DTIC ADA570434: Transport of Bacillus thuringiensis var. Kurstaki Via Fomites

2.2 MB·English

by  Defense Technical Information Center

BiosecurityandBioterrorism: Biodefense Strategy, Practice, andScience
Volume9,Number 3,2011ªMary AnnLiebert,Inc.
DOI:10.1089/bsp.2010.0073
Special Feature: Remediation
Transport of Bacillus Thuringiensis var.
Kurstaki Via Fomites
Sheila Van Cuyk, Lee Ann B. Veal, Beverley Simpson, and Kristin M. Omberg
Theintentionalandcontrolledreleaseofanaerosolizedbacteriumprovidesanopportunitytoinvestigatetheimplications
ofabiologicalattack.Since2006,LosAlamosNationalLaboratoryhasworkedwithseveralurbanareas,includingFairfax
County, VA, to design experiments to evaluate biodefense concepts of operations using routine spraying of Bacillus
thuringiensisvar.kurstaki(Btk).Btkisdispersedinlargequantitiesasaslurrytocontrolthegypsymoth,Lymantriadispar.
Understandingwhetherpersonnelandequipmentpickupresidualcontaminationduringsamplingactivitiesandtransport
ittootherareasiscriticalfortheformulationofappropriateresponseandrecoveryplans.Whilethereisagrowingbodyof
literaturesurroundingthetransmissionofviraldiseasesviafomites,thereislimitedinformationonthetransportofBacillus
species via this route. In 2008, LANL investigated whether field sampling activities conducted near sprayed areas, post-
spray,resultedinmeasurablecross-contaminationofsamplingpersonnel,equipment,vehicles,andhotelrooms.ViableBtk
wasdetectedinallsampletypes,indicatingtransportoftheagentoccurredviafomites.
Thegypsymoth,Lymantriadispar,isamajorforest scenarioinvolvingadry,weaponizedpowder,analystshave
pestalongtheeasternseaboardandinthemidwestern noted it is a ‘‘technically feasible’’ and effective (albeit
United States. Over the past 20 years, many thousands of simplistic) means of producing inhalational exposures.1
acresoflandhavebeensprayedwithpesticidestosuppress Since 2006, Los Alamos National Laboratory (LANL) has
or eradicate gypsy moth populations. The most common workedwithseveralurbanareas,includingFairfaxCounty,
pesticide used is a water-based slurry containing Bacillus VA,todesignexperimentstoevaluatebiodefenseconcepts
thuringiensisvar.kurstaki(Btk).Thisbacteriumproducesa of operationsusingnormalBtkspray activities.
toxin thatis lethal to gypsy moth caterpillars. Informulatingappropriateresponseandrecoveryplans,
The intentional and controlled release of an aerosolized it is critical to understand whether personnel and equip-
bacterium provides an opportunity to investigate the im- ment pick up residual contamination during sampling ac-
plications of a biological attack. While the application of tivitiesandtransferittootherareas.Thereisagrowingbody
Btk does not resemble a typical state-sponsored release of literature surrounding the transmission of viral diseases
Sheila Van Cuyk, PhD, is a Scientist, Systems Engineering and Integration, and Kristin M. Omberg, PhD, is Deputy Division
Director, Decision Applications; both are at Los Alamos National Laboratory, Los Alamos, New Mexico. Beverley Simpson,
MT(ASCP),MBA,isLaboratoryDirector,LosAlamosMedicalCenter,LosAlamos,NM.LeeAnnB.Veal,BS,isDirector,Center
for Radiological Emergency Response, Radiation ProtectionDivision, U.S. Environmental Protection Agency,Washington, DC.
288
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1. REPORT DATE 2. REPORT TYPE 3. DATES COVERED
01 NOV 2011 Final 01 Feb 2011 - 30 Oct 2012
4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER
Wide Area Recovery and Resiliency Program (WARRP) Transport of
5b. GRANT NUMBER
Bacillus Thuringiensis var. Kurstaki Via Fomites
5c. PROGRAM ELEMENT NUMBER
6. AUTHOR(S) 5d. PROJECT NUMBER
Van Cuyk, Sheila Veal, Lee Ann B. Simpson, Beverley Omberg, Kristin
5e. TASK NUMBER
M.
