EPA/600/R-12/620 | October 2012 | www.epa.gov/ord
United States
Environmental Protection
Agency
Method development for
optimum recovery of
Yersinia pestis from
transport media and swabs
Final Study Report
Office of Research and Development
National Homeland Security Research Center
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EPA/600/R-12/620
Method development for optimum recovery of
Yersinia pestis from transport media and swabs
Final Study Report
Centers for Disease Control and Prevention
National Center for Emerging and Zoonotic Infectious Diseases
Atlanta, Georgia 30329
United States Environmental Protection Agency
Office of Research and Development
National Homeland Security Research Center
Cincinnati, Ohio 45268
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Disclaimer:
The U.S. Environmental Protection Agency (EPA), National Homeland Security Research
Center and the Centers for Disease Control and Prevention (CDC), National Center for
Emerging and Zoonotic Infectious Diseases, under interagency agreement IA#DW-75-922597-
01-0 (CDC IA# CI08-002, CI08-014, CI10-001, and CI10-030), collaborated in the development
of the analysis procedure described here.
This report has been peer and administratively reviewed and has been approved for publication
as a joint EPA and CDC document. Note that approval does not signify that the contents
necessarily reflect the views of either the CDC or EPA. Reference herein to any specific
commercial product, process, or service by trade name, trademark, manufacturer, or
otherwise does not necessarily constitute or imply its endorsement, recommendation, or
favoring by the United States government. The views and opinions expressed herein do
not necessarily state or reflect those of the United States government and shall not be
used for advertising or product endorsement purposes.
Questions concerning this document or its application should be addressed to:
Erin Silvestri, MPH
Project Officer
U.S. Environmental Protection Agency
Office of Research and Development
National Homeland Security Research Center
26 W. Martin Luther King Drive, MS NG16
Cincinnati, OH 45268
513-569-7619
Silvestri.Erin@epa.gov
Laura Rose, MS
Centers for Disease Control and Prevention
National Center for Emerging and Zoonotic Infectious Diseases
Division of Healthcare Quality Promotion
Clinical and Environmental Microbiology Branch
1600 Clifton Avenue
Atlanta, GA 30329
404-639-2161
Lmr8@cdc.gov
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Acknowledgements:
The following individuals and organizations served as members of the Project Team and
contributed to the development of this project are acknowledged:
U.S. Department of Health and Human Services
Centers for Disease Control and Prevention (CDC)
Sarah E. Gilbert
Laura J. Rose
Judith Noble-Wang
Matthew Arduino
Michele Howard
K. Allison Perry
Meranda Bradley
Heather O'Connell
Alicia Shams
Stephen Morse
U.S. Environmental Protection Agency (EPA)
Office of Research and Development
National Homeland Security Research Center
Sanjiv Shah
Erin Silvestri
Sarah Perkins (formerly EPA)
Frank Schaefer
Eugene Rice
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Table of Contents
Disclaimer i
Acknowledgements ii
List of Figures v
List of Tables vii
List of Acronyms ix
Executive Summary x
1.0 Introduction 1
2.0 Materials and Methods 3
2.1 Culture preparations for Yersinia pestis and Francisella tularensis 3
2.1.1 Yersinia pestis 3
2.1.2 Francisella tularensis 3
2.2 Preliminary Study: Survival of Y. pestis in liquid transport media 4
2.3 Growth Curves 5
2.4 Phase I: Evaluation of swab extraction methods 5
2.5 Phase II and III: Evaluation of sample storage parameters for both Y. pestis strains
and sterile swabs 6
2.6 Phase IV: Evaluation of sample storage parameters for both Y. pestis strains and "dirty" swabs 6
2.7 Statistical Analysis 7
3.0 Results and Discussion 8
3.1 Preliminary Study: Survival of Y. pestis in various liquid transport media 8
3.1.1 Amies medium without Charcoal 8
3.1.2 Amies medium with Charcoal 9
3.1.3 Gary and Blairmod medium 10
3.1.4 Phosphate buffered saline with 0.05% Triton X-100 11
3.1.5 Neutralizing Buffer 12
3.1.6 Stuart, Toshach and Patsula medium 13
3.1.7 Selection of transport media for the following study phases 14
3.2 Growth curves 16
3.2.1 Yersinia pestis 16
3.2.2 Francisella tularensis 19
3.3 Phase I: Evaluation of swab extraction methods 20
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3.4 Phase II and III: Evaluation of sample storage parameters for both strains and sterile swabs 21
3.4.1 Phase II: Low-virulent strain, Yersinia pestis A1122 21
3.4.1.1 Macrofoam Swabs 22
3.4.1.2 Rayon Swabs 24
3.4.2 Phase III: Virulent strain, Yersinia pestis CO92 26
3.4.2.1 Macrofoam Swabs 26
3.4.2.2 Rayon Swabs 29
3.5 Phase IV: Evaluation of sample storage parameters for both Y. pestis strains
inoculated on "dirty swabs" 32
3.5.1 Low-virulent strain, Yersinia pestis A1122 32
3.5.1.1 Macrofoam Swabs 32
3.5.1.2 Rayon Swabs 35
3.5.2 Virulent strain, Yersinia pestis CO92 38
3.5.2.1 Macrofoam Swabs 38
3.5.2.2 Rayon Swabs 41
3.6 Statistical Analysis 44
4.0 Conclusions 46
5.0 References 48
6.0 Appendix 40
IV
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List of Figures:
Figure 1: Survival of Y. pestis A1122 over storage time in Amies without Charcoal liquid medium. The
bars represent the mean Iog10 CFU/mL recovered over time. The error bars represent the standard
deviation (n=5) 8
Figure 2 : Survival of Yersinia pestis A1122 over storage time in Amies with Charcoal liquid medium. The
bars represent the mean Iog10 CFU/mL recovered over time. The error bars represent the standard
deviation (n=5) 9
Figure 3: Survival of Yersinia pestis A1122 over storage time in Gary and Blairmod liquid medium. The bars
represent the mean logic CFU/mL recovered over time. The error bars represent the standard deviation
(n=5) 10
Figure 4: Survival of Yersinia pestis A1122 over storage time in PBSTX liquid medium. The bars represent
the mean logic CFU/mL recovered over time. The error bars represent the standard deviation (n=5).... 11
Figure 5: Survival of Yersinia pestis A1122 over storage time in NB liquid medium. The bars represent the
mean Iog10 CFU/mL recovered over time. The error bars represent the standard deviation (n=5) 12
Figure 6: Survival of Yersinia pestis A1122 over storage time in Stuart, Toshach and Patsula liquid
medium 13
Figure 7: Growth curve for Yersinia pestis A1122 17
Figure 8: Growth curve for Yersinia pestis Harbin 17
Figure 9: Growth curve for Yersinia pestis CO92 18
Figure 10: Growth curve for Francisella tularensis LVS 19
Figure 11: Growth curve for Francisella tularensis Schu S4 20
Figure 12: Mean % recovery of Y. pestis A1122 (104 CFU/ swab) from macrofoam swabs over storage
time. Bars represent the 95% confidence interval of the mean % recovery (n=10) 22
Figure 13: Mean % recovery of Y. pestis A1122 (104 CFU/ swab) from rayon swabs over storage time.
Bars represent the 95% confidence interval of the mean recovery (n=10) 24
Figure 14: Mean % recovery of Y. pestis CO92 (104 CFU/ swab) from macrofoam swabs over storage
time. Bars represent the 95% confidence interval of the mean recovery (n=10) 27
Figure 15: Mean % recovery of Y. pestis CO92 (104 CFU/ swab) from rayon swabs over storage time.
Bars represent the 95% confidence interval of the mean recovery (n=10) 30
Figure 16: Mean % recovery of Y. pestis A1122 (104 CFU/ swab) from dirty macrofoam swabs over
storage time. Bars represent the 95% confidence interval of the mean recovery (n=10) 33
Figure 17: Mean % recovery of Y. pestis A1122 (104 CFU/ swab) from dirty rayon swabs over storage
time. Bars represent the 95% confidence interval of the mean recovery (n=10) 36
Figure 18: Mean % recovery of Y. pestis CO92 (104 CFU/ swab) from dirty macrofoam swabs over storage
time. Bars represent the 95% confidence interval of the mean recovery (n=10) 39
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Figure 19: Mean % recovery of Y. pestis CO92 (104 CPU/ swab) from dirty rayon swabs over storage time.
Bars represent the 95% confidence interval of the mean recovery (n=10) 42
VI
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List of Tables:
Table 1. Percent of Y. pestis samples with recoveries of < 0.5 logic change relative to T0for storage times
of 12 to 72 hours (n=60) in various transport media 14
Table 2. Percent of Y. pestis samples with recoveries of < 0.3 logic change from T0for storage times of 12
to 72 hours (n=60) in various transport media 16
Table 3. Mean percent recovery for each swab material, pre-moistening liquid, and extraction method
(n=10) 21
Table 4: Comparison of mean % recovery of Y. pestis A1122 from macrofoam swabs stored for 12, 18
and 24 hours, (n=30 for each experiment) using Tukey HSD analysis 23
Table 5: Comparison of mean % recovery of Y. pestis A1122 from macrofoam swabs stored for 12, 18, 24
and 48 (n=40 for each experiment) using Tukey HSD analysis 23
Table 6: Comparison of mean % recovery of Y. pestis A1122 from rayon swabs stored for 12, 18 and 24
hours, (n=30 for each experiment) using Tukey HSD analysis 25
Table 7: Comparison of mean % recovery of Y. pestis A1122 from rayon swabs stored for 12, 18, 24 and
48 hours (n=40 for each experiment) using Tukey HSD analysis 26
Table 8: Comparison of mean % recovery of Y. pestis CO92 from macrofoam swabs stored for 12, 18 and
24 hours (n=30 for each experiment) using Tukey HSD analysis 28
Table 9: Comparison of mean % recovery of Y. pestis CO92 from macrofoam swabs stored for 12, 18, 24
and 48 hours (n = 40 for each experiment) using Tukey HSD analysis 29
Table 10: Comparison of mean % recovery of Y. pestis CO92 from rayon swabs after storage for 12, 18
and 24 hours (n=30 for each experiment) using Tukey HSD analysis 31
Table 11: Comparison of mean % recovery of Y. pestis CO92 from rayon swabs after storage of 12, 18, 24
and 48 hours (n=40 for each experiment) using Tukey HSD analysis 31
Table 12: Comparison of mean % recovery of Y. pestis A1122 from dirty macrofoam swabs after storage
for 12 and 24 hours (n=20 for each experiment) using Tukey HSD analysis 34
Table 13: ANOVA of Y. pestis A1122 mean % recovery from dirty macrofoam swabs after storage for 12,
24 and 48 hours (n=30 for each experiment) using Tukey HSD analysis 35
Table 14: Comparison of mean % recovery of Y. pestis A1122 from dirty rayon swabs after storage for 12
and 24 hours (n=20 for each experiment) using Tukey HSD analysis 37
Table 15: Comparison of mean % recovery of Y. pestis A1122 from dirty rayon swabs after storage at 12,
24 and 48 hours (n=30 for each experiment) using Tukey HSD analysis 38
Table 16: Comparison of mean % recovery of /. pestis CO92 from dirty macrofoam swabs after storage
at 12 and 24 hours (n=20 for each experiment) using Tukey HSD analysis 39
Table 17: Comparison of mean % recovery of Y. pestis CO92 from dirty macrofoam swabs after storage
for 12, 24 and 48 hours (n=30 for each experiment) using Tukey HSD analysis 41
VII
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Table 18: Comparison of mean % recovery of Y. pestis CO92 from dirty rayon swabs after storage for 12
and 24 hours (n=20 for each experiment) using Tukey HSD analysis 43
Table 19: Comparison of mean % recovery of Y. pestis CO92 from dirty rayon swabs after storage for 12,
24 and 48 hours (n=30 for each experiment) using Tukey HSD analysis 44
Table 20: Optimum recovery of low-virulent (A1122) and virulent (CO92) Y. pestis from each swab type
at the specified condition 45
VIII
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List of Acronyms:
|j| microliter
ANOVA analysis of variance
AID Arizona test dust
BB Butterfield's buffer
BHIB brain heart infusion broth
C&B Gary and Blair
CDC Centers for Disease Control and Prevention
CPU colony forming units
CHOC chocolate agar plates
Cl confidence interval
EPA U.S. Environmental Protection Agency
g gravity
hms hemin storage locus
hr hour
kb kilo base pairs
LOD limit of detection
mg milligram
ml_ milliliter
n number
NB neutralizing buffer
NHSRC National Homeland Security Research Center
PBS phosphate buffered saline
PBST phosphate buffered saline with 0.02%Tween® 80
PBSTX phosphate buffered saline with 0.05% Triton™ X-100
PCR polymerase chain reaction
pgm pigmentation locus
rpm revolutions per minute
sd standard deviation
SPSS Statistical Package for the Social Sciences
TSAII trypticase soy agar + 5 % Sheep's Blood
TSB+ Isovitalex trypticase soy agar + Isovitalex™
IX
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Executive Summary;
The GAO report investigating the surface sampling methods used during the 2001 mail
contamination with Bacillus anthracis brought to light certain knowledge gaps that existed
regarding environmental sampling [1, 2]. Since then, the Centers for Disease Control and
Prevention (CDC) developed and validated sample preparation methods for environmental
samples for use in a B. anthracis investigation. Should a contamination event occur involving
non-spore forming biological select agents, surface sample collection material and transport
media specific to those agents will be needed. Under a collaborative interagency agreement
between the U.S. Environmental Protection Agency (EPA) and the CDC, CDC executed a
research project to evaluate surface sampling materials, transport media, and processing
methods for potential bioterrorism agents. EPA's National Homeland Security Research Center
(NHSRC) funded the project. Work began with Francisella tularensis and Yersinia pestis, the
causative agents for tularemia and plague, respectively. However, because of continued
difficulties obtaining consistent, reproducible F. tularensis growth, the study continued with Y.
