthehumanindigenousmicrobialecosystem.Dr.RelmanadvisestheU.S.government aswell as nongovernmental organizations inmatters pertaining tomicrobiology,emerginginfectiousdiseases,andbiosecurity.Hecurrentlyservesas Chair of the Institute ofMedicine’s Forum onMicrobial Threats (NationalAcademyofSciences), amemberof theNationalScienceAdvisoryBoard forBiosecurity (NSABB), and a member of the Physical and Life SciencesDirectorate Review Committee for Lawrence Livermore National Laboratory,

and advises several U.S. government departments and agencies on mattersrelatedtopathogendiversity,thefuturelifescienceslandscape,andthenatureofpresent and future biological threats. He has served as Chair of the Board ofScientific Counselors of the National Institute of Dental and CraniofacialResearch (National Institutes of Health [NIH]) and member of the Board ofDirectors,InfectiousDiseasesSocietyofAmerica(IDSA).Dr.Relmancochairedathree-yearNationalResearchCouncilstudythatproducedawidelycitedreportentitledGlobalization,Biosecurity, and theFutureof theLifeSciences (2006).HeisaFellowoftheAmericanAcademyofMicrobiologyandamemberoftheAssociation of American Physicians. Dr. Relman received the Squibb AwardfromtheIDSAin2001andwastherecipientofboththeNIHDirector’sPioneerAward and the Distinguished Clinical Scientist Award from the Doris DukeCharitableFoundationin2006.

ArturoCasadevallistheLeoandJuliaForchheimerProfessorofMicrobiologyandImmunologyandChairoftheDepartmentofMicrobiologyandImmunologyat the Albert Einstein College of Medicine. He is also a Professor in theDepartment ofMedicine. He received his B.A. fromQueens College, CUNY,andM.S.,M.D., andPh.D.degrees fromNewYorkUniversity.His laboratoryhas a multidisciplinary research program spanning several areas of basicimmunology and microbiology to address general questions in these areas,resulting in over 460 publications. His laboratory studies are focused on twomicrobes: the fungus Cryptococcus neoformans, a ubiquitous environmentalmicrobethatisafrequentcauseofdiseaseinimmunocompromisedindividuals,andBacillus anthracis, which is amajor agent of biological warfare. He is afellowof theAmericanAcademyofMicrobiologyandhasbeenelected to theAmerican Society for Clinical Investigation, to the American Association ofPhysicians, andasa fellowofAAAS.Dr.Casadevallhas servedonnumerousNIH advisory committees including study sections, strategic planning for theNational Institute of Allergy and Infectious Diseases (NIAID), and the blueribbon panel on response to bioterrorism. He currently cochairs the NIAIDBoard of Scientific Counselors and is a member of the NSABB. He is thefounding editor of the firstAmericanSociety ofMicrobiology (ASM) generaljournal,mBio,servesontheeditorialboardsofseveraljournals,andhasbeentherecipient of numerous awards,most recently the SolomonA. BersonMedicalAlumniAchievementAward inBasicScience-NYUSchoolofMedicine2005,IDSAKassLecturerin2008,andtheASMWilliamHintonAwardformentoringscientistsfromunderrepresentedgroups.

NancyD.Connell isprofessorofmedicineat theUniversityofMedicineandDentistry of New Jersey (UMDNJ)-New Jersey Medical School. She is alsodirectorof theUMDNJCenter forBioDefense,whichwasestablished in1999for research into the detection and diagnosis of biological warfare agents andbiodefense preparedness.Dr.Connell also is director of theBiosafetyLevel 3Facility of UMDNJ’s Center for the Study of Emerging and Re-emergingPathogensandchairs theuniversity’s InstitutionalBiosafetyCommittee.She ispast chair of NIH’s Center for Scientific Review Study Section HIBP (HostInteractions with Bacterial Pathogens), which reviews bacterial pathogenesissubmissions toNIAID.She is current chair of theF13 infectiousdiseases andmicrobiologyfellowshippanel.Dr.Connell’sinvolvementinbiologicalweaponscontrolbeganin1984,whenshewaschairoftheCommitteeontheMilitaryUseofBiologicalResearch,asubcommitteeoftheCouncilforResponsibleGenetics,based inCambridge,Massachusetts.Shehasworkedwithseveral internationalprograms on dual use research issues and served on variousNRC committeeswith her expertise in select agentmicrobiology, dual use, and biocontainment.Dr.Connell receivedherPh.D. inmicrobialgenetics fromHarvardUniversity.HermajorresearchfocusistheinteractionbetweenMycobacteriumtuberculosisandthemacrophage.

