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MICROBIAL FORENSICS THIRDEDITION

Editedby BruceBudowle

StevenSchutzer

StephenMorse

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Contributors

M.W.Allard DivisionofMicrobiology,Ofﬁceof RegulatoryScience,CFSAN,USFDA,College Park,MD,UnitedStates

JonathanAllen LawrenceLivermoreNationalLaboratory,Livermore,CA,UnitedStates

PhilipS.Amara CollegeofPublicHealth,UniversityofNebraskaMedicalCenter,Omaha,NE, UnitedStates

NeelG.Barnaby FBILaboratory,Quantico,VA, UnitedStates

JohnR.Barr CentersforDiseaseControlandPrevention,NationalCenterforEnvironmentalHealth, DivisionofLaboratorySciences,Atlanta,GA, UnitedStates

AerielBelk AnimalSciences,ColoradoStateUniversity,FortCollins,CO,UnitedStates

NicholasH.Bergman NationalBioforensicand AnalysisCenter,Frederick,MD,UnitedStates

ChristopherA.Bidwell FederationofAmerican Scientists,Washington,DC,UnitedStates

DawnBirdsell ThePathogenandMicrobiomeInstitute,NorthernArizonaUniversity,Flagstaff,AZ, UnitedStates

ThomasA.Blake CentersforDiseaseControland Prevention,NationalCenterforEnvironmental Health,DivisionofLaboratorySciences,Atlanta, GA,UnitedStates

E.W.Brown DivisionofMicrobiology,OfﬁceofRegulatoryScience,CFSAN,USFDA,CollegePark, MD,UnitedStates

SibylBucheli DepartmentofBiologicalSciences, SamHoustonStateUniversity,Huntsville,TX, UnitedStates

BruceBudowle CenterforHumanIdentiﬁcation, DepartmentofMicrobiology,Immunologyand Genetics,UniversityofNorthTexasHealthScience Center,FortWorth,TX,UnitedStates

RobertL.Bull FBILaboratory,Quantico,VA, UnitedStates

JamesBurans NationalBioforensicandAnalysis Center,Frederick,MD,UnitedStates

DavidO.Carter LaboratoryofForensic Taphonomy,ForensicSciencesUnit,Divisionof NaturalSciencesandMathematics,Chaminade UniversityofHonolulu,Honolulu,HI,United States

AngelaChoi DepartmentofMicrobiology,Icahn SchoolofMedicineatMountSinai,NewYork, NY,UnitedStates

BrookeL.DeatherageKaiser PaciﬁcNorthwest NationalLaboratory,Richland,WA,UnitedStates

R.ScottDecker Bio-LogicSecurity,LLC,LasVegas, NV,UnitedStates

HeatherDeel AnimalSciences,ColoradoStateUniversity,FortCollins,CO,UnitedStates

DavidM.Engelthaler TGenNorth,Translational GenomicsResearchInstitute,Flagstaff,AZ,United States

HectorF.Espitia-Navarro SchoolofBiologicalSciences,GeorgiaInstituteofTechnology,Atlanta, GA,UnitedStates;PanAmericanBioinformatics Institute,Cali,ValledelCauca,Colombia

JacquelineFletcher DepartmentofEntomology & PlantPathology,NationalInstituteforMicrobial Forensics & FoodandAgriculturalBiosecurity, OklahomaStateUniversity,Stillwater,OK,United States

JeffreyT.Foster ThePathogenandMicrobiome Institute,NorthernArizonaUniversity,Flagstaff, AZ,UnitedStates

AdolfoGarcía-Sastre DepartmentofMicrobiology, IcahnSchoolofMedicineatMountSinai,New York,NY,UnitedStates;DepartmentofMedicine, IcahnSchoolofMedicineatMountSinai,NewYork,

NY,UnitedStates;GlobalHealthandEmerging PathogensInstitute,IcahnSchoolofMedicineat MountSinai,NewYork,NY,UnitedStates;The TischCancerInstitute,IcahnSchoolofMedicineat MountSinai,NewYork,NY,UnitedStates

JenniferS.Goodrich NationalBioforensicand AnalysisCenter,Frederick,MD,UnitedStates

BradleyHart LawrenceLivermoreNationalLaboratory,Livermore,CA,UnitedStates

DavidR.Hodge DepartmentofHomelandSecurity, Washington,DC,UnitedStates

PaulJ.Jackson AdjunctProfessor,Nonproliferation andTerrorismStudies,JamesMartinCenterfor NonproliferationStudies,MiddleburyInstituteof InternationalStudiesatMonterey,Monterey,California,UnitedStates

CrystalJaing LawrenceLivermoreNationalLaboratory,Livermore,CA,UnitedStates

RudolphC.Johnson CentersforDiseaseControl andPrevention,NationalCenterforEnvironmental Health,DivisionofLaboratorySciences,Atlanta, GA,UnitedStates

I.KingJordan SchoolofBiologicalSciences,GeorgiaInstituteofTechnology,Atlanta,GA,United States;PanAmericanBioinformaticsInstitute, Cali,ValledelCauca,Colombia;AppliedBioinformaticsLaboratory,Atlanta,GA,UnitedStates

SuzanneR.Kalb CentersforDiseaseControland Prevention,NationalCenterforEnvironmental Health,DivisionofLaboratorySciences,Atlanta, GA,UnitedStates

ArnoldF.Kaufmann UnitedStatesPublicHealth Service,StoneMountain,GA,UnitedStates

PaulKeim ThePathogenandMicrobiomeInstitute, NorthernArizonaUniversity,Flagstaff,AZ,United States

TerryL.Kerns Bio-LogicSecurity,LLC,LasVegas, NV,UnitedStates

AliS.Khan CollegeofPublicHealth,Universityof NebraskaMedicalCenter,Omaha,NE,United States

RachelE.Kieser CenterforHumanIdentiﬁcation, GraduateSchoolofBiomedicalSciences,University ofNorthTexasHealthScienceCenter,FortWorth, TX,UnitedStates

RobKnight DepartmentofPediatrics,Department ofComputerScienceandEngineering,Microbiome InnovationCenter,UniversityofCalifornia,San Diego,CA,UnitedStates

AsjaKorajkic U.S.EnvironmentalProtection Agency,OfﬁceofResearchandDevelopment, Cincinnati,OH,UnitedStates

StevenB.Lee InternationalForensicResearchInstitute,FloridaInternationalUniversity,Miami,FL, UnitedStates;ForensicScienceProgram,DepartmentofJusticeStudies,SanJoseStateUniversity, SanJose,CA,UnitedStates

AnastasiaP.Litvintseva MycoticDiseasesBranch, CentersforDiseaseControlandPrevention, Atlanta,GA,UnitedStates

DouglasG.Luster USDAARS,ForeignDiseaseWeedScienceResearchUnit,FortDetrick,MD, UnitedStates

AaronLynne DepartmentofBiologicalSciences, SamHoustonStateUniversity,Huntsville,TX, UnitedStates

LeonardW.Mayer PanAmericanBioinformatics Institute,Cali,ValledelCauca,Colombia;Applied BioinformaticsLaboratory,Atlanta,GA,United States

UlrichMelcher OklahomaStateUniversity,DepartmentofBiochemistry & MolecularBiology,Stillwater,OK,UnitedStates

EricD.Merkley PaciﬁcNorthwestNationalLaboratory,Richland,WA,UnitedStates

JessicaL.Metcalf AnimalSciences,ColoradoState University,FortCollins,CO,UnitedStates

DeEttaK.Mills InternationalForensicResearch Institute,FloridaInternationalUniversity,Miami, FL,UnitedStates

StephenA.Morse IHRC,Inc.,Atlanta,GA,United States

TedD.Mullins AmericanTypeCultureCollection (ATCC),Manassas,VA,UnitedStates

RandallMurch VirginiaPolytechnicInstituteand StateUniversity,Arlington,VA,UnitedStates

ForrestW.Nutter,Jr. IowaStateUniversity,DepartmentofPlantPathologyandMicrobiology,Ames, IA,UnitedStates

FranciscoM.OchoaCorona DepartmentofEntomology & PlantPathology,NationalInstitutefor MicrobialForensics & FoodandAgriculturalBiosecurity,OklahomaStateUniversity,Stillwater, OK,UnitedStates

SamOgden AnimalSciences,ColoradoStateUniversity,FortCollins,CO,UnitedStates

RichardT.Okinaka ThePathogenandMicrobiome Institute,NorthernArizonaUniversity,Flagstaff, AZ,UnitedStates

TalimaPearson ThePathogenandMicrobiome Institute,NorthernArizonaUniversity,Flagstaff, AZ,UnitedStates

BernardR.Quigley IHRC,Inc.,Atlanta,GA,United States

LavanyaRishishwar SchoolofBiologicalSciences, GeorgiaInstituteofTechnology,Atlanta,GA, UnitedStates;PanAmericanBioinformaticsInstitute,Cali,ValledelCauca,Colombia;Applied BioinformaticsLaboratory,Atlanta,GA,United States

JasonW.Sahl ThePathogenandMicrobiomeInstitute,NorthernArizonaUniversity,Flagstaff,AZ, UnitedStates

StephenA.Saltzburg WallaceandBeverleyUniversityProfessor,TheGeorgeWashingtonUniversity LawSchool,UnitedStates

HaraldScherm UniversityofGeorgia,Department ofPlantPathology,Athens,GA,UnitedStates

DavidG.Schmale,III VirginiaTechUniversity, SchoolofPlantandEnvironmentalSciences,Blacksburg,VA,UnitedStates

SarahE.Schmedes AssociationofPublicHealth Laboratories,SilverSpring,MD,UnitedStates

StevenE.Schutzer DepartmentofMedicine, RutgersNewJerseyMedicalSchool,Newark,NJ, UnitedStates