5f. WORK UNIT NUMBER
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION
Los Alamos National Lab Systems Engineering & Integration Group PO REPORT NUMBER
Box 1663, MS F607 Los Alamos, NM 87544
9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S)
Lori Miller Department of Homeland Security Science and Technology DHS
Directorate Washington, DC 20538
11. SPONSOR/MONITOR’S REPORT
NUMBER(S)
4.5.0
12. DISTRIBUTION/AVAILABILITY STATEMENT
Approved for public release, distribution unlimited
13. SUPPLEMENTARY NOTES
The original document contains color images.
14. ABSTRACT
The intentional and controlled release of an aerosolized bacterium provides an opportunity to investigate
the implications of a biological attack. Since 2006, Los Alamos National Laboratory has worked with
several urban areas, including Fairfax County, VA, to design experiments to evaluate biodefense concepts
of operations using routine spraying of Bacillus thuringiensis var. kurstaki (Btk). Btk is dispersed in large
quantities as a slurry to control the gypsy moth, Lymantria dispar. Understanding whether personnel and
equipment pick up residual contamination during sampling activities and transport it to other areas is
critical for the formulation of appropriate response and recovery plans. While there is a growing body of
literature surrounding the transmission of viral diseases via fomites, there is limited information on the
transport of Bacillus species via this route. In 2008, LANL investigated whether field sampling activities
conducted near sprayed areas, postspray, resulted in measurable cross-contamination of sampling
personnel, equipment, vehicles, and hotel rooms. Viable Btk was detected in all sample types, indicating
transport of the agent occurred via fomites.
15. SUBJECT TERMS
WARRP, Biological Attack, Fomites, Cross-contamination
16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF 18. NUMBER 19a. NAME OF
ABSTRACT OF PAGES RESPONSIBLE PERSON

a. REPORT b. ABSTRACT c. THIS PAGE UU 13
unclassified unclassified unclassified
Standard Form 298 (Rev. 8-98)
Prescribed by ANSI Std Z39-18
VANCUYK ETAL.
via fomites. Boone and Gerba have reported ‘‘proven’’ or contaminates personnel, accessories, and secondary spaces
‘‘accepted’’ transmission of respiratory syncytial virus, rhi- atdetectable levels.
novirus, influenza virus, rotavirus, hepatitis A virus, and
adenovirusviafomites.2Ledermanandcolleaguesreported
Experimental Conditions
the suspected transmission of vaccinia virus via fomites.3
However,thereislimitedinformationinthepeer-reviewed
literatureonthetransportofBacillusspeciesviathisroute. In 2008, Fairfax County sprayed 46 blocks with Btk, cov-
In a 2008 review of the persistence of Category A select ering3,500totalacres(Figure1).Aerialsprayingbeganon
agents, Sinclair and colleagues noted, ‘‘Knowledge of the May 2 and continued through May 10. LANL conducted
survival of B. anthracis on fomites is fairly limited,’’4(p558) an experiment in Mason District Park, immediately adja-
citingonly2papersinthepeer-reviewedliterature.4 cent to Spray Block 20 (Figure 2), between April 30 and
Toaddressthisgap,inconjunctionwithBtksprayingin May 8 to determine whether field sampling activities con-
Fairfax County, VA, in 2008, LANL collected samples ducted in close proximity to sprayed areas resulted in
fromthebackpacks,vehicles,andhotelroomsofpersonnel measurable cross-contamination of sampling personnel,
who performed aerosol sampling near a spray area after backpacks,vehicles,andhotelrooms.Incoordinationwith
spraying had occurred, as well as dry gauze that had been FairfaxCounty,SprayBlock20wasthefirstblocksprayed
attached to their persons, to determine whether viable Btk onMay 2, 2008.
Figure 1. Map ofFairfax County2008BtkAerial Spray Blocks.Counties are outlined ingreen; Fairfax Countyspray blocks
are outlinedin pink.