ThomasV.Inglesby isCEOandDirectorof theCenter forBiosecurityof theUniversity of PittsburghMedical Center andAssociate Professor ofMedicineand Public Health at the University of Pittsburgh Schools of Medicine andPublicHealth.Heisaninfectiousdiseasephysicianbytraining.HeisCoeditorinChiefofthepeer-reviewedjournalBiosecurityandBioterrorism:BiodefenseStrategy, Practice, and Science and has authored a number of widely citedpublications on anthrax, smallpox, plague, and biosecurity issues related tomedicineandhospitalpreparedness,publichealth,science,pandemicplanning,and national security. He is a principal editor of the Journal of the AmericanMedical Association book entitled Bioterrorism: Guidelines for Medical andPublicHealthManagement.Dr. Inglesbywas aprincipaldesigner, author, andcontrollerof thewidelyrecognizedAtlanticStormexerciseof2005andof theDark Winter smallpox exercise of 2001. He has served in advisory andconsultativecapacitiesforgovernment,scientificorganizations,andacademiaonissues related to biosecurity, providing briefings for officials in theadministrationandforcongressionalmembersandstaff;servingonataskforceoftheDefenseScienceBoardoftheDepartmentofDefenseandacommitteeoftheUSNationalResearchCouncil;andparticipatinginanadvisorycapacitytotheCentersforDiseaseControlandPrevention,NIH,theDepartmentofHealth

and Human Services, the Department of Homeland Security, the DefenseAdvanced Research Projects Agency (DARPA), and the Defense IntelligenceAgency(DIA).PriortohelpingestablishtheCenterforBiosecurityin2003,Dr.Inglesby was one of the founding members of the Johns Hopkins Center forCivilianBiodefenseStrategies,whereheservedasDeputyDirectorfrom2001to2003.HewasalsoafacultymemberoftheJohnsHopkinsSchoolofMedicinefrom 1999 to 2003.Dr. Inglesby is Board-certified in InfectiousDiseases.Hereceived a B.A. in 1988 from Georgetown University and an M.D. from theColumbiaUniversityCollegeofPhysiciansandSurgeonsin1992.Hecompletedhis internalmedicine residency and InfectiousDiseases Fellowship training attheJohnsHopkinsSchoolofMedicine,andservedasAssistantChiefofServiceintheJohnsHopkinsDepartmentofMedicinein1996and1997.

MurrayV.JohnstonisProfessorofChemistryintheDepartmentofChemistryandBiochemistryattheUniversityofDelaware.HereceivedaB.S.inchemistryfromBucknellUniversityandPh.D.inanalyticalchemistryfromtheUniversityof Wisconsin, Madison. He is the recipient of a Center for Advanced Studyfellowship in1999, theOutstandingScholarAward in theCollegeofArtsandSciences in 2001, the Delaware Section Award of the American ChemicalSociety in 2003, and the Benjamin Y.H. Liu Award from the AmericanAssociation forAerosolResearch in2008. In2007,he servedon theNationalResearch Council panel on Testing and Evaluation of Biological StandoffDetection Systems. Dr. Johnston’s research includes applications of massspectrometry toawidearrayofmaterials, fromairborneparticles tobiologicaland polymeric macromolecules. He has used real-time single-particle massspectrometry to studymicrochemical reactionswithin particles, heterogeneousreactionsbetweengas-phaseandparticulate-phasespecies,andambientparticlesatvariousurbansites.Hiscurrentworkemphasizes theuseofphotoionizationaerosol mass spectrometry to characterize organic components of combustionandambientaerosols,nanoaerosolmassspectrometrytocharacterizeindividualnanoparticlesandmacromoleculessmaller thanabout30nm,andconventionalmass spectrometry tocharacterizeoligomericcompounds in secondaryorganicaerosols. Dr. Johnston is a member of the editorial board of the journalAnalyticalChemistry and theBoard ofDirectors of theAmericanAssociationforAerosolResearch.HehasservedasanadhocmemberofseveralNIHreviewpanelsassociatedwithbiologicalandenvironmentalmassspectrometry.

KarenKafadarisJamesH.RudyProfessorofStatisticsandPhysicsatIndianaUniversity.ShereceivedherB.S.andM.S.degreesfromStanfordandherPh.D.

in Statistics from Princeton under John Tukey. Her research focuses onexploratory data analysis, robust methods, characterization of uncertainty inquantitativestudies,andanalysisofexperimentaldatainthephysical,chemical,biological, and engineering sciences. Prior to Indiana University, she wasProfessor and Chancellor’s Scholar in the Departments of MathematicalSciencesandPreventiveMedicineandBiometricsattheUniversityofColorado-Denver;FellowattheNationalCancerInstitute(cancerscreeningsection);andMathematical Statistician at Hewlett Packard Company (R&D laboratory forRF/microwave test equipment) and at the National Institute of Standards andTechnology (where she continues as Guest Faculty Visitor on problems ofmeasurement accuracy, experimental design, and data analysis). Previousengagements include consultancies in industry and government as well asvisiting appointments at University of Bath, Virginia Tech, and Iowa StateUniversity. She has served on previous NRC committees and chaired theCommitteeonAppliedandTheoreticalStatistics.ShealsoservesontheeditorialboardsforseveralprofessionaljournalsasEditororAssociateEditorandonthegoverning boards for the American Statistical Association, the Institute ofMathematical Statistics, and the International Statistical Institute. She is anElected Fellow of the American Statistical Association and the InternationalStatisticalInstitute,hasauthoredover90journalarticlesandbookchapters,andhasadvisednumerousM.S.andPh.D.students.