pestis only. The study subsequently focused on investigating the recovery of virulent and low
virulent strains of Y. pestis from four different swab types (polyester, macrofoam, rayon, and
cotton) pre-moistened in various buffers and stored in various transport media.
Preliminary work in which six transport media were inoculated with the low virulence strain Y.
pestis A1122 and held for 7 days at 4°, 25°, and 35°C, revealed that Gary and Blair (C&B)
transport medium (modified formulation without calcium chloride [CaCI2] and agar, C&Bmod) and
the 0.05 % phosphate buffered saline with Triton™ X-100 surfactant (PBSTX) transport medium
provided the most stable Y. pestis viability. The most favorable sample storage temperature was
found to be 4°C, and the remainder of the study was conducted at this temperature.
Phase I of the study involved evaluation of four swab materials; cotton, polyester, rayon, and
macrofoam. Each swab type was pre-moistened with either Neutralizing Buffer (NB) or PBSTX,
inoculated with 104 Y. pestis A1122 cells, then the cells were extracted by one of three
extraction methods; vortexing, sonicating, or a combination of both vortexing and sonicating.
Percent recovery (% recovery) was determined relative to the inoculum. Conditions consisting of
macrofoam swabs pre-moistened with NB and vortexed for 3 minutes (% recovery 93.9%,
standard deviation [sd] 13.1%), and rayon swabs pre-moistened with NB and sonicated for 3
minutes (% recovery 77.0%, sd 14.4%) were chosen as the two best swab, premoistening
medium, and processing method combinations because of the higher % recovery obtained, and
these swab conditions were used for the remaining phases of the study.
Phase II of the study investigated the optimum swab conditions over various storage times.
Macrofoam and rayon swabs were pre-moistened with NB, inoculated with 104 Y. pestis A1122
cells, held for 1 hour, and then placed in one of the selected transport media (C&Bmod or
PBSTX). The swabs were held at 4°C, with 10 swabs being removed from storage and
processed to extract the cells at 0, 12, 18, 24, 48 and 72 hours.
When the rayon swab data from all time points (12 to 72 hours) are combined within each test
condition (premoistening agent and transport medium combination), the condition providing the
greatest mean % recovery was if pre-moistened with PBSTX and stored in PBSTX as a
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transport medium (103.7%, sd 17.2%). When rayon swabs were processed by sonication within
24 or 48 hours, no significant difference was seen between this optimum condition and another
condition; pre-moistened with NB and stored in PBSTX. When all macrofoam swab data for (12
to 72 hours) are combined within each test condition (premoistening agent and transport
medium combination), the optimum mean % recovery was seen when swabs were pre-
moistened with NB and stored in C&Bmodas a transport medium (99.6%, sd 10.9%). When
macrofoam swabs were processed within 24 hours (99.2%, sd 11.6%) by vortexing, no
significant difference in % recovery was found between this optimum condition and two other
conditions (Premoistening agent/transport media: PBSTX/PBSTX and PBSTX/C&Bmod).
Phase III of the study involved conducting the same evaluations as in phase II, but with the
virulent Y. pestis CO 92 strain. The virulent strain behaved somewhat differently than the low
virulent strain in that more decline in recovery was seen after 24 hours if macrofoam swabs
were pre-moistened with NB, and held in C&Bmod. When all phase III macrofoam swab data are
combined (12 to 72 hours) within each test condition, the highest mean % recovery was seen
when pre-moistened with PBSTX and stored in C&Bmod(101.8%, sd 10.0%). The same
conditions provided the optimum % recovery at 24 and 48 hours. When all phase III rayon swab
data are combined (12 to 72 hours) within each test condition, the highest mean % recovery
was seen if pre-moistened with NB and stored in PBSTX (110.1%, sd 21%). If processed at 24
hours, no significant difference was seen between these optimum conditions and two other
conditions; PBSTX/PBSTX and NB/C&Bmod.
After some work with Y. pestis CO92 was completed, it was found that the C&B liquid medium
formulation was altered to exclude CaCI2 because it precipitated out of solution, a problem not
seen when the media was prepared as the intended semi-solid formulation. CaCI2 was shown to
be a critical ingredient for maintaining the virulence of Y. pestis in growth media [3, 4], though no
information is available regarding the need for CaCI2 in transport media. Though the modified
formulation of the liquid C&B medium did provide good stability, the modified C&B would be
difficult to obtain quickly during an investigation because it is not commercially available. We
therefore decided that the altered C&B should be replaced with the next-best transport medium,
NB, for phase IV of the study.
The recovery of both low virulent and virulent Y. pestis strains from swabs in the presence of
simulated dust material containing other viable organisms was determined using sixteen
combinations of the two optimum swabs, two pre-moistening agents, two transport media and
two extraction methods. A mixture of Arizona Test Dust (ATD; Powder Technology Inc.,
Burnsville, MN) and PBSTX or NB was created and used as the pre-moistening agent before
inoculating the swab with 104 CPU of Y. pestis. Holding and processing was performed as
described above.
When ATD was present on macrofoam swabs, the highest mean % recovery of Y. pestis A1122
was achieved when the swabs were pre-moistened with PBSTX and held in NB as a transport
medium. The % recovery was 106.6%, 106.1%, and 105.1% for the holding times of 24, 48 and
72 hours, respectively. The of Y. pestis CO92 was also seen with the same condition
XI
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(PBSTX/NB), though the % recovery was somewhat lower than the A1122 strain at 87.7%,
85.4%, or 85.6% for 24, 48, or 72 hours, respectively.
When AID was present on rayon swabs, the highest mean % recovery of low-virulent Y. pestis
A1122 was achieved when the swabs were pre-moistened with PBSTX and held in PBSTX as a
transport medium: 105.3%, 107.8%, or 107.2% for 24, 48, or 72 hours, respectively. The highest
% of the virulent strain CO92 from rayon swabs was slightly lower than A1122, and was seen
when the swabs were pre-moistened with NB and held in PBSTX as a transport medium:
82.5%, 84.3%, or 83.7% for 24, 48, or 72 hours, respectively.
Since the optimum pre-moistening agent, transport medium and hold time was not consistent
across both strains (Y. pestis A1122 and CO92) and selected swabs (rayon and macrofoam),
the data were separated according to swab type and strain, and then compared. Within these
categories (swab type and strain), data were combined for all storage times from 12 to 72 hours.
In a worst case scenario, where sample processing and analyses couldn't occur until 72 hours
after sampling, macrofoam swabs pre-moistened with PBSTX and stored in NB as a transport
medium performed significantly better than all other conditions, regardless of strain tested
(96.0%, p<0.001). In the same scenario, rayon swabs had two optimum premoistening
medium/transport medium combinations: PBSTX/PBSTX and NB/PBSTX (94.4% and 93.6%,
respectively). These two combinations performed equally well, and significantly better than the
other two conditions (p<0.001).
This research should be considered preliminary, as additional research will be needed to
optimize sampling, transport, and extraction protocols for recovering these biothreat agents from
surfaces. The results of this research showed that the best recovery of Y. pestis from the
swabs was obtained using either macrofoam or rayon swabs. Based on the highest % recovery
when dust was present (ATD), the optimal conditions for macrofoam swabs were obtained when
premoistening with PBSTX and transporting in NB. The optimum recovery of Y. pestis A1122
strain and CO92 strain from rayon swabs occurred when the swabs were pre-moistened with
either PBSTX or NB, and then placed in tubes containing PBSTX as a transport medium.
Regardless of which swab was chosen, the optimum temperature for transport was determined
to be 4°C. Additional research will be necessary to apply these findings to a "real world"
scenario in which Y. pestis is extracted from swabs used to sample from varying surface types
and environmental conditions.
XII
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1.0 Introduction
After the anthrax attacks in the fall of 2001, the sampling and processing methods used
during the investigation were found to be less than optimum. The methods were not well
characterized or standardized, therefore post-decontamination sampling data offered little
confidence that the buildings were safe to re-occupy [1, 2]. Several ways that the country
could better prepare itself in the event of a terrorist attack were identified. One critical need
was for validated sampling methods that could be used by all laboratories in the event of a
homeland security incident. The U.S. Environmental Protection Agency's Selected
Analytical Methods for Environmental Remediation and Recovery[5] contains suggested
methods for use by laboratories tasked with performing analysis of environmental samples
following a homeland security event.
Sample preparation methods need to be optimized for environmental samples containing
biological pathogens. Much effort and resources have been allocated to the development of
molecular assays and culture techniques; however, the initial sample collection and
preparation lags behind in development. Sample preparation involves recovery of the
biological agent from the sampling device, and remains the limiting step in the detection
techniques, whether those techniques use non-culture methods (e.g., polymerase chain
reaction [PCR]), or culture-based methods. Extracting and recovering pathogens and
biotoxins from environmental matrices (e.g., air, soil, and water) and sampling devices
(swab, wipe, or vacuum device) present challenges, because the matrices and devices are
composed of non-target biological and chemical analytes that may inhibit or interfere
(compete) with the extraction and detection of the target analyte.
The Centers for Disease Control and Prevention (CDC), part of the Department of Health
and Human Services, has extensive knowledge of, and experience with, developing
methods for potential bioterrorism agents. The EPA and CDC collaborated to improve and
develop methods for sample collection, sample preparation, and sample analysis for
biological agents. In this preliminary study, previously developed CDC methods for recovery
of Bacillus anthracis from non-porous surfaces were investigated for their application to
Yersinia pestis and Francisella tularensis [6, 7]. Y. pestis and F. tularensis are the causative
agents for plague and tularemia, respectively. The primary goal of this project was to
determine the best combination of sampling swab, pre-moistening agent, transport media,
and extraction method for a high efficiency recovery of Y. pestis and F. tularensis vegetative
cells. The study did not investigate recovery of cells from environmental surfaces. Two
strains of Francisella tularensis were initially selected as a second pathogen for evaluation
during this study, however, it proved to be difficult to obtain consistent growth with either
strain, and the organism was subsequently omitted from the study (see section 2.3 and
3.2.2). The limit of detection (LOD) of an analytical method depends on optimized materials,
protocols, and conditions that can best maintain the integrity of the sample, and the efficient
recovery of the target biological agent from the sampling tools. A secondary goal was to
determine the % recovery of these bacteria after they have been held in transport media for
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different time intervals (e.g., 24, 48, or 72 hours). In an actual contamination event, it could
take from 24 to 72 hours before the sample is processed and analyzed. This research
should be considered preliminary, and additional research may be needed to develop
sampling, transport, and extraction protocols for recovering these biothreat agents from
swabs during an event for use by first responders, EPA's Environmental Response
Laboratory Network (ERLN), and the biodefense community as a whole.
