Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 1 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Signature Page
Author’s Signature:
Trevor Brasel PhD__________________
Typed Name of Author
Acceptance by Subcontracting Institution:
Robert Sherwood_PhD______________
Typed Name of Subcontracting PI
Acceptance by the University of New Mexico:
__C. Rick Lyons MD. PhD__________
Principal Investigator
Acceptance by NIAID:
_Patrick Sanz______3/5/10_________
Typed Name of NIAID Project Officer
Heidi Holley ___________________
Typed Name of NIAID Contract Officer
__3/9/2010___
Date Accepted
Page 1 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 2 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Table of Contents
1 Milestone Summary ...................................................................................................................................................2
2 Milestone Objectives..................................................................................................................................................3
3 Methods, Critical Reagents and SOPs........................................................................................................................3
3.1
Methods Summary......................................................................................................................................3
3.2
Critical Reagents ...................................................................................................................................... 17
3.3
Standard Operating Procedures ................................................................................................................ 17
4 Salient Original Data, Results, Interpretation, Quality Control ............................................................................... 18
4.1
Original Data and Results (Rationale, Tables/Figures with legends and location annotations) ............... 18
4.2
Interpretation ............................................................................................................................................ 18
4.3
Quality Control ......................................................................................................................................... 39
5 Deliverables Completed ........................................................................................................................................... 46
6 Appendices ............................................................................................................................................................... 47
6.1
Appendix 1: Original Data Tables and Figures ....................................................................................... 47
6.2
Appendix 2: Quality Assessment of Milestone Completion and Report ................................................. 53
6.3
Appendix 3: Additional Data/Figures not included in the Text of the Milestone Completion Report
(Section 4) .............................................................................................................................................. 56
1
Milestone Summary
The goal of Milestone #3 was to optimize Francisella tularensis (F. tularensis) bioaerosol
procedures, including bacterial proliferation and aerosol generation/collection techniques.
Completion of these tasks was critical in that all future vaccine efficacy testing in nonhuman
primates (as described under the Tularemia Vaccine Development Contract) will be dependent
upon an optimal means of F. tularensis bioaerosol challenge. Cumulative results from live vaccine
(LVS) and SCHU S4 strain testing demonstrated that an optimal F. tularensis challenge bioaerosol
was achieved under the following parameters: (1) challenge material prepared from a 24h
subculture of F. tularensis in Chamberlain’s broth subcultured from 48h growth on blood cysteine
glucose agar (BCGA) (2) bacterial suspension aerosolized using the Collison 3-jet nebulizer at a
normalized flow rate of 7.5 L/min (3) relative humidity in the aerosol chamber maintained at 6090% (4) bioaerosol material collected into brain heart infusion broth (BHIB) plus antifoam A using
the AGI-4 all-glass impinger (5) bacterial culture for purity and titer performed on BCGA.
Because of the observed repeatability, bacterial viability retention, and spray factor efficiencies,
these procedures will be incorporated into all future F. tularensis nonhuman primate aerosol
challenge studies.
Page 2 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Version: 2.0
Page 3 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
2
Accepted Date:4/23/09
Milestone Objectives
Bioaerosol technique selected and optimized. This milestone consisted of testing of a variety of
selected aerosol generation devices as agreed upon by LBERI and UNM in order to optimize
aerosol delivery of bacteria to mammals. Minimal endpoints included determination of the
conditions that optimize bacterial survival during aerosolization (pre- and post-), reproducibility
and accuracy of targeted inoculums, and size of droplets generated. This Milestone Completion
Report (MSCR) for Milestone 3 summarizes the data generated.
3
Methods, Critical Reagents and SOPs
3.1
3.1.1
Methods Summary
NOTE: Because F. tularensis is a select agent, all manipulations were performed under ABSL-3
conditions using aseptic techniques in a Class II biological safety cabinet.
Comparison of Solid and Liquid Media for the Culture of F. tularensis
Prior to initiation of MS3, growth conditions of F. tularensis LVS were optimized on solid agar
and in liquid broth media.
The following nine solid agar media types were tested: Chamberlain’s agar supplemented with
hemoglobin (CAH), cysteine heart agar supplemented with 2 g/L hemoglobin (CHAH-2),
cysteine heart agar supplemented with 10 g/L hemoglobin (CHAH-10), cysteine heart agar
(CHA), blood cysteine glucose agar (BCGA), cysteine heart agar with blood (CHAB),
supplemented BCGA (SBCGA), glucose cysteine agar with thiamine (GCAT), and
Chamberlain’s agar (CA). Components of each agar are detailed in Table A1 in Appendix 3.
For each agar type, a frozen LVS working stock vial (see Section 3.1.2) was thawed and used to
inoculate a 90-mm agar plate via the standard quadrant streak method as depicted in Figure 1.
The plates were allowed to incubate in a 37°C ± 2°C incubator for 24 or 48h after which purity
and phenotype were evaluated. Additionally, as a positive control, a 48h culture of LVS grown
in Chamberlain’s broth (procedure detailed below) was serially diluted and plated onto eight of
the nine types of agar media (GCAT was removed from this portion of the study). Plates were
allowed to incubate in a 37°C ± 2°C incubator for 72h after which colonies were enumerated
(Table 6). Relative recoveries of LVS for each media type were compared.
The following five broth media types were tested as well: modified cysteine partial hydrolysate
(MCPH), modified Mueller-Hinton broth (MMHB), Chamberlain’s broth (CB), Chamberlain’s
broth supplemented with hemoglobin (10 g/L; CBH), and MCPH with isovitale-X (MCPHI).
Components of each broth are detailed in Table A2 in Appendix 3. Broth cultures were grown
with shaking (225 rpm) in a 37°C ± 2°C incubator for 48h in 250- or 500-mL Erlenmeyer
flasks. The lower volumes were inoculated with 20 μL of LVS working stock while 500-mL
flasks were inoculated with 40 μL. Bacterial suspensions were serially diluted and cultured
Page 3 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 4 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
onto 90-mm CHAB plates. The plates were allowed to incubate in a 37°C ± 2°C incubator for
72h after which purity, phenotype, and colony counts were determined.
3.1.2
Preparation of Francisella tularensis Seed and Working Stocks
F. tularensis seed and working stocks were prepared according to SOP LBERI-1.1, Preparation
of Francisella tularensis Seed and Working Stocks. Bacterial seed stocks were prepared using
the following primary stock vials:
 LVS Lot 703-0303-016 (13DEC05, Fisher Biosciences)
 SCHU S4 Submaster Project 110419.2 (9MAR06, Fisher Biosciences)
o SCHU S4 Sublot#1 was received at UNM 9MAR06 and transferred to LBERI
o SCHU S4 Sublot#1 (NOV 05 to FEB 06, Midwest Research Institute) to Fisher
o SCHU S4 parent strain originated from the Salk Institute in Swiftwater, PA, Lot
623-42 (6SEP86)
o The Salk Institute received the seed stock from Fort Detrick, MD.
Briefly, upon receipt, the vial was thawed and used to inoculate a 90-mm BCGA plate via the
standard quadrant streak method as depicted in Figure 1 below:
Figure 1. Standard microbiological quadrant streak method for bacterial cultures.
The plates were allowed to incubate in a 37°C ± 2°C incubator for 48-72h after which purity and
phenotype were determined.
After 48-72h of growth on BCGA, F. tularensis LVS and SCHU S4 colonies are 3-4 mm in
diameter, gray-white to slightly blue and opaque, demonstrate little or no hemolysis, present a
raised, convex appearance, and have a wet, shiny surface as depicted in Figure 2 below:
Page 4 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 5 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Figure 2. Francisella tularensis LVS growth on BCGA. This image represents colony
morphology after 48h of incubation at 37°C (see method summary in section 3.1.1) Data
location: \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY06\FY06-078_TUL-03\LVS Growth
characterization\48h on BCGA.jpeg
Upon verification of purity and phenotype, one isolated colony was picked from the BCGA plate
and aseptically inoculated into 100 mL of sterile Chamberlain’s broth medium in a sterile, plastic
250-mL Erlenmeyer flask. The broth was incubated with shaking at 37°C ± 2°C, 225 ± 25 rpm
for 48h ± 8h. Following incubation, the culture was aseptically aliquoted into two sterile 50-mL
centrifuge tubes in a Class II biosafety cabinet (BSC). Suspensions were centrifuged in a sealed
rotor at 2,400 x g, 4-6°C, for 20 min after which the supernatant was decanted from each tube.
To each bacterial pellet, 20-25 mL of sterile Chamberlain’s broth was added (depending on the
desired number of seed stock vials). The solutions were mixed thoroughly using a vortex mixer
to ensure complete resuspension of the culture. An equal volume of 20% (w/v) sucrose was then
added to each tube resulting in a final sucrose concentration of 10%. Aliquots (0.5 mL) of this
suspension were aseptically added to 1.8 mL capacity cryovials and stored in a -80°C freezer.
These cryovials served as the F. tularensis seed stock.
The working stock was prepared in the same manner using 100 L of bacterial suspension from a
seed stock vial for the initial Chamberlain’s broth inoculum. These stocks, at a working volume
of 1.0 mL, were stored in a -80°C freezer for a maximum of six months due to significant titer
decrease observed after this time period.
3.1.3
Generator and Impingement Solution Optimization
In addition to growth media, generator and impingement solutions were tested in order to
determine the optimal aerosolization and collection media for F. tularensis. Focus for this study
Page 5 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 6 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
was placed on the media type and bacterial viability as a function of time and temperature. The
following four media types were evaluated: Chamberlain’s broth, phosphate buffered saline
(PBS), brain-heart infusion broth (BHIB), and 1% (w/v) peptone (See Figure 9). Each solution
was inoculated with F. tularensis LVS from frozen working stock to a final concentration of 2.0
or 3.0 x 107 CFU/mL in 20 mL. Suspensions were allowed to sit at room temperature (20°C) or
4°C for four hours during which a 500 L aliquot was removed hourly, serially diluted, and
cultured onto 90-mm CHAB plates. We have observed previously with other bacterial agents that
storage temperature significantly impacts bioaerosol stability. For example, with Yersinia pestis,
bioaerosol stability (i.e., bacterial viability) greatly decreases if the generator suspension is not
stored cold. For F. tularensis, this variable was unknown thus requiring the need to test the two
temperatures stated above. Following inoculation, plates were allowed to incubate in a 37°C ±
2°C incubator for 72h after which purity, phenotype, and colony counts were determined.