Volume9,Number3,2011 289
TRANSPORTOF BACILLUSTHURINGIENSIS VAR.KURSTAKI VIAFOMITES
Figure2. Mason DistrictPark (outlined ingreen) andSpray Block 20(outlined inpink).
A sample of the spray suspension (Foray 76B, Valent FairfaxCountyappliestheForay76Bviasprayingfroma
Biosciences) was obtained from the Fairfax County Gypsy helicopter at a rate of 0.5 gallons per acre. Based on the
Moth Suppression Program authorities for analysis using measured concentration of viable agent, this equates to
culture methods. (The manufacturer optimizes the suspen- 2.5·1012 CFU applied per acre. Spray Block 20 is ap-
sion for toxin activity and does not perform direct mea- proximately 63 acres; an estimated 1.6·1014 total CFU
surements of B. thuringiensis concentration.) An aliquot of were therefore applied to Block 20. Using a value of
the sample was diluted 1,000-fold in phosphate buffer so- 3.3·10-11 grams/wet spore,5 an estimated 5.1kg viable
lution (PBS, Fisher Scientific) and heat-treatedat80(cid:2)C for BtkwereappliedinBlock20.Thematerialisdistributedin
10mins.Serialdilutionsofthispreparation(100mLaliquots) large droplets using a rotary atomizer and is intended to
in PBS were plated onto trypticase soy agar (Becton Dick- stick to the foliage; measured at the canopy height, the
insonandCo.,FisherScientific)withcycloheximide(50mg/ droplets have a median volume diameter of 125mm and
L, Sigma-Aldrich), and colony counts were obtained after mediannumberdiameterof80mm.Thedistributionofthe
overnightincubationat37(cid:2)C.Triplicatemeasurementsgave droplet sizes, however, can be reasonably approximated as
an average viable Btk density of 1.3·109 colony-forming log-normal, which means at least a portion of droplets
units(CFU)permlsuspensionwithastandarddeviationof produced are in therespirable (1-10mm) range.
5·108.AdditionalstudieswithForay76Bdemonstratedno Figure3showsthe24-hourprecipitationlevelsmeasured
significant difference in viable Btk density with or without atRonaldReaganNationalAirport,locatedapproximately
heat treatment, indicating the presence of vegetative (ie, 7mileseastofBlock20,andDullesInternationalAirport,
nonsporulated)Btkinthespraysuspensionisnegligible. located approximately 16 miles northwest of Block 20,
290 BiosecurityandBioterrorism:BiodefenseStrategy,Practice,andScience
VANCUYK ETAL.
Figure 3. 24-hour integrated precipitation (measured at 7:52ameach day) over the experimentalperiod. Colorimages available
online at www.liebertonline.com/bsp
during the study period. Precipitation for each 24-hour transported to the sample collection area. Four of these
period was measured at 7:52 am local time, and the value weresitedaroundtheamphitheater,andthefifthunitwas
shown(ininches)isforaccumulationovertheprevious24 sited near the restroom (Figure 6). The number and con-
hours.Theexperimentalperiodwasgenerallydry,butthere figuration of the collectors were constrained by both the
was heavy rain on the last day of the study. needtominimizetheexperiment’simpactonroutineuseof
Figure 4 shows the wind rose measured at Ronald thepark as well as availability of electricity.
Reagan National Airport over the period May 2-9, 2008. Aerosol samples were collected using the Portable Sam-
Winds were typically from the southwest during the ex- plingUnit-2(PSU-2;Hi-QEnvironmentalProducts).The
perimentalperiod,indicatingahighprobabilityofairborne PSU-2 is equipped with a size-selective PM inlet, which
10
transport of Btk from the spray zone to Mason District excludes 50% or more of all particles greater than 10 mi-
Park,wheresampling activities wereconducted. However, cronsindiameter,therebyselectivelycollectingparticlesin
a significant occurrence of winds from the northwest is the respirable range. Each sampler was adjusted to a flow
also observed. This pattern of winds is typical for this re- rate of 100 liters per minute at the start of the sampling
gionandtimeperiod,asseeninFigure5,the9-yearaverage period. Aerosol samples were collected in 24-hour incre-
wind rose. ments onto a 47-mm Fluoropore membrane filter (Milli-
pore). The filters were enclosed in sealed aluminum
holders.Theholderswereplacedinachainofcustodybag.