OrinC.Shanks U.S.EnvironmentalProtection Agency,OfﬁceofResearchandDevelopment, Cincinnati,OH,UnitedStates

FrankP.Simione AmericanTypeCultureCollection(ATCC),Manassas,VA,UnitedStates

TomSlezak LawrenceLivermoreNationalLaboratory,Livermore,CA,UnitedStates

JeniferA.L.Smith DistrictofColumbiaDepartment ofForensicSciences,Washington,DC,UnitedStates

CarlaS.Thomas UniversityofCalifornia,Plant PathologyDepartment,Davis,CA,UnitedStates

StephanP.Velsko LawrenceLivermoreNational Laboratory,Livermore,CA,UnitedStates

RichardR.Vines AmericanTypeCultureCollection (ATCC),Manassas,VA,UnitedStates

AmyVogler ThePathogenandMicrobiomeInstitute,NorthernArizonaUniversity,Flagstaff,AZ, UnitedStates

DavidM.Wagner ThePathogenandMicrobiome Institute,NorthernArizonaUniversity,Flagstaff, AZ,UnitedStates

KarenL.Wahl PaciﬁcNorthwestNationalLaboratory,Richland,WA,UnitedStates

CharlesH.Williamson ThePathogenandMicrobiomeInstitute,NorthernArizonaUniversity, Flagstaff,AZ,UnitedStates

MarkWilson MRWAnalytics,LLC,Fredericksburg,VA,UnitedStates

AugustWoerner CenterforHumanIdenti ﬁcation, UniversityofNorthTexasHealthScienceCenter, FortWorth,TX,UnitedStates;Departmentof Microbiology,ImmunologyandGenetics,UniversityofNorthTexasHealthScienceCenter,Fort Worth,TX,UnitedStates

DavidS.Wunschel PaciﬁcNorthwestNational Laboratory,Richland,WA,UnitedStates

Foreword

Iamhonoredthattheauthorshaveaskedme tocontributetheforewordtothethirdeditionof MicrobialForensics. Myconnectiontothebook andtothe ﬁeldingeneralwasasafacilitator ratherthanasaresearcher,anditwasinthe distantpast,20yearsago!

MicrobialForensics receivedinputanddirection inaseriesofmeetingsheldattheBanburyCenter, aconferencecenteratColdSpringHarborLaboratory.Indeed,somemeetingsheldattheBanbury,whichpredatetheemergenceofMicrobial Forensicsasaformalizeddiscipline,laidsome ofthegroundworkforthismust-neededforensic ﬁeld.Banburyhasaninternationalreputationfor intensivediscussionmeetingswithfewerthan30 participantsoftopicsinthelifesciencesthat wouldbeneﬁtfromcriticalreview,topicsthat arefrequentlycontroversialandofteninvolve issuesofpolicyaswellasscience.

In1999,SuzanneVernonandBillReevesof theCentersforDiseaseControl(CDC) approachedmeaboutameetingtobefunded byCDCtodiscusshowitwouldbepossibleto detectanddeveloptestsforunknownpathogens.CDCwasinterestedbecauseithadbeen proposedthatchronicfatiguesyndrome(CFS) wasaninfectiousdisease,butthepathogen detectiontechniquesusedbyCDC serology, culture,PCR hadfailedtoidentifyaninfectiousagentassociatedwithCFS.By1999,new techniqueswerecomingonline,andCDC wantedtoreviewhowthesemightbeusedto ﬁndnovelorpreviouslyuncharacterizedpathogensassociatedwithCFS.

Banburyalreadyhadheldaseriesofmeetings onwhatwasstilla “newlyemerged” disease, Lymedisease . The firstmeetingwasheldin 1991,onlynineyearsafterWillyBurgdorfer hadidenti fiedthepreviouslyunknownpathogen,thespirochete, Borreliaburgdorferi.Iput Suzanneintouchwiththeorganizersofthose meetings,SteveSchutzerandBenLuft,andthe threeorganizedtheApril2000meeting “StrategiesforDetectionandIdentificationofUnknown Pathogens.”

Theprimaryfocusofthismeetingwas health,andtheword “forensic” didnotappear. However,oneoftheparticipantswasRoger Breeze,thenwiththeUSDepartmentofAgriculture.Whilethetitleofthemeetingheorganized inOctober2000hadtheratherinnocuoustitle “TheChallengeofInfectiousDiseasesinthe 21stCentury,” hisviewofunrecognizedinfectiousdiseasewasquitedeﬁnitelyforensic;the invitationreferredtotheneedforstrategiesto dealwithnewandemergingthreats “ ofwhich biologicalterrorismisthelatest.”

Finally,inNovember2002,Schutzerand BreezewithBruceBudowle(thenwiththeFBI LaboratoryinQuantico)organizedthe ﬁrst “MicrobialForensics” meeting,followedbyadditionalfourmeetings.The ﬁnalmeetingin2010 wason “MicrobialForensicsintheEraofGenomics,” whichreviewedtechniquesforDNA sequencingandbioinformaticsthatwereunfathomablemereeightyearsearlier.

Despitetheseandotherdramaticadvancesin technology,itisnotsurprisingthatthesame

broadissuesremain,someofwhichareidentiﬁedintheauthors’ introduction,forexample, thedifferingneedsoftheworldsofpublichealth andforensics;thelatterrequiredmeetingthe legalstandardsforevidencethatmightbeused inthecourt.

Theauthorsrefertotheissueofwhethermicrobialforensicswillbecentralized,forexample, attheNationalBioforensicsAnalysisCenter,or becomedistributed.Ifthelatter,adequate resources grainingandinfrastructure willbe needed,butthismaybehardtojustifyfor whatonehopeswillberareevents.

Insomeways,Iamremindedoftheearly daysofforensicDNA ﬁngerprinting.Thetechniquesandassociatedpopulationgeneticanalyseshadbeendevelopedinacademic laboratories,andthereweremanyobstaclesto beovercomebeforeDNA ﬁngerprintingwas accepted.Itrequiredtrainingintechniquesand theory,aswellasanacceptancebythepublic, lawenforcement,anddefenseattorneys,before

itbecameapowerfultoolforthejusticesystem. IamproudtorecallthataBanburyCenter meetingin1988(attendedbyBruceBudowle) contributedtocementingthistechnologyasthe goldstandardoftheforensicsciences.

Finally,Ifullyendorsetheauthors’ closing remarks.Scientificevidencecarriesgreatauthorityoverandaboveitsevidentiaryvalue.This responsibilityrequiresthatthoseemploying scientifictechniquesintheserviceofforensicsciencedosotothehigheststandardspossible.It tookseveralyearsforDNA fingerprintingto reachthatposition,anditwilltakethesame commitmentformicrobialforensicstoattain thesamelevelofcredibilityandacceptance. Butwithoutadoubt,itwillbewellworthwhile asthisburgeoningdisciplinecontinuestofulfill itspromise.

JanAWitkowskiPhD Professor ColdSpringHarborLaboratory

Introduction

StephenA.Morse1,BruceBudowle2,StevenE.Schutzer3

1IHRC,Inc.,Atlanta,GA,UnitedStates; 2CenterforHumanIdenti ﬁcation,UniversityofNorthTexas HealthSciencesCenter,FortWorth,TX,UnitedStates; 3DepartmentofMedicine,RutgersNewJersey MedicalSchool,Newark,NJ,UnitedStates

Deliberatedisseminationofabiologicalagent viaseveraldifferentroutes,includingair,water, food,infectedvectors,andmanmadeavenues, presentsachallengetoglobalhealthandsecurity.Theabilitytoidentifytheperpetrator(s)in anexpeditiousandeffectivemannerisofparamountimportanceasbothadeterrentandfor gauginganappropriateresponse.Eliminating thoseindividualswhoarefalselyassociated withevidenceoracrimeisofequalimportance.

Thepastdecadehasseenmajoradvancesin thescienti ficdisciplineofmicrobialforensics, whosecoreobjectiveisattribution:theinvestigativeprocessaimedatidentifyingtheperpetratorsofabiowarfare,bioterrorism,or biocrimeeventandbringingthemtojustice. Thishighlyinterdisciplinary field,whichhas largelyemergedthroughUSinitiatives(Tucker andKoblentz,2009;U.S.,2010),encompasses traditionallawenforcementandforensicinvestigativemethodsaswellasthedetailedcharacterizationofsamplescontainingbiologicalagents. Moreover,withtheadventofhighthroughput technologies,suchasmassivelyparallel sequencing,microbialforensicsasadiscipline hasexpandedbeyonditsprimaryfocuson microbesandtheirproductsinvolvedinbioterrorismandbiocrimestootherapplicationssuch asexploitingthehumanmicrobiomeforhuman identification,humangeolocation,anddeterminingpostmorteminterval.

Microbialforensicinvestigationsandmolecularepidemiologicinvestigationssharemany aspectsbutareperformedfordifferentreasons. Anepidemiologicinvestigationaimstoidentify thesourceofanoutbreakandtounderstand routesoftransmissioninordertopreventfurther spreadandreducetheriskoffutureoutbreaks througheffectivepreventivemeasures.Amicrobialforensicinvestigationisundertakentoidentifytheperpetratorofacrimeandtogatherhigh qualityevidencethatcanbeusedforindictment andconvictionofperpetratorsortohelpdecisionmakersprepareanappropriateresponse toanattack.Amicrobialforensicinvestigation hasbeendescribedasconsistingofthreeinterrelatedstages:identiﬁcationofthebiological agent(s)responsibleforanevent;characterizationoftheeventasintentional,unintentional (e.g.,duetonegligenceorcarelessness),ornaturallyoccurring;and,iftheeventisdeemed illegitimate(i.e.,intentionalorunintentional), attributionofusetoaspeci ﬁcperpetrator(s) (KoblentzandTucker,2010).Additionally, analysesmaybeusedtodeterminetheprocess(es)usedtoproduceabioweapon,which canprovideadditionalforensicevidenceand insightintothesophisticationoftheproduction process.Regardlessifaneventwasintentional oraccidental,animmediategoalistoprevent additionalattacksandprotectthepublic.