3.1.4
Generator Suspension Preparation
Throughout the milestone testing period, various methods of F. tularensis growth were tested for
use as the generator suspension material. Of these, two methods were finalized: one for frozen
material and one for fresh bacterial growth. The following sections describe these optimized
procedures.
Frozen F. tularensis
Separate bacterial suspensions were prepared for each bioaerosol run. Because of an observed
titer decrease over time, all working stock vials used for frozen F. tularensis bioaerosol testing
were less than or equal to six months in age. Briefly, a working stock cryovial of confirmed titer
(i.e., culture was performed shortly before testing to obtain the viable bacterial titer) was removed
from frozen storage, thawed and used to directly prepare the suspensions needed for the day of
aerosol testing. Bioaerosol material was prepared in sterile BHIB to optimal working volumes
(based on manufacturer recommendations and reservoir capacity) of 5 mL for the Aeromist
nebulizer, 10 mL for the Collison and Micropump generators, and 20 mL for the sparging
generator reservoir. Prior to delivery to the Aerosol laboratory, bacterial suspensions were
weighed in order to establish a baseline for post-bioaerosol volume calculations.
Fresh F. tularensis
Separate bacterial suspensions were prepared for each bioaerosol run. Briefly, a working stock
cryovial was removed from frozen storage, thawed and used to inoculate three BCGA plates
using the standard bacterial quadrant streak method. Plates were allowed to incubate at 37°C for
48h or until the colonies were 3-4 mm in diameter. Following incubation, plates were removed
and purity was verified. Using a 1-L inoculating loop, colonies (generally n=10) were removed
from the BCGA and inoculated into 4.5 mL of CB. This suspension was mixed thoroughly using
Page 6 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 7 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
a vortex mixer and analyzed at 600 nm (OD600) using a spectrophotometer. Data were used to
normalize the bacterial suspension to a final OD600 value of 0.100 ± 0.01. Three new, sterile 500mL baffled flasks containing 100 mL of CB were each inoculated with 400 L of the normalized
suspension. Flasks, sealed with filtered stoppers, were allowed to incubate in a 37°C incubator
with shaking (200 RPM) in the dark for 24h. Using a growth curve prepared from similarly
prepared material, bioaerosol material was diluted from the 24h culture in sterile BHIB to final
volumes of 5 mL for the Aeromist nebulizer and 10 mL for the Collison and Hospitak generators.
Prior to delivery to the Aerosol laboratory, bacterial suspensions were weighed in order to
establish a baseline for post-bioaerosol volume calculations. (See LBERI SOP 2.1)
3.1.5
Impingement Solution Preparation
Impingement solutions were prepared according to SOP LBERI-2.1, Preparation of Francisella
tularensis for Bioaerosol Studies. In brief, 20 mL of sterile BHIB were aseptically dispensed into
sterile, plastic 50 mL centrifuge tubes (n=1 per bioaerosol run). To minimize foaming, one drop
(10-20 L) of Antifoam A was added to each tube prior to delivery to the Aerosol laboratory. Allglass impinger (AGI) solutions were then weighed in order to establish a baseline for postbioaerosol volume calculations.
3.1.6
Aerosol Exposure System and Bioaerosol Generator Operation
The LBERI NHP head-only exposure system (Figure 3) was used for the F. tularensis bioaerosol
runs conducted throughout MS3. The main components of the system were as follows:

The bioaerosol generators, operated using HEPA-filtered compressed air at selected
delivered pressures, were used to aerosolize the F. tularensis at stable, controlled flow
rates.

The dilution/delivery line was used to add dilution air to the bioaerosol at a controlled
rate and to deliver the agent to the head-only chamber. Dilution air was humidified using
an ultrasonic humidification chamber. Humidity was maintained between 60% and 90%.

A head-only exposure chamber provided primary containment of the aerosolized F.
tularensis and ensured a homogenous bioaerosol.

Mass flow controllers (MFCs) were used to monitor and control the aerosol generator,
dilution makeup air, impingers, and exposure chamber exhaust.

A RH/temperature sensor and a percent oxygen transducer were used to measure the
relative humidity and temperature and oxygen concentration, respectively, with the
exposure chamber.

The AGI sampling system was used to collect the bioaerosol from the exposure chamber
during each run in order to assess the aerosol concentration and other critical parameters.
Page 7 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 8 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10

A DustTrak aerosol monitor was used to indicate the relative real-time chamber
concentration of the test aerosol.

An Aerodynamic Particle Spectrometer (APS) was used to measure the particle size
distribution of a representative sample during each exposure period.

A Class III biosafety cabinet provided secondary containment of the exposure system.
All manipulations were performed therein.

The LabVIEW monitoring/control system was used to electronically control and monitor
aerosol parameters including nebulizer and AGI flows, humidity, DustTrak data, exhaust
flow and to display summary data of the bioaerosol conditions.
Figure 3. LBERI NHP Head-Only Exposure System. Data location: \\Saturn\absl3\Agent and
Study Specific Data and Miscellaneous Documents\STUDY SPECIFIC
DATA\FY06\FY06078_TUL-03\TUL-03 data files\Exposure line schematic.ppt
Page 8 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 9 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
All F. tularensis testing was conducted using the Aeromist, Collison 3-jet, Aeroneb® Lab
Micropump, and Hospitak Nebulizers, and/or the sparging generator. Operating procedures for
each unit are described in SOP LBERI 3.1 and in the following sections.
Page 9 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 10 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Aeromist Nebulizer
The Aeromist Nebulizer is depicted in Figure 4:
Figure 4. Aeromist Nebulizer. In addition to the Collison and Hospitak nebulizers, the Aeromist
was one of the three bioaerosol generators down-selected for extensive LVS and SCHU S4
testing.
The Aeromist Nebulizer was filled with 4.5-5.0 mL of the F. tularensis suspension (preparation
procedures described above) and was operated by delivering compressed air at 5-20 pounds per
square inch, depending on the objectives of the study. This provided a nebulizer flow rate of 3.99.0 L/min. The bacterial aerosol was diluted with humidified air and delivered to the head-only
chamber. Total chamber flow was regulated at 16 L/min and main exhaust was adjusted to
maintain chamber pressure slightly negative (-0.1 to -0.3 inches water column) with respect to the
Class III biosafety cabinet (a means to prevent inadvertent release of the aerosol). Samples were
extracted from the head-only chamber to quantify the bioaerosol concentration (via AGI-4
sampling), size distribution (via 30-sec APS measurement) and relative particle concentration (via
continuous DustTrak sampling). AGI sampling was performed at flows of ranging from 5.1 to
5.4 L/min during the aerosol trials. Following aerosol trials, AGI suspensions were transferred to
the Microbiology laboratory for quantitative analyses as described below (Determination of F.
tularensis Bioaerosol Parameters). The nebulizer and AGI flowrates were calibrated on the day
of and prior to all of the aerosol trials.
Page 10 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 11 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Collison 3-Jet Nebulizer
The Collison 3-Jet Nebulizer is depicted in Figure 5:
Figure 5. Collison 3-Jet Nebulizer. In addition to the Aeromist and Hospitak nebulizers, the
Collison was one of the three bioaerosol generators down-selected for extensive LVS and SCHU
S4 testing. It is considered to be the standard generator throughout the bioaerosol field.
The Collison nebulizer (BGI, Inc., Waltham, MA) was filled with 10 mL of the F. tularensis
suspension (preparation procedures described above) and was operated by delivering compressed
air at 24.5 to 26.0 pounds per square inch. This provided a nebulizer flow rate of 7.5 ± 0.1 L/min.
The bacterial aerosol was diluted with humidified air and delivered to the head-only chamber.
Total chamber flow was regulated at 16 ± 0.2 L/min and main exhaust was adjusted to maintain
chamber pressure slightly negative (-0.1 to -0.3 inches water column) with respect to the Class III
biosafety cabinet (a means to prevent inadvertent release of the aerosol). Samples were extracted
from the head-only chamber to quantify the bioaerosol concentration (via AGI-4 sampling), size
distribution (via 30-sec APS measurement) and relative particle concentration (via continuous
DustTrak sampling). AGI sampling was performed at flows of ranging from 5.3 to 5.5 L/min
during the aerosol trials. Following aerosol trials, AGI suspensions were transferred to the
Microbiology laboratory for quantitative analyses as described below (Determination of F.
tularensis Bioaerosol Parameters). The nebulizer and AGI flowrates were calibrated on the day
of and prior to all of the aerosol trials.
Page 11 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 12 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Aeroneb® Lab Micropump Nebulizer
The Micropump Nebulizer is depicted in Figure 6:
Figure 6. Aeroneb® Lab Micropump Nebulizer. The Micropump nebulizer was used only in
LVS testing throughout the milestone study period.
The Aeroneb® Lab Micropump Nebulizer (Aerogen, Inc., Galway, Ireland) was filled with 10
mL of the F. tularensis suspension (preparation procedures described above) and was operated by
delivering compressed air at a flow rate of 7.5-10.0 L/min. The bacterial aerosol was diluted
with humidified air and delivered to the head-only chamber. Total chamber flow was regulated at
16 ± 0.2 L/min and main exhaust was adjusted to maintain chamber pressure slightly negative (0.1 to -0.3 inches water column) with respect to the Class III biosafety cabinet (a means to
prevent inadvertent release of the aerosol). Samples were extracted from the head-only chamber
to quantify the bioaerosol concentration (via AGI-4 sampling), size distribution (via 30-sec APS
measurement) and relative particle concentration (via continuous DustTrak sampling). AGI
sampling was performed at flows of ranging from 5.2 to 5.3 L/min during the aerosol trials.
Following aerosol trials, AGI suspensions were transferred to the Microbiology laboratory for
quantitative analyses as described below (Determination of F. tularensis Bioaerosol Parameters).
The nebulizer and AGI flowrates were calibrated on the day of and prior to all of the aerosol
trials.