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2.0 Materials and Methods
2.1 Culture preparations for Yersinia pestis and Francisella tularensis
2.1.1 Yersinia pestis
Preparation of frozen stock: Y. pestis Harbin, Y. pestis CO92 and Y.
pestis A1122 were obtained from CDC's Division of Vector Borne
Diseases, Ft. Collins, CO, and stored at
-70°C in cryovials with beads (PL170, Prolab Diagnostics, Austin, TX).
One bead was removed from a freezer vial and cultured onto trypticase
soy agar with 5% sheep blood (ISAM; BD Diagnostic Systems, Sparks,
MD) and incubated at 25°C for 48 hours. After 48 hours of incubation,
purity of culture was assessed, and then a 0.5 McFarland standard
suspension (108 colony forming units [CPU] per ml_) of the culture was
prepared in Butterfield's buffer (BB; Becton Dickinson, Franklin Lakes,
NJ). Ten-fold serial dilutions were prepared in BB and spread plated onto
ISAM plates in triplicate to determine the concentration of cells. The
plates were incubated at 25°C for 48 hours. From the 10"4 dilution tube
(104 CFU/mL), 1 ml_ was removed and placed in a 250 ml_ flask
containing 99 ml_ brain heart infusion broth (BHIB; Becton Dickinson, #
237500) and allowed to incubate on a shaker table (100 rpm) at 25°C for
48 hours. After incubation, 1 ml_ of the culture was placed into a 50 mL
glass tube containing 30 ml of BHIB with 10% glycerol. This mixture was
vortexed, then dispensed into 1.2 mL volumes in 2 ml_ Cryovial®tubes
(Thermo Scientific, Rochester, NY) and frozen at -80°C.
Preparation of the working suspension: From the frozen stock, 1 mL was
added to a 100 mL flask containing 30 mL of BHIB. This was incubated
at 25°C, in a shaker-incubator, for 26-30 hours to reach late log phase
growth (as determined from the growth curve, refer to section 2.2.1). Cells
were harvested by centrifugation at 3000 x gt 4°c, for 10 minutes. The
supernatant was decanted and the pellet resuspended in 25 mL of sterile
phosphate buffered saline (PBS). This wash step was repeated two
additional times. After the third wash and re-suspension, a 0.5 McFarland
standard suspension was prepared (108 CFU/mL), diluted in series, and
plated onto TSAII plates to determine the concentration of the cells.
Plates were incubated at 25°C for 48 hours. One mL of the 10"3 dilution
tube (10s CFU/mL) was used to inoculate the 9 mL of transport media
(resulting in 104CFU/mL). One hundred uL of the 10'3 dilution tube (105
CFU/mL) was used to inoculate the swabs (resulting in 104 CFU/swab).
2.1.2 Francisella tularensis
Preparation of frozen stock: F. tularensis subsp. holartica LVS and F.
tularensis subsp. tularensis Schu S4 were obtained from CDC's Division
of Vector Borne Diseases, Ft. Collins, CO, and stored at -70°C in
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cryovials with beads. One bead from a previously frozen vial of F.
tularensis was removed and cultured on chocolate agar (CHOC; BD
Diagnostic Systems,) and incubated at 35°C for 48 hours. After 48 hours,
the purity of the culture was ascertained and a 0.5 McFarland standard
suspension (~108 CPU per ml_) of the culture prepared in BB. Ten-fold
serial dilutions were prepared in BB and spread plated onto CHOC plates
in triplicate to check the titer. The plates were incubated at 35°C for 48
hours. From the 10"4 dilution tube (104 CFU/mL), 1 ml_ was removed and
placed in a 250 ml_ flask containing 99 ml_ trypticase soy broth (TSB)
containing 2% Isovitalex™ (Becton, Dickinson and Company, Franklin
Lakes, NJ) and incubated on a shaker table (100 rpm) at 35°C for 48
hours. After incubation, 1 ml_ of the culture was placed in a 50 ml_ glass
tube containing 30 ml_ of BHIB supplemented with 10% glycerol. This
mixture was vortexed and dispensed into 1.2 ml_ volumes in 2 mL
Cryovial tubes and frozen at -80°C.
Preparation of the working suspension: From a frozen stock, 1 mLwas
added to a 100 ml_ flask containing 30 ml_ of TSB+ Isovitalex. This was
incubated at 35°C, in a shaker-incubator, until late log phase growth was
acquired. After incubation, cells were harvested by centrifuging at 3000 x
g for 10 minutes at 4°C. The supernatant was then decanted and the
pellet resuspended in 25 ml_ of sterile PBS. These wash steps were
repeated two additional times, and the final cell pellet was resuspended in
25 ml_ of PBS. A 0.5 McFarland standard suspension of the culture was
prepared, for a final titer of 108 CFU/mL. The titer was checked by
performing ten-fold serial dilutions and spread plating onto CHOC. Plates
were incubated at 35°C for 48 hours. One mL of the 10"3 dilution tube (10s
CFU/mL) was used to inoculate the 9 mL of transport media (resulting in
104 CFU/mL). One hundred uL of the 10'3 dilution tube (105 CFU/mL) was
used to inoculate the swabs (resulting in 104 CFU/swab).
2.2 Preliminary Study: Survival of Y. pestis in liquid transport media
A preliminary study was conducted to investigate the survival of a low virulence
strain (Y. pestis A1122, CDC, Ft. Collins, CO) in various transport media. Six liquid
transport media were evaluated: (1) Stuart, Toshach, and Patsula medium, (2) Gary
and Blair without Calcium chloride (CaCI2) (C&Bmod) medium, (3) Amies medium
without charcoal, (4) Amies medium with charcoal, (5) phosphate buffered saline with
0.05% Triton X-100 (PBSTX) surfactant, and 6) neutralizing buffer (NB). Transport
media 1-4 are traditional clinical transport media, meant to be prepared with 0.2 %
agar to form a semi-solid mass in a transport tube. Traditionally, the clinical swabs
are submerged in the soft media to preserve the cells on the swab. In this
application, quantitation is not a goal, but simply preserving any quantity of cell for
isolation in the lab in order to identify the organism. For the purposes of this study,
however, we required quantitation of cell recovered from the swabs, and found that
-------
significant numbers of cells remained in the semi-solid media upon removing the
swab and processing in a separate tube of extraction fluid. We therefore omitted the
agar from all formulations to create liquid transport media. In order to evaluate the
best transport media, a bacterial suspension of Y. pestis A1122 was created in
Butterfield buffer (BB; Becton Dickinson, Sparks, MD) from 48 hour plate growth. The
suspension was diluted in series to a concentration of 10s CFU/mL. Five replicate
tubes (9 ml_ per tube) of each transport medium were inoculated with 1 ml_ of the 10s
CFU/mL bacterial suspension. The tubes were held at 4°C, 25°C, or 35°C over a 72-
hour holding period, with intermittent sampling over the hold time (see Appendix,
Table 1 for test matrix). Tubes were vortexed immediately before samples were
taken at 0, 12, 18, 24, 48, and 72 hours. Traditional plate culture methods were
used to detect the presence of viable organisms. Samples were plated onto TSAII,
incubated at 25°C for 48 hours and colonies counted. The total CFU recovered for
the sample was determined and the results were reported as Log10 CFU recovered
at each time point. Based on the results of the preliminary study (see section 3.1),
two transport media were selected for the remainder of the study.
2.3 Growth Curves
For both organisms, 99 ml_ of broth (BHIB for Y. pestis and TSB +lsovitalex for F.
tularensis) was inoculated with 1 ml_ from a working stock suspension and allowed to
shake at 100 rpm, 25°C (Y. pestis) or 35°C (F. tularensis). Two strains of each
organism were tested, the low-virulence strains Y. pestis A1122 and F. tularensis
LVS, and the virulent strains Y. pestis CO92 (initially Y. pestis Harbin until found to
be lacking one of its virulence plasmids) and F. tularensis Schu S4. The titer of the
stock suspension was checked by diluting in series and plating in triplicate on the
appropriate media. The flask was allowed to shake at 100 rpm for 24 hours and then
sampled periodically to monitor growth. The optical density and the titer were
checked at each time point, and a growth curve generated for each isolate. The time
required to achieve late logarithmic growth was determined, and all cells used in the
study were harvested at that time.
2.4 Phase I: Evaluation of swab extraction methods
Phase 1 was conducted to evaluate the optimum swab materials and extraction
methods. Table 2 in the appendix shows the matrix of tests conducted. Four types
of swab materials were evaluated: cotton (Baxter Healthcare Corp., Deerfield, IL cat
#A5002-5), polyester (Falcon™ #220690, BD, Franklin Lakes, NJ), macrofoam
(Puritan Medical, Guilford, ME, # 25-1607 1PF SC), and rayon (Puritan, # 25-806 1
WR). Two premoistening solutions were evaluated, PBSTX and NB. The swabs
were pre-moistened by submerging them in a tube with one of the premoistening
liquids for 10 minutes. The swabs were then pressed against the inside wall of the
tube as they were removed, to express any excess premoistening liquid. The pre-
moistened swabs were directly inoculated with 100 uL of 10s CFU/mL working
suspension of the test organism, then placed in a 15 mL conical tube to hold for 1
hour at room temperature. After the one hour hold, the swabs were placed into
-------
tubes containing 5 ml_ of phosphate buffered saline with 0.02% Tween®80 (PBST)
(Sigma-Aldrich, St. Louis, MO). Preliminary work demonstrated that no significant
number of cells were lost during the transfer of the swabs from one tube to another
after the 1 hour hold. The swabs were then processed by one of four methods: (1)
vortexing for 3 minutes (VX-2500 multi-tube vortexer set on the highest speed, VWR,
Sewanee, GA, (2) sonicating for 3 minutes (FS 20, a 40-KHz sonic cleaner; Fisher
Scientific, Pittsburg, PA), (3) vortexing and sonicating for 30 seconds each -
repeated three times for a total of 3 minutes, and (4) no extraction method
(submersion in PBST only). After the extraction, the swabs were removed from the
transport medium, the excess liquid was expressed from the swab heads by pushing
the swabs against the inside wall of the tube. The PBST containing the extracted
cells was diluted in series, and each dilution plated in triplicate on TSAII. Positive
controls consisted of inoculating 5 ml_ of PBST (no swab) with the same quantity of
cells and processing alongside the swabs. The PBS containing cells was diluted in
series, and each dilution plated onto ten plates per dilution. The plates were
incubated for 48 hours at 25°C. Colonies were counted and the percent of viable
cells recovered was determined, relative to the positive controls, for each test
parameter. Each swab material and premoistening solution combination was tested
with ten replicate swabs and one positive control.
2.5 Phase II and III: Evaluation of sample storage parameters for both Y. pestis
strains and sterile swabs
The two best performing transport media and the two best swab materials from the
phase I preliminary study were selected for use for the remainder of the study.
Various storage conditions, such as swab transport media (top two performers from
preliminary studies), temperature (best survival temperature from preliminary
studies) and holding time, were evaluated for survival and % recovery of Y. pestis.
Tables 3 and 4 show the matrices of tests performed in phases II and III. The
swabs were pre-moistened with either PBSTX or NB, inoculated with a known
concentration of low-virulent Y. pestis A1122 (phase II) or virulent Y. pestis CO92
(phase III) as described in the methods for phase I, then placed into one of the two
best transport media chosen in phase I. The swabs were held at 4°C (found to be
optimum temperature in preliminary phase), with 10 swab samples being removed
and processed at each of the following time points: 0, 12, 18, 24, 48 and 72 hours.
Swabs were processed by the optimum method for each, as determined in phase I.
2.6 Phase IV: Evaluation of sample storage parameters for both Y. pestis strains
and "dirty" swabs
In order to prepare the "dirty" swabs, a slurry of pre-characterized Arizona test dust
(ATD, A-3 Medium, Powder Technology Inc., Burnsville, MN) was prepared to pre-
moisten the swabs for experiments which called for "dirty" swabs. One gram of ATD
was added to 10 ml_ of the premoistening agent to achieve a concentration of 100
mg/mL. The slurry was diluted once more, by adding 10 mL to 90 mL of the pre-
moistening agent (PBST or NB), creating a final concentration of 10 mg ATD/mL in
-------
the premoistening solution. This slurry was stored at 4°C for up to one week before
use. The slurry was re-suspended by vortexing 1 min, then swabs were dipped into
the slurry before direct inoculation with the test organism and processed as
described in previously mentioned methods in section 2.4 and 2.5, using the
transport media selected in phase I. Tables 5 and 6 show the matrices of tests
performed in phase IV.