Manyofthequestionsaskedduringthe first twostagesofamicrobialforensicinvestigation areidenticaltothoseofanepidemiologicinvestigation,andthesamemethodsandtechnologies aregenerallyusedtoanswerthem(Sjodinetal., 2013).However,theattributionstepisuniqueto microbialforensics.Inadditiontotraditional forensicanalysesofrecoveredevidencefrom thecrimescene(analysisofhumanDNA, fingerprints, fibers,etc.),detailedanalysesareconductedoftheresponsiblebiologicagent (bacterium,virus,fungus,ortoxin).Itispossible thattheresultsofanepidemiologicinvestigation maysuggestadeliberateorunintentionalrelease ofabiologicalagentandthusleadtotheinitiationofamicrobialforensicinvestigation.The intentionalreleaseofabiologicalagentcanbe doneovertly,inwhichcasetheperpetrators willannouncetheirresponsibility,orcovertly, inwhichcasetheywillnotcallattentiontothe releaseofabiologicalagent(Budowleetal., 2005).Itislikelythatmostunintentionalreleases willbecovert-likeinnature(ZimmerandBurke, 2009).

Sincethepublicationofthe firsteditionofthis bookin2005,the fieldofmicrobialforensicshas evolvedsubstantiallyinitsabilitytoattributethe sourceofmicroorganismsandtoxinsthathave beenusedincasesofbioterrorismandinbiocrimes.The2001anthraxmailattack(often referredtoas “Amerithrax”)fortunatelyremains theworstandbest-knownexampleofsuccessful bioterrorismonUSsoil(Chapters2and3).At thetime,theUSwasillpreparedtoinvestigate theuniquetypesofmicrobiologicalevidence forattributionthatweregeneratedbythatinvestigation,althoughtraditionaltypesofforensic evidencecouldreadilybecollected,analyzed, andinterpretedwithintheframeworkofastatisticallysoundscientificfoundation.The first editionofthisbookhadonlylimitedreference totheanthraxmailattackbecausemuchofthe scienti ficexaminationwasstillinprogressand methodologieswerestillunderdevelopment. Earlycapabilitieswereinitiallylimitedto

detectionandidentificationanddidnotinclude detailedcharacterizationandcomparativeanalyses.Thus,the firsteditionwasdevelopedasa foundationaltexttostimulatescientists,legal experts,anddecisionmakersresponsiblefor analyzingandinterpretingevidencefroman actofbiologicalwarfare,bioterrorism,biocrime, orinadvertentreleaseofamicroorganismor toxinforattributionpurposesandtodescribethe discipline.Inaddition,theinitialeditionofthis bookdiscussedsomeofthefutureopportunities andchallengesforthemicrobialforensic fi eld.

Thesecondeditionofthisbook,publishedin 2011,containedseveralchaptersthatbroughtthe Amerithraxinvestigationtoaconclusion.The anthraxmailinvestigation,oneofthemost intenseandexpansiveinvestigationstodateby theFederalBureauofInvestigationandUS PostalService(http://www.fbi.gov/antrhrax/ amerithraxlinks.htm),isnowconsideredclosed. However,thethirdeditionofthisbookrevisits thiscaseandprovidesa ﬁrst-handaccountof theinvestigationaswellaslessonslearnedon whatshouldbedoneinmicrobialforensicsto supportaninvestigationandequallyimportant whatshouldbeavoided.

Manyoftheelementsdiscussedinthe ﬁrst andsecondeditionsofthisbookareechoedin the2009USNationalResearchandDevelopmentStrategyforMicrobialForensics(U.S., 2010),whichidentiﬁesthreatawareness,preventionandprotection,surveillanceanddetection, andresponseandrecoveryastheessentialpillarsofUSnationalbiodefensepolicy. “Attribution” theinvestigativeprocessby whichtheUSgovernmentlinkstheidentityof aperpetratororperpetratorsofillicitactivity andthepathwayleadingtocriminalactivity is partofthesurveillanceanddetectionpillar. Makingadeterminationofattributionfora plannedoractualperpetratedbiologicalattack wouldbetheculminationofacomplexinvestigativeprocessdrawingonmanydifferentsourcesofinformation,includingtraditional forensicanalysisofmaterialevidencecollected

duringaninvestigationofaplannedattackor materialevidenceresultingfromanattackand relevantsubjectmatterexperts.Oneofthekey sourcesofattributioninformationinabiological attackistheresultofamicrobialforensic investigation.

Futureinvestigationsofbiologicalwarfare, bioterrorismevents,orbiocrimesarelikelyto beasmultifacetedastheAmerithraxinvestigation,ifnotmoreso,andwilldemandmoreintegrationandbettercommunicationamong governmentagencies.Fortunately,thetechnologiestodayprovidemuchbettercapabilitiesto respondwithastrongmicrobialforensicprogram.Atthesametime,microbialforensicsis stilladeveloping fieldfacingnumerousscientific challenges.Fortheseandotherreasons,governmentsaremakinginvestments,oftenforpublic healthpurposes,toprovidearobustcapability todetect,identify,andcharacterizebiological agents.Advancesinthisareahavebeensuf ficientlyexcitingandsignificanttomakethisthird editionof MicrobialForensics bothtimelyand necessary.Thereisgrowinginterestandapplicationofmicrobialforensicprinciplesinfood safety,waterquality,andbioinformatics,and thusthiseditioncontainschapterstocovera broaderaudienceinterestthanthatofnational security.Werecognizethattraditionaldetective investigation,coupledwithscientificanalysis ofevidenceandfrequentlyadditionalongoing andnovelexperimentation,cangenerateinvestigativeleads.Thus,chaptersonmicrobial forensicapproachestohumanidentification andthedeterminationofpostmorteminterval havebeenincluded.

The ﬁrsteditionofthisbookwasdeveloped forindividualsenteringthe ﬁeldofmicrobialforensicswhowerelookingforasinglesourcefor initialguidanceandinformation.Thesecond editionsupportedthatneedwithacombination ofbasictextsandchaptersonmoresophisticated technologies,suchasnonbiologicalanalytical toolsandnext-generationsequencingmethods. Therapidadvanceintechnologyandits

applicationhaveledtothethirdedition.This editionrecognizesthecombinationofdiverse disciplinesthatmustbeexploitedtoanalyzeevidence,includingbiology,microbiology,medicine,chemistry,physics,statistics,population genetics,computerscience,andcomputational biology.Newtechniquesmustbedeveloped andemployedtoextractthemostinformation fromforensicevidenceobtainedfromterrorist andcriminalevents,especiallywhenmoretraditionalformsofevidenceareeithernotavailable orverylimitedincontent.Yetmicrobial forensics amaturingdiscipline depends veryheavilyonsometraditionalandfrequently overlookedscienti ﬁcvalues:willingnesstoshare oftenpricelesssampleswithothersacrossinternationalboundaries,rigorouscurationofmicrobialrepositorysamplesoverdecades,and organizationandexecutionofinternational collaborativestudieswithrecognitionofall involved.

Overthepastcentury,sciencehasplayedan increasinglygreaterroleincriminalinvestigation(Budowleetal.,2008,2009).Microbial forensicsciencewillcontinuethistradition.But scientiﬁcanalysisisonlypartoftheprocess. Forensicscienceresultsmustbeintegratedwith otherinformation,andattentionmustbepaid tostepsthatwillassurethequalityofresultsas wellassupporttheiradmissibilityinacourtof law(seeChapter23 27).Westressqualitycontrolandqualityassuranceasthemeanstoensure integrityoftheevidence.Practicessuchasadherencetochainofcustodyprocedures,documentationofactivities,andtheuseofvalidated reagents,calibratedequipment,negativeand knownpositivecontrolsamples,validatedprocedures,standardoperatingprocedures,andso onaretheessenceofreliabilityandconﬁdence. Thesecriteriainturnensureadmissibilityin court.

Thescienti ﬁcfoundationsofmicrobialforensicswillbestrengthened,builtupon,andlikely remodeledbyourpresentandfuturecolleagues. Theiraccomplishmentsoverthepasteightyears

haveledtothisnewedition.Welookforwardto theircontinuedinput,interaction,andinsight.

References

Budowle,B.,Bottrell,M.C.,Bunch,S.G.,Fram,R., Harrison,D.,Meagher,S.,etal.,2009.Aperspectiveonerrors,bias,andinterpretationintheforensicsciencesand directionforcontinuingadvancement.J.ForensicSci. 54,798 809.

Budowle,B.,Schutzer,S.E.,Ascher,M.S.,Atlas,R.M., Burans,J.P.,Chakraborty,R.,etal.,2005.Towardasystemofmicrobialforensics:fromsamplecollectiontointerpretationofevidence.Appl.Environ.Microbiol.71, 2209 2213.

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TheKameidoanthraxincident:a microbialforensiccasestudy

ArnoldF.Kaufmann1,PaulKeim2

1UnitedStatesPublicHealthService,StoneMountain,GA,UnitedStates; 2ThePathogenandMicrobiome Institute,NorthernArizonaUniversity,Flagstaff,AZ,UnitedStates

Introduction

TheAumShinrikyo,anapocalypticreligious sectbasedinJapan, ﬁrstcametoworldwideattentionin1995astheresultoftheirdeadlysaringas attackontheTokyosubwaysystem(Hudson, 1999;Smithson,2000;Tu,2002).Subsequent investigationsrevealedthattheAumShinrikyo hadlaunchedearlierattackswithbothchemical andbiologicalagents.Thebiologicalattacksutilizing Bacillusanthracis sporesandbotulinum neurotoxinwerenotablyunsuccessful,withfailuretoproduceanycasualtiesinatleastseven allegedattemptsoverseveralyearsbeginningin 1990.Inthischapter,anattackin1993thatwas launchedfromtheAumShinrikyoheadquarters buildingthenlocatedinKameido,aTokyosuburb,willbediscussed,withemphasisonlaboratoryandepidemiologicalstudies(Takahashi etal.,2004;Keimetal.,2001).