RichardE.LenskiistheJohnA.HannahDistinguishedProfessorofMicrobialEcology at Michigan State University. His research explores the geneticmechanisms and ecological processes that underlie evolution. While mostevolutionary research uses the comparative method, Lenski pursues anexperimentalapproachusingbacteria.Inanexperimentstartedin1988,Lenskiandhisteamhavewatched12populationsofE.colievolveinthelabformorethan50,000generations to investigate thephenotypicandgeneticdynamicsofadaptation and diversification. Lenski and his students have performed otherexperiments with microbes on the dynamics of host-parasite interactions, theevolution of mutation rates, and even social interactions. Lenski alsocollaborates with an interdisciplinary team on experiments using digitalorganisms—computerprogramsthatreplicate,mutate,compete,andevolve—toinvestigatetheevolutionofcomplexity.ProfessorLenskireceivedaMacArthurFoundation Fellowship in 1996 and was elected to the National Academy ofSciencesin2006.

RichardM.Losick istheMariaMoorsCabotProfessorofBiology,aHarvard

College Professor, and a Howard Hughes Medical Institute Professor in theFaculty of Arts and Sciences at Harvard University. He received his A.B. inChemistry at Princeton University and his Ph.D. in Biochemistry at theMassachusetts InstituteofTechnology.Uponcompletionofhisgraduatework,ProfessorLosickwasnamedaJuniorFellowoftheHarvardSocietyofFellowswhen he began his studies on RNA polymerase and the regulation of genetranscriptioninbacteria.ProfessorLosickisapastChairmanoftheDepartmentsofCellularandDevelopmentalBiologyandMolecularandCellularBiologyatHarvard University. He received the Camille and Henry Dreyfuss Teacher-ScholarAwardandisamemberoftheNationalAcademyofSciences,aFellowof the American Academy of Arts and Sciences, a member of the AmericanPhilosophical Society, a Fellow of the American Association for theAdvancementofScience,aFellowoftheAmericanAcademyofMicrobiology,and a formerVisiting Scholar of the Phi BetaKappa Society.He is the 2007recipient of the Selman A. Waksman Award of the National Academy ofSciencesanda2009recipientoftheCanadaGairdnerAward.

AliceC.Mignereyisanuclearchemistwithresearchprogramsinbasicnuclearscience and in applications of the nuclear analytical technique of acceleratormass spectrometry (AMS) to environmental problems. Professor Mignerey’sbasic nuclear research is focused on understanding the behavior of nuclearmatter under conditions of extreme density (pressure) and temperature. Theseconditions are postulated to have existed just after the Big Bang, when theprotonsandneutronshadnotyet formedfromtheirconstituentquarksand thegluons that hold them together. This so-called quark-gluon plasma has beenpredicted to be accessible through heavy ion reactions at high energies. Theexperimental program is centered at the Brookhaven National LaboratoryRelativistic Heavy Ion Collider (RHIC) accelerator where colliding beams ofnuclei reach center-of-mass energies of 200 AGeV, producing conditionsmimicking thoseof theearlyuniverse.ProfessorMignerey isamemberof thePhobosandPHENIXCollaborationsatRHICandtheCMSHeavyIonGroupatthe CERN Large Hadron Collider (LHC). The research program in AMS hasconcentratedontheusesofthecosmogenicnuclides,suchasC-14andCl-36,tostudygroundwaterandsoilsystems.Techniquedevelopment iscurrentlybeingcarried out with researchers at the Naval Research Laboratory Trace ElementAMSfacility(TEAMS)toallowthedatingofseparateorganicfractions in theorganicC-14carbonpool.

DavidL.Popham is a professor in theDepartment ofBiological Sciences at

VirginiaTech.Heteachesintheareasofmicrobialgeneticsandphysiology.Hedirectsaresearchprogramintheareasofbacterialendosporestructure,content,germination,andresistanceproperties.Dr.PophamhasaPh.D.inmicrobiologyfromtheUniversityofCalifornia-Davis.Heheldpostdoctoralresearchpositionsat the Institut deBiologie Physico-Chimique in Paris and at theUniversity ofConnecticutHealth ScienceCenter before joining theVirginiaTech faculty in1996.He has over 20 years of experience in research onBacillussubtilis cellwall synthesis, spore formation, and spore resistanceproperties.More recentlyhisresearchhasexpandedintothecontent,structure,andgerminationofsporesproducedbyB.anthracis,Clostridiumdifficile,and

C.perfringens.Dr.PophamisamemberoftheeditorialboardsoftheJournalofBacteriology andMolecularMicrobiology and has served as amember of sixNIH grant review panels. He has served on the Environmental ProtectionAgencyFederalInsecticide,Fungicide,andRodenticideActScientificAdvisoryPanelforthedevelopmentofguidelinesfortheapprovalofsporicidalproducts.