2.7 Statistical Analysis
The preliminary study data are presented as the mean Iog10 CPU recovered at each
temperature, time point, transport medium and organism evaluated. In the
preliminary phase, it was necessary to identify the condition(s) that allowed for the
least amount of change in cell number (growth or death). To identify the
condition(s), the percent of samples with < 0.5 Iog10and < 0.3 Iog10 CPU change in
recovered cells, relative to the T0 (time zero) CPU recovery was calculated for each
transport medium, swab type, and temperature. In phase I, the % recovery of cells
after each extraction method was calculated relative to the recovery from the
positive control. (Positive control was 5 ml_ of PBS, same inocula and treatment, but
with no swab.) In phases II, III, and IV - in order to normalize the % recovery data
when comparing storage temperatures, premoistening agents and transport media
over several time periods - the % recoveries were calculated relative to the T0 CPU
recovery. The statistical analysis for phases II, III, and IV were SPSS software,
version 18 (IBM, Armonk, NY). Each data set was tested for normality. If a normal
distribution was found, then ANOVA were performed between various combinations
of the two selected swabs, premoistening agents and transport media when
grouped 12 to 24 hours, 12 to 48 hours, and 12 to 72 hours (a < 0.05). Tukey
Highest Significant Difference test was performed as a Post Hoc Test. If the data
was found to be non-parametric, the Kruskal-Wallis and Mann-Whitney Tests were
performed.
-------
3.0 Results and Discussion
3.1 Preliminary Study: Survival of Y. pestis in various liquid transport media
3.1.1 Amies medium without Charcoal
The recovery of Y. pestis A1122 in Amies without Charcoal medium
(Figure 1) was maintained within 0.52 Iog10 of the T0 (4.22 log10CFU/mL)
for all time points at 4°C and 25°C. After 48 hours, viable cell count
declined when stored at 35°C. No significant difference in recovery was
noted between T0 and cells held at 4°C for 24 hours (p=0.76). Although
an increase in cells occurred if the medium were held at 4°C or 25°C for
96 hours, the counts were still within the same order of magnitude as T0.
It appears that 4°C is the better holding temperature for this medium at 24
hours (p<0.001), though no significant difference was seen in recovery
between 4° and 25°C at the 48 and 72 hour hold times.
Amies without Charcoal Transport Medium
Y.pestfsA1122
24 48
Time (Hours)
72
96
D4°C •25°C D35°C
Figure 1: Survival of Y. pestis A1122 over storage time in Amies without Charcoal liquid medium. The
bars represent the mean logic CFU/mL recovered over time. The error bars represent the standard
deviation (n=5).
-------
3.1.2 Amies medium with Charcoal
The optimal storage temperature for Y. pestis A1122 in Amies with
Charcoal medium (Figure 2) was 4°C. If held at 4°C or 25°C, recovery at
all sample points remained within 0.9 Iog10 of T0 (4.6 Iog10 CFU/mL),
though 4°C provided significantly more viable cells than 25°C after
storage for 24 and 48 hours (p<0.05). When stored at 35°C, a significant
decline in viable cells was seen after 24 hours, and by 96 hours, no
detectable cells were found, representing greater than 4.3 Iog10 loss of
cells (the limit of detection of the assay was 0.33 log10CFU/ml).
O
O)
O
Amies with Charcoal Transport Medium
Y. pesf/sA1122
12
24 48
Time (Hours)
96
D4°C •25°C D35°C Q35°C (below detection)
Figure 2 : Survival of Yersinia pestis A1122 over storage time in Amies with Charcoal liquid medium. The
bars represent the mean logic CFU/mL recovered over time. The error bars represent the standard
deviation (n=5).
-------
3.1.3 Gary and Blairm0d medium
There was no more than a 0.35 Iog10 change relative to T0 (5.16 Iog10
CFU/ml) of cells held at either 4° or 25°C at any time point (Figure 3). The
optimal temperature for Y. pestis in C&Bmod medium was 4°C. When data
for all time points are combined, there is no significant difference in the
recovery of viable cells between at 4° or 25°C. However, if analyzed at
each time period, 4°C provided significantly better recovery at except at
72 hours (when no difference was noted). A decline of viable cells was
observed at 35°C with a 2 Iog10 reduction relative to T0 by 96 hours.
O
O)
O
5
4
3
2
1
0
Gary and Blairmod Transport Medium
Y.pesf/sA1122
11111
0
12
24 48
Time (Hours)
72
96
I4°C B25°C D35°C
Figure 3: Survival of Yersinia pestis A1122 over storage time in Gary and Blairmod liquid medium. The bars
represent the mean Iog10 CFU/mL recovered over time. The error bars represent the standard deviation
(n=5).
10
-------
3.1.4 Phosphate buffered saline with 0.05% Triton X-100
When Y. pestis A1122 was held in PBSTX medium in at 4°C or 25°C, less
than a 0.1 Iog10 change in recovery was seen relative to T0 (3.67 Iog10
CFU/mL) (Figure 4). When data for all time points are combined, 25°C
appears to be slightly better in Iog10 recovery than 4°C, though no
significant differences were seen at 24 and 48 hours (p>0.05). There was
significant loss if held at 35°C at all time periods, and as much as a 3
Iog10 reduction in recovery by 96 hours.
PBSTX Transport Medium
Y. pesf/sA1122
5
4
1 3
O
o
6) 1
o
~" 0
-i-
-±-
-I-
-i-
-±-
-Jn
0
12
24 48
Time (Hours)
96
D4°C B25°C D35°C
Figure 4: Survival of Yersinia pestis A1122 over storage time in PBSTX liquid medium. The bars represent
the mean logic CFU/mL recovered over time. The error bars represent the standard deviation (n=5).
11
-------
3.1.5 Neutralizing Buffer
Holding cells in NB at 4°C was significantly better than holding at 25°C or
35°C for any period of time (p<0.05), with less than a 0.23 Iog10 decline
from the T0 concentration (4.77 log10CFU/mL) after 96 hours (Figure 5).
When held at 25° and 35°C, a 0.6 Iog10 and 1.9 Iog10 decline was seen by
96 hours, respectively.
5
I 4
2 3
O
? 2
O)
° 1
0
Neutralizing Buffer Transport Medium
Y.pesf/sA1122
0
12
24 48
Time (Hours)
72
96
D4°C
I25°C D35°C
Figure 5: Survival of Yersinia pestis A1122 over storage time in NB liquid medium. The bars represent the
mean Iog10 CFU/mL recovered over time. The error bars represent the standard deviation (n=5).
12
-------
3.1.6 Stuart, Toshach and Patsula medium
When using the Stuart, Toshach and Patsula medium (Figure 6) growth
occurred within 12 hours at 25°C or 35°C. When held at 4°C, no
significant differences, relative to T0(4.64 Iog10 CFU/mL) were observed
in cell recovery (p>0.05) across all time points. A greater than 3 Iog10
increase in cell numbers was observed by 96 hours if held at 35°C, and a
1.8 Iog10 increase occurred by 96 hours if held at 25°C.
Q
a
E 7
""• R
"^ D
0 4
t
° 0
g> 1
O Z
_l H
n
Transport Medium: Stuart, Toshach, & Patsula
Y. pesf/sA1122
• T .
J^
rn
in
0
Illl
12
I
^rT
n
I I
24 48
Time (hours)
D4°C B25°C D35°C
R
n
72
|
96
Figure 6: Survival of Yersinia pestis A1122 over storage time in Stuart, Toshach and Patsula liquid
medium.
The bars represent the mean Iog10 CFU/mL recovered over time. The error bars represent the standard
deviation (n=5).
13
-------
3.1.7 Selection of transport media for the following study phases
The data for each medium was compiled by temperature from 12 to 72
hours. Though 72 hours is beyond what might be expected for shipment
or storage of samples, samples could get lost in transit or be held until
supplies or personnel arrived for processing. We must consider how cell
viability would be affected beyond optimum storage times. Though there
was some variability in inocula between transport media tests, this
variability is unlikely to have influenced the results, since the selection
criteria was from log transformed data, and the changes compared were
determined relative to the recovery at T0 for each medium. The percent
of transport tubes with cells recovered that remained within 0.5 Iog10 of
the recovery at the zero time point was calculated and is presented in
Table 1. These data indicate that with three transport media (Amies no
charcoal, C&Bmod, and PBSTX) 100% of samples yielded recoveries
within 0.5 Iog10 of the T0 recovery. Neutralizing buffer was a close fourth
choice, with 100% and 68% of samples yielding recoveries within 0.5
log10at4°and25°C.
Table 1. Percent of Y. pestis samples with recoveries of < 0.5 Iog10change relative to T0for
storage times of 12 to 72 hours (n=60) in various transport media
Storage
temp
4°C
25°C
Transport Media
Amies
with
charcoal
77
55
Amies no
charcoal
100
100
Cary&
Blair
100
100
Neutralizing
Buffer
100
68
Stuart,
Toshach
& Patsula
100
0
PBSTX
100
100
The data were then re-examined so that the percent of samples with cell
recovery that remained within 0.3 Iog10 of the recovery at T0 was
calculated. These data are presented in Table 2, and indicate that C&Bmod
and PBSTX provide the higher percent of swabs with recoveries within
0.3 Iog10 of the T0 recovery. PBSTX yielded 92% and 100% of swabs
within 0.3 Iog10 of the T0 recovery at 4°C and 25°C, respectively, C&Bmod
yielded 97% and 77% of samples within 0.3 Iog10of the T0 recovery at 4°C
and 25°C, respectively. Based on results from the preliminary study,
C&Bmod and PBSTX were chosen as the two transport media that
maintained cell viability well at both temperatures. The optimum storage
temperature was found to be 4°C for the transport media chosen.
Subsequent evaluations were conducted at this temperature and in these
two transport media.
14
-------
After phase III of the study, the formulation for C&Bmod was found to have
been altered by omitting the CaCI2. C&Bmod was replaced with NB
because it provided comparable results to C&Bmod at 4° (already chosen
as the optimal temperature by the time this altered formulation was
discovered) when examining the < 0.3 Iog10 change data (Table 2), but
also because it is immediately commercially available and would be easily
obtained without special formulation (as would be needed if the next best
choice, Amies without charcoal, was chosen).
15
-------
Table 2. Percent of Y. pestis samples with recoveries of < 0.3 Iog10change from T0for storage
times of 12 to 72 hours (n=60) in various transport media
Storage
temp
0
4C
0
25 C
Transport Media
Amies with
charcoal
67
0
Amies no
charcoal
71
85
Gary &
Blair
97
77
Neutralizing
Buffer
77
0
Stuart,
Toshach &
Patsula
97
0
PBSTX
92
100
3.2 Growth curves
3.2.1 Yersinia pestis
The growth curves for Y. pestis A1122 (low virulence), and virulent Harbin
and CO92 strains are shown in Figures 7, 8 and 9, respectively. After the
growth curve was completed for the Y. pestis Harbin strain, testing
revealed that this isolate had lost one of the virulence plasmids before we
acquired it. Based on these results, CDC and EPA jointly decided to
instead use Y. pestis CO92 for work calling for a virulent strain. Y. pestis
CO92 was confirmed as virulent by the Congo red pigment test [8]
(indicating the presence of the hemin storage [hms] and pigmentation
[pgm] loci) and by confirmation of the presence of the three virulence
plasmids (110, 70, and 9.5 kb) as carried out by CDC's Division of Vector
Borne Diseases in Ft Collins, CO. Figure 7 shows the growth curve for Y.
pestis A1122 strain, indicating 60 hours was required to reach the late log
phase when starting with 104CFU/ml_ in BHIB. The Y. pestis Harbin
strain curve is shown in Figure 8, and late log phase was reached around
42 hours. The Y. pestis CO92 strain, seen in Figure 9, reached late log
phase by 26 hours.
16
-------
Y. pestis A1122 growth curve
48 72
Time (hours)
96
120
Figure 7: Growth curve for Yersinia pestis A1122.
_l
E
D
Li.
o
0
d>
o
10
8
6
4,
2
0
G
Y. pestis Harbin growth curve
• •••• ••!!•
m mmm mmmi ^
_>
^
-—
24 48 72 96 120
Time (hours)
Figure 8: Growth curve for Yersinia pestis Harbin.