TheAumShinrikyo:abriefhistory

ToputtheKameidoincidentintocontext,a briefhistoryoftheAumShinrikyoanditsfounder,

primarilyadaptedfromthereviewsbyHudson, Smithson,andTu,isuseful(Hudson,1999; Smithson,2000;Tu,2002).TheAumShinrikyo wasfoundedbyShokoAsahara,whosebirth namewasChizuoMatsumoto.Bornintopoverty in1955andsufferingseverevisualimpairment duetoinfantileglaucoma,Matsumotowassent atayoungagetoagovernment-subsidized boardingschoolfortheblind.Hepurportedly feltabandonedbyhisfamily,whichmayhave laterledtoanAumShinrikyorulethatfollowers weretocutoffrelationshipswiththeirparentsto attainthesupremetruth.

Havinglimitedvisioninoneeye,Matsumoto developedinﬂuenceovertheotherblindstudents,whopaidhimforvariousservices.During hisstudentyears,hedevelopedareputationasa bullyandconartist.Afterhighschoolgraduation in1975,Matsumotoestablishedasuccessful acupunctureclinic,buthehadtomovetoTokyo in1977duetohisinvolvementina ﬁghtthat resultedininjurytoseveralpersons.Aboutthis time,hisstatedambitionsincludedbecoming theleaderofarobotkingdomorthePrime MinisterofJapan.

InTokyo,hefoundworkasanacupuncturist andenrolledinapreparatoryschoolfortheJapanesecollegeentranceexamination,withagoal ofqualifyingformatriculationinTokyoUniversity.Failingtheentranceexamination,Matsumotomarriedandestablishedanacupuncture clinicandanaturalfoodsshop.Asasideline, heconcoctedanalcoholextractoftangerine skinsthathemarketedasamiracledrugfor weightlossandavarietyofotherconditions. Hissuccessinsalesofthisproductattracted theattentionofthepoliceandsubsequentarrest andimprisonmentforviolatingtheJapanese CosmeticsandMedicalInstrumentsAct.This experiencemayhavecontributedtohisanimositytowardestablishedauthority.

AfterhismovetoTokyo,Matsumotobecame interestedinreligionand,in1981,joinedAgon Shu,anewreligionbasedonBuddhismand yoga.In1984,MatsumotoquittheAgonShu andestablishedAumShinsen,ayogaclubwhich rapidlygrewfrom15tomorethan1000 members.HealsochangedhisnametoShoko AsaharaorBrightLightinJapanese.Following atriptoIndiain1986 87,Asaharachangedthe nameoftheyogaclubtoAumShinrikyo.Aum isaHindusyllablerepresentingthespoken essenceoftheuniverse,andShinrikyoisderived fromtheJapanesewordsfor “supremetruth.” In 1989,AumShinrikyowasofﬁciallyrecognizedas areligioussectinJapan,givingthesecttax advantagesaswellastheabilitytoclaimthe members’ workinthesect’svariousenterprises asvoluntary.Thesect’sgrowthcontinuedwith spreadtoothercountries,includingtheUnited States,Germany,andRussia.

SectmemberslivedaSpartanlifeandwereexpectedtocutoffallassociationsfromtheirpast life,totakeachastityvow,andtoturnoverall theirassetstotheAumShinrikyo.Theyweresubjectedtoaheavydietoftheirmaster’ s “wisdom,” oftensimultaneouslyundergoingfoodandsleep deprivation.Memberswereexpectedtovoluntarilylaborinthesect’svariouscommercialenterprises,suchassalesofherbalteasandnatural

medications,operationofnoodleshops,health clubsandbaby-sittingservices,andcomputerrelatedservices.Thosewhobalkedweredriven everharder,druggedandconﬁned.Insome extremecases,defectorsweremurdered.

Tocarryoutitsactivities,theAumShinrikyo developedacomplexorganizationalstructure consistingof22ministriesplustheOfﬁcesofReligiousMembers.Thelatterwaschargedwith recruitmentofpersonshavingneededskills, suchasmembersoftheJapaneseSelfDefense Forcesandscientists.Ineffect,theAumShinrikyo assumedtheformofashadowgovernment whichcouldsupplanttheexistentJapanesegovernmentifAsahara’sambitionswerefulﬁlled.

By1990,withmembershipinthetensofthousandsspreadoversixcountriesandanestimated 300milliontoabilliondollarsavailable,the AumShinrikyowaswellpositionedtofurther Asahara’sambitionsanddelusions.TheAum Shinrikyoinitiallyattemptedaconventional approachtopoliticalpowerby ﬁeldingaslate of25candidatesforthenationalelectionsthat year.Noneofthecandidates,includingAsahara, wereevenmodestlysuccessful.Asahara believedthattheJapanesegovernmenthad cheatedhimratherthanthattheelectoratewas putoffbythedoomsdayovertonesofthecandidate’sspeeches.Hisbeliefwasreinforcedbythe factthatthenumberofvotesreceivedbyall thecandidateswasfarfewerthanthenumber ofAumShinrikyomembersinJapan.

Basedonapasticheofapocalypticscenarios drawnfromvariousreligions,Asaharapreached thatJapanwasdestinedtosufferanumberof overwhelmingcatastrophes,includingapoison gasattackbytheUnitedStates.Asaharaand hisfollowerswouldsurvivetheloomingArmageddonandevolveintoasuperracedominating theworld.Hebecamemorevocalinexpressing thisbeliefafterthehumiliatingelectoraldefeat in1990.Notcontenttoallowthecatastrophes tooccurintheirowntime,Asaharainitiated developmentofchemicalandbiological weaponstospeeduptheprocess.

Onlythechemicalweaponsprogramhad somesuccess.Afterovercominginitialproductionproblems,theAumlaunchedanattack withsaringasinMatsumotoCityinJune1994. Theattacktargetedjudgespresidingoveraland usedisputebetweentheAumShinrikyoanda localrealestateagent.Suspectingthatthejudges wouldmakeadecisionunfavorabletothecult, Asaharaorderedtheirassassination.Thisgave anopportunitytotesttheeffectivenessoftheir sarinonhumans.Thesarinreleaseutilizeda spraydeviceandresultedin311knowncasualties,with58hospitalized,including7deaths. Thejudgeswereunharmed.

InvestigationoftheMatsumotoCityattack proceededslowly,withoutdefinitiveevidence linkingthecrimetoaspeci ficindividualor group.TheAumShinrikyowassuspected,and asarindegradationproductwasdetectedin soilnearabuildinginanAumShinrikyocompound,theSeventhSatayan,inKamakiuishiki. Thepolicedidnotseekawarranttosearchthe facilitybecauseofaconservativeinterpretation ofpertinentlaws.Inanunrelatedkidnapping case,however,thepolicefound fingerprintevidencethatanAumShinrikyomemberwas involved.Thisgaveajusti ficationforobtaining awarranttoinvestigatethefacility.InMarch 1995,Asaharalearnedoftheplansforapolice raidfromAummemberswithintheJapanese SelfDefenseForces.Inaploytodistractthe policeandbuytime,Asaharadecidedtorelease sarinintheTokyosubwaysystem.Twodays later,theattackwascarriedout,resultingin severalthousandcasualties,including12fatalities.Duringthesameperiod(1994 95)they alsoproduced100 200gofVX,whichwas usedtoattackthreepersons(onedied).Hence, theAum’schemicalweaponsprogramwas moreextensivethansarin.

TheAumShinrikyoculthadbeenthoughtto beanoddgroupandevenanuisanceonoccasion.InvestigationsaftertheTokyosarinattack revealedamoresinisteraspectofthesectand itsleader.Inadditiontothechemicalweapons

developmentprogram,theAumShinrikyowas foundtohavebeenactivelypursuingbiological weapons,albeitwithoutsuccessduetoincompetence.Inparticular,theineffectivereleaseof B.anthracis sporesinKameidowasdiscovered, leadingtotheinvestigationsdiscussedinthis chapter.

TheKameidoanthraxincident

OnJune29 30,1993,complaintsaboutfoul odorswereregisteredwithlocalenvironmental healthauthoritiesinKameidointheTokyo metropolitanarea(Takahashietal.,2004).The odorsoriginatedfromtheeight-storyheadquartersbuildingoftheAumShinrikyo.Someofthe exposedpersonsreportedappetiteloss,nausea, andvomiting.Birdsandpetswerealsoreportedlyill,butthenatureoftheseillnesseswasnot defined.Theenvironmentalhealthofficials requestedpermissiontoinspecttheheadquarters building,butAumShinrikyomembersatthe scenerefused.Theofficialscheckedthebuilding’ s exterior,collectedairsamples,andbegansurveillanceofactivitiesatthebuilding.Otherthanthe nuisanceposedbytheodor,definitivehuman healthriskscouldnotbeidentified.

OnthemorningofJuly1,neighborsbeganto complainaboutloudnoisesandanintermittent mistoriginatingfromoneoftwostructureson theroofwhichwerethoughttobecoolingtowers (Fig.1.1).Asthedayprogressed,118complaints aboutfoulodorswerereceivedfromnearbyresidents,primarilytothesouthofthebuilding. Winds(2 4m/s)thatdayblewfromanortheasterlydirection(Takahashietal.,2004).Lightrain (7mmtotal)fellintheearlymorning,withcloudy conditionsthereafter.Thetemperatureranged from16.9to19.9 Cthroughtheday.