Page 12 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 13 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
HospitakTM Nebulizer
The Hospitak Nebulizer is depicted in Figure 7:
Figure 7. Hospitak Nebulizer. In addition to the Collison nebulizer, the Hospitak was one of the
two bioaerosol generators down-selected for extensive LVS and SCHU S4 testing.
The Hospitak Nebulizer (Unomedical, a/s, Birkeroed, Denmark) was filled with 10.0 mL of the F.
tularensis suspension (preparation procedures described above) and was operated by delivering
compressed air at 10 pounds per square inch. This provided a nebulizer flow rate of 4.8-5.2
L/min. The bacterial aerosol was diluted with humidified air and delivered to the head-only
chamber. Total chamber flow was regulated at 16 L/min and main exhaust was adjusted to
maintain chamber pressure slightly negative (-0.1 to -0.3 inches water column) with respect to the
Class III biosafety cabinet (a means to prevent inadvertent release of the aerosol). Samples were
extracted from the head-only chamber to quantify the bioaerosol concentration (via AGI-4
sampling), size distribution (via 30-sec APS measurement) and relative particle concentration (via
continuous DustTrak sampling). AGI sampling was performed at a flow rate of 5.0 ± 0.5 L/min
during the aerosol trials. Following aerosol trials, AGI suspensions were transferred to the
Microbiology laboratory for quantitative analyses as described below (Determination of F.
tularensis Bioaerosol Parameters). The nebulizer and AGI flowrates were calibrated on the day
of and prior to all of the aerosol trials.
Page 13 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 14 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Sparging Generator
The sparging generator is depicted in Figure 8:
Figure 8. Sparging Generator. The sparging generator, similar in design to the Collison
nebulizer, was used only in LVS testing throughout the milestone study period.
The Sparging Generator (CH Technologies, Inc., Westwood, NJ) was delivered a F. tularensis
suspension (preparation procedures described above) at a rate of 1.0 ± 0.1 L/min and operated by
providing a carrier air flow of 8.0 L/min. This provided a generator flow rate of 8.0 ± 0.1 L/min.
The bacterial aerosol was diluted with humidified air and delivered to the head-only chamber.
Total chamber flow was regulated at 16 ± 0.2 L/min and main exhaust was adjusted to maintain
chamber pressure slightly negative (-0.1 to -0.3 inches water column) with respect to the Class III
biosafety cabinet (a means to prevent inadvertent release of the aerosol). Samples were extracted
from the head-only chamber to quantify the bioaerosol concentration (via AGI-4 sampling), size
distribution (via 30-sec APS measurement) and relative particle concentration (via continuous
DustTrak sampling). AGI sampling was performed at flows of ranging from 5.1 to 5.2 L/min
during the aerosol trials. Following aerosol trials, AGI suspensions were transferred to the
Microbiology laboratory for quantitative analyses as described below (Determination of F.
tularensis Bioaerosol Parameters). The generator and AGI flowrates were calibrated on the day
of and prior to all of the aerosol trials.
3.1.7
Comparison of Pre- and Post-Spray F. tularensis Generator Suspension
Concentrations
In order to assess bacterial viability within the generator suspension, bacterial concentrations
were analyzed prior to the bioaerosol run and compared to the titer in the residual generator
Page 14 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 15 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
suspension following the test. This was performed only for the Aeromist, Hospitak, and Collison
nebulizers. For each sample tested in this manner, suspensions were diluted into sterile 1%
peptone to three final culture dilutions (dependent upon the initial generator concentration) using
standard ten-fold microbial dilution procedures. Aliquots from each dilution (100 L) were
plated in triplicate onto BCGA and incubated at 37°C ± 2°C for 48h ± 8h after which purity was
determined and F. tularensis colonies were counted. Post-spray analyses were performed
similarly and are described in the following section.
3.1.8
Determination of F. tularensis Bioaerosol Parameters
Following each bioaerosol exposure, generator and impinger suspensions were aseptically
aliquoted into 50 mL centrifuge tubes and transferred to the Microbiology laboratory for
immediate processing. Briefly, suspensions were weighed to assess the volume generated during
the bioaerosol run and diluted into sterile 1% peptone to three final culture dilutions (dependent
upon the initial generator concentration) using standard ten-fold microbial dilution procedures.
Aliquots from each dilution (100 L) were plated in triplicate onto BCGA and incubated at 37°C
± 2°C for 48h ± 8h after which purity was determined and F. tularensis colonies were counted.
Using colony enumeration data, relevant data, including CFU/L of air and spray factors, were
determined. Total CFU sprayed was determined using Equation 1:
CFU Aerosolized = CFU/mL (post-bioaerosol generator suspension)*Vol sprayed (mL)
CFU/mL (post-bioaerosol generator suspension) was calculated using Equation 2:
CFU/mL = (Mean of plate CFU counts)*dilution factor
The dilution factor was inclusive of the 10-fold dilution resulting from the 100 L plating
volume.
Volume sprayed (mL) was calculated using Equation 3:
Vol sprayed (mL) = Initial weight (g) – Final weight (g)
It was assumed that the specific gravity of the suspension fluid was equal to that of water.
The CFU sprayed data were used to indicate the accuracy of the suspension preparation,
concentration of total CFU sprayed during the bioaerosol run, and to provide insight into the
bioaerosol stability during aerosolization.
Page 15 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 16 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
CFU/L of air was calculated using Equation 4:
CFU/L of air =
total CFU recovered in the AGI
(sample time [min]*flow rate [L/min]to the AGI)
The total CFU recovered in the AGI was calculated using Equation 5:
Total CFU recovered in AGI = Final AGI vol (mL)*CFU/mL
CFU/mL represents the calculated CFU/mL of the AGI suspension following the bioaerosol run
and was calculated using Equation 2.
The final AGI volume (mL) was calculated using Equation 6:
Final AGI vol (mL) = Initial vol (mL)-(Initial weight [g] – Final weight [g])
For each bioaerosol run, the initial volume aliquoted into the AGI was 20 mL. It was assumed
that the specific gravity of the suspension fluid was equal to that of water. Sample times and flow
rates to the AGI were recorded electronically using the LabView software. CFU/L data were
used to establish the bacterial atmosphere concentration.
The spray factor (SF), a unitless measurement defined as the ratio of the aerosol concentration to
the starting concentration (in the generator suspension) was calculated using Equation 7:
SF = (CFU/L)/(CFU/mL [post-bioaerosol generator suspension]*1000)
The spray factor is primarily a function of the generator, the agent being tested and the system
flow and is used to compare efficacy between bioaerosol runs and, for this milestone, generator
types.
Page 16 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Version: 2.0
Page 17 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
3.2
Critical Reagents
Preparation of the critical reagents listed below are described in detail in SOP LBERI 4.1.
Reagent
Manufacturer
Catalog #
Agar
n/a (components
below)
Remel
n/a (components
below)
451012
Peptone
BD/Difco
211677
Meat Extract
EMD/Merck
1.03979.0500
Sodium Chloride
Sigma Aldrich
S3014-500G
L-histidine
Sigma Aldrich
H8000-25G
Glucose
Sigma Aldrich
G5767-500G
L-cysteine
Sigma Aldrich
168149-25G
Sterile Defibrinated
Sheep Blood
PML
A0407
Sucrose
Sigma Aldrich
S0389-500G
Peptone
BD/Difco
211677
Antifoam A
Sigma
A5633-100G
Brain Heart Infusion
Broth
Remel
R452472
Chamberlains Medium
Teknova
C0711
BCGA
3.3
Accepted Date:4/23/09
Standard Operating Procedures
SOP Number1
SOP Title
LBERI-1.1
Preparation of Francisella tularensis Seed and Working
Stocks
LBERI-2.1
Preparation of Francisella tularensis for Bioaerosol Studies
LBERI-3.1
Operation of the Aeromist, Hospitak, Collison 3-jet,
Aeroneb® Micropump, and Sparging Generators During
Francisella tularensis Bioaerosol Studies
Page 17 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Version: 2.0
Page 18 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
SOP Number1
LBERI-4.1
Accepted Date:4/23/09
SOP Title
Preparation of Reagents and Media for Francisella
tularensis Studies
1
Individual Standard Operating Procedures will be reviewed separately and accepted by the Subcontracting PI and UNM
PI
4
Salient Original Data, Results, Interpretation, Quality Control
4.1
4.1.1
Original Data and Results (Rationale, Tables/Figures with legends and location
annotations)
Comparison of Solid and Liquid Media for the Growth of F. tularensis
Results from solid and liquid media optimization analyses using LVS are summarized in Tables
1-3:
Table 1. Qualitative Comparison of Francisella tularensis LVS Growth on
Various Solid Agar Media
Media
Qualitative
Analysis
Quadrant
Colony Description
CAH
Poor growth
1
Circular, raised, entire, glistening,
smooth, opaque, grey/white
CHAH-2
Good growth
2
Circular, raised, entire, glistening,
smooth, opaque, white
CHAH-10
Fair growth
2
Circular, raised, entire, glistening,
smooth, opaque, white/green/pink
CHA
Fair growth
1
Circular, raised, entire, glistening,
smooth, opaque, white
BCGA
Fair growth
2
Circular, raised, entire, glistening,
smooth, opaque, grey/white
CHAB
Good growth
3
Circular, raised, entire, glistening,
smooth, opaque, grey/blue/white
SBCGA
Fair growth
2
Circular, raised, entire, glistening,
smooth, opaque, grey/blue/white
GCAT
No growth
n/a
n/a
CA
No growth
n/a
n/a
Page 18 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 19 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Table 1. Qualitative Comparison of Francisella tularensis LVS Growth on Various
Solid Agar Media (see method summary in section 3.1.1). Results demonstrated that
F. tularensis LVS qualitatively grew best on CHAB and CHAH-2, though fair growth
was also noted on CHAH-10, CHA, BCGA and SBCGA. Colony morphologies were
similar among the different agars. The predominant colony morphology difference
between the media, when present, was in colony color. No growth was observed on
GCAT or CA. From these data, the decision was made to proceed with consistent use of
CHAB and secondary use of BCGA as the solid media for growth of LVS, the latter
due to the ease of preparation versus other media types.