17
-------
Li.
o
O)
O
Y. pestis CO 92 growth curve
12
10
8
24
Time (hours)
48
72
Figure 9: Growth curve for Yersinia pestis CO92.
18
-------
3.2.2 Francisella tularensis
The growth curves for F. tularensis LVS and Schu S4 strains were found
to both require 60 hours to reach late the log phase when starting with
104CFU/ml_ in trypticase soy broth + 2% Isovitalex (Figures 10 and 11,
respectively).
While performing these growth curve studies, problems were encountered
with getting consistent growth with the F. tularensis strains. F. tularensis is
known to be a fastidious organism, especially when grown in liquid media3
with specific nutritional requirements and high inocula required. Though a
well characterized medium was used, several times the organisms simply
did not grow, or did not grow to the expected titer after extremely long
incubation times. These problems set the timeline for the project back
significantly. Therefore, after considering time and budget constraints,
CDC and EPA jointly decided that all subsequent work would focus only on
Yersinia pestis.
10
I 8
D
o
o
d>
o
F. tularensis LVS growth curve
24
48 72
Time (hours)
96
120
Figure 10: Growth curve for Francisella tularensis LVS.
19
-------
F. tularensis Schu S4 growth curve
m -,
I U
8 i
= 6
E °
S A '
o 4
4, 4
f
-I 0 _
^^
__^-«*«« • mmMm^
r~"
0 24 48 72 96
Time (hours)
Figure 11: Growth curve for Francisella tularensis Schu S4.
3.3 Phase I: Evaluation of swab extraction methods
Table 2 in the appendix shows a matrix of all tests performed in Phase I. Macrofoam
swabs pre-moistened with NB and inoculated with Y. pestis A1122 that were
processed by vortexing only or by sonication and vortexing together yielded the
highest % recovery, relative to the positive control (inoculated PBST, no swab) at
93.9% or 93.5%, respectively (Table 3, shaded cells). Since no significant difference
was seen between these two processing methods (p=0.94), the method with the
fewer steps and simpler equipment (vortexing only) was chosen for subsequent
evaluations. Using the no extraction method was the least efficient, with only 68.7%
recovered from the swab.
Rayon swabs pre-moistened with NB and inoculated with Y. pestis A1122 that were
processed using sonication only yielded the highest % recovery at 77.0% (Table 3,
shaded cell). Combining sonication and vortexing yielded a % recovery of 73%. The
vortex-only method was less efficient for rayon swabs than for macrofoam swabs, at
55.2%. Polyester swabs yielded the lowest % recoveries of all the swab types and
were discontinued for future testing. Although the optimal condition for cotton (pre-
moistened with PBSTX and vortexed and sonicated) yielded a statistically equivalent
% recovery to rayon's optimum (79% and 77%, respectively), cotton swabs were
excluded from subsequent evaluations because of the concerns for potential PCR
inhibition.
As a result of these findings, subsequent phases of the project focused on the use of
macrofoam swabs processed by vortexing (3 minutes) and rayon swabs processed
by sonication only (3 minutes).
20
-------
Table 3. Mean percent recovery for each swab material, pre-moistening liquid, and extraction
method (n=10).
Extraction Method*
Premoistening
agent
Vortex Only
NB
PBSTX
p-value
Sonicate Only
NB
PBSTX
p-value
Vortex & Sonicate
NB
PBSTX
p-value
None
NB
PBSTX
p-value
Swab Material
Cotton
60.9 (8.6)
69.04 (2.03)
0.009
53.7 (8.8)
69.43 (2.31)
0.000
53.6 (11.0)
79.42 (3.08)
0.000
40.4 (5.0)
48.42 (2.50)
0.000
Macrofoam
93.9(13.1)**
86.53 (2.59)
0.099
89.0 (12.7)
84.78 (3.89)
0.330
93.5 (9.4)
86.33 (3.24)
0.035
68.7 (9.2)
62.45 (3.86)
0.065
Polyester
44.6(7.1)
55.54 (1.86)
0.000
44.6 (11.7)
50.57 (2.73)
0.132
53.7 (11.0)
53.65 (3.59)
0.989
25.1(5.0)
46.40 (1.43)
0.000
Rayon
55.2(12.2)
68.7 (5.20)
0.005
77.0 (14.4)
64.6 (5.0)
0.019
73.0(15.7)
67.8 (4.6)
0.328
28.6 (7.8)
29.80(3.1)
0.641
*Extractions were performed one hour after swab inoculation. All extractions were performed in
PBS+0.02%Tween®80.
** Shading indicates the best extraction methods for the two best swab materials
3.4 Phase II and III: Evaluation of sample storage parameters for both strains and
sterile swabs
3.4.1 Phase II: Low-virulent strain, Yersinia pestis A1122
The recovery efficiency of low virulent Y. pestis (strain A1122) from
macrofoam swabs and rayon swabs was evaluated. Tables 3 and 4 in
the appendix show the matrices of tests performed in phases II and III.
Both swab materials were inoculated directly with 100 ul of a 10s
CFU/mL suspension (104 CFU/swab) and held at room temperature for
one hour before placing the swabs in one of the two optimum liquid
transport media, either the C&Bmod or the phosphate buffered saline with
0.05% Triton X-100 (PBSTX). The swabs were held in the media at 4°C
for the following times: 0, 12, 18, 24, 48, and 72 hours before processing.
Two swab preparations (pre-moistened with either PBSTX or NB) and two
methods of swab processing (vortexing only for macrofoam swabs and
sonication only for rayon swabs) were evaluated. Percent recovery
relative to T0 was determined. We subsequently adopted the convention
of denoting each specific condition as pre-moistening medium / transport
medium.
21
-------
140.0-
£=> 120.0-
o
%
14
01
100.0-
30.0-
0 60.0-
0
40.0-
20.0-
o.o-
3.4.1.1 Macrofoam Swabs
Macrofoam swabs yielded a % recovery range of 46.9-106.2% (Figure
12). When all time points from the 12 to 72 hour storage periods were
averaged for each condition (premoistening agent/transport medium), the
highest mean % recovery from macrofoam swabs was 99.6%, (sd=10.9,
n=49) when pre-moistened with NB and stored at 4°C in C&Bmodas the
transport medium (denoted as NB/C&Bmod). When swabs were
processed within 24 hours, no significant difference was seen between
this optimum condition and two other conditions, PBSTX/PBSTX and
PBSTX/C&Bmod (p>0.05). Swabs pre-moistened with NB and stored in
PBSTX (NB/PBSTX) yielded significantly lower % recoveries (Table 4).
When swabs were processed within 48 hours, condition NB/C&Bmod
yielded significantly higher recoveries than two other conditions,
PBSTX/C&Bmod and NB/PBSTX (p<0.04) (Table 5).
Premoistening Agent
NB
PBSTX
{T U , f
1 F ffl
i
12
18 24
i
48
72
i
12
i
18
i
24
Transport
Medium
I CSB
: PBSTX
48 72
Time (hours)
Figure 12: Mean % recovery of Y. pestis A1122 (104 CPU/ swab) from macrofoam swabs over storage
time. Bars represent the 95% confidence interval of the mean % recovery (n=10).
22
-------
Table 4: Comparison of mean % recovery of Y. pestis A1122 from macrofoam swabs stored for 12, 18
and 24 hours, (n=30 for each experiment) using Tukey HSD analysis.
Reference
Experiment a
1
2
3
4
Comparative
Experiment
2
3
4
1
3
4
1
2
4
1
2
3
Mean
Difference13
4.8433
19.6167
-1.7651
-4.8433
14.7733
-6.6084
-19.6167
*
-14.7733
-21.3817
1.7651
6.6084
21.3817
Std. Error
2.7614
2.7614
2.7851
2.7614
2.7614
2.7851
2.7614
2.7614
2.7851
2.7851
2.7851
2.7851
P
.301
.000
.921
.301
.000
.088
.000
.000
.000
.921
.088
.000
a Experiments (premoistening agent/transport media) were as follows: 1=PBSTX/PBSTX,
2=PBSTX/C&Bmod, 3=NB/PBSTX, 4=NB/C&Bmod.
b mean % recovery of reference experiment minus the mean % recovery of the
comparative experiment.
* indicates that the mean difference is significant at the 0.05 level
Table 5: Comparison of mean % recovery of Y. pestis A1122 from macrofoam swabs stored for 12, 18, 24
and 48 (n=40 for each experiment) using Tukey HSD analysis.
Reference
Experiment a
1
2
3
4
Comparative
Experiment
2
3
4
1
3
4
1
2
4
1
2
3
Mean Difference
b
4.7850
23.6350
-3.4848
-4.7850
*
18.8500
-8.2698
-23.6350
*
-18.8500
-27.1198
3.4848
8.2698
27.1198
Std. Error
3.0859
3.0859
3.1056
3.0859
3.0859
3.1056
3.0859
3.0859
3.1056
3.1056
3.1056
3.1056
P
.410
.000
.676
.410
.000
.042
.000
.000
.000
.676
.042
.000
23
-------
Experiments (premoistening agent/transport media) were as follows: 1=PBSTX/PBSTX,
2=PBSTX/C&Bmod, 3=NB/PBSTX, 4=NB/C&Bmod.
b mean % recovery of reference experiment minus the mean % recovery of the
comparative experiment.
* indicates that the mean difference is significant at the 0.05 level
3.4.1.2 Rayon Swabs
Rayon swabs inoculated with Y. pestis A1122 yielded a % recovery range
of 73.6-118.1% (Figure 13). When all time points within the 12 to 72 hour
storage periods were averaged for each condition (premoistening
agent/transport medium), the highest mean % recovery was from rayon
swabs (103.7%, sd=17.1, n=50) pre-moistened with PBSTX, stored at4°C
in PBSTX as a transport medium. When swabs were processed within 12
to 24 hours or within 12 to 48 hours, no significant difference was seen
between this optimum condition (PBSTX/PBSTX) and the NB/PBSTX
condition (Tables 6 and 7). The other two conditions (PBSTX/C&Bmod or
NB/C&Bmod) yielded significantly lower % recoveries whether processed
within 12 to 24 hours (p<0.03) or 12 to 48 hours (p<0.004).
Premoistening Agent
NB
PBSTX
o
55
10
0>
o
4>
4*
O
4>
O
i_
-------
Table 6: Comparison of mean % recovery of Y. pestis A1122 from rayon swabs stored for 12, 18 and 24
hours, (n=30 for each experiment) using Tukey HSD analysis.
Reference
Experiment a
1
2
3
4
Comparative
Experiment
2
3
4
1
3
4
1
2
4
1
2
3
Mean Difference
b
13.1567*
4.2167
18.6130*
-13.1567*
-8.9400
5.4563
-4.2167
8.9400
14.3963*
-18.6130*
-5.4563
-14.3963*
Std. Error
4.6354
4.6354
4.6752
4.6354
4.6354
4.6752
4.6354
4.6354
4.6752
4.6752
4.6752
4.6752
p
.027
.800
.001
.027
.222
.649
.800
.222
.014
.001
.649
.014
a Experiments (premoistening agent/transport media) were as follows: 1=PBSTX/PBSTX,
2=PBSTX/C&Bmod, 3=NB/PBSTX, 4=NB/C&Bmod.
b mean % recovery of reference experiment minus the mean % recovery of the
comparative experiment.
* indicates that the mean difference is significant at the 0.05 level
25
-------
Table 7: Comparison of mean % recovery of Y. pestis A1122 from rayon swabs stored for 12, 18, 24 and
48 hours (n=40 for each experiment) using Tukey HSD analysis.
Reference
Experiment a
1
2
3
4
Comparative
Experiment
2
3
4
1
3
4
1
2
4
1
2
3
Mean Difference
13.5275*
4.7075
18.7040*
-13.5275*
-8.8200
5.1765
-4.7075
8.8200
13.9965*
-18.7040*
-5.1765
-13.9965*
Std. Error
3.9419
3.9419
3.9671
3.9419
3.9419
3.9671
3.9419
3.9419
3.9671
3.9671
3.9671
3.9671
p
.004
.631
.000
.004
.118
.561
.631
.118
.003
.000
.561
.003
a Experiments (premoistening agent/transport media) were as follows: 1=PBSTX/PBSTX,
2=PBSTX/C&Bmod, 3=NB/PBSTX, 4=NB/C&Bmod.
b mean % recovery of reference experiment minus the mean % recovery of the
comparative experiment.