JedS.RakoffhasbeenaUnitedStatesDistrictJudgefortheSouthernDistrictofNewYork since 1996. Prior to his appointment, hewas a partner at Fried,Frank,Harris,Shriver&JacobsonLLP.From1980to1990,hewasapartneratMudge,Rose,Guthrie,Alexander&FerdonLLP.JudgeRakoffwasanAssistantU.S. Attorney for the SouthernDistrict of NewYork from 1973 to 1980 andchiefoftheBusinessandSecuritiesFraudProsecutionsUnitfrom1978to1980.Before joining the U.S. Attorney’s Office, Judge Rakoff spent two years inprivate practice as an associate attorney at Debevoise & Plimpton LLP. Heserved as a law clerk to theHonorableAbrahamL. Freedman,U.S. Court ofAppeals, 3rdCircuit, in 1969-70. JudgeRakoff is coauthor of five books andauthorofmore than100publishedarticles,more than300speeches,andmorethan 650 judicial opinions. He has been a lecturer in law at Columbia LawSchool since 1988.Hewas amember of theBoard ofManagers, SwarthmoreCollege,from2004to2008.JudgeRakoffcurrentlyservesasaTrusteefortheWilliamNelsonCromwellFoundationandamemberoftheGovernanceBoardfor the MacArthur Foundation Initiative on Law and Neuroscience. He is amemberoftheNationalResearchCouncilCommitteeontheDevelopmentoftheThirdEditionof theReferenceManualonScientificEvidenceandchairof theCriminal JusticeAdvisory Board, SouthernDistrict of NewYork; the SecondCircuit Bankruptcy Committee; and the Honors Committee of the New YorkCityBarAssociation.HeisaJudicialFellowat theAmericanCollegeofTrialLawyersandwaschairoftheDownstateNewYorkChapterin1993

94. Judge Rakoff is the former director of theNewYork Council ofDefenseLawyersandformerchairoftheCriminalLawCommittee,NewYorkCityBarAssociation.HehasbeenaJudicialFellowat theAmericanBoardofCriminalLawyerssince1995.JudgeRakoffreceivedaB.A.fromSwarthmoreCollegein1964,anM.Phil.fromOxfordUniversityin1966,andaJ.D.fromHarvardLawSchoolin1969.HewasawardedhonoraryLL.D.sfromSwarthmoreCollegein2003andSt.FrancisUniversityin2005.

Robert C. Shaler obtained a doctoral degree in Biochemistry from thePennsylvania State University in 1968 and then worked at the University ofPittsburghasaprofessorofchemistryandatthePittsburghCrimeLaboratoryasacriminalist.Hisresearchresulted in thedevelopmentofabloodstainanalysissystem,thedefactostandardinforensiclaboratoriesuntiltheearly1990s.TheNew York City Office of Chief Medical Examiner beckoned in 1978. Hedirected the forensic serology laboratory and performed and directed forensicbiologicalanalysesinallNewYorkCityhomicideinvestigations.InthewakeoftheWorldTradeCenter(WTC)attacksonSeptember11,2001,heassumedtheresponsibilityfor identifyingthepeoplewhoperished.Hedesigned,organized,and implemented theDNAtestingstrategy thatbecame thecornerstonefor themajorityoftheidentifiedvictims.WhentheNewYorkCityOfficeoftheChiefMedical Examiner effort to identify the WTC victims paused, he accepted aprofessorship in theBiochemistry andMolecularBiologyDepartment and thedirectorship of the forensic science program at the Pennsylvania StateUniversity.

Elizabeth A. Thompson is a professor in the Department of Statistics andadjunctprofessorinthedepartmentsofBiostatisticsandofGenomeSciencesatthe University of Washington, and Director of an Interdisciplinary GraduateCertificateprograminStatisticalGenetics.ShereceivedherB.A.inmathematicsand Ph.D. inmathematical statistics fromCambridgeUniversity,UK, and didpostdoctoral work in the Department of Genetics, Stanford University, beforetakingupapositiononthefacultyoftheDepartmentofPureMathematicsandMathematicalStatisticsat theUniversityofCambridgein1976.Shejoinedthefaculty of the University ofWashington in December 1985 as a professor ofstatistics and served as chair 1989-1994. Dr. Thompson’s research is in thedevelopmentofmethodsformodel-basedlikelihoodinferencefromgeneticdata,particularlyfromdataobservedonlargeandcomplexpedigreestructuresbothofhumans and of other species, and including inference of relationships amongindividualsandamongpopulations.Dr.ThompsonisarecipientofaDoctorof

SciencedegreefromtheUniversityofCambridge, theJeromeSacksawardforcross-disciplinaryresearchfromtheNationalInstituteforStatisticalScience,theWeldon Prize for contributions to Biometric Science fromOxford University,UK,andaGuggenheimfellowship.ShehasservedontheNRCCommitteeonAppliedandTheoreticalStatisticsandontheScientificAdvisoryBoardsofthePacific Institute for Mathematical Sciences, the Banff International ResearchStation,andtheInstituteforPureandAppliedMathematics.Shealsoservesonseveral committees of the International Biometric Society, including as amember of Council. Dr. Thompson is an electedmember of the InternationalStatistical Institute, the American Academy of Arts and Sciences, and theNationalAcademyofSciences.