A “gelatin-like,oily,gray-to-black” fluidfrom themistcomingoffthe “coolingtowers ” collectedonthesideofthebuilding(Takahashi etal.,2004).Samplesofthe fluidwerecollected bytheenvironmentalhealthofficialsandstored inarefrigerator(4 C)forlatertesting.

FIGURE1.1 MistbeingdispersedfromspraydeviceontheroofoftheAumShinrikyoheadquartersbuilding,Kameido, Japan,July1,2006(Takahashietal.,2004).

Thenextday,July2,ShokoAsaharaagreedto stopusingtherooftopdevicesandtocleanand vacatethebuilding(Takahashietal.,2004).An environmentalinspectionfoundnoequipment, includingtherooftopdevices,remaininginthe building,andblackstainsonthewallswere theonlynotableobservation.

Theproblem,apparentlybeingresolved,was largelyforgottenuntil1996.Policeinvestigations ofthesarinattackontheTokyosubwaysystem revealedthattheAumShinrikyowasalso involvedinbioterrorism.FollowingtheconservativeJapanesepolicyofnotrevealingcriminal evidenceuntilthetimeoftrialincourt,thetruenatureoftheKameidoincidentwas ﬁrstdisclosedto thepublicinMay1996whenAsaharawas arraigned(Smithson,2000;Takahashietal., 2004).AumShinrikyomembershadconfessed thattheodorsresultedfromeffortstoaerosolize aliquidsuspensionof B.anthracis spores.The motivewastotriggeraninhalationalanthrax epidemicandasubsequentworldwar.The warwouldculminateintheAumShinrikyo membersbecomingasuperracewhichwould ruletheworldinaccordwithAsahara’spreaching(Takahashietal.,2004).

Manyquestionsabouttheincidentremained unanswered.Forexample,didtheattackactually occur?Ifso,were B.anthracis sporesutilized? Couldthespeciﬁc B.anthracis strainanditsorigin beidentiﬁed?Wasthestrainvirulent?Whydid theattackapparentlyfail?Hadillnessesoccurred butgoneundetected?Investigationswereinitiatedtobettercharacterizetheallegedattackand itsconsequences.

Microbialforensicinvestigation

Fluid,thathadbeencollectedfromtheAum ShinrikyoheadquartersbuildinginJuly1993 andsubsequentlystoredat4 C,wasexamined inJanuary2000forbacterialcontent(Keim etal.,2001).Polymerasechainreaction(PCR) screeningofthe fluidwaspositivefor B.anthracis.Microscopicexaminationofthe fluid afterstainingwithmalachitegreenandsafranin revealedspores,nonspecificdebris,andbacterial cellsotherthanlargebacilli.The fluidwas culturedbyspreadingonsheepbloodagarplates andincubatingat37 CunderambientCO2 concentration(Fig.1.2).Basedonthenumberof

bacterialcoloniesobservedontheplatesafterincubation,the ﬂuidcontainedapproximately 4 104 bacterialcolony-formingunits(CFUs) permL.Mostcoloniesgrewonlyweaklyand weremorphologicallyinconsistentwithnormal B.anthracis characteristicswhengrownunder theseconditions.Thepoorlygrowingbacteria werenotfurthercharacterized.

About10%ofthecoloniesontheplateswere typicalof B.anthracis ,beinglargeandhavinga nonhemolytic, “graygroundglass” appearance (Fig.1.2).Thenumberofobservedcolonies consistentwith B.anthracis wasconsistentwith aconcentrationof4 103 CFUpermLofthe fluid.Forty-eightofthesecolonieswerepurified bysingle-colonystreakingandsubjectedtothe 8-locusmultiple-locusvariable-numbertandem repeatanalysis(MLVA)(Keimetal.,2000).All 48colonieswere B.anthracis andhadanidentical MLVAgenotype,thoughtheVNTRmarkeron thepX02plasmidfailedtoamplify.Thegenotype ofall48strainswasvrrA,313bp;vrrB1,229bp; vrrB2,162bp;vrrC1;583bp;vrrC2;532bp;CG3, 158bp;pX01-aat,129bp;pX02-at,noamplification.ThelackofPCRamplificationatthepX02 markersisconsistentwithstrainsthataremissing

thepX02plasmidentirely.Ampliﬁcationofthese locicanoccurincloselyrelated Bacilluscereus strains,buttheactualampliconsizeshadbeen previouslyobservedonlyin B.anthracis.The MLVAgenotypeobservedwasconsistentwith resultsobtainedwiththeSterneanthraxvaccine strain(Fig.1.3).TheSternestrainisamemberof theA3.bdiversitycluster,andinastudyof419 isolatesonlyfournaturallyoccurring B.anthracis strainsintheelectronicdatabaseofworldwide isolateshadthesameseven-markergenotype, thoughthesestrainswerepX02positive(Keim etal.,2000).

Astheabilitytoperformwholegenome sequencinghasimproved,oneisolatecollected

FIGURE1.3 Multiple-locus,variable-numbertandem repeatanalysisofaKameidoisolateandtheSternestrainof Bacillusanthracis (Takahashietal.,2004).

FIGURE1.2 Bloodagarplateofthesamplingfromthe Kameidosite(Takahashietal.,2004).

atthereleasesitewasDNAsequencedusing shortreadIlluminatechnology(Keim,unpublisheddata).TheresultingsequencewasidenticaltothatoftheSternegenomeavailableat NCBI.Thissuggeststhatthecultdirectlyused acommercialstockwithoutmanipulationor extensivelaboratorygrowth.

TheSterne34F2 anthraxvaccinestrainis commerciallyavailableinJapanforveterinary use.Ithadbeenpreviouslyreportedthatthe AumShinrikyohadobtainedaveterinaryvaccinestrainof B.anthracis whichmayhavebeen usedbythemforbioterroristattacks(Hudson, 1999).Ourresultsareconsistentwiththispreviouslyunsubstantiatedreport.TheSternestrain haslowvirulenceduetolackofthepXO2 plasmid,whichisthelocationofgenescoding forthepoly-D-glutamatecapsule,amajorvirulencefactorof B.anthracis

Epidemiologicinvestigation

Culture-con ﬁrmedanthraxisanationally notiﬁablediseaseinJapan,withphysiciansbeing requiredtoreportallcases(Takahashietal., 2004).Onlyfourhumananthraxcaseswere reportedduringthe1990s,withasinglecasebeingreportedinTokyo.TheTokyocasewasdiagnosedinamanwhowasinhiseightiesand residedinSumidawardwhichisadjacentto Kameidoward.ThecaseoccurredinAugust 1994andhadnoobviousassociationwiththe 1993Kameidoincident.

Couldadditionalanthraxcasesfromthe1993 attackhavegoneunrecognizedorunreported?A retrospectivecase-detectionsurveywasconductedin1999toaddressthisquestion(Takahashi etal.,2004).Usingtheofﬁcialregistryof “foul odor” complaints,theresidencesofthe118complainantsweremappedtoidentifytheareaof presumedhighestrisk.The0.33km2 high-risk areadeterminedbythisapproachcontained approximately3400householdsand7000 residents.Atelephonesurveywasconductedof

physiciansat39medicalfacilities(15internal medicine,7dermatology,and15other specialties)servingthearea.Noneofthesephysicianshadtreatedcasesofanthrax,unexplained seriousrespiratoryillnesses,orhemorrhagicmeningitis,acommoncomplicationofsystemic anthraxinresidentsofthehigh-riskarea(Holty etal.,2006).

Discussion

Severallessonscanbelearnedfromthe Kameidoincident.Theinvestigationsuffered fromafailuretodetecttheincidentatthetime. TheAumShinrikyodidnotattractmuchofficial attentionuntilthesarinattackinMatsumoto Cityin1994.TheJapanesecultureisverytolerant ofvaryingreligiousbeliefs,anattitudereflecting Japaneseconstitutionalguaranteesofreligious freedom.TheAumShinrikyowasbutoneof morethan180,000minorreligionsactiveinJapan (Hudson,1999).Thepolicepolicyofconservative interpretationofpertinentlawswasanotherfactor.Inaddition,thepolicyofnotrevealingdetails aboutacriminalinvestigationuntilthetimecourt proceduresareinitiatedslowedcommunications betweenpertinentagencies,delayinginvestigationevenaftertheAumShinrikyo’sattemptto utilizebiologicweapons firstbecameknownduringtheinvestigationofthe1995sarinattackon theTokyosubway(Tu,2002).Awarenessof potentialthreats,alowthresholdofsuspicion, andactivesharingofinformationbetween governmentalagencieshavingpertinentexpertise and/orauthorityaresomekeycomponentsin earlydetectionofterrorismincidents.

EarlycharacterizationoftheAumShinrikyo biologicalweaponsprogramwasprimarily basedonstatementsbytheperpetratorsand expertopinions(Hudson,1999;Smithson,2000; Tu,2002).Physicalevidenceandindependent corroborationoftheclaimswerenotablyabsent. Fortunately,healthofﬁcialshadcollectedaspecimenof ﬂuidfromthebuildingatthetimeofthe

Kameidoincidentalthoughitwasnotbeen analyzedatthetime.The ﬂuidwaskeptas potentialevidenceintheeventdiseasecouldbe associatedwiththeincident.Apolicyofkeeping evidenceforaperiodofyearsalloweditsexaminationin2000,morethan6yearsaftertheincident.Fromaforensicperspective,however,the lackofformalchain-of-custodydocumentation mightbeanissueinsomejurisdictions.