Methods Priortotransporttotheanalyticallaboratory,thechainof
custody bag was wiped with a hospital-grade bleach wipe
(Dispatch brand, Medline), allowed to air-dry,and placed
Aerosol Sample Collection
in aclean secondary container.
Five aerosol samplers were placed in Mason District Park, Onesetofsampleswascollectedbeforesprayingbeganas
justnorthofthesprayblock,todeterminewhetherBtkwas background. Post-spray, samples were collected each day
Volume9,Number3,2011 291
TRANSPORTOF BACILLUSTHURINGIENSIS VAR.KURSTAKI VIAFOMITES
Figure4. Wind rosemeasured atRonald Reagan National Airport, May2-9,2008. Colorimages available online at www.
liebertonline.com/bsp
for 1 week, May 3-9, 2008. All samples were collected by indicatingtape,andplacedinachainofcustodybag.Prior
personnel wearing clean, disposable nitrile gloves and to transport to the analytical laboratory, the chain of cus-
booties. Gloves were changed after collection of each tody bag was wiped with a hospital-grade bleach wipe
sample.Sampledatawererecordedonlogsheetsandinan (Dispatch brand, Medline),allowed to air-dry, andplaced
electronicdatabase. in a clean secondary container. One set of samples was
EachPSU-2wascoveredwithalargeplasticbagduring collectedbeforesprayingbeganasbackground.Post-spray,
Btksprayingtominimizedepositionontheunit.Thebags sampleswerecollectedeachdayfor1week,May3-9,2008.
were removed on May 2 after aerial spraying was com- All samples were collected by personnel wearing clean,
pleted. The size-selective PM inlets were replaced at the disposablenitrile gloves andbooties. Gloves were changed
10
start of each sampling cycle to reduce the likelihood of aftercollectionofeachsample.Sampledatawererecorded
samplecontamination from prior deposition. on log sheets and in an electronicdatabase.
Vacuum Sample Collection Swipe Sample Collection
Vacuumsampleswerecollectedfromthevehicleandfrom Swipe samples were collected using sterile noncotton gauze
hotelroomsusingthe3MTraceEvidenceCollectionFilter wipes(3in·3in)moistenedwith500mLphosphatebuffer
(3M) inserted into a HEPA Dirt Devil Classic handheld solution(Fisher).A1-m2areawaswiped,making2passesof
vacuum cleaner.Team memberscollected 1-2tablespoons the entire sampling area using vertical then horizontal S-
ofvacuumeddebrisovera1-m2surface,making2passesof strokes.Theswipewasplacedinasterile50-mLconicalvial,
theentiresamplingareaataslowrate(12inper5sec)using which was capped and sealed with Parafilm. The sealed vial
verticalthenhorizontalS-strokes.The3MTraceEvidence wasplacedinachainofcustodybag.Priortotransporttothe
Collection Filters were recapped, taped shut with tamper- analyticallaboratory,thechainofcustodybagwaswipedwith
292 BiosecurityandBioterrorism:BiodefenseStrategy,Practice,andScience
VANCUYK ETAL.
Figure 5. Wind rosemeasured at Ronald Reagan National Airport,9-year average(1984-1992), May1-15, 2008.Colorimages
available online at www.liebertonline.com/bsp
a hospital-grade bleach wipe(Dispatch brand, Medline), al- Onesetofsampleswascollectedbeforesprayingbeganas
lowedtoair-dry,andplacedinacleansecondarycontainer. background.Post-spray,sampleswerecollectedeachdayfor1
Onesetofsampleswascollectedbeforesprayingbeganas week,May3-9,2008.Allsampleswerecollectedbypersonnel
background.Post-spray,sampleswerecollectedeachdayfor1 wearing clean, disposable nitrile gloves and booties. Gloves
week,May3-9,2008.Allsampleswerecollectedbypersonnel were changed after collection of each sample. Sample data
wearing clean, disposable nitrile gloves and booties. Gloves wererecordedonlogsheetsandinanelectronicdatabase.
were changed after collection of each sample. Sample data
wererecordedonlogsheetsandinanelectronicdatabase.