KasthuriVenkateswaranisaseniorresearchscientistattheCaliforniaInstituteofTechnology’sJetPropulsionLaboratory.His32yearsofresearchencompassmarine, food, and environmentalmicrobiology.He has applied his research inmolecular microbial analysis to better understand the ecological aspects ofmicrobes,whileconducting fieldstudies inseveralextremeenvironmentssuchas deep sea (2,500 m), pristine caves (3,000 m altitude), spacecraft (MarsOdyssey,Genesis,MER,MarsExpress,Phoenix,MSL)assemblyfacilitycleanrooms (various NASA and European Space Agency facilities), as well as thespace environment in Earth orbit (International Space Station). Of particularinterest are microbe-environment interactions with emphasis on theenvironmentallimitsinwhichorganismscanlive.Theresultsareusedtomodelmicrobe-environment interactions with respect to microbial detection and thetechnologies to rapidly monitor them without cultivation. The bioinformaticsdatabases generated by Dr. Venkateswaran are extremely useful in thedevelopmentofbiosensors.Further,thesemodelsorinformationindatabasesareextrapolated to what is known about the spacecraft surfaces and enclosedhabitats in an attempt to determine forward contamination aswell as developcountermeasures (develop cleaning and sterilization technologies) to controlproblematic microbial species. Specifically, his research into the analysis ofcleanroomenvironmentsusingstate-of-the-artmolecularanalysiscoupledwithnucleicacidandprotein-basedmicroarrayswillenableaccurateinterpretationofdata and implementation of planetary protection policies of present missions,helpingtosetstandardsforfuturelife-detectionmissions.

DavidR.WaltisRobinsonProfessorofChemistryandProfessorofBiomedicalEngineering at Tufts University and is a Howard Hughes Medical InstituteProfessor.HereceivedaB.S.inChemistryfromtheUniversityofMichiganand

aPh.D.inChemicalBiologyfromSUNYatStonyBrook.Hislaboratoryappliesmicro-andnanotechnologytourgentbiologicalproblemssuchastheanalysisofgeneticvariationandthebehaviorofsinglecells,singlemoleculedetection,aswell as the practical application of arrays to the detection of explosives,chemicalandbiologicalwarfareagents,andfoodandwaterbornepathogens.Dr.WaltistheScientificFounderandaDirectorofbothIlluminaInc.andQuanterixCorp.Hehas receivednumerousnational and international awards andhonorsforhisfundamentalandappliedworkinthefieldofopticalsensorsandarrays.He is a member of the National Academy of Engineering, a fellow of theAmerican Institute forMedicalandBiologicalEngineeringand theAAAS.HehasservedonanumberofNRCcommitteesincludingtheCommitteeonReviewandEvaluationMethodologyforBiologicalPointDetectors.

Staff

Anne-MarieMazza is theDirector of theCommittee on Science, Technology,and Law. Dr. Mazza joined the National Research Council in 1995. She hasserved as Senior Program Officer with both the Committee on Science,Engineering, and Public Policy and the Government-University-IndustryResearchRoundtable.In1999shewasnamedthefirstdirectoroftheCommitteeon Science, Technology, and Law, a newly created activity designed to fostercommunication and analysis among scientists, engineers, andmembers of thelegalcommunity.Dr.MazzahasbeenthestudydirectoronnumerousAcademyreports including Managing University Intellectual Property in the PublicInterest (2010); Strengthening Forensic Science in the United States: A PathForward (2009); Science and Security in a Post-9/11 World (2007);DaubertStandards:SummaryofMeetings(2006);ReapingtheBenefitsofGenomicandProteomicResearch:IntellectualPropertyRights,Innovation,andPublicHealth(2005); Intentional Human Dosing Studies for EPA Regulatory Purposes:ScientificandEthicalIssues(2004);EnsuringtheQualityofDataDisseminatedby theFederalGovernment (2003).Dr.Mazza receivedanNRCdistinguishedserviceaward in2008. In1999-2000,Dr.Mazzadividedher timebetween theNational Academies and theWhite House Office of Science and TechnologyPolicy (OSTP), where she served as a Senior Policy Analyst responsible forissues associated with a Presidential Review Directive on the government-university researchpartnership.Before joining theAcademy,Dr.Mazzawas aSenior Consultant with Resource Planning Corporation. Dr.Mazza received aB.A.,M.A.,andPh.D.fromtheGeorgeWashingtonUniversity.

Frances E. Sharples has served as the Director of the National ResearchCouncil’sBoardonLifeSciencessinceOctober2000.Immediatelypriortothisposition, shewasaSeniorPolicyAnalyst for theEnvironmentDivisionof theWhiteHouseOfficeofScienceandTechnologyPolicy (OSTP) for fouryears.Dr.SharplescametoOSTPfromtheOakRidgeNationalLaboratory,wheresheserved in various positions in the Environmental Sciences Division between1978and1996,mostrecentlyasaResearchandDevelopmentSectionHead.Dr.SharplesreceivedherB.A.inBiologyfromBarnardCollegeandherM.A.andPh.D. in Zoology from the University of California, Davis. She served as anAAAS Environmental Science and Engineering Fellow at EPA during thesummerof1981andasaAAASCongressionalScienceandEngineeringFellowin the office of Senator Al Gore in 1984-85. She was a member of NIH’sRecombinant DNA Advisory Committee in the mid-1980s and was elected aFellowoftheAAASin1992.