Data location:
\\Saturn\absl3\Agent and Study Specific Data and Miscellaneous Documents\STUDY
SPECIFIC DATA\FY06-078_TUL-03\TUL-03 data files\Working Group\F. tularensis
media comparison.pdf
Table 2. Quantitative Comparison of Francisella tularensis LVS Growth on Various Solid Agar Media
% Recovery
Media Type
Mean
CFU/mL
SBCGA
CHAH-2
CAH
CHA
BCGA
CHAB
CHAH-10
1.05E+08
2.00E+08
1.02E+08
2.50E+08
2.00E+08
2.00E+08
5.35E+07
SBCGA
1.05E+08
100%
53%
103%
42%
53%
53%
196%
CHAH-2
2.00E+08
190%
100%
197%
80%
100%
100%
374%
CAH
1.02E+08
97%
51%
100%
41%
51%
51%
190%
CHA
2.50E+08
238%
125%
246%
100%
125%
125%
467%
BCGA
2.00E+08
190%
100%
197%
80%
100%
100%
374%
CHAB
2.00E+08
190%
100%
197%
80%
100%
100%
374%
CHAH-10
5.35E+07
51%
27%
53%
21%
27%
27%
100%
Table 2. Quantitative comparison of Francisella tularensis LVS growth on various solid agar
media (see method summary in section 3.1.1). Here, each media type is compared as row
versus column (numerator versus denominator, respectively). For instance, the percent recovery
of LVS on SBCGA versus CHAH-2 was 53%, indicating that LVS quantitatively grew better on
CHAH-2 among these two media types. Overall, peak recoveries were noted on CHAH-2,
Page 19 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 20 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
CHA, BCGA, and CHAB. GCAT and CA were excluded from these tests as qualitative
analyses demonstrated the absence of growth (Table 1). These data supported our decision to
move forward with consistent use of CHAB and secondary use of BCGA as the solid media for
growth of LVS.
Data location: \\Saturn\absl3\Agent and Study Specific Data and
Miscellaneous Documents\STUDY SPECIFIC DATA\FY06-078_TUL-03\TUL-03 data
files\Working Group\F. tularensis media comparison.pdf
Table 3. Quantitative Comparison of Francisella tularensis LVS Growth in Various Nutrient
Broth Media
CFU/mL
Broth Medium
Flask #1
Flask #2
Average
MCPH
0 (at 1:1000 dilution)
0 (at 1:1000 dilution)
0 (at 1:1000 dilution)
MMHB
3.00E+05
1.20E+05
2.10E+05
CB
2.00E+07
2.00E+07
2.00E+07
CBH
3.00E+07
Not tested
3.00E+07
MCPHI
0 (at 1:1000 dilution)
2.00E+07
Inconclusive
Table 3. Quantitative comparison of F. tularensis LVS growth in various liquid broth media
(See method summary in section 3.1.1). Data demonstrated that bacteria grew optimally in
CBH. However, this testing was not repeated and it was noted that CBH was difficult to
prepare. Because of this, CB, the next best liquid broth medium based on quantitative LVS
growth, was chosen for all subsequent testing. Though growth was noted in MMHB, and
MCPHI, inconsistencies were observed and/or titers were significantly lower when compared to
CB. No growth was observed in MCPH following 72h of incubation at 37°C. All broth
suspensions were serially diluted and sub-cultured onto CHAB. Data location:
\\Saturn\absl3\Agent and Study Specific Data and Miscellaneous Documents\STUDY
SPECIFIC DATA\FY06-078_TUL-03\TUL-03 data files\Working Group\F. tularensis media
comparison.pdf
4.1.2
Generator and Impingement Solution Optimization
Results from generator and impingement solution optimization testing using LVS are
summarized in Figure 9:
Page 20 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 21 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Francisella tularensis LVS Stability Testing: Effect of Time,
Temperature, and Media on Viability and Culturability
CFU/mL (log10)
9.5
9.0
8.5
8.0
7.5
7.0
0
1
2
3
4
Time (h)
Chamberlains, RT
Chamberlains, 4 deg C
PBS, RT
PBS, 4 deg C
BHIB, RT
BHIB, 4 deg C
1% peptone, RT
1% peptone, 4 deg C
Figure 9. Effect of time, temperature, and media on F. tularensis LVS viability/culturability
(See method summary in section 3.1.3). Bacterial suspensions in various liquid media were
allowed to sit at room temperature or refrigerated for up to four hours. Aliquots were cultured
hourly and the resulting data were used to evaluate the effects of media, time and temperature
on LVS viability/culturability. Though results were similar among the groups, BHIB at room
temperature demonstrated optimal viability and culturability maintenance. It was for this reason
that BHIB was chosen as the final medium for bioaerosol material resuspension. All samples
presented an increase in titer over the first two hours indicating bacterial proliferation upon
initiation of the testing. Data location: \\Saturn\absl3\Agent and Study Specific Data and
Miscellaneous Documents\STUDY SPECIFIC DATA\FY06-078_TUL-03\LVS Growth
characterization\Stability\Effect of time and temp on LVS recovery_18April07.xls
4.1.3
Comparison of Pre- and Post-Spray F. tularensis Generator Suspension
Concentrations
Results from pre- and post-bioaerosol run generator suspension analyses using the Aeromist,
Hospitak and Collison Nebulizers are presented in Figures 10-13 below:
Page 21 of 62
5
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 22 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Figure 10. Effect of delivered pressure to the Aeromist Nebulizer on F. tularensis LVS
viability/culturability (see method summary in section 3.1.6 and 3.1.7). During select bioaerosol
runs, varied pressures ranging from 5.0 to 20.0 psig were delivered to the Aeromist while testing
frozen and fresh LVS cultures. Qualitatively, results indicated that as pressure delivered to the
generator increased, viability/culturability decreased following the bioaerosol runs. Statistically
(paired t-test performed on log-transformed data), there was no difference between pre- and posttest generator suspensions using frozen LVS at 5.0 or 10.0 psig; however, due to poor bioaerosol
output at 5.0 psig, 10.0 psig was chosen as the final delivered pressure parameter. Remaining
data, consisting of fresh LVS tested at 10.0, 15.0, and 20.0 psig, could not be statistically
analyzed due to insufficient replicates, though data suggested little or no difference between
frozen and fresh bacteria. Continued testing was halted with LVS because of our clearance to
work with SCHU S4, a critical step for this milestone. All NHP challenges were planned with
SCHU S4 aerosols; however, LBERI staff started aerosol testing with LVS because they were
waiting for USAMRIID to provide the LVS vaccinations to the LBERI staff. LBERI could only
prepare LVS aerosols until LBERI staff were LVS vaccinated as another layer of protection from
Page 22 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 23 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
SCHU S4 infection. After the LBERI staff were LVS vaccinated, the LBERI management
allowed the LBERI staff to switch to SCHU S4 aerosols. Switching to SCHU S4 aerosols was
more important than generating statistically significant aerosol data with LVS. Results obtained
with the experiments presented above served as a baseline to initiate the SCHU S4 aerosol
optimization. Data location: \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY06-078_TUL-03\TUL-03 data files\FY06-078_TUL03_Cumulative MS3 data.xls
Page 23 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 24 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Figure 11. Effect of delivered pressure to the Aeromist Nebulizer on F. tularensis SCHU S4
viability/culturability (see method summary in sections 3.1.6 and 3.1.7). During select bioaerosol
runs, varied pressures (either 7.5 or 10.0 psig) were delivered to the Aeromist while testing frozen
and fresh SCHU S4 cultures. SCHU S4 testing was performed following all LVS testing. We
knew with LVS, that delivered pressures of 15 and 20 psi were too high and likely would destroy
SCHU S4 as well. We also knew that 5 psi was too low; the low pressure was very gentle on the
bacteria, but not enough to actually generate the aerosol. For LVS, we concluded 10 psi was
optimal. We tested 7.5 psi for SCHU S4 because it was desirable to attempt to be slightly gentler
to the SCHU S4 bacteria, but that pressure, like 5 psi, was not high enough to generate the
aerosol. Fresh SCHU S4 was tested at 10 psi because that was concluded to be the optimal
delivered pressure for aerosol generation and bioaerosol stability. Essentially, output was better
with 10 psig; 7.5 psig was simply not enough to generate sufficient numbers of particulates.
Page 24 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 25 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Statistically (paired t-test performed on log-transformed data), there was a difference between
pre- and post-spray SCHU S4 suspensions (frozen and fresh) when tested at 7.5 or 10.0 psig, with
values demonstrated better recovery (i.e., increased viability retention) when a fresh bacterial
culture was used. From these data, we concluded that optimal SCHU S4 bioaerosols were
achieved with the Aeromist using fresh bacteria and a delivered pressure of 10 psig. Animal
exposures for ensuing studies could be conducted while observing these optimized procedures.
Please note: Initial target CFU/mL values were purposely varied to see the effect of concentration
on viability during the aerosol testing.
Data location: \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY06-078_TUL-03\TUL-03 data files\FY06-078_TUL03_Cumulative MS3 data.xls
Page 25 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 26 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Figure 12. Effect of aerosol generation using the Hospitak Nebulizer on F. tularensis SCHU S4
viability/culturability (see method summary in section 3.1.6 and 3.1.7). The X-axis represents replicates
performed on seven separate days. The Hospitak was viewed as a potential replacement for the Aeromist
since production of the Aeromist was ceased by the manufacturer during the course of this milestone.
During all bioaerosol runs, a standard pressure of 10.0 psig (to mimic the optimized procedures
established for the Aeromist) was delivered to the Hospitak while testing fresh SCHU S4 cultures.
Statistically (paired t-test performed on log-transformed data), there was no difference between pre- and
post-test generator suspensions using fresh SCHU S4 at 10.0 psig. Following these experiments, it was
decided that the Hospitak behaved very similarly to the Aeromist and could be used since LBERI could
no longer purchase the Aeromist. Data location: \Saturn\absl3\Agent and Study Specific Data \STUDY
SPECIFIC DATA\FY06-078_TUL-03\TUL-03 data files\FY06-078_TUL-03_Cumulative MS3 data.xls
Page 26 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 27 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Figure 13a. Effect of delivered pressure to the Collison Nebulizer on F. tularensis LVS and SCHU S4
viability/culturability (see method summary in section 3.1.6 and 3.1.7). X axis represents replicates.