TheMLVAanalysisofthe B.anthracis strain isolatedfromthe ﬂuidfromthebuildingrevealed agenotypeconsistentwiththatoftheSterne34F2 anthraxvaccinestrain.Adirectcomparison,however,wasnotmadewiththeSterne34F2 strain usedtoproduceanthraxvaccineinJapan.Inaddition,theMLVAgenotypicmatchwasnotunique toSterneasthepublisheddatabasecontained additionalsampleswithidentical(excluding pXO2)genotypes.NosamplesfromJapanhad everbeencharacterizedusingMLVAand,as such,thenaturalbackground B.anthracis was unknown.Asnotedabove,wholegenome sequencinghasbeenrecentlyperformedandthe exactidentitybetweentheKameidoisolateand thecommercialSternestraingenomestronglysupportsthepositionthatthisiswhatthecultused.In addition,noeffortwasmadetoisolate B.anthracis fromthebuilding’senvironmentoradjoining areasofKameidotoprovideevidencethatthe strainisolatedfromthe ﬂuidhadbeendispersed andtheextentofthedispersal.Theabilityof B.anthracis tosurvivemanyyearsincontaminated environmentmakessuchaneffortpotentiallyproductive(Turnbull,2008).

Whydidnodiseaseresultfromtheattack? Onepossibilityisasmallinhaleddoseofspores forexposedpersons.Althoughpresumably straindependent,theminimuminhaleddosage capableofcausinghumandiseaseisunclear. However,unknownssuchasaerosoldispersal deviceefficiencyingeneratinga fineparticle aerosol,concentrationofsporesinthe fluidbeing dispersed,andaerosolcloudmovementprecludemakingdefensibleestimatesofinhaled dosageforpersonsexposedduringtheattack.

AnotherpossiblefactorinlackofdetecteddiseaseisthelowvirulenceoftheSternestrainfor humans.Despitefrequentaccidentalinoculationsofthevaccineduringimmunizationof domesticanimals,nodocumentedcasesofassociateddiseasehavebeenreported.TheSterne strainlacksacapsule,andotherstrainsof B.anthracis lackingacapsulerarelyproduce humanillness.Onlythreereportsofillnessassociatedwithnonencapsulatedstrainshavebeen made,withonecasebeinginanimmunocompromisedpersonandthecause effectrelationshipoftheisolatetotheillnessobservedinthe othertwocasesbeinguncertain.

Serologicstudieswerenotdoneatthetimeof theeventbutcouldhavebeenpotentiallyuseful. Forexample,antibodyandcell-mediatedimmuneresponsesinpersonswhowereexposed inthe2001bioterrorismattackattheUnited Statescapitolwereusedtodemonstrateinfection withoutresultantdisease(Doolanetal.,2007).If asimilarstudyhadbeendoneinKameidoatthe timeoftheattack,thequestionofwhetherinfectionhadoccurredinabsenceofdiseasecould havebeenaddressed.ThelonglapseoftimebetweentheKameidoeventandtheepidemiologic studywouldcompromisethevalidityofsuch studiesatthistime.

Theisolationof B.anthracis andtheresultsof theMLVAtestingmighthaveprovenusefulin prosecutingsomeAumShinrikyomembers, butthiswillhavetoremainconjectural.Criminal chargesrelatedtothebiologicweaponsdevelopmentandattackswerenotmade,and,hence,the microbiologicalevidencewasnevertestedin court.However,13AumShinrikyomembers, includingAsahara,havebeenconvictedfor perpetratingthesarinattackontheTokyosubwayandcondemnedtodeath.Threeofthe AumShinrrikyomembersincludingAsahara wereexecutedbyhangingin2018afterappeals hadbeenexhaustedforalloftheaccused.

Despitetheconvictionsofmanyleaders,the AumShinrikyocontinuestoexist,havingbeen renamedAleph,the ﬁrstletterofthePhoenician

alphabet.Asplintergroup,HikarinoWaorRing ofLight,brokeofffromAlephfollowingdiscord withintheAlephleadership.Surveillanceofthe twogroupsforpotentialterroristactivitieshas beenmaintained,andtheyhavebeenformally designatedterroristorganizationsbyseveral countries,includingtheEuropeanUnion,Russia, Canada,Kazakhstan,andtheUnitedStates.

Challenges

TheKameidoincidentunderscoredanumber ofessentialconceptsinmicrobialforensics.Implementingtheseconceptsmustbeahighpriority forlawenforcementjurisdictions.Earlycommunicationandinformationsharingbetweenappropriategovernmentalagencies,particularlylaw enforcementandpublichealth,areessentialto anoptimaloutcomeofmicrobialforensicstudies. Publichealthagencieshaveamajorroleinthe investigationofsuspectedanddocumentedbioterrorismincidents,butthesimilaritiesanddifferencesinpublichealthandlawenforcement investigationsmustbeunderstoodandcoordinatedsothatbothcanbemoreeffective(Butler etal.,2002).Investigationsmust,ofnecessity,be multidisciplinaryanddrawonthebestavailable expertisewhetherornotlocatedinagovernmentalagency.Specimentestingmustfollow establishedchain-of-custodyprocedures,andall involvedgroupsmustbetrainedintheseprocedures.Strainsubtypingisapowerfulinvestigative toolfortracingtheoriginsofmicrobialagents,but theproceduresmustbevalidatedandhaveyetto beacceptedbythecourts.Epidemiologicstudies areessentialtoputtinglaboratory ﬁndingsinto thecontextofabioterroristevent.

References

Butler,J.C.,Cohen,M.L.,Friedman,C.R.,Scripp,R.M., Watz,C.G.,2002.Collaborationbetweenpublichealth andlawenforcement:newparadigmsandpartnerships forbioterrorismplanningandresponse.Emerg.Infect. Dis.8(10),1152 1156.

Doolan,D.L.,Freilich,D.A.,Brice,G.T.,Burgess,T.H., Berzins,M.P.,Bull,R.L.,Graber,N.L.,Dabbs,J.L., Shatney,L.L.,Blazes,D.L.,Bebris,L.M.,Malone,M.F., Eisold,J.F.,Mateczun,A.J.,Martin,G.J.,2007.TheUS capitolbioterrorismanthraxexposures:clinicalepidemiologicalandimmunologicalcharacteristics.J.Infect.Dis. 195(2),174 184.

Holty,J.E.C.,Bravata,D.M.,Liu,H.,Olshen,R.A., McDonald,K.M.,Owens,D.K.,2006.Systemicreview:a centuryofinhalationalanthraxcasesfrom1900to2005. Ann.Intern.Med.144(4),270 280.

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Takahashi,H.,Keim,P.,Kaufmann,A.F.,Keys,C., Smith,K.L.,Taniguchi,K.,Inouye,S.,Kurata,T.,2004. Bacillusanthracis incident,Kameido,Tokyo,1993.Emerg. Infect.Dis.10(1),117 120.

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TheFBI’sAmerithraxTaskForceandthe adventofmicrobialforensics

R.ScottDecker,TerryL.Kerns

Bio-LogicSecurity,LLC,LasVegas,NV,UnitedStates

Concernforthefuture

The1990ssawadramaticriseinawarenessof theuseofbiologicalweaponsandterrorism. SecretaryofDefenseWilliamCohenstartled thenationoneSundaymorningbyequating theamountofanthrax1 powderwithabagof sugaranditscapabilitytokillone-halfofWashington,D.C.’spopulation;UnitedNation’ s inspectorsuncoveredtheextentofIraq’sbiologicalweaponsprogram;RichardPreston’sbestsellingbook, TheHotZone,followedby The CobraEvent broughtmicrobesgoneawrytothe public’seye(Decker,2018).

Thoseingovernmentcirclestooknoteand realizedthenationwasunpreparedforabiologicalattacktheyfearedwasloomingonthehorizon.IntheFederalBureauofInvestigation(FBI) LaboratoryDivision,DeputyAssistantDirector RandallS.Murchstoodupanewunit,the HazardousMaterialsResponseUnit(HMRU). Itsmissionwastorespondtocrimescenes involvinguseofaweaponofmassdestruction,

collectandpreservetheevidence,andcoordinateitsforensicexploitation.AttheCentersfor DiseaseControlandPrevention(CDC),US PublicHealthServicemedicalofﬁcerScottLillibridgewiththehelpofmicrobiologistStephen A.MorseinitiatedtheBioterrorismPreparedness andResponseProgram(BPRP).Richard F.MeyerfromPlumIslandandRichardB.KelloggsoonjoinedMorse,andtheybeganidentifyingstate-of-the-arttechnologyfor characterizingmicrobes.

LaboratoryResponseNetwork

InadditiontobuildingtheCDC’sBPRPand theFBI’sHMRU,MorseandMurchdeveloped theideaforanationalLaboratoryResponse Network(LRN) aconceptsimple,elegant, andnovel highlyeffectiveandefﬁcient.The CDC’sheadquartersinAtlantahostedthe January1999kick-offmeeting.Attendees includedrepresentativesfromtheDepartments

1 Anthraxisdeﬁnedasthediseasecausedbythebacterium, Bacillusanthracis.However,inconversationalusage, anthraxand Bacillusanthracis areoftenusedinterchangeably.

oftheArmyandNavyandtheAmericanPublic HealthAssociation.Toppublichealthofﬁcials fromaroundthecountryandtheFBI’sLaboratoryDivisionjoinedthem.Thatmeetingbegan effortsforthestandardizationofmethodsand useofequipmentforthedetectionandidentiﬁcationofmicrobesusedinbioterroristandcriminalevents.