Ericka D.MartinMcGowan is program officer with the National ResearchCouncil Board on Chemical Sciences and Technology (BCST), where shecontributestoscientificpolicystudiesrelatedtothedetectionofbiologicalandchemical warfare agents as well as issues at the interface of chemistry andbiology.SincejoiningtheNRCin2004,Mrs.McGowanhasbeeninvolvedwiththe following NRC studies and reports: BioWatch and Public HealthSurveillance:EvaluatingSystems for theEarlyDetectionofBiologicalThreats(2010); Test and Evaluation of Biological Standoff Detection Systems (2008);Protecting BuildingOccupants andOperations formBiological andChemicalAirborne Threats: A Framework for Decision Making (2007); ExploringOpportunities in Green Chemistry and Engineering Education: A WorkshopSummary to the Chemical Sciences Roundtable (2007);Measuring RespiratorUse in the Workplace (2007) Terrorism and the Chemical Infrastructure:ProtectingPeopleandReducingVulnerabilities(2006).Mrs.McGowanreceiveda B.S. in Biology, minor in Chemistry, from Southern University and A&MCollege, and anM.S. in PublicHealthMicrobiology andEmerging InfectiousDiseasesfromtheGeorgeWashingtonUniversity.

StevenKendall is Associate Program Officer for the Committee on Science,Technology,andLaw.HeisaPh.D.candidateintheDepartmentoftheHistoryofArtandArchitectureattheUniversityofCalifornia,SantaBarbara,whereheis completing a dissertation on 19th century British painting. Mr. KendallreceivedhisM.A.inVictorianArtandArchitectureattheUniversityofLondon.PriortojoiningtheNRCin2007,heworkedat theSmithsonianAmericanArt

MuseumandtheHuntingtoninSanMarino,California.

AmandaClineisanadministrativeassistantontheBoardonChemicalSciencesand Technology. She joined the NRC in 2007, after receiving her B.S. inenvironmental studies fromBucknellUniversity.Ms.Cline hasworked in thereportreviewofficeoftheDivisiononEarthandLifeStudiesandasaprogramassistant for the Board on Life Sciences, where she supported the HumanEmbryonicStemCellAdvisoryCommittee,theInterstateAllianceonStemCellResearch,theCommitteeonaNewBiologyforthe21stCentury,theCommitteeonEcologicalImpactsofClimateChange,andotherNRCactivities.

Kathi E. Hanna has over 25 years of experience in science, health, andeducationpolicyasananalyst,writer,andeditor.Inthe1990sDr.Hannaservedas Research Director and Editorial Consultant to President Clinton’s NationalBioethics Advisory Commission. She also served as Senior Advisor onReproductive Toxicology to the President’sAdvisoryCommittee onGulfWarVeteransIllnesses.ShewastheleadanalystandauthorforPresidentBush’sTaskForcetoImproveHealthCareDeliveryforOurNation’sVeteransandservedinasimilarcapacityfor theTaskForceontheFutureofMilitaryHealthCare.Inthe1980sand1990s,Dr.HannawasaSeniorAnalystatthecongressionalOfficeof Technology Assessment, contributing to numerous science policy studiesrequestedbycongressionalcommitteesonscienceeducation,researchfunding,science and economic development, biotechnology, women’s health, mentalhealth, children’s health, human genetics, bioethics, cancer biology, andreproductivetechnologies.Inthepasttwodecadesshehasservedasananalystandeditorial consultant to theHowardHughesMedical Institute, theNIH, theNRC, theU.S.OfficeforHumanResearchProtections,FasterCures, theLanceArmstrong Foundation, the American Heart Association, the BurroughsWellcome Fund, the March of Dimes, the U.S. Anti-Doping Agency, andbiotechnology companies. She has been a consultant and lead author onNIHstrategicplans;adhoccommitteesof theAdvisoryCommittee to theDirector,NIH;theSecretary’sAdvisoryCommitteeonGenetics,Health,andSociety;andtheNIHOfficeofBehavioralandSocialScienceResearch.Shehasauthoredorcoauthored over 40 reports of studies of children and environmental health,obesity, immunization, genetics, emergency care, epilepsy, cancer, forensicscience, and general health and science policy. Before moving to theWashington, D.C., area, she was the Genetics Coordinator/Counselor atChildren’sMemorialHospitalinChicago,whereshedirectedclinicalcounselingin pediatric genetics and coordinated an international research program in

prenatal diagnosis. Dr. Hanna received an A.B. in biology from LafayetteCollege,anM.S.inhumangeneticsfromSarahLawrenceCollege,andaPh.D.ingovernment andhealth services administration from theSchoolofBusinessandPublicManagementatGeorgeWashingtonUniversity.

Cameron H. Fletcher is the Managing Editor of the ILAR Journal, thequarterly,peer-reviewedofficialpublicationoftheNationalResearchCouncil’sInstitute forLaboratoryAnimalResearch. Inher25years at theNRCshehaseditednumerousreportsfortheDivisiononEarthandLifeStudies,DivisiononBehavioral and Social Sciences and Education, Division on Engineering andPhysicalSciences,andPolicyandGlobalAffairs.ShehasalsoeditedreportsandotherpublicationsforthePewNationalCommissiononIndustrialFarmAnimalProduction,thePetersonInstituteforInternationalEconomics,theCongressionalBudget Office, the International Association of Oil and Gas Producers, andColumbiaUniversity.BeforehertenureattheNRCshetaughtFrench,Spanish,Latin, and English at private schools in Connecticut and Rhode Island. ShereceivedherABcumlaudefromBrynMawrCollege.