During all bioaerosol runs, pressure delivered to the Collison was maintained between 20 and 30 psig (as
required per the Collison manufacturer) to establish a flow rate of 7.5 L/min. Pre- and post-generator
suspension analyses were performed at a delivered pressure of approximately 25 psig. Statistically
(paired t-test performed on log-transformed data), there was no difference between pre- and post-test
generator suspensions using frozen LVS or SCHU S4 at 25.0 psig (though it should be noted that there
were only three replicates of each). Analyses did, however, demonstrate a statistical difference between
pre- and post-spray values using fresh SCHU S4. Data location: \\Saturn\absl3\Agent and Study Specific
Data\STUDY SPECIFIC DATA\FY06-078_TUL-03\TUL-03 data files\FY06-078_TUL-03_Cumulative
MS3 data.xls
NOTE: The Collison required 20-30 psig per the manufacturer; however, LBERI tested lower psig for
other aerosol generators with the goal of increasing bacterial viability with lower pressures. The
Collison’s potential weaknesses were variability between different Collison units, the potential for the 3
Page 27 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 28 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
jets to become blocked, the glass construction as a safety risk in BSL3 environment, and potential
suboptimal bacterial viability due to the higher required pressures.
Figure 13b (continuation of 13a). Effect of delivered pressure to the Collison Nebulizer on fresh F.
tularensis SCHU S4 viability/culturability (see method summary in section 3.1.6 and 3.1.7). During all
bioaerosol runs, pressure delivered to the Collison was maintained between 20 and 30 psig to establish a
flow rate of 7.5 L/min. Pre- and post-generator suspension analyses were performed at a delivered
pressure of approximately 25 psig. Statistical analyses are described above under Figure 13a. From the
experimental data presented in Figures 13a and 13b, it was decided that the Collison, while operated
under manufacturer specifications using fresh SCHU S4, produced results similar to those optimal for the
Aeromist and Hospitak. Essentially, based on viability retention alone (i.e., pre- versus post-bioaerosol
generator suspension cultures), the Aeromist and Hospitak operated at a delivered pressure of 10 psig and
Page 28 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 29 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
the Collison operated at a delivered pressure of 25 psig demonstrated no significant differences with fresh
SCHU S4 testing. Thus, after months of testing multiple aerosol generators, LBERI in consultation with
NIAID and UNM chose the field standard Collison aerosol generator for all future NHP challenges with
fresh SCHU S4. Data location: \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY06-078_TUL-03\TUL-03 data files\FY06-078_TUL03_Cumulative MS3 data.xls
4.1.4
Bioaerosol Data
All F. tularensis bioaerosol optimization tests were performed first with LVS under the premise
that LVS data would linearly predict the SCHU S4 data (i.e., it was hypothesized that the two
strains would behave the same as bioaerosols). Also, LBERI initially could work only with LVS
and not SCHU S4, until the LBERI staff received the LVS vaccination. LBERI management
would not allow LBERI to work with SCHU S4 aerosols, until the LBERI staff were vaccinated.
UNM, LBERI and NIAID agreed that LBERI should start with LVS aerosols until SCHU S4
aerosols were permitted by the LBERI management.Results from these initial studies were used
to down-select to optimal conditions for SCHU S4 bioaerosol testing. These data are summarized
in following graphs and tables.
Frozen F. tularensis LVS
Page 29 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 30 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Spray Factors vs CFU/L: Frozen Francisella tularensis LVS Testing
with the Aeromist, Collison, Micropump and Sparging Nebulizers
6.0
CFU/L (log10)
5.0
4.0
3.0
2.0
1.0
0.0
-4.5
-1.0
-5.5
-6.5
-7.5
-8.5
-9.5
-10.5
Spray Factor (log10)
Aeromist, 5 psi
Aeromist, 10 psi
Aeromist, 15 psi
Aeromist, 20 psi
Collison, 20 psi
Collison, 25 psi
Micropump, 20 psi
Sparging
Figure 14. Cumulative spray factor versus aerosol concentration data using frozen F. tularensis LVS and
the Aeromist, Collison, Micropump and Sparging Nebulizers (see method summary in sections 3.1.6,
3.1.7, and 3.1.8). Data are presented as log10 values. Tested nebulizers and associated delivered
pressures are presented in the accompanying legend. A decrease in spray factor is proportional to a
decrease in bioaerosol efficiency (i.e., bioaerosol efficiency decreases from left to right). These data
demonstrate that the Aeromist operated at a delivered pressure of 10-20 psi performed most consistently
and efficiently with frozen LVS among the four tested nebulizers. The Collison operated under normal
operating conditions (20-25 psi) performed well overall, but presented with some poor spray factors. The
Micropump and Sparging generators were least efficient with frozen LVS under the specified operating
parameters.
Data location: \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY06-078_TUL-03\TUL-03 data files\FY06-078_TUL03_Cumulative MS3 data.xls
Page 30 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 31 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Table 4. Frozen LVS Testing with the Aeromist, Collison, Micropump, and Sparging
Nebulizers: Relationship between Delivered Pressure, Aerosol Concentration, and
Spray Factor
Generator
Pressure
psig
N
CFU/L
Spray Factor
Geo Mean
Geo SD
Geo Mean
Geo SD
Aeromist
5
6
3.26E+02
9.78E+00
5.98E-08
1.58E+00
Aeromist
10
3
1.08E+03
1.50E+00
3.38E-07
1.28E+00
Aeromist
15
2
1.07E+03
2.11E+00
2.00E-06
2.54E+00
Aeromist
20
30
1.70E+02
9.26E+00
4.61E-07
2.62E+00
Collison
20
8
4.80E+02
4.78E+01
4.61E-07
1.78E+00
Collison
25
18
7.96E+01
3.60E+01
1.96E-07
1.44E+01
Micropump
20
11
2.67E+01
6.21E+00
3.34E-08
1.48E+01
Sparging
n/a**
12
6.55E+01
8.98E+00
6.01E-09
8.17E+00
*N, number of replicates
**n/a, not applicable as carrier air was supplied to the Sparging for bioaerosol generation
Table 4. Summary spray factor and aerosol concentration data using frozen F. tularensis LVS
and the Aeromist, Collison, Micropump and Sparging Nebulizers. Data are presented as
geometric means and standard deviations (see method summary in section 3.1.6, 3.1.7, 3.1.8).
These data are presented graphically in Figure 14. For this milestone, adjustments based on
available data were made by the TVDC and LBERI teams in real-time as to how to proceed.
Replicates were not originally planned with statistical analyses in mind, rather, decisions were
made to conduct further testing on generator/pressure combinations that demonstrated promising
results with respect to bioaerosol optimization. It should be noted here, that further testing was
ceased with LVS following LBERI’s clearance to work with SCHU S4 (again, a critical step for
this milestone); this is reflected in the small number of LVS replicates at 5, 10, and 15 psig
delivered for the Aeromist as well as 20 psig delivered to the Collison. Once the LBERI staff
were allowed to use SCHU S4 aerosols, LVS aerosol testing ceased Data location:
\\Saturn\absl3\Agent and Study Specific Data and Miscellaneous Documents\STUDY SPECIFIC
DATA\FY06-078_TUL-03\TUL-03 data files\FY06-078_TUL-03_Cumulative MS3 data.xls
Page 31 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 32 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Fresh F. tularensis LVS
Spray Factors vs CFU/L: Fresh Francisella tularensis LVS Testing with
the Aeromist, Collison, and Micropump Nebulizers
6.0
CFU/L (log10)
5.0
4.0
3.0
2.0
1.0
0.0
-4.5
-5.0
-5.5
-6.0
-6.5
-7.0
-7.5
-8.0
-8.5
-9.0
Spray Factor (log10)
Aeromist, 10 psi
Aeromist, 15 psi
Collison, 30 psi
Micropump, 20 psi
Aeromist, 20 psi
Collison, 20 psi
Collison, 25 psi
Figure 15. Cumulative spray factor versus aerosol concentration data using fresh F. tularensis
LVS and the Aeromist, Collison, and Micropump Nebulizers (see method summary in section
3.1.6, 3.1.7, 3.1.8). The Sparging generator was removed from further testing shortly following
frozen LVS experiments due to poor results in conjunction with the difficulties encountered
during setup in the LBERI ABSL-3 Facility; because of these factors, it was not pursued for
further fresh LVS testing. Setting up the Sparging generator made it technically difficult for the
personnel working the constrained space in the ABSL- 3 conditions so LBERI did not pursue
the Sparging generator with fresh LVS. Data are presented as log10 values. Tested nebulizers
and associated delivered pressures are presented in the accompanying legend. A decrease in
spray factor is proportional to a decrease in bioaerosol efficiency (i.e., bioaerosol efficiency
decreases from left to right). These data demonstrate that the Aeromist operated at a delivered
pressure of 10-20 psi performed most consistently and efficiently with fresh VS among the three
tested nebulizers. The Collison operated under normal operating conditions (20-30 psi)
performed consistently, but was less efficient than the Aeromist. The Micropump performed
poorly with fresh LVS under the specified operating parameters.