TheDepartmentofHealthandHuman ServicesthroughtheCDCrebuiltthenation’ s publichealthinfrastructurebyprovidingfunds tostateandlargecountypublichealthlabsfor thepurchaseofstate-of-the-artequipmentfor microbiologicalidentiﬁcation.Atthesame time,scientistsfromnationalinstitutionssuch astheUnitedStatesArmyMedicalResearch InstituteofInfectiousDiseases(USAMRIID), theNavalMedicalResearchLaboratory,and CDC’sRichMeyerwoulddevelop,standardize, andvalidateprotocols.Standardizedreagents andtrainingwouldalsobeprovided.Itwasan ambitiousundertaking,butonesoondesperatelyneeded.Inthemonthsfollowing9/11,the LRNwouldprovideinvaluableresourcesand rescuethenationfromnear-panicoverwhite powderlettersandhoaxes.

Whilethepublichealthlabswerewell attunedtoprocessingpublichealthsamples, theynowlearnedtoprocessevidentiary samples.InthePhoenixFBIDivision,for example,agentsaddedchainofcustodydocumentationtotheexistingpublichealthaccession paperworkandprocessesweredevelopedfor cross-referencingevidentiarysampleswith publichealthcatalogingsystems.Onarrivalat anLRNmemberlab,theFBI’sevidencewould bestoredinadedicatedandsecurearea. Followingtestingforbiologicalthreatagents, laboratorypersonnelwouldtransferthe evidencetoasecond,secureareatoavoid cross-contaminationwithnewincoming samples.Entrytosecureareaswaslimitedand recordsofingress/egressmaintained.Following

intakeofsuspectedsamples,theLRNlabswould communicatedailywiththesubmittingFBI ﬁeld of ﬁce;testresultswererelayedimmediatelyto victimsandtheirdesignatedhealthcare professionals.

TheequipmentprovidedtotheLRNwas availableforday-to-daypublichealthwork, andintheinstanceofasuspectedbiological attackorthreat,availabletoanalyzeevidence collectedthroughthecoordinationofFBIJoint TerrorismTaskForces.Evidencethattestedpositiveforapathogenortoxin(e.g.,ricin)wouldbe transferredtoanational-levelfacility CDCora Washington,DCareaDepartmentofDefense lab forconﬁrmationandforensicanalysis. Specializedtesting,suchasgenomeanalysis, wouldtakeplaceatpartnerlaboratoriessuch asPaulKeim’slaboratoryatNorthernArizona University.

Theprimaryadvantageofusinglocalfacilities wastimelyturnaround.In1998,anythreatening material,suchasanenvelope ﬁlledwithapowder,wastransportedtoanational-levellabfor testing,takingdaysbeforeananswer,usually negative,couldbegiventovictims,risking unnecessaryadministrationofantibioticsorin thecaseofapositivetest,costlydelay.Should thecountrybedelugedwithanthraxhoaxletters asin1998,thevolumeoftestingwouldthreaten tooverwhelmnationalassets.

Beginningin1998,theFBIaddedmicrobialforensicstoitslistofavailableinvestigative approaches.Itbuiltandstrengthenedrelationshipswiththecountry’smicrobiologyexperts andwasabletoproviderudimentarytestingto its ﬁeldof ﬁcesaroundtheUnitedStates. ContractsandMemorandaofAgreementwere putinplaceasvehiclesforfundingandreimbursement.Microbialforensicswouldnotbea stand-alonetooltoproveinnocenceorguilt, butoneofmanyavailabletogenerateinvestigativeleads(Fig.2.1).

FIGURE2.1 Flowchartforinvestigationofuseofabiologicalweaponofmassdestructionaspresentedto ﬁrstresponders andpublichealthpersonnelbytheauthorswhileassignedtotheFBI’sHazardousMaterialsResponseUnit(c.1998 2001).

Post-9/11:thesecondwaveofattack

TheeventsofSeptember11,2001,areoneof thosedaysinhistorythateveryonewill rememberwheretheywere.ThenearsimultaneousairlinerattacksontheWorldTradeCenter towers(Fig.2.2)andthePentagon,alongwith thedivertedattacktoacoalﬁeldinPennsylvania, remainscorchedinournationalcollectiveness alongwithJapan’s1941attackonPearlHarbor andthe1963assassinationofPresidentJohnF. Kennedy.

MembersoftheFBI’sHMRUrespondedtoall threeofthe9/11crimescenes.InNewYorkCity, epidemiologistsfromtheCDCbeganmonitoringareahospitalsformysteriousillnesses.

FIGURE2.2 TheGroundZeroPile.ACaterpillarexcavatorclearssteelandconcretefromthewreckageofManhattan’ s WorldTradeCentercomplexfollowinganattackbyhijackedcommercialairlinersonSeptember11,2001.

Concernranthroughgovernmentofabiological bombaboardoneoftheairliners.Officials worriedaboutabiologicalattackcomingon theheelsof9/11.AroundWashington,D.C., theDepartmentofEnergydeployeditsBiologicalAerosolSentryandInformationSystem (BASIS)technologytosampletheairforbiologicalaerosols, Bacillusanthracis atthetopoftheir list.USAMRIIDtestedthesmall filterscollected hourlyfromBASISmonitoringstations deployedthroughouttheNationalCapital region.Whilethelocationsofthestationswere confidential,USAMRIIDstaffwereawareof thetesting,anditgeneratedspeculationabout whatwascomingnext.

Threeweeksafter9/11,earlyinthemorning, RobertStevensofsouthFloridaadmittedhimself totheJFKMedicalCenterlocatedbetweenWest PalmBeachandBocaRaton.Hehadbeen vomitingandincoherentduringthenight.By thetimehearrivedatJFK,deliriumhadtaken over.Initially,doctorssuspectedbacterialmeningitisandadministeredmultipleantibiotics. Sixhourslaterhesufferedaseizureandwas placedonaventilator(Cole,2003).Farther south,Stevens ’ coworker,ErnestoBlanco,a mailroomworkeratAmericanMedia,Incorporated(AMI)checkedhimselfintoMiami’sCedarsMedicalCenterashebecameincreasingly disorientated.Themedicalstaffsoonadministeredintravenousantibioticsandhookedhim uptoamechanicalrespirator.AdeﬁnitivediagnosisforBlancowouldeludethemfordays.

AnexaminationofStevens’ cerebrospinal fluidrevealedgram-positivebacilliandaFlorida PublicHealthlaboratory,usingthenewLRN protocols,identi fiedthebacteriaas B.anthracis, 44hafterStevenswasadmittedtoJFK.ThebacteriaweresenttoAtlantawheretheCDC’sBPRP confirmedthediagnosisofanthrax.BPRPstaff inoculatedsamplesofStevens’ B.anthracis onto anagarslantand flewittoNorthernArizona University.There,workingthroughthenight, PaulKeimandhisstaffidenti fiedthe B.anthracis astheAmesstrain,aresearchstrain

usedtoassessvaccineef ficacy.Atthesame time,theCDC’sAlexHoffmaster,usingthe MultipleLocusVariableNumberTandem Repeat(VNTR)Analysis(MLVA)developedby theKeimlab,confirmedtheAmesidenti fication (Keimetal.,1997,2000).FindingthatalaboratorystraininfectedStevensandnotanunknown environmentalstrainpointedtowardanintentionalact potentiallythe firstactofdeadly bioterrorismintheUnitedStates.OnOctober 5,2daysafterenteringJFKMedicalCenter,Stevenssuccumbed.Hewasthe firstcaseoflethal inhalationalanthraxintheUnitedStatessince 1976.

Blanco’sdiagnosiswasnotasstraightforward.RepeatedattemptstoidentifyviablebacteriainBlancofailed.TheCDCcasedefinition requiredviable B.anthracis foradiagnosisof anthrax.However,inhisRapidResponseand AdvancedTechnologylabatCDC,RichMeyer hadvalidatedanewassay,ahighlysensitive andquantitativeimmunoassayhetermed Time-ResolvedFluorescence(TRF).Applying TRFtoBlanco’spleural fluid,Meyerdetected highlevelsofinert B.anthracis antigens, convincingMeyerthatBlancohadinhalational anthrax.ButCDCexecutiveleadershipneeded moreconvincing.Afterseveraldaysoftensionfilledmeetingsandtersediscussions,theCDC frontofficeagreedthatErnestoBlancowasthe secondinhalationalanthraxvictimsince1976 (Blanco’scasewouldleadtotheCDCrevising theircriteriaforanthraxdiagnosis).Whilethe extraordinaryhighlevelsofintravenousantibioticsmayhaveconfoundedanearlydiagnosis, theyundoubtedlysavedBlanco’slife.Hewould goontoafullrecovery(UCLA,FieldingSchool ofPublicHealth).

OnFriday,October12,2001,9daysafterStevens ’ initialdiagnosis,suspicionsofa bioterroristattackwerecon ﬁrmed.Afteracircuitoussearch,aletteraddressedtoNBCnews anchorTomBrokawinManhattanwasrecoveredcontainingdry B.anthracis spores.The envelopewaspostmarked “Trenton,NJ.” Three

dayslater,asecondspore-filledletterwasrecoveredintheCapitolHillof ficesofUSSenatorTom Daschle thehandwritingandeagleprestampedenvelopeidenticaltotheBrokawletter. However,theDaschleletter,alsopostmarked “Trenton,NJ,” boreaNewJerseyreturn address whichprovedtobefake.OneweekafterrecoveringtheBrokawletter,a B.anthracis filledletterwasrecoveredaddressedtotheeditorofthe NewYorkPost inManhattan.Inearly November,aletter filledwithsporeswasrecoveredinquarantinedmail.Thisonewas addressedtoUSSenatorPatrickLeahy.Four letterswererecoveredinall,the Post andLeahy letterswereunopened.RecoveryoftheLeahy letterallowedforarangeofdestructiveforensic analyses.