Index

A

agar,89,113,167AmericanMedia,Inc.(AMI),26,56–57,60,62,65,67,76AmericanSocietyofMicrobiology(ASM)BioDefenseMeetingPresentations,178Amerithraxinvestigations,25–26analyticaltechniquesusedonevidentiarymaterial,57,58–59t

collectionandanalysisofclinicalandenvironmentalsamplesandcrosscontamination,60,176–77

clinicalandepidemiologicalsamples,60–62,64

crimesceneenvironmentalsamples,64–66

lettermaterialandcrosscontamination,67–70

samplesfromoverseassiteidentifiedbyintelligence,66–67

federalcoordinatedresponseandassignmentoflaboratorywork,55–57

Seealsospecifictopics

AmesAncestorsequence,103

AmesstrainB.anthracis,6–7,31,32,44,103,129,169

AmesstrainDNA,8

Amesstrainidentification,FBIdocumentsregarding,162–63

Amesstrainsamples,subpoenaprotocolforcollectionandsubmissionof,126–30,132,144–47

amplifiedfragmentlengthpolymorphism(AFLP),98

anthraxmailings,40–41sizeandgranularityofmaterialinletters,79–80trajectoryandoutcomesof,61–62,62f

anthraxmailingscasebackground,25–26chronologyof,26,30–31

timelineofkeyevents,28–30t

timelineofscientificeventsin,48–52tanthraxprogram,66ArmedForcesInstituteofPathology(AFIP),

56,57,66,79,81–84,95

B

Bacillussp.

B.anthracis,1,37,44–45,97asbiologicalweapon,40–41biology,37–38,44–45chemicalandphysicalproperties,177clinicalaspects,38–40earlyhistoryofAmesstrainof,44identificationofB.anthracisstrain,

97–100isolates(seemorphotypeisolates)modesoftransmission,39phylogeny,

41–44worldwidedistributionoflineagesof,

43,43f

B.cereus,42,84,88

B.subtilis,84,96,121–22,169–70contaminationofNewYorksampleswith,65,104–6

geneticdiversityandphylogeneticcharacterizationof,171

205

inNewYorkPostletter,96,105,121–22wholegenomeassemblyofB.subtilisisolate,170–71

B.subtilisscreening,171–72bacterialgrowthconditionsandprocessing

methods,featuresof,87–89bioterrorisminvestigations,53,54Blanco,Ernesto,26bloodagar,89,113,167Brokaw,Tom,26

C

carbon-14(14C)dating,165–66.Seealsoradiocarbondating

CenterforAcceleratorMassSpectrometry(CAMS),90

CentersforDiseaseControlandPrevention(CDC),126

Chemical,Biological,Radiological,andNuclear(CBRN)SciencesUnit/ChemicalBiologicalScienceUnit(CBSU),71

chemicalanalysiscommitteefindings,93–96methodsfor,81,81tSeealsospecifictopics

ChemistryUnit,FBILaboratory,165

CommitteeonReviewoftheScientificApproachesUsedDuringtheFBI’sInvestigationofthe2001BacillusanthracisMailingsbiographicalinformationon,193–204chargeto,27committeeprocess,33–35findings,4–23t,70–74,121–23formation,xirecommendations,10,70–74

Seealsospecifictopicscrosscontamination.SeeunderAmerithraxinvestigations

D

Daschle,Tom,letterreceivedby,26,28t,30–31,58–59t,68,79–80,82,87,113–22.Seealsospecifictopics

“deepsequencing,”101,150

DefenceResearchEstablishmentSuffield(DRES),69

DepartmentofJustice(DOJ)AmerithraxInvestigativeSummary,93,147scientificconclusions,11–23t,32–33

diatrizoate,38detectionof,77,87–89,96dilutionexperiments,188–89DNA,8,102DugwayProvingGround(DPG),78,95–96,

168–69

E

edemafactor(EF),39Ekaterinburg.SeeSverdlovskoutbreakenvelopemeasurements,92–93enzymesproducedbyB.anthracis

F

FBI(FederalBureauofInvestigation),9documentsprovidedby,161–79declassifiedreports,177scientific

conclusionsandcommittee

findings,4–23t,32–33scientificinvestigations,31–32(seealsoAmerithraxinvestigations)


FBIhazardousmaterials(HAZMAT)teams,64,68FBIRepository(FBIR),32creationof,126–30FBIRepository(FBIR)samplescomparisonofmaterialinletterswith,125–26

analysesbasedonresamplingRMR1029andinterpretationofresults,140–44

committeefindings,144–51


statisticalinterpretationoftheevidenceandanalysesof,132–34

G

geneticengineering,100,102–4,163geneticmarkersinNewYorkPostletter(powder),115t,116–22,148–49

genomeassemblyofB.subtilisisolate,170–71

genomesequencing,101,163–64ofmorphotypeisolates,114–19SeealsoInstituteofGenomicResearch

genotypes,139,139tA1andA3,119,172–73BandD,119–20,173–75developmentandapplicationofassaysfor,

119–21E,120–21,175–76geneticassaystotestforthefour,130mutation,inFBIRsamples,133–34,133t,