* indicates that the mean difference is significant at the 0.05 level
3.4.2 Phase III: Virulent strain, Yersinia pestis CO92
The recovery efficiency of virulent Y. pestis (strain CO92) from
macrofoam swabs and rayon swabs was evaluated. Both swab materials
were inoculated directly with 100 ul of a 10s CFU/mL suspension and held
at room temperature for one hour before placing the swabs in either of the
two optimum liquid transport media: C&Bmod or PBSTX. The swabs were
stored in the medium at 4°C for the following times: 0, 12, 24, 48, and 72
hours before processing. Two swab preparations (pre-moistened with
either PBSTX or NB) and two methods of swab processing (vortex only
for macrofoam swabs, sonicate only for rayon swabs) were evaluated.
3.4.2.1 Macrofoam Swabs
Macrofoam swabs yielded a % recovery range of 2.3-114.3% for the
virulent cells (Figure 14). When all storage periods from 12 to 72 hours
were averaged for each condition (premoistening agent/transport
medium), the highest mean %% recovery (101.8%, sd=18.0, n=49) was
for macrofoam swabs stored at 4°C for the PBSTX/C&Bmod condition.
Whether processed within 24 hours or 48 hours, the optimum condition
26
-------
140.00-
S- 120.00-
u
in
01
- 100.00-
«
^ 80.00-
01
o
-------
Table 8: Comparison of mean % recovery of Y. pestis CO92 from macrofoam swabs stored for 12, 18 and
24 hours (n=30 for each experiment) using Tukey HSD analysis.
Reference
Experiment a
1
2
3
4
Comparative
Experiment
2
3
4
1
3
4
1
2
4
1
2
3
Mean
Difference b
-48.4103*
-30.2377*
.5040
48.4103*
18.1726*
48.9143*
30.2377*
-18.1726*
30.7417*
-.5040
-48.9143*
-30.7417*
Std. Error
3.3701
3.3419
3.7300
3.3701
3.3419
3.7300
3.3419
3.3419
3.7045
3.7300
3.7300
3.7045
p
.000
.000
.999
.000
.000
.000
.000
.000
.000
.999
.000
.000
a Experiments (premoistening agent/transport media) were as follows: 1=PBSTX/PBSTX,
2=PBSTX/C&Bmod, 3=NB/PBSTX, 4=NB/C&Bmod.
b mean % recovery of reference experiment minus the mean % recovery of the
comparative experiment.
* indicates that the mean difference is significant at the 0.05 level
28
-------
Table 9: Comparison of mean % recovery of Y. pestis CO92 from macrofoam swabs stored for 12, 18, 24
and 48 hours (n = 40 for each experiment) using Tukey HSD analysis.
Reference
Experiment a
1
2
3
4
Comparative
Experiment
2
3
4
1
3
4
1
2
4
1
2
3
Mean
Difference b
-47.8026*
-30.6796*
12.6046*
47.8026*
17.1230*
60.4072*
30.6796*
-17.1230*
43.2842*
-12.6046*
-60.4072*
-43.2842*
Std. Error
3.8081
3.7842
4.0837
3.8081
3.7842
4.0837
3.7842
3.7842
4.0615
4.0837
4.0837
4.0615
p
.000
.000
.013
.000
.000
.000
.000
.000
.000
.013
.000
.000
a Experiments (premoistening agent/transport media) were as follows: 1=PBSTX/PBSTX,
2=PBSTX/C&Bmod, 3=NB/PBSTX, 4=NB/C&Bmod.
b mean % recovery of reference experiment minus the mean % recovery of the
comparative experiment.
* indicates that the mean difference is significant at the 0.05 level
3.4.2.2 Rayon Swabs
Rayon swabs yielded a % recovery range of 74.7-126.5% for all storage
time points, pre-moistening agents and transport media for the virulent
cells (Figure 15). When all time points within the 12 to 72 hour storage
period were averaged for each condition (premoistening agent/transport
medium), the highest mean recovery (110.1%, sd=21.6, n=40) was from
rayon swabs stored at 4°C for the NB/PBSTX condition. When processed
after a storage time of 12 or 24 hours, no significant difference was seen
between this optimum condition and two others, PBSTX/PBSTX and
PBSTX/C&Bmod (Table 10). When processed after a storage time of up to
48 hours, no significant difference was seen between this optimum
condition (NB/PBSTX) and the PBSTX/C&Bmod condition (P>0.06) (Table
11). This optimum condition (NB/PBSTX) yielded significantly higher %
recoveries than the PBSTX/PBSTX and NB/C&Bmod conditions, when
processed after a storage time of up to 48 hours (p=0.01).
29
-------
Premoistening Agent
NB PBSTX
140.00-
^ 120.00-
O)
£ 100.00-
M
5
^ 80.00-
01
0
« 60.00-
o:
4J
01 40.00-
u
*
Q.
20.00-
o.oo-
ll 1
U 1
1 1
>
1
t
)
1 (
}1
)
*;
J
;.
.:
(
1
1
)
(
)
t,t ,
M i:
i
i
Transport
Medium
I C3B
I PBSTX
12 24 48 72 0 12
Time (hours)
24
48
72
Figure 15: Mean % recovery of Y. pestis CO92 (104 CPU/ swab) from rayon swabs over storage time.
Bars represent the 95% confidence interval of the mean recovery (n=10).
30
-------
Table 10: Comparison of mean % recovery of Y. pestis CO92 from rayon swabs after storage for 12, 18
and 24 hours (n=30 for each experiment) using Tukey HSD analysis.
Reference
Experiment a
1
2
3
4
Comparative
Experiment
2
3
4
1
3
4
1
2
4
1
2
3
Mean
Difference b
-9.8450
-16.7750
3.5600
9.8450
-6.9300
13.4050
16.7750
6.9300
20.3350*
-3.5600
-13.4050
-20.3350*
Std. Error
6.4382
6.4382
6.4382
6.4382
6.4382
6.4382
6.4382
6.4382
6.4382
6.4382
6.4382
6.4382
P
.425
.053
.945
.425
.705
.168
.053
.705
.012
.945
.168
.012
a Experiments (premoistening agent/transport media) were as follows: 1=PBSTX/PBSTX,
2=PBSTX/C&Bmod, 3=NB/PBSTX, 4=NB/C&Bmod.
b mean % recovery of reference experiment minus the mean % recovery of the
comparative experiment.
* indicates that the mean difference is significant at the 0.05 level
Table 11: Comparison of mean % recovery of Y. pestis CO92 from rayon swabs after storage of 12, 18, 24
and 48 hours (n=40 for each experiment) using Tukey HSD analysis.
Reference
Experiment a
1
2
3
4
Comparative
Experiment
2
3
4
1
3
4
1
2
4
1
2
3
Mean
Difference b
-3.1700
-15.9767*
.1400
3.1700
-12.8067
3.3100
15.9767*
12.8067
16.1167*
-.1400
-3.3100
-16.1167*
Std. Error
5.0699
5.0699
5.0699
5.0699
5.0699
5.0699
5.0699
5.0699
5.0699
5.0699
5.0699
5.0699
p
.924
.011
1.000
.924
.061
.914
.011
.061
.010
1.000
.914
.010
a Experiments (premoistening agent/transport media) were as follows: 1=PBSTX/PBSTX,
2=PBSTX/C&Bmod, 3=NB/PBSTX, 4=NB/C&Bmod.
31
-------
b mean % recovery of reference experiment minus the mean % recovery of the
comparative experiment.
* indicates that the mean difference is significant at the 0.05 level
During this phase of the study, it was discovered that the C&Bmod
transport media was altered more than simply removing the agar. When
the agar was omitted, the calcium chloride was also omitted because it
fell out of solution. CaCI2 is an important nutrient for growth media to
maintain the virulence of Y. pestis [3, 4]. Though no data is available
indicating CaCI2 is important for transport media, we were not comfortable
continuing to use C&Bmod with this change in formulation. Because of our
uncertainty as to the consequences of omitting the CaCI2, and because
the altered formulation is not currently commercially available, the use of
C&Bmod transport medium was discontinued and replaced with NB when
conducting the experiments in phase IV. NB was chosen because it was
considered the "next best" performing transport media to C&B (that was
immediately commercially available) in the preliminary study, based on
4°C data (Table 2).
3.5 Phase IV: Evaluation of sample storage parameters for both Y. pestis strains
inoculated on "dirty swabs"
3.5.1 Low-virulent strain, Yersinia pestis A1122
The recovery efficiency of Y. pestis A1122 from "dirty" macrofoam swabs
and rayon swabs was evaluated. Refer to Table 5 in the appendix for a
matrix of tests performed. Both swab materials were inoculated directly
with a 100 ul of a 104 CFU/mL suspension and held at room temperature
for one hour before placing the swabs in one of the two optimum liquid
transport media, either NB or PBSTX. The swabs were held in the
respective medium at 4°C for before the following processing times: 0,
12, 24, 48, and 72 hours. Two swab preparations (pre-moistened with
either PBSTX or NB both with a slurry of Arizona Test Dust (ATD) and
two methods of swab processing (vortex only for macrofoam swabs,
sonicate only for rayon swabs) were evaluated.
3.5.1.1 Macrofoam Swabs
Macrofoam swabs yielded a % recovery range of 65.8-111.6% of the low-
virulent cells recovered for all storage time points, pre-moistening agents
and transport media (Figure 16). When all time points within the 12 to 72
hour storage period were averaged for each condition (premoistening
agent/transport medium), the highest mean % recovery (105.1%,
sd=11.7, n=40) was from macrofoam swabs pre-moistened with PBSTX
and stored at 4°C in NB as the transport medium (PBSTX/NB). For
storage times of 24 hours or less, the optimum condition (PBSTX/NB)
yielded significantly higher % recoveries than PBSTX/PBSTX and NB/NB
conditions (p<0.001) (Table 12). For storage times of 48 hours or less,
32
-------
the optimum condition (PBSTX/NB) yielded significantly higher %
recoveries than all other conditions (p<0.025) (Table 13).
Premoistening Agent
NB PBSTX
o
10
0)
•V
u
1_
0>
CL
140.0-
120.0-
100 .o-|
£, 80.0-
0)
o 60.0-
4)
40.0-
20.0-
0.0-
I
12 24
I
48
I
72
12 24
I
48
I
72
Transport
Medium
I NB
: PBSTX
Time (hours)
Figure 16: Mean % recovery of Y. pestis A1122 (104 CPU/ swab) from dirty macrofoam swabs over
storage time. Bars represent the 95% confidence interval of the mean recovery (n=10).
33
-------
Table 12: Comparison of mean % recovery of Y. pestis A1122 from dirty macrofoam swabs after storage
for 12 and 24 hours (n=20 for each experiment) using Tukey HSD analysis.
Reference
Experiment a
1
2
3
4
Comparative
Experiment
2
3
4
1
3
4
1
2
4
1
2
3
Mean
Difference b
-21.9500*
-15.9750*
-2.5500
21.9500*
5.9750
19.4000*
15.9750*
-5.9750
13.4250*
2.5500
-19.4000*
-13.4250*
Std. Error
4.5156
4.5156
4.5156
4.5156
4.5156
4.5156
4.5156
4.5156
4.5156
4.5156
4.5156
4.5156
p
.000
.004
.942
.000
.551
.000
.004
.551
.020
.942
.000
.020
a Experiments (premoistening agent/transport media) were as follows: 1=PBSTX/PBSTX,
2=PBSTX/NB, 3=NB/PBSTX, 4=NB/NB
b mean % recovery of reference experiment minus the mean % recovery of the
comparative experiment.
* indicates that the mean difference is significant at the 0.05 level
34
-------
Table 13: ANOVA of Y. pestis A1122 mean % recovery from dirty macrofoam swabs after storage for 12,
24 and 48 hours (n=30 for each experiment) using Tukey HSD analysis.
Reference
Experiment a
1
2
3
4
Comparative
Experiment
2
3
4
1
3
4
1
2
4
1
2
3
Mean
Difference b
-24.8633*
-15.2133*
-4.9967
24.8633*
9.6500*
19.8667*
15.2133*
-9.6500*
10.2167*
4.9967
-19.8667*
-10.2167*
Std. Error
3.3572
3.3572
3.3572
3.3572
3.3572
3.3572
3.3572
3.3572
3.3572
3.3572
3.3572
3.3572
p
.000
.000
.448
.000
.025
.000
.000
.025
.015
.448
.000
.015
a Experiments (premoistening agent/transport media) were as follows: 1=PBSTX/PBSTX,
2=PBSTX/NB, 3=NB/PBSTX, 4=NB/NB
b mean % recovery of reference experiment minus the mean % recovery of the
comparative experiment.