Data location:
Page 32 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 33 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
\\Saturn\absl3\Agent and Study Specific Data and Miscellaneous Documents\STUDY SPECIFIC
DATA\FY06-078_TUL-03\TUL-03 data files\FY06-078_TUL-03_Cumulative MS3 data.xls
Page 33 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 34 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Table 5. Fresh LVS Testing with the Aeromist, Collison, and Micropump Nebulizers:
Relationship between Delivered Pressure, Aerosol Concentration, and Spray Factor
Generator
Pressure
psig
N*
CFU/L
Spray Factor
Geo Mean
Geo SD
Geo Mean
Geo SD
2
2.38E+03
1.32E+00
7.36E-07
1.70E+00
Aeromist
10
Aeromist
15
2
1.67E+03
1.56E+00
1.71E-06
1.80E+00
Aeromist
20
20
6.62E+02
5.55E+00
3.79E-06
2.59E+00
Collison
20
3
5.96E+03
1.43E+00
3.75E-07
1.32E+00
Collison
25
21
1.29E+03
7.48E+00
4.16E-07
2.44E+00
Collison
30
9
4.11E+00
2.17E+00
2.09E-07
2.14E+00
Micropump
20
12
9.37E+01
1.06E+01
2.67E-08
1.02E+01
*N, number of replicates
Table 5. Summary spray factor and aerosol concentration data using fresh F. tularensis LVS and
the Aeromist, Collison, and Micropump Nebulizers. Data are presented as geometric means and
standard deviations (see method summary in section 3.1.6, 3.1.7, 3.1.8). These data are presented
graphically in Figure 15. For this milestone, adjustments based on available data were made by
the TVDC and LBERI teams in real-time as to how to proceed. Replicates were not originally
planned with statistical analyses in mind, rather, decisions were made to conduct further testing
on generator/pressure combinations that demonstrated promising results with respect to
bioaerosol optimization. It should be noted here, that further testing was ceased with LVS
following LBERI’s clearance to work with SCHU S4 (again, a critical step for this milestone);
this is reflected in the small number of replicates at 10 and 15 psig delivered for the Aeromist as
well as 20 psig delivered to the Collison. Data location: \\Saturn\absl3\Agent and Study Specific
Data and Miscellaneous Documents\STUDY SPECIFIC DATA\FY06-078_TUL-03\TUL-03 data
files\FY06-078_TUL-03_Cumulative MS3 data.xls
Frozen F. tularensis SCHU S4
Page 34 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 35 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Spray Factors vs CFU/L: Frozen Francisella tularensis SCHU S4
Testing with the Aeromist and Collison Nebulizer
6.0
CFU/L (log10)
5.0
4.0
3.0
2.0
1.0
0.0
-4.5
-5.0
-5.5
-6.0
-6.5
-7.0
-7.5
-8.0
Spray Factor (log10)
Aeromist, 7.5 psi
Aeromist, 10 psi
Collison, 25 psi
Figure 16. Cumulative spray factor versus aerosol concentration data using frozen F. tularensis
SCHU S4 and the Aeromist and Collison Nebulizers. Data are presented as log10 values (see
method summary in section 3.1.6, 3.1.7, 3.1.8). Tested nebulizers and associated delivered
pressures are presented in the accompanying legend. A decrease in spray factor is proportional to
a decrease in bioaerosol efficiency (i.e., bioaerosol efficiency decreases from left to right). These
data demonstrate that the Aeromist operated at a delivered pressure of 10 psi performed best with
frozen SCHU S4 among the four tested nebulizers. The Collison operated under a normal
operating condition of 25 psi performed similar to the Aeromist at 10 psi. The Aeromist operated
at a delivered pressure of 7.5 psi performed poorly with frozen SCHU S4 likely due to the
inability to effectively generate particles at this low pressure. Replicates were minimal for frozen
SCHU S4 testing based on data from frozen LVS testing and due to the knowledge that fresh
cultures were chosen for future bioaerosol optimization. Data location: \Saturn\absl3\Agent and
Study Specific Data and Miscellaneous Documents\STUDY SPECIFIC DATA\FY06-078_TUL03\TUL-03 data files\FY06-078_TUL-03_Cumulative MS3 data.xls
Page 35 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 36 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Table 6. Frozen SCHU S4 Testing with the Aeromist and Collison Nebulizers:
Relationship between Delivered Pressure, Aerosol Concentration, and Spray Factor
Generator
Pressure
psig
N*
CFU/L
Spray Factor
Geo Mean
Geo SD
Geo Mean
Geo SD
Aeromist
7.5
3
1.86E+01
1.51E+00
1.02E-07
1.45E+00
Aeromist
10
3
1.74E+02
1.78E+00
1.09E-06
1.82E+00
Collison
25
3
9.63E+01
1.56E+00
6.22E-07
1.86E+00
*N, number of replicates
Table 6. Summary spray factor and aerosol concentration data using frozen F. tularensis SCHU
S4 and the Aeromist and Collison Nebulizers. At this point in the milestone, TVDC and LBERI
teams narrowed further bioaerosol testing down to the Aeromist and Collison nebulizers. This
decision was based on the overall good viability retention of both units in conjunction with
efficient spray factors. Here, the number of replicates (n=3) was small because it was concluded
previously that frozen bacteria as a starting generator suspension material was suboptimal for
bioaerosols. Data are presented as geometric means and standard deviations (see method
summary in section 3.1.6, 3.1.7, 3.1.8). These data are presented graphically in Figure 16. Data
location: \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous Documents\STUDY
SPECIFIC DATA\FY06-078_TUL-03\TUL-03 data files\FY06-078_TUL-03_Cumulative MS3
data.xls
Page 36 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 37 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Fresh F. tularensis SCHU S4
Figure 17. Cumulative spray factor versus aerosol concentration data using fresh F. tularensis SCHU S4
and the Aeromist, Hospitak, and Collison Nebulizers. Data are presented as log10 values (see method
summary in section 3.1.6, 3.1.7, 3.1.8).
Tested nebulizers and associated delivered pressures are
presented in the accompanying legend. A decrease in spray factor is proportional to a decrease in
bioaerosol efficiency (i.e., bioaerosol efficiency decreases from left to right). Here, testing with the
Hospitak nebulizer was initiated due to the termination of Aeromist production by the manufacturer
(described previously). These data demonstrate that that the Aeromist, Hospitak and Collison nebulizers
performed consistently with one another, a result that, based on stability data, was expected. Data
location: \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous Documents\STUDY
SPECIFIC DATA\FY06-078_TUL-03\TUL-03 data files\FY06-078_TUL-03_Cumulative MS3 data.xls
Page 37 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 38 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Fresh SCHU S4 Testing with the Aeromist, Hospitak, and Collison Nebulizers:
Relationship between Delivered Pressure, Aerosol Concentration, and Spray Factor
Generator
Pressure
psig
N*
CFU/L
Spray Factor
Geo Mean
Geo SD
Geo Mean
Geo SD
Aeromist
10
13
1.21E+02
8.89E+00
1.14E-07
2.27E+00
Hospitak
10
27
1.73E+03
1.60E+01
1.11E-07
4.03E+00
Collison
25
35
1.03E+03
1.54E+01
1.08E-07
2.41E+00
*N, number of replicates
Table 7. Summary spray factor and aerosol concentration data using fresh F. tularensis SCHU
S4 and the Aeromist, Hospitak, and Collison Nebulizers. Data are presented as geometric means
and standard deviations (see method summary in section 3.1.6, 3.1.7, 3.1.8). These data are
presented graphically in Figure 17. Replicates for the Aeromist operated at a delivered pressure
of 10 psig were minimal due to ceased production of the unit by the manufacturer. From these
results, the decision was made that either the Hospitak or Collison could be used for animal
challenges, though more weight was placed on the Collison due to its historical use, published
literature, and consistent product availability over time from the manufacturer. Data location:
\\Saturn\absl3\Agent and Study Specific Data and Miscellaneous Documents\STUDY SPECIFIC
DATA\FY06-078_TUL-03\TUL-03 data files\FY06-078_TUL-03_Cumulative MS3 data.xls
Particle Size Data
Table 8 summarizes particle size data, as determined using the APS, for the Aeromist, Collison,
and Micropump nebulizers. Due to poor aerosol production, similar data were not obtained for
the sparging generator.
Note: Particle size is inherent to the device; delivered pressure has very little effect on the size,
particularly at the pressure ranges utilized. Additionally, most of the particles generated are from
the diluent itself and not the bacterium; therefore, particle size is not dependent on the use of LVS
vs. SCHU S4.
Table 8. Particle Size Data for the Aeromist, Hospitak,
Collison, and Micropump Nebulizers
MMAD
Nebulizer
m 
GSD
Page 38 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Version: 2.0
Page 39 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Aeromist
1.60
1.56
Hospitak
2.22
1.71
Collison
1.49
1.57
Micropump
1.49
1.67
Accepted Date:4/23/09
Table 8. Particle size data for the Aeromist, Hospitak, Collison, and
Micropump Nebulizers. During bacterial aerosol generation, representative
samples were measured using an aerodynamic particle sizer (APS), a device
that, through a sophisticated laser system, measures the drop sizes produced
by the generator. Briefly, during testing, a 30-second sample from the
exposure line was pulled through the instrument; readings were automatic
and the device was calibrated using monodisperse polystyrene latex particles
prior to use. Results demonstrate similar performance between the four
nebulizers, though the Hospitak generated slightly larger particles (P<0.05, ttest). For our purposes, the acceptable size was defined as < 4 m MMAD
since particles in this range demonstrate high deep lung deposition rates. For
these types of tests, it should be noted that particle size is inherent to the
device; delivered pressure has very little effect on the size, particularly at the
pressure ranges we worked within. Additionally, most of the particles
generated are from the diluent itself and not the bacterial presence; therefore,
particle size during these tests was not dependent on the use of LVS versus
SCHU S4. MMAD, mass median aerodynamic diameter; GSD, geometric
standard deviation. Data location: \\Saturn\absl3\Agent and Study Specific
Data and Miscellaneous Documents\STUDY SPECIFIC DATA\FY06078_TUL-03\TUL-03 data files\FY06-078_TUL-03_Summary particle size
data.xls
4.2
4.2.1
Interpretation
Comparison of Solid and Liquid Media for the Culture of F. tularensis
Two solid media failed to support the growth of LVS (GCAT and CA). CAH supported only
poor growth of LVS. Several media, CHAH-2, CHAH-10, CHA, and BCGAH) presented fair
to good growth of LVS (qualitatively based on morphology, size, and concentration), but the
colony morphology was atypical primarily due to coloration; specifically, colonies were small,
in few number, and grew pink, green, and brown rather than the expected blue/gray
pigmentation. Several solid media provided fair to good growth of LVS with expected colony
morphology (BCGA, SBCGA, and CHAB). SBCGA, BCGA, and CHAB also supported good
percent recovery of LVS when compared to other media types.
Page 39 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 40 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Based on these studies alone, it was concluded that CHAB was the optimal solid agar medium
for F. tularensis LVS growth. In studies conducted post-optimization of agar medium,
however, it was noted that F. tularensis growth on CHAB was inconsistent, when present, and
growth took a significant amount of time (minimum of 72h). Certain lots of CHAB supported
growth when others would not, even though preparation and quality control procedures were
consistent. Taken together, CHAB was concluded to be sub-optimal in context of data
management. Contrary to this, growth on BCGA was consistently present following 48h of
incubation (Figure 18). Based on these observations, LBERI decided to use BCGA for the
majority of the bioaerosol optimization testing (including bacterial proliferation and
generator/AGI suspension cultures).