Additional Sampling Precautions
The hotel sampled was located in Merrifield, VA. The dis-
Personnel Sample Collection
tancefromthehoteltothenearestsprayblock(block37in
Three dry gauze wipes (3 in·3 in) were secured to sam- Figure1,locatednorthwestofthehotel)was1.3miles.The
plingpersonnel(1onthearm,1ontheknee,and1onthe distancefromthehoteltothenextnearestsprayblock(block
back between the shoulder blades; Figure 7) using tape 39inFigure1,locatednorthofthehotel)was1.4miles.No
priortoaerosolsamplecollectionactivitiesinBlock20.At sprayblockswerelocatedsouthorsouthwestofthehotel.As
the end of sampling activities, each wipe was placed in a indicatedinFigure4,thewindsduringtheexperimentwere
sterile 50-mL conical vial, which was capped and sealed predominantly from the south and southwest, with non-
with Parafilm. The sealed vial was placed in a chain of negligiblecontributionsfromthenorth-northwest.
custodybag.Priortotransporttotheanalyticallaboratory, LANLnegotiatedwiththeirlodgingprovidertosamplea
the chain of custody bag was wiped with a hospital-grade ‘‘new’’(ie,previouslyunusedbysamplingpersonnel)hotel
bleachwipe(Dispatchbrand,Medline),allowedtoair-dry, room each day to minimize the possibility that material
and placed in aclean secondary container. collected wasdeposited by theprevious day’sactivities.
Volume9,Number3,2011 293
TRANSPORTOF BACILLUSTHURINGIENSIS VAR.KURSTAKI VIAFOMITES
Figure6. Location ofaerosol samplers inMason DistrictPark (red icons). Spray Block20is outlined inpink.
As noted above, sample collection personnel wore clean All sample media were extracted using PET buffer
gloves and nitrile booties while collecting all samples. No (100mM sodium phosphate, 10mM EDTA, 0.01% v/v
other personal protective equipment was worn, and no Tween-20, pH 7.4). A 100-mL aliquot of the extract was
specialprecautionsweretakenwhenenteringorleavingthe plated onto trypticase soy agar (Beckton Dickinson and
sampling area (Mason DistrictPark). Co.,FisherScientific)withcycloheximide(50mg/L,Sigma-
Of the total number of samples, 5% were taken as field Aldrich), and colony counts were obtained after overnight
blanks.Atleast1fieldblankforeachmediumwasdeliveredto incubation at 37(cid:2)C. Colonies exhibiting B. thuringiensis
theanalyticallabeachdaytoensurenocross-contamination morphologywereanalyzedusingpolymerasechainreaction
ofsamplesoccurred. (PCR) to confirm their identity as Btk.
Fivepercentofthetotalnumberofsamples,andatleast
1 of each type of medium per day, were analyzed as field
Sample Analysis blanks. All field blanks were negative for viable Btk.
Upon receipt at the analytical laboratory, all sample bags
were bleach-wiped and allowed to air-dry. The bags were
Results
cut open with a sterile scalpel, and the primary sample
containerswereremovedbypersonnelwearingcleannitrile
gloves.Thecontainerswerethenbleach-wipedandallowed Table 1 presents aerosol sample results. One of 5 back-
to air-dry. groundaerosolsamples(collectedonMay1,2008,priorto
294 BiosecurityandBioterrorism:BiodefenseStrategy,Practice,andScience
VANCUYK ETAL.
Table 2 presents the results from the dry gauze wipes
attached to sampling personnel. Four of 31 background
samples (collected on April 30, 2008, prior to spraying)
tested positive by culture (13%); 27 of 31 background
sampleswerenegativebyculture.Whileitisnotpossibleto
unambiguously determine the source of the material that
resultedin4backgroundgauzepositives,itisnotlikelydue
to cross-contamination as allblankstested were negative.