* indicates that the mean difference is significant at the 0.05 level
3.5.1.2 Rayon Swabs
Rayon swabs yielded a % recovery range of 79.1-112.8% for the low-
virulent cells (Figure 17) from all storage times, pre-moistening agents
and transport media. When all time points within the 12 to 72 hour
storage period were averaged for each condition (premoistening
agent/transport medium),, the highest mean recovery (107.2%, sd=14.3,
n=40) from rayon swabs was obtained when pre-moistened with PBSTX
and stored at 4°C in PBSTX as the transport medium. This optimum
condition (PBSTX/PBSTX) yielded significantly higher % recoveries than
all other conditions for storage times of 24 hours (p<0.02) or 48 hours
(p<0.001) (Tables 14 and 15).
35
-------
140.0-
120.0-
o
m
o
o
80 .OH
60.0-
40.0-
20.0-
0.0-
Premoistening Agent
NB
PBSTX
Transport
Medium
I NB
: PBSTX
12
24
72 0
Time (hours)
12
24
48
72
Figure 17: Mean % recovery of Y. pestis A1122 (104 CPU/ swab) from dirty rayon swabs over storage
time. Bars represent the 95% confidence interval of the mean recovery (n=10).
36
-------
Table 14: Comparison of mean % recovery of Y. pestis A1122 from dirty rayon swabs after storage for 12
and 24 hours (n=20 for each experiment) using Tukey HSD analysis.
Reference
Experiment a
1
2
3
4
Comparative
Experiment
2
3
4
1
3
4
1
2
4
1
2
3
Mean
Difference b
12.3450*
11.3650*
12.7100*
-12.3450*
-.9800
.3650
-11.3650*
.9800
1.3450
-12.7100*
-.3650
-1.3450
Std. Error
3.8618
3.8618
3.8618
3.8618
3.8618
3.8618
3.8618
3.8618
3.8618
3.8618
3.8618
3.8618
p
.011
.022
.008
.011
.994
1.000
.022
.994
.985
.008
1.000
.985
a Experiments (premoistening agent/transport media) were as follows: 1=PBSTX/PBSTX,
2=PBSTX/C&Bmod, 3=NB/PBSTX, 4=NB/C&Bmod.
b mean % recovery of reference experiment minus the mean % recovery of the
comparative experiment.
* indicates that the mean difference is significant at the 0.05 level
37
-------
Table 15: Comparison of mean % recovery of Y. pestis A1122 from dirty rayon swabs after storage at 12,
24 and 48 hours (n=30 for each experiment) using Tukey HSD analysis.
Reference
Experiment a
1
2
3
4
Comparative
Experiment
2
3
4
1
3
4
1
2
4
1
2
3
Mean
Difference b
12.8333*
14.9333*
16.4367*
-12.8333*
2.1000
3.6033
-14.9333*
-2.1000
1.5033
-16.4367*
-3.6033
-1.5033
Std. Error
2.9779
2.9779
2.9779
2.9779
2.9779
2.9779
2.9779
2.9779
2.9779
2.9779
2.9779
2.9779
p
.000
.000
.000
.000
.895
.622
.000
.895
.958
.000
.622
.958
a Experiments (premoistening agent/transport media) were as follows: 1=PBSTX/PBSTX,
2=PBSTX/C&Bmod, 3=NB/PBSTX, 4=NB/C&Bmod.
b mean % recovery of reference experiment minus the mean % recovery of the
comparative experiment.
* indicates that the mean difference is significant at the 0.05 level
3.5.2 Virulent strain, Yersinia pestis CO92
The recovery efficiency of Y. pestis strain CO92 from "dirty" macrofoam
swabs and rayon swabs was evaluated. Refer to Table 6 in the appendix
for a matrix of tests performed. Both swab materials were inoculated
directly with 100 ul of a 104 CFU/mL suspensions and held at room
temperature for one hour before placing the swabs in one of the two
optimum liquid transport media, either NB or PBSTX. The swabs were
held in the respective medium at 4°C for the following times: 0, 12, 24,
48, and 72 hours before processing. Two swab preparations (pre-
moistened with either PBSTX or NB both with a slurry of ATD) and two
methods of swab processing (vortex only for macrofoam swabs, sonicate
only for rayon swabs) were evaluated.
3.5.2.1 Macrofoam Swabs
Macrofoam swabs yielded a % recovery range of 58.0-100% of the
virulent cells (Figure 18) from all storage times, pre-moistening agents
and transport media. When all time points within the 12 to 72 hour
storage periods were averaged for each condition (premoistening
38
-------
agent/transport medium), three of the conditions yielded similar
recoveries. The optimum condition for macrofoam swabs was NB/PBSTX
(85.9%, sd=10.6, n=40). No significant difference was seen between this
optimum condition (NB/PBSTX) and two other conditions (PBSTX/NB and
PBSTX/PBSTX) (Tables 16 and 17). The other condition (NB/NB) yielded
a significantly lower % recovery than the optimum condition (NB/PBSTX),
whether processed within 24, 48, or 72 hours (p<0.022) (Table 16).
0>
140.0-
120.0-
100.0-
a, 80.0-
01
>
o
o 60.0-
£
01
i_
-------
Reference
Experiment a
1
2
3
4
Comparative
Experiment
2
3
4
1
3
4
1
2
4
1
2
3
Mean
Difference b
-5.7550
-2.2800
10.9800
5.7550
3.4750
16.7350*
2.2800
-3.4750
13.2600*
-10.9800
-16.7350*
-13.2600*
Std. Error
4.5050
4.5050
4.5050
4.5050
4.5050
4.5050
4.5050
4.5050
4.5050
4.5050
4.5050
4.5050
P
.580
.957
.079
.580
.867
.002
.957
.867
.022
.079
.002
.022
a Experiments (premoistening agent/transport media) were as follows: 1=PBSTX/PBSTX,
2=PBSTX/NB, 3=NB/PBSTX, 4=NB/NB
b mean % recovery of reference experiment minus the mean % recovery of the
comparative experiment.
* indicates that the mean difference is significant at the 0.05 level
40
-------
Table 17: Comparison of mean % recovery of Y. pestis CO92 from dirty macrofoam swabs after storage
for 12, 24 and 48 hours (n=30 for each experiment) using Tukey HSD analysis.
Reference
Experiment a
1
2
3
4
Comparative
Experiment
2
3
4
1
3
4
1
2
4
1
2
3
Mean
Difference b
-1.7167
-1.6267
14.0600*
1.7167
.0900
15.7767*
1.6267
-.0900
15.6867*
-14.0600*
-15.7767*
-15.6867*
Std. Error
3.9966
3.9966
3.9966
3.9966
3.9966
3.9966
3.9966
3.9966
3.9966
3.9966
3.9966
3.9966
p
.973
.977
.003
.973
1.000
.001
.977
1.000
.001
.003
.001
.001
a Experiments (premoistening agent/transport media) were as follows:l=PBSTX/PBSTX,
2=PBSTX/NB, 3=NB/PBSTX, 4=NB/NB
b mean % recovery of reference experiment minus the mean % recovery of the
comparative experiment.
* indicates that the mean difference is significant at the 0.05 level
3.5.2.2 Rayon Swabs
Rayon swabs yielded a % recovery range of 34.3-100% of the virulent
cells (Figure 19) from all storage times, pre-moistening agents and
transport media. When all time points within the 12 to 72 hour storage
times were averaged for each condition (premoistening agent/transport
medium),, the highest mean recovery (83.7%, sd=14.3, n=40) from rayon
swabs was obtained at 4°C for the NB/PBSTX condition. This optimum
condition (NB/PBSTX) yielded significantly higher % recoveries than
PBSTX/NB, for storage times of 24 hours or 48 hours (p<0.001). No
significant differences were seen between this optimum condition and two
others (PBSTX/PBSTX and NB/PBSTX) for storage times up to 24 or up
to 48 hours (Tables 18 and 19).
41
-------
Premoistening Agent
Transport
Medium
NB PBSTX
140.0-
o" 120.0-
8
c- 100.0-
M
S
"^ 80.0-
01
0
5
* 40.0-
01
Q.
20.0-
0.0-
(
1 (
0
)
1*1
1 J
12
<
~>
T
24
I I
I !
I
t)
I
i
C
>
* 1
I
I I — I — 1 1
48 72 0 12 24
)
(
)
1
I T J
T
48 72
: NB
: PBSTX
Time (hours)
Figure 19: Mean % recovery of Y. pestis CO92 (104 CPU/ swab) from dirty rayon swabs over storage time.
Bars represent the 95% confidence interval of the mean recovery (n=10).
42
-------
Table 18: Comparison of mean % recovery of Y. pestis CO92 from dirty rayon swabs after storage for 12
and 24 hours (n=20 for each experiment) using Tukey HSD analysis.
Reference
Experiment a
1
2
3
4
Comparative
Experiment
2
3
4
1
3
4
1
2
4
1
2
3
Mean
Difference b
41.3450*
-.6450
7.5500
-41.3450*
-41.9900*
-33.7950*
.6450
41.9900*
8.1950
-7.5500
33.7950*
-8.1950
Std. Error
5.9671
5.9671
5.9671
5.9671
5.9671
5.9671
5.9671
5.9671
5.9671
5.9671
5.9671
5.9671
p
.000
1.000
.588
.000
.000
.000
1.000
.000
.520
.588
.000
.520
a Experiments (premoistening agent/transport media) were as follows:l=PBSTX/PBSTX,
2=PBSTX/NB, 3=NB/PBSTX, 4=NB/NB
b mean % recovery of reference experiment minus the mean % recovery of the
comparative experiment.
* indicates that the mean difference is significant at the 0.05 level
43
-------
Table 19: Comparison of mean % recovery of Y. pestis CO92 from dirty rayon swabs after storage for 12,
24 and 48 hours (n=30 for each experiment) using Tukey HSD analysis.
Experiment
1
2
3
4
Experiment
2
3
4
1
3
4
1
2
4
1
2
3
Mean Difference
37.9733*
-6.6367
5.7633
-37.9733*
-44.6100*
-32.2100*
6.6367
44.6100*
12.4000
-5.7633
32.2100*
-12.4000
Std. Error
4.8860
4.8860
4.8860
4.8860
4.8860
4.8860
4.8860
4.8860
4.8860
4.8860
4.8860
4.8860
P
.000
.528
.641
.000
.000
.000
.528
.000
.059
.641
.000
.059
a Experiments (premoistening agent/transport media) were as follows:l=PBSTX/PBSTX,
2=PBSTX/NB, 3=NB/PBSTX, 4=NB/NB
b mean % recovery of reference experiment minus the mean % recovery of the
comparative experiment.
* indicates that the mean difference is significant at the 0.05 level
3.6 Statistical Analysis
When recovery of Y. pestis was compared from all conditions tested (clean or dirty,
premoistening agent and transport medium), no specific condition was identified as
the best for recovery from both macrofoam and rayon swabs (Table 20). Therefore,
another approach to analyzing the data was considered. The data for each swab
type were combined; that included both strains, both clean and dirty, and data for all
time points up to 72 hours (assumes a worst case scenario of a processing time of
72 hours). For these analyses, the data for the substituted transport media (C&B
and NB) were treated as one data set (PBSTX/C&B data pooled with PBSTX/NB,
and NB/C&B pooled with NB/NB). The combined data for each swab type were not
normally distributed, and therefore analyzed using the Kruskal-Wallis and Mann-
Whitney tests. The results of these tests show that macrofoam swabs had one
condition that resulted in statistically higher recoveries than the others; the
PBSTX/C&Bmod (data pooled with PBSTX/NB) (p<0.000, data not shown). Since the
standard C&B formulation was found to be unstable in liquid form and was not
commercially available, PBSTX/NB was chosen as the optimum premoistening
agent/transport medium for macrofoam swabs. When the data was grouped
similarly for rayon swabs (both strains, dirty and clean, all time points), the test
results show that the optimum premoistening agent/transport media combinations
44
-------
were PBSTX/PBSTX (p<0.000, data not shown) and NB/PBSTX (p<0.000, data not
shown).