Figure 18. Growth of F. tularensis LVS on CHAB (left) and BCGA (right). As depicted here,
LVS growth was optimal on BCGA after 48h of incubation (see method summary in section
3.1.1) Data location: \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY06\FY06-078_TUL-03\LVS Growth
characterization\48h on CHAB.jpeg and \48h on BCGA.jpeg
MCPH broth provided poor growth of LVS; less than 1000 CFU/mL was observed. MMHB
grew LVS to approximately 2.1 x 105 CFU/mL. Bacterial titers in three of the five media (CB,
CBH, and MCPHI) were greater than 1.0 x 107 CFU/mL. One flask of MCPHI demonstrated
no growth while the second presented a bacterial titer greater than 1.0 x 107 CFU/mL. The
reasons for this were inconclusive. Because of the ease of preparation for CB and CBH as
Page 40 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 41 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
compared to MCPHI (i.e., one or two components, respectively, versus the seven needed for
MCPHI), the use of CB or CBH was concluded to be optimal. CB and CBH supported near
identical F. tularensis LVS growth characteristics; however, CB appeared as a clear solution
which allowed for easy optical density titer characterization. It was therefore decided that CB
was the optimal broth medium for F. tularensis LVS culture and CB would be used for SCHU
S4 growth, until SCHU S4 growth optimization is completed under Milestone 9, the
Qualification of SCHU S4 bioaerosols.
4.2.2
Generator and Impingement Solution Optimization
Based on LVS viability and culturability, data from these studies demonstrated that the optimal
medium and storage conditions for use during F. tularensis (LVS tested; SCHU S4 assumed)
bioaerosol studies was brain heart infusion broth at room temperature.(see Figure 9) BHIB is a
nutrient-rich medium capable of maintaining bacterial viability more than minimal media such
as PBS and 1% peptone. Though Chamberlain’s broth contains sufficient nutrients to allow for
bacterial growth, it did not demonstrate the capacity to stabilize a F. tularensis suspension
under sub-optimal conditions. During bacterial aerosolization, the liquid droplet in which a
bacterium is contained significantly desiccates to the point where the resulting particulate
delivered to the exposure chamber is likely composed only of the organism and the dried
remnants of the suspension medium. It is hypothesized that a nutrient-rich medium such as
BHIB forms a protective coating on the bacterium upon desiccation. Solutions such as PBS and
1% peptone may not do this to the same degree. BHIB was chosen and was used for the
generator and impinger solutions for all ensuing bioaerosol tests throughout Milestone 3.
4.2.3
Comparison of Pre- and Post-Spray F. tularensis Generator Suspension
Concentrations
Production of a bioaerosol via compressed air generation, as was performed throughout these
studies, places a high degree of stress on bacteria within a suspension. During aerosolization,
significant shear forces are generated as a result of collisions between bacterial particles and
between bacteria and the exposure system (i.e., the generator walls, exposure line, etc.).
Though these forces are unavoidable, our hypothesis was that they could be minimized in order
to optimize bacterial viability and, consequently, the bioaerosol. The objective was to screen
multiple bioaerosol generators for reproducible, bacterial viability in the pre- and post-sprays.
The decision to conduct these experiments in Milestone 3 was not finalized until generator
down-selection for SCHU S4 bioaerosol optimization was made to the Aeromist, Hospitak, and
Collison Nebulizers. This decision was based primarily on spray factors, repeatability and ease
of use as discussed in the sections below. Results from these tests demonstrated that bacterial
viability/culturability in the Aeromist suspensions was highly dependant upon pressure
delivered to the generator; specifically, as delivered pressure increased, bacterial viability in the
Page 41 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 42 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
suspension decreased. The Hospitak was tested only at a delivered pressure of 10 psig;
however, because of its similarity to the Aeromist, it can be hypothesized that results would be
similar at higher delivered pressures. This phenomenon was not observed in the Collison for
which a relatively high pressure (25 psig) was tested. Though bacterial viability and
culturability decreased after each Collison run, it was not as marked as observed with the
Aeromist even though a higher pressure was used to generate the bioaerosol with the Collison (a
requirement as specified by the Collison manufacturer). The observed difference between the
two types of generators was likely due to the manufacturing of the units; the Aeromist and
Hospitak nebulizers utilized one jet for delivery of compressed air whereas the Collison evenly
distributed the supplied air across three jets. This distribution decreased the shear forces during
operation. To clarify, each jet of the 3-jet Collison MRE exhibited 1/3 of the total pressure
provided to the unit. Results from these tests demonstrated that decreased pressure delivery to
the aerosol generators increased bacterial viability. Though valuable information, this attribute
cannot predict how the bioaerosol will performed since many other factors such as come into
play following bacterial nebulization. This is discussed in the following section.
4.2.4
Bioaerosol Optimization
Frozen F. tularensis LVS
Based on spray factor values, data from these studies demonstrated that optimal bioaerosol
performance for frozen LVS was established with the Aeromist operating at a delivered
pressure of 15 psig. The geometric mean calculated spray factor under these parameters was
2.00 x 10-6. Further interpretation, however, indicated that these data were misleading. As
discussed in the previous section, a significant decrease in viability/culturability was observed
in the Aeromist generator suspension at a delivered pressure of 15 psig. The spray factor is
calculated using post-bioaerosol run concentrations; a decreased titer falsely improves the spray
factor that would not otherwise be noted if very little or no change in titer were observed.
Though the calculated spray factor was optimal, the viability of the bioaerosol was sub-optimal.
Taking immune responses into consideration, it was undesirable to generate a bioaerosol
containing an increased concentration of non-viable (and potentially immune-stimulating)
bacteria at the expense of a better spray factor. The Aeromist operating at a delivered pressure
of 10 psig did not demonstrate this significant decrease in bacterial viability and presented spray
factor values comparable to the Collison at 20-25 psig. Poor bioaerosol parameters were
observed when testing the Aeromist at 5 psig. The geometric mean spray factor for these
experiments was 5.98 x 10-8. Though bacterial viability was maintained in the generator
suspension, the delivered pressure was not sufficient to physically aerosolize the agent, thereby
negating this approach for further F. tularensis bioaerosol optimization testing. The
Micropump and Sparging generators demonstrated poor bioaerosol efficacy when testing frozen
LVS. Geometric mean spray factor values were 3.34 x 10-8 and 6.01 x 10-9 for the two units,
respectively. Taken together, data indicated that the Aeromist operating under a delivered
Page 42 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 43 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
pressure of 10 psig was optimal for generation of frozen LVS. The Collison was comparable,
but several poor spray factors were noted for unknown reasons. In addition, we noticed early on
in the milestone that the 3-jet nozzle of the Collison required routine cleaning to prevent
clogging, an aspect that was deemed as less desirable for optimized testing.
Fresh F. tularensis LVS
Overall, data from these studies demonstrated that fresh LVS performed more efficiently as a
bioaerosol when compared to spray suspensions prepared from frozen LVS stock vials. Based
on spray factor values, the optimal bioaerosol performance was established with the Aeromist
operating at a delivered pressure of 20 psig. However, as discussed previously, this pressure
was concluded to be unacceptable due to the significant drop in viability/culturability in the
generator suspension. As was observed with frozen LVS testing, the Aeromist at 10 psig and
the Collison at 20-25 psig resulted in improved bioaerosol parameters (i.e., spray factors were
more efficient and viability retention was increased ). Geometric mean spray factor values were
7.36 x 10-7 and 4.16 x 10-7 for the two units, respectively. The Micropump nebulizer performed
poorly demonstrating a geometric mean spray factor of 2.67 x 10-8. Taken together, data
indicated that the Aeromist and Collison nebulizers operating under delivered pressures of 10
and 25 psig respectively were optimal for generation of fresh LVS.
There are no defined criteria for what is considered to be an optimal/acceptable spray factor.
LBERI considers any spray factor above 1 x 10-7 to be acceptable; values greater than or equal
to 1 x 10-6 could be considered optimal, though this is subjective and will vary between
microbes and bioaerosol procedures. Values less than 1 x 10-7 (i.e., in the 1 x 10-8 range) are
generally considered to be poor spray factors, again depending on the organism and aerosol
parameters.
Frozen F. tularensis SCHU S4
Results from these studies demonstrated that frozen SCHU S4 performed more efficiently
(based on spray factor values) as a bioaerosol when compared to frozen LVS. Optimal
bioaerosol performance was established with the Aeromist operating at a delivered pressure of
10 psig. The geometric mean spray factor value was 1.09 x 10-6. When a lower delivered
pressure was tested (7.5 psig), the performance decreased significantly from a geometric mean
spray factor of 1.09 x 10-6 to 1.02 x 10-7. This was due to the same reason that poor spray
factors were observed when testing frozen LVS at 5 psig: efficient bacterial aerosolization was
not established at this low-end parameter. The Collison, at 25 psig, performed slightly less
efficiently than the Aeromist with respect to spray factors, though better than frozen LVS under
the same conditions. The associated geometric mean spray factor value was 6.22 x 10-7.
Page 43 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 44 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Fresh F. tularensis SCHU S4
Results from these studies demonstrated that fresh SCHU S4 performed less optimally (i.e.,
spray factors were lower overall) as a bioaerosol when compared to fresh LVS. The reasons for
this are currently unknown, but can be hypothesized to be due to structural differences between
the two strains. Based on spray factor values, there was no difference between the Aeromist,
Hospitak and Collison nebulizers operated at 10, 10, and 25 psig, respectively, with fresh
SCHU S4. Additionally, data demonstrated consistency between the three units under these
operating conditions. The geometric mean spray factor for all three units was approximately 1.1
x 10-7.
Particle Size Data
Particle size data indicated that for all tested nebulizers, the droplet size generated (1.49 to 2.2
m) during bioaerosol runs was well within the respirable range (less than 4.0 m MMAD).