134t

observedandexpecteddistributionofpositivesignaturesforthefour,137,138t

inRMR1029,125,130–32,138,139t,140–42,145–48

H

hazardousmaterials(HAZMAT)teams,64,68heme,89,167

I

inductivelycoupledplasma-opticalemissionspectroscopy(ICP-OES),81–83,94inhalationalanthrax,26,28–29t,30,31,39,40,44–45,60–62,64,97InstituteofGenomicResearch(TIGR),32,102–5,115,117–20

InstituteofInfectiousDiseases.SeeU.S.ArmyResearchInstituteofInfectiousDiseases

Ivins,Bruce,26,140–42,145

J

JusticeDepartment,U.S.SeeDepartmentofJustice

K

Keim,Paul,99–100

L

LawrenceLivermoreNationalLaboratory(LLNL),79,86,90Leahy,Patrick,letterreceivedby,30,68,69,

76–80,88–92,96,109,113–22powderon,63fsiliconcontent,82–84,85f,87,94,95

lethalfactor(LF),39lettermaterial,siliconandotherelementsin,80elementalanalysis,81–84

letterpowderLeahy,62,63f,64NewYorkPost,62,63f,64

LosAlamosNationalLaboratory(LANL),100

M

massspectrometry(MS),88–90mediacomponentanalysis,89meglumine,38

detectionof,77,87–89,96

morphologicalvariantsinevidentiarymaterial,identificationandcharacterizationofcommitteefindings,121–23

developmentandapplicationofassaysforgenotypes,119–21selectioncriteriaforgeneticvariationsusedinscreening,113–14Seealsomorphotypesmorphotypeisolates,wholegenomesequencingof,114–19

morphotypes,5–6backgroundinformationon,107–9defined,106detectionandcharacterizationof,109–13

phenotypiccharacteristics,113,113tgeneticcharacterizationof,116,116treasonsFBIwasinterestedin,106–7

multiple-locusVNTRanalysis(MLVA),98,99

N

nanotime-of-flightsecondaryionmass

spectrometry(nano-SIMS),86NationalAcademies,xii–xiii,35NationalAcademyofSciences(NAS),xi,56NationalResearchCouncil(NRC),xi,1,26

NewYorkCityletters,26,60–62.Seealso

AmericanMedia,Inc.

NewYorkPostletter(powder),62,63f,64,68,85f,94–95

B.subtilisin,96,105,121–22geneticmarkersin,115t,116–22,148–49SEM-EDXanalysisof,83–85,94–96

P

plasmids,39polymerasechainreaction(PCR)technique,102

polymorphism(s)amplifiedfragmentlength,98singlenucleotide,115–18

postalworkers,61protectiveantigen(PA),39

R

radiocarbondating,181–82ofB.anthracissamples,90ofletterreceivedbyPatrickLeahy,95–96Seealsocarbon-14(14C)dating

RenoCal,88,168RMR1029(spore-containingflask),32,74,77,

85,88,96,149,150analysesbasedonresampling,140–44genotypesin,125,130–32,138,139t,

140–42,145–48resultsobtainedbyresamplingfrom,142,

143tRMR1029spores,derivationof,130–32RMR-1030(spore-containingflask),85n

S

SandiaNationalLaboratories(SNL),83,84,

164–65scanningelectronmicroscope.SeeSEMscience

FBI’susesof,35–36qualifiersofcertaintyinbiologicalsciences,53,55

andscientificinvestigation,aspartoflawenforcementinvestigation,47,53–55

“SelectAgents”program,126SEM(scanningelectronmicroscope),79,81,85f

SEM-EDX(scanningelectronmicroscopewithenergy-dispersiveX-ray)analysis,79,81–86

ofLeahypowder,85f

ofNewYorkPostletter,83–85,94–96Senateletters,26,30–31siliconanalysis,7–8,12t,84–87,94–96.See

alsounderlettermaterialsiliconmeasurementsinevidentiaryandsurrogatesamples,82,82tsinglenucleotidepolymorphisms(SNPs),

115–18SovietUnion.SeeSverdlovskoutbreakspatiallyresolvedelementalanalysis,83–84spo0Agene,108–9spo0Fgene,117sporepreparation

estimatesofmediavolumerequiredfor,

77,77tandpurification,75–78timeneededfor,8

sporesbiology,37–38,44–45derivationofRMR1029,130–32estimatedrangesoftotalnumberof,76,

76tresilience,37–38stableisotopeanalysis,90–93,166–67forensicspotential,183–84

StableIsotopeRatioFacilityforEnvironmentalResearch(SIRFER),90–93

StatisticalAnalysisReport(FBI),135–36committeeassessmentof,185–91representativeness,randomness,and

independence,136–40,185–86Stevens,Robert,26,28t,60subpoenaprotocolforcollectionand

submissionofAmesstrainsamples,

126–30,132,144–47surrogatepreparationandpurification,78–79Sverdlovskoutbreak,41

T

TaqMantechnique,105,106TechnicalReviewPanels,56

UV

U.S.ArmyResearchInstituteofInfectiousvariablenumbertandemrepeat(VNTR)Diseases(USAMRIID),56–57,66,analysis,98,99109,131,140–41,161–62volatileorganicchemicals(VOCs),89–90

USDOJ.SeeDepartmentofJustice

W

watersamples,stableisotopeanalysisof,92

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