Table 20: Optimum recovery of low-virulent (A1122) and virulent (CO92) Y. pestisirom each swab type
at the specified condition.
Strain
YPA1122
YPA1122
YP CO92
YP CO92
YPA1122
YPA1122
YP C092
YP C092
Swab Type
Macro-foam
Macro-foam
Macro-foam
Macro-foam
Rayon
Rayon
Rayon
Rayon
Clean
or
Dirty
Clean
Dirty
Clean
Dirty
Clean
Dirty
Clean
Dirty
Pre-
moistening
Agent
NB
PBSTX
PBSTX
NB
PBSTX
PBSTX
NB
NB
Transport
Medium
C&Bmod
NB
C&Bmod
PBSTX
PBSTX
PBSTX
PBSTX
PBSTX
Percent
Recovery
(24hrs)t
101.9%
106.7%
110.6%
84.2%
101.0%
105.3%
120.3%
82.5%
Percent
Recovery
(48hrs)i
99.2%
106.1%
106.1%
85.3%
100.1%
107.8%
115.9%
84.3%
Percent
Recovery
(72hrs)=
99.6%
105.1%
101.8%
85.9%
103.7%
107.2%
110.1%
83.7%
t24 hrs represents the mean percent recovery for 12hr and 24hr combined.
J48 hrs represents the mean percent recovery for 12hr, 24hr, and 48hr combined.
= 72 hrs represents the mean percent recovery for 12hr, 24hr, 48hr, and 72hr combined.
45
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4.0 Conclusions:
Six liquid transport media (no swabs) were evaluated using the low-virulent strain of Y.
pestis A1122 and holding time of up to 72 hours at 4°, 25°, or 35°C. The most consistent
recovery of cells was found when the storage temperature was maintained at 4°C for all
transport media. C&Bmod and PBSTX were found to be the best of the six transport media
evaluated, and were selected for use in subsequent evaluations. It was later found that the
C&B liquid formulation was prepared without calcium chloride, an ingredient that influences
the virulence factors of Y. pestis in growth media [3, 4] so the next best commercially
available transport medium, NB, was substituted after phase III.
Of the four swabs evaluated, rayon and macrofoam were chosen as the best two swabs
because they were found to release Y. pestis cells significantly better than polyester swabs
and they did not have the PCR reagent inhibition concerns associated with cotton swabs.
Rayon swab recoveries were higher when processed by sonication for three minutes, and
macrofoam swab recoveries were higher when processed by vortexing for three minutes.
The pre-moistening agent and transport medium combinations were analyzed for each
strain and swab, dirty or clean, and at each storage time. No single pre-moistening
agent/transport media condition stood out as the best for all combinations (both swabs, both
isolates, clean and dirty).
The data were separated by swab type and then the results were combined for both strains
for both dirty and clean combinations. Considering a worse case storage time of 72 hours,
all data from all time points were also combined for these analyses.
The results showed that macrofoam swabs pre-moistened with PBSTX and stored in NB or
C&Bmod (PBSTX/NB or PBSTX/C&Bmod) provided significantly higher % recovery of Y. pestis
than any other combinations of premoistening agent and transport media tested. The C&B
medium was discontinued after phase III because of the aforementioned formulation
problems, so NB was chosen as the optimum transport medium for macrofoam swabs.
When all data for rayon swabs (both strains, dirty and clean, all time points) were combined
and analyzed, two combinations of pre-moistening agent/transport medium, PBSTX/PBSTX
and NB/PBSTX , stood out as significantly better than the other two. Rayon swab
recoveries were higher when clean than when dirty with these two optimum combinations
(p<0.001). The combination of PBSTX/PBSTX was optimum for the YP A1122 and
NB/PBSTX worked best for the YP CO92 strain.
Collection of cells from surfaces was not evaluated in this study. The choice of
premoistening agent may affect the collection efficiency of cells from the surface.
It should be noted that in phase III and IV, the swabs were stored and processed in the
given transport medium, so the differences in preferred transport medium for each swab
may reflect the differences in the need for the surfactant to release the cells during
46
-------
processing. Each swab has unique electrochemical properties that interact with the cells and
influence adherence [9]. This project did not address these properties.
47
-------
5.0 References
1. Observations and lessons learned from Anthrax responses, interim report, draft, N.R. Team,
Editor 2001, National Response Team: Washington, D.C.
2. Agencies need to validate sampling activities in order to increase confidence in negative results,
GAO, Editor 2005, U.S. Government Accountability Office: Washington, D.C.
3. Bearden, S.W., et al., Attenuated enzootic (pestoides) isolates of Yersinia pestis express active
aspartase. Microbiology, 2009. 155(Pt 1): p. 198-209.
4. Higuchi, K. and J.L. Smith, Studies on the nutrition and physiology ofPasteurella pestis. VI. A
differential plating medium for the estimation of the mutation rate to avirulence. J Bacteriol,
1961. 81: p. 605-8.
5. Selected analytical methods for environmental remediation and recovery N.H.S.R. Center, Editor
2012, US Environmental Protection Agency: http://epa.gov/sam/.
6. Hodges, L.R., et al., Evaluation of a macrofoam swab protocol for the recovery of Bacillus
anthracis spores from a steel surface. Appl Environ Microbiol, 2006. 72(6): p. 4429-30.
7. Rose, L, et al., Swab materials and Bacillus anthracis spore recovery from nonporous surfaces.
Emerg Infect Dis, 2004. 10(6): p. 1023-9.
8. Surgalla, M.J. and E.D. Beesley, Congo red-agar plating medium for detecting pigmentation in
Pasteurella pestis. Appl Microbiol, 1969. 18(5): p. 834-7.
9. Da Silva, S.M., J.J. Filliben, and J.B. Morrow, Parameters affecting spore recovery from wipes
used in biological surface sampling. Appl Environ Microbiol, 2011. 77(7): p. 2374-80.
48
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6.0 Appendix: Study Matrix Tables
1. Preliminary Study: Survival of Y. pestis in liquid transport media
Table 1. Preliminary study matrix: Transport media, temperature, holding times evaluated
Transport media
Stuart, Toscach and Patsula
Gary and Blair
Amies without charcoal
Amies with charcoal
PBS +0.05% Triton X-100
Neutralizing buffer
Temperature (°C)
4,25,35
4,25,35
4,25,35
4,25,35
4,25,35
4,25,35
Holding time (h)
0, 12, 18, 24, 48, 72
0, 12, 18, 24, 48, 72
0, 12, 18, 24, 48, 72
0, 12, 18, 24, 48, 72
0, 12, 18, 24, 48, 72
0, 12, 18, 24, 48, 72
2. Phase I: Evaluation of swab extraction methods
Table 2. Phase I study matrix: Swab material, premoistening agent and extraction methods
evaluated. All extractions performed with Y. pestis A1122 and all processing performed in PBS
+ 0.02% Tween 80. N= 10 per extraction method
Swab material
Rayon
Polyester
Macrofoam
Cotton
Rayon
Polyester
Macrofoam
Cotton
Premoistening agent
Triton X-100
Triton X-100
Triton X-100
Triton X-100
Neutralizing buffer
Neutralizing buffer
Neutralizing buffer
Neutralizing buffer
Extraction method a
V, S, VS, N
V, S, VS, N
V, S, VS, N
V, S, VS, N
V, S, VS, N
V, S, VS, N
V, S, VS, N
V, S, VS, N
aV=vortexing 3 min, S = sonicating 3 min , VS = vortexing and sonicating 30 seconds each,
repeated three times for a total of 3 min, N=no extraction method.
49
-------
3. Phase II: Evaluation of sample storage parameters for Y. pestis A1122 on sterile
swabs
Table 3. Phase II study matrix; YP strain, swab material, premoistening agent, transport media
and holding times evaluated. All swabs were held at 4°C (found to be optimum temperature in
preliminary study).
Organism
Phase II
YP A1122
Swab material3
macrofoam
macrofoam
macrofoam
macrofoam
rayon
rayon
rayon
rayon
Premoistening agent
Triton X-100
Triton X-100
Neutralizing buffer
Neutralizing buffer
Triton X-100
Triton X-100
Neutralizing buffer
Neutralizing buffer
Transport
media b
PBSTX
C&Bmod
PBSTX
C&Bmod
PBSTX
C&Bmod
PBSTX
C&Bmod
Holding Time (h)c
0, 12, 18, 24, 48, 72
0, 12, 18, 24, 48, 72
0, 12, 18, 24, 48, 72
0, 12, 18, 24, 48, 72
0, 12, 18, 24, 48, 72
0, 12, 18, 24, 48, 72
0, 12, 18, 24, 48, 72
0, 12, 18, 24, 48, 72
a macrofoam swabs processed by vortexing 3 min, rayon swabs processed by
sonicating 3 min (optimum conditions determined for each swab type in phase I).
b optimum transport media determined in preliminary study
c 10 swabs per holding time
4. Phase III: Evaluation of sample storage parameters for Y. pestis CO92 on sterile swabs
Table 4. Phase III study matrix: YP strain, swab material, premoistening agent, transport media
and holding times evaluated. All swabs were held at 4°C.
Organism
Phase III
YP CO92
Swab material3
macrofoam
macrofoam
macrofoam
macrofoam
rayon
rayon
rayon
rayon
Premoistening agent
Triton X-100
Triton X-100
Neutralizing buffer
Neutralizing buffer
Triton X-100
Triton X-100
Neutralizing buffer
Neutralizing buffer
Transport media
PBSTX
C&Bmod
PBSTX
C&Bmod
PBSTX
C&Bmod
PBSTX
C&Bmod
Holding Time (h)b
0, 12, 18, 24, 48, 72
0, 12, 18, 24, 48, 72
0, 12, 18, 24, 48, 72
0, 12, 18, 24, 48, 72
0, 12, 24, 48, 72
0, 12, 24, 48, 72
0, 12, 24, 48, 72
0, 12, 24, 48, 72
a macrofoam swabs processed by vortexing 3 min, rayon swabs processed by
sonicating 3 min (optimum conditions found for each swab type in phase I).
b 10 swabs per holding time, the 18 h holding time dropped after macrofoam
evaluations complete.
50
-------
5. Phase IV: Evaluation of sample storage parameters for both Y. pestis strains on "dirty"
swabs
Table 5. Phase IV study matrix: YP A1122, swab material, premoistening agent, transport media
and holding times evaluated. All swabs were held at 4°C. Arizona Test Dust was added to the
swabs to simulate background dust and organisms prior to inoculation with YP.
Organism
Phase IV
YP A1122
Swab material
macrofoam
macrofoam
macrofoam
macrofoam
rayon
rayon
rayon
rayon
Premoistening agent
Triton X- 100
Triton X-100
Neutralizing buffer
Neutralizing buffer
Triton X-100
Triton X-100
Neutralizing buffer
Neutralizing buffer
Transport
media
PBSTX
NB
PBSTX
NB
PBSTX
NB
PBSTX
NB
Holding Time (h)a
0, 12, 24, 48, 72
0, 12, 24, 48, 72
0, 12, 24, 48, 72
0, 12, 24, 48, 72
0, 12, 24, 48, 72
0, 12, 24, 48, 72
0, 12, 24, 48, 72
0, 12, 24, 48, 72
10 swabs per holding time
Table 6. Phase IV study matrix: YP CO92, swab material, premoistening agent, transport media
and holding times evaluated. All swabs were held at 4°C. Arizona Test Dust was added to the
swabs to simulate background dust and organisms prior to inoculation with YP.
Organism
Phase IV
YP CO92
Swab material
macrofoam
macrofoam
macrofoam
macrofoam
rayon
rayon
rayon
rayon
Premoistening agent
Triton X-100
Triton X-100
Neutralizing buffer
Neutralizing buffer
Triton X-100
Triton X-100
Neutralizing buffer
Neutralizing buffer
Transport media
PBSTX
NB
PBSTX
NB
PBSTX
NB
PBSTX
NB
Holding Time (h)a
0, 12, 24, 48, 72
0, 12, 24, 48, 72
0, 12, 24, 48, 72
0, 12,24, 48, 72
0, 12, 24, 48, 72
0, 12, 24, 48, 72
0, 12, 24, 48, 72
0, 12, 24, 48, 72
10 swabs per holding time
51
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United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
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