ThetwoletterssenttoNewYorkCity containedgranulardarkbrowndriedspores mixedwithdebris;thetwoCapitolHillletters senttoDaschleandLeahycontainedlightgray powder,very ﬁneandlittletonodebris (Fig.2.3).Thedriedpowderintheseletters consistedoftinyclumpsofsporesandsingle

spores.WhentheDaschleletterwasopenedin hisoffice,thepowderbehavedasanaerosol andspreadthroughofficebuildingsonCapitol Hill.Cleanuptookmonths.InNewJersey, NewYork,Maryland,Washington,D.C.,Florida,andConnecticut,victimspresentedwith cutaneousandinhalationalanthrax.Bytheend ofNovember2001,therewere22totalcasesof anthrax,11inhalationaland11cutaneous.The inhalationalformclaimed fivelives,onein Florida,oneinConnecticut,twoinMaryland, andoneinNewYorkCity.Seventeenofthe victimsrecoveredfromtheirinfection;many remaindisabledordis figured.

Qualityofsporematerial behavioral assessment

TheSenateletterswereofthegreatestinterest intermsofpurityandconcentration.Estimates arethat0.8 1.0gofpowder ﬁlledtheletter mailedtoSenatorLeahy,withadensityof 2 1012 sporespergram.The NYPost letter materialwasofconsiderablylesserpuritywith estimatesof2 1011 sporespergram.Forthe twoCapitolHillletters,thetwoNewYorkletters andassumingoneletterattheAMIbuilding,the FBILaboratoryestimatedtheattackermailed approximately3.7 1012 spores.Conclusions abouttheamountoftimerequiredtoproduce thematerialvariedwidely,from2to3daysto months,assumingliquidcultureinshaking ﬂasksorafermenter(Gastetal.,2011).

StudiesofthematerialintheDaschleand Leahylettersrevealedabimodalparticledistribution.Thepowderconsistedofsinglespores witha1.5umdiameterthatrepresented1%of thetotalsporemass.Theremainderconsisted ofclumpsapproximately20umindiameter. Thesemeasurementswereconductedwithan Aerosizer(TSI,Inc.)followingaerosolizationof thepowder.Scanningelectronmicroscopy (SEM)conﬁrmedabimodalparticledistribution:

FIGURE2.3 Powderofdried Bacillusanthracis spores recoveredfromenvelopeandletteraddressedtoUSSenator Patrick.

singleparticleswithamorphologyconsistent with B.anthracis sporesandclustersofspores. However,thepowderwassubjectedtoautoclavingbeforeSEManalysisandthatmayhave affectedsizedistribution(Gast).

Thelevelofpurityprovidedaforensicclueto thesophisticationofthematerial’sproducer(s). ThetransitionofthecrudeNewYorkletter materialwithin3weekstothe ﬁnepowderin theCapitolHilllettersindicatedaquicklearner andastutemicrobiologist.Thisinformation wasfactoredintothebehavioralassessment beingdevelopedbyFBIproﬁlersinQuantico.

AmerithraxTaskForce

AnthraxvictimswerelocatedinsixFBIdivisions,butFBIheadquartersrecognizedthe attackswerelikelytheworkofonepersonora singlegroupandconsolidatedthegrowing investigation.TheWashingtonFieldOfﬁce (WFO)inWashington,D.C.wasdesignated OfﬁceofOrigin(OO).InFBIparlance,theOO wouldbearresponsibilityforsolvingthecase.

TheWFOquicklyformedataskforce;headquartersgavetheinvestigationacodename, AMERITHRAX(AMX).ItincludedFBIagents withsciencebackgroundsandUnitedStates PostalInspectionServiceinspectors.Together withscientistsfromtheFBILaboratory’ s ForensicScienceResearchUnit,thescience agentsbeganbrainstormingsessions.Originally, itwashopedthatforensicmethodsalreadyin placeattheFBIwouldprovideinvestigative leads,butthosehopesquicklydissipated.The envelopeshadbeentapedshut,andtherewere nostampswithwater-solubleadhesive,leaving littlehopeforrecoveringhumanDNA.Nor werelatentimpressionsfoundthatcouldbe fedintotheAutomatedFingerprintIdentiﬁcationSystem.Examinersquicklydetermined thattheinkusedtowritetheenvelopes’ addresseswascommonandofnovalueforattribution.Neitherlettersnorenvelopescontained

indentedwriting.Themailboxusedtopostthe letterswaseventuallylocatedinPrinceton,NJ, butasearchoftheareaturnedupnosurveillance cameras.Trafﬁccitationsissuedintheareawere ofnovalueandnearbyshopownersandresidentsprovidednoclues.

Thebrainstormingsessionsidentifiednovel scienceapplications.Consideredwerestable isotopeanalysisforattributiontoageographic area;thepresenceofhemoglobinasanindicationbloodagarhadbeenusedtoculturethe microorganism;originofthesiliconidentified inthesporecoat;datingthesporeswith carbon-14;analysisofplasmidDNAformutationsspecifictothemailed B.anthracis .Ofthe techniquesconsidered,threeprovidedleadsor corroboratedinvestigativeconclusions.

ColonymorphologyandDNAsequencing

Asoftenhappensinscience,greatdiscoveries beginasaccidents,followedbyastuteobservations.Suchwasthebiggestleadintheanthrax investigation.Whilecharacterizingthespores fromtheDaschleand NYPost letters(mostof thepowderintheBrokawletterwaslostbefore itstransportationtotheCDC),USAMRIID microbiologistTerryAbshiresetupculture platesanddilutedthesporestoallowsingle colonypropagation.Ononeoccasion,shelet theplatesremainintheincubatorat37 C,rediscoveringthemseveraldayslater.Shenoticeda minorityofthecolonieshadmorphologies differentthanthepredominantwild-type B.anthracis.Basedontheirexperience,Abshire andhersupervisor,JohnEzzell,suspectedthe variantsmightbeduetomutationsinthesporulationpathway.

PatriciaWorsham,alsoaUSAMRIIDmicrobiologist,wastheinstitution’sexpertonbacillus sporulation.ShebeganworkingwiththeAmerithraxTaskForcetocharacterizethemorphology variants soonknownas “morphs.” Shefound theminallthreeletters,Daschle,Leahy,and

NYPost.Thevariantswerestableinculture,an indicationofbeingtheresultofDNAmutation. SheisolatedseveralanddesignatedthemA1, A2,A3,B,C,D,andE,estimatingthey accountedfor10%ofthetotalcolonies.

Luck,fueledbypersistenceanddeliberation, comesintoplayduringlawenforcementinvestigationsaswellasthoseofscience.Duringaconsentsearchofrefrigeratorsinconjunctionwith interviewsofUSAMRIIDpersonnel,theoriginal agarslantoftheAmesisolatewasdiscovered.Its tubewasdatedFebruary1981andviablespores remained.Findingthe1981AmesgavetheFBI anidealcandidatetoprovideabaseline,unalteredDNAsequenceofthegenomefromthe originalsample(Raveletal.,2009).Apioneer inwholegenomesequencing,TheInstitutefor GenomicResearch(TIGR)ofRockville,MD, wassoonunderagreementtoperformthework.

Beforetheisolationandinitialcharacterizationofthemorphologyvariantsandwhole genomesequencingofthe1981Ames,theFBI hadbegunanambitiousproject.Beginning withselectagenttransferregulationrecords fromtheCDC,coupledwithliteratureandpatentreviews,and ﬁeldinterviews,theFBItracked downlocationsoflaboratoriespossessingthe Amesstrainof B.anthracis.Theeffortwould takeagentsto16laboratoriesintheUnited States,laboratoriesinCanada,theUnited Kingdom,andSweden;theinvolvementofat leastoneforeignembassyandtwotripsacross theAtlanticbyprivateaircraft.Collecting Amessamplesbeganinthefallof2001and wascompletedduringfall2006(Fig.2.4).

Followingcompletionofthe1981Ames’ genomeDNAsequence,TIGRbegansequencing DNAisolatedfromthemorphologyvariants. WorshamisolatedandpropagatedA1-3,B,C, D,andEcolonies;thebacteriaweretransferred toPaulKeimforDNAisolation(TIGRwasnot approvedtoworkwithliveselectagents);the DNAwassenttoTIGRforgenomesequencing; andlastly,samplescontainingputativemutationsreturnedtoKeimforconﬁrmation.All

FIGURE2.4 Oneof1082samplesof Bacillusanthracis Amesstraincollectedworldwide.EachsamplewassubmittedtotheFBIrepositoryinduplicate.Oneoftheduplicates sampleswassenttoPaulKeimattheNorthernArizona Universityforstrainconﬁrmationandemergencybackup storageincaseoflossofpower, ﬁre,waterlinebreak,etc., atUnitedStatesArmyMedicalResearchInstituteofInfectiousDiseases. PhotocourtesyofTomReynolds,NEXTMolecularAnalytics,LLC.

scientistsreceivedcodedsamplesanddidnot knowtheiridentityororigin.

During2003,theeffortsoftheFBI,Worsham, Keim,andTIGRbegantopayoff.TheBvariant’ s morphologywasattributedtoasinglepoint mutation thiswouldproveunrealisticfor developingascreeningassaywithtechnology thatwasavailable.However,anAvariantwas foundtocontaina2024basepair(bp)insertion inoneoftheeleven16sRNAgenes.Thisinsertionwasamenabletodevelopingascreening assay.Mutationswerealsoidenti ﬁedinadditionalA,andC,D,andEvariants(Raskoetal., 2011).Foracompletelist,seethe2011report fromtheNationalResearchCouncilofthe NationalAcademyofSciences(Gast).

TheFBIcontractedthecomparisonofthe evidentiarymorphologyvariantsandtheAmes exemplarsintheirrepositorytoCommonwealth Biotechnologies,Incorporated(CBI).Withthe identiﬁcationofthemorphologymutations, CBIbegandevelopingTaqmanreal-time polymerasechainreaction(PCR)assays.The FBIimposedstrictspeci ﬁcations.Eachsample initsAmesrepositorywouldbescreenedin