Post-spray,between17%and78%ofeachday’ssamples
werepositivebyculture,withviabilityinCFUstypically1
to2ordersofmagnitudehigherthantheconcentrationof
thepositive backgroundsamples.
Table 3 presents the results from wet swipes obtained
from samplers’ backpacks. Both of the 2 background
samples (collected on April 30, 2008, prior to spraying)
tested negative by culture. Post-spray, the majority of
samples were positive by culture, with viability results
ranging from 2·102 to 4.4·103 CFUs.
Table4presentstheresultsfromwetswipesandvacuum
samples obtained from the vehicle used as primary trans-
port for the sampling team and their supplies. One of 6
Figure 7. Photograph ofagauzewipe attached toasampler’s backgroundsamples(collectedonApril30,2008,priorto
arm.Colorimagesavailableonlineatwww.liebertonline.com/bsp spraying)testedpositivebyculture;itwasavacuumsample
collected from the rear compartment of the sport utility
vehicle. While it is not possible to unambiguously deter-
spraying)tested positive byculture. Fourof5background
mine the source of the material that resulted in 4 back-
aerosol samples were negative by culture. While it is not
ground gauze positives, it is not likely due to cross-
possible to unambiguously determine the source of the
contamination as all blanks tested were negative. Post-
materialthatresultedin1backgroundaerosolpositive,itis
spray,themajorityofsampleswerepositivebyculture,with
not likely due to cross-contamination as all blanks tested viability results ranging from 1·102 to 4.5·104 CFUs
werenegative.FairfaxCountyhashadanactivegypsymoth
(duetoaprocessingerror,nosamplescollectedonMay4,
suppression program since 1982, and Btk has been exten-
2008,weretestedforviability).Thefinalsample,aswipeof
sively disseminated in the county, including areas near
the vehicle roof collected on May 8, 2008, was positive
MasonDistrictPark.RecentstudiesindicateBtkcanpersist
despite being collectedin therain.
for 4 years in urban environments,6 so it would not be
Table 5 presents the results from vacuum samples ob-
unexpected if background Btk was detected from previous
tained from sample collectors’ hotel rooms. A ‘‘new’’ (ie,
sprays. Post-spray, between 4 and 5 (of 5 total) samples
previouslyunusedbyLANLstaff)hotelroomwassampled
each daytested positive byculture, with viability in CFUs
eachdaytominimizethepossibilitythatmaterialcollected
typically 1 to 2 orders of magnitude higher than the con-
was deposited by the previous day’s operations. Hotel
centration of thepositive backgroundsample.
vacuum samples were collected from the carpet, starting
fromtheentrancetotheroomtoasofathatwaslocatedin
the approximate middle of the room. One (of 1) back-
Table 1. AerosolSampleResults
ground sample (collected on April 30, 2008, prior to
spraying) was negative by culture. Post-spray, all samples
Number
Aerosol but2werepositivebyculture,withviabilityresultsranging
Samples % Aerosol from 4.0·102 to 6.2·103 CFUs.
Collection Positive Samples
Date (N=5) Positive CFURange
Discussion
5/1/2008 1 20 4.0·102
(background)
5/3/2008 5 100 1.3·103–9.2·103 MasonDistrictPark,wheretheexperimentwasconducted,
5/4/2008 5 100 9.8·103–5.7·104 is located just north of Fairfax County’s 2008 spray block
5/5/2008 5 100 1.8·103–2.9·104 number 20 (Figure 2). The expected and observed wind
5/6/2008 5 100 1.5·103–4.1·104 conditions(Figures4and5)indicateahighprobabilityof
5/7/2008 4 80 2.0·102–2.4·103 transport of Btk from the spray block to the park. The
5/8/2008 5 100 8.0·102–5.8·103
aerosolsampleresultsareconsistentwithtransportofBtkto
Volume9,Number3,2011 295