Conclusions
Based on cumulative results from LVS and SCHU S4 testing, the following parameters were
concluded to be optimal for F. tularensis SCHU S4 growth, bioaerosol generation, delivery,
maintenance, collection, and analysis:
 Challenge material (SCHU S4) will be cultured in Chamberlain’s broth medium as
follows: 24h at 37°C, 200 rpm, in the dark. The broth will be inoculated with a
normalized suspension (OD600 = 0.100 ± 0.010) of harvested SCHU S4 from a 48h
BCGA culture grown at 37°C. Because a significant titer drop (> 1log10) was
observed in the frozen stocks after six months of -80°C storage, the use of fresh
cultures was optimal for bioaerosol studies.
 The Collison 3-jet Nebulizer will be used to generate F. tularensis SCHU S4 in
challenge studies.
o Bacteria will be diluted into sterile BHIB for the generator suspension
o An output flowrate of 7.5 L/min will be maintained during bioaerosol runs.
Data from Milestone 3 demonstrated that this was, in general, established at a
delivered pressure of 25 psig for the Collison generator. Significant deviation
from this pressure may be indicative of a defective/clogged aerosol jet; if such a
deviation is observed, a new jet or new Collison unit will be employed.
Page 44 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 45 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
o



4.3
When compared to the Aeromist and Hospitak nebulizers, the Collison proved
to be slightly less efficient and less cost-effective, but overall, more consistent
and reliable. Additionally, the availability of historical data for the Collison far
outweighs bioaerosol optimization data for the Aeromist and Hospitak
nebulizers. Furthermore, the Aeromist is no longer commercially available.
For nonhuman primate exposures, F. tularensis bioaerosols will be delivered to the
head-only chamber via a 2-inch diameter, 24-inch length stainless steel inlet line as
originally described by Henderson, 1952. Relative humidity of the exposure system
will be maintained at 60-90%.
The bioaerosol will be collected into BHIB plus antifoam A using the AGI-4 (All
Glass Impinger). The flow to the impinger will be maintained at 5.0-5.5 liters per
minute.
Following bioaerosol runs, bacterial suspensions will be cultured onto BCGA plates to
enumerate the viable CFU. F. tularensis SCHU S4 proved to grow more consistently
and in a shorter period time on this medium versus other solid agar such as CHAB.
Quality Control


Use of Controls
o Bioaerosol runs: Prior to generation of bacteria for each day of testing, sterile
BHIB was aerosolized for 10 minutes and collected as described throughout
this report in order to verify sterility of the exposure line and aerosol equipment
(i.e., nebulizers, delivery lines, AGIs, etc.). This was defined as the negative
control.
Microbiological Media
o Quality control was performed on all prepared media according to SOP LBERI
4.1. Briefly, a representative sample of each media batch was allowed to
incubate at 37°C in a dark, humidified incubator for a minimum of 48h.
Following the allotted time, media were qualitatively assessed for the presence
of contaminants (e.g., turbidity, mycelia formation, etc.). All testing included
the following:
 In-house prepared culture plates
 Broth
 Dilution blanks
 Water
 Antifoam A
Page 45 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 46 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
o

5
Certificates of Analysis were received and filed for purchased media. Any
batch suspected of being compromised (i.e., cracked plates), were not used.
The manufacturer was immediately notified of such findings.
Replicates and Statistical Analysis
o When possible, bioaerosol testing with the different aerosol generator/F.
tularensis strain (i.e., LVS or SCHU S4) combinations was replicated a
minimum of three times over separate days between different scientists.
Instances for which this approach was not followed were due to time and/or
supply constraints, personnel availability, and/or at the request of the Study
Director. For the latter, the Study Director made decisions primarily based on
poor testing results or other factors such as difficulty in aerosol generator
handling.
o Statistical analyses were performed using Prism (GraphPad Software, Version
5.01). When necessary, data were transformed (e.g., log10 transformation)
such that parametric calculations could be performed. Reproducible
bioaerosols were defined as having no significant (p<0.05) difference between
replicates with respect to aerosol concentrations and spray factors.
o Acceptable bioaerosol criteria for GLP studies have yet to be defined. This will
be addressed in Milestone 9 of the Tularemia Vaccine Development Contract in
which F. tularensis SCHU S4 bioaerosols generated and sampled in the LBERI
head-only primate exposure system will be validated.
Deliverables Completed
There are no reagent deliverables associated with Milestone 3.
The MS#3 MSCR and 4 protocols (LBERI 1.1, LBERI 2.1, LBERI 3.1 and LBERI 4.1) for
generating optimal aerosolization are the deliverables.
Page 46 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Version: 2.0
Page 47 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
6
6.1
Accepted Date:4/23/09
Appendices
Appendix 1: Original Data Tables and Figures
Table/
Figure1
Title
Notebook
Location2
(Notebook #
and page
numbers)
Electronic Location2 (Full
Path & File Name)
T-1
Qualitative Comparison of Francisella
tularensis LVS Growth on Various Solid
Agar Media
n/a
\\Saturn\absl3\Agent and Study
Specific Data and Miscellaneous
Documents\STUDY SPECIFIC
DATA\FY06-078_TUL-03\TUL03 data files\Working Group\F.
tularensis media comparison.pdf
T-2
Quantitative Comparison of Francisella
tularensis LVS Growth on Various Solid
Agar Media
n/a
\\Saturn\absl3\Agent and Study
Specific Data and Miscellaneous
Documents\STUDY SPECIFIC
DATA\FY06-078_TUL-03\TUL03 data files\Working Group\F.
tularensis media comparison.pdf
Page 47 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Version: 2.0
Page 48 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Table/
Figure1
Title
Notebook
Location2
(Notebook #
and page
numbers)
Accepted Date:4/23/09
Electronic Location2 (Full
Path & File Name)
T-3
Quantitative Comparison of Francisella
tularensis LVS Growth in Various
Nutrient Broth Media
n/a
\\Saturn\absl3\Agent and Study
Specific Data\STUDY SPECIFIC
DATA\FY06-078_TUL-03\TUL03 data files\Working Group\F.
tularensis media comparison.pdf
T-4
Summary spray factor and aerosol
concentration data using frozen F.
tularensis LVS and the Aeromist,
Collison, Micropump and Sparging
Nebulizers
n/a
\\Saturn\absl3\Agent and Study
Specific Data \STUDY SPECIFIC
DATA\FY06-078_TUL-03\TUL03 data files\FY06-078_TUL03_Cumulative MS3 data.xls
[Worksheet entitled “Summary
Tables”]
T-5
Summary spray factor and aerosol
concentration data using fresh F.
tularensis LVS and the Aeromist,
Collison, and Micropump Nebulizers
n/a
\\Saturn\absl3\Agent and Study
Specific Data \STUDY SPECIFIC
DATA\FY06-078_TUL-03\TUL03 data files\FY06-078_TUL03_Cumulative MS3 data.xls
[Worksheet entitled “Summary
Tables”]
T-6
Summary spray factor and aerosol
concentration data using frozen F.
tularensis SCHU S4 and the Aeromist
and Collison Nebulizers
n/a
\\Saturn\absl3\Agent and Study
Specific Data \STUDY SPECIFIC
DATA\FY06-078_TUL-03\TUL03 data files\FY06-078_TUL03_Cumulative MS3 data.xls
[Worksheet entitled “Summary
Tables”]
T-7
Summary spray factor and aerosol
concentration data using fresh F.
tularensis SCHU S4 and the Aeromist,
Hospitak, and Collison Nebulizers
n/a
\\Saturn\absl3\Agent and Study
Specific Data \STUDY SPECIFIC
DATA\FY06-078_TUL-03\TUL03 data files\FY06-078_TUL03_Cumulative MS3 data.xls
[Worksheet entitled “Summary
Tables”]
T-8
Particle size data for the Aeromist,
Hospitak, Collison, and Micropump
n/a
\\Saturn\absl3\Agent and Study
Specific Data \STUDY SPECIFIC
Page 48 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Version: 2.0
Page 49 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Table/
Figure1
Title
Notebook
Location2
(Notebook #
and page
numbers)
Nebulizers
Accepted Date:4/23/09
Electronic Location2 (Full
Path & File Name)
DATA\FY06-078_TUL-03\TUL03 data files\FY06-078_TUL03_Summary particle size data.xls
F-1
Standard microbiological quadrant streak
method for bacterial cultures
n/a
\\Saturn\absl3\Agent and Study
Specific Data \STUDY SPECIFIC
DATA\FY06-078_TUL03\Quadrant_streak_method_imag
e
F-2
Francisella tularensis LVS growth on
BCGA
n/a
\\Saturn\absl3\Agent and Study
Specific Data and Miscellaneous
Documents\STUDY SPECIFIC
DATA\FY06\FY06-078_TUL03\LVS Growth
characterization\48h on
BCGA.jpeg
F-3
LBERI NHP Head-Only Exposure
System
n/a
\\Saturn\absl3\Agent and Study
Specific Data and Miscellaneous
Documents\STUDY SPECIFIC
DATA\FY06\FY06-078_TUL03\TUL-03 data files\Exposure
line schematic.ppt
F-4
Aeromist Nebulizer
n/a
\\Saturn\absl3\Agent and Study
Specific Data and Miscellaneous
Documents\STUDY SPECIFIC
DATA\FY06\FY06-078_TUL03\TUL-03 data files\Aeromist
nebulizer\Aeromist_nebulizer_ima
ge
F-5
Collison 3-jet Nebulizer
n/a
\\Saturn\absl3\Agent and Study
Specific Data and Miscellaneous
Documents\STUDY SPECIFIC
DATA\FY06\FY06-078_TUL03\TUL-03 data files\Collison
generator\Collison_nebulizer_ima
ge
F-6
Aeroneb® Lab Micropump Nebulizer
n/a
\\Saturn\absl3\Agent and Study
Page 49 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Version: 2.0
Page 50 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Table/
Figure1
Title
Notebook
Location2
(Notebook #
and page
numbers)
Accepted Date:4/23/09
Electronic Location2 (Full
Path & File Name)
Specific Data and Miscellaneous
Documents\STUDY SPECIFIC
DATA\FY06\FY06-078_TUL03\TUL-03 data files\Micro
Pump\Micropump_nebulizer_imag
e
F-7
Hospitak Nebulizer
n/a
\\Saturn\absl3\Agent and Study
Specific Data and Miscellaneous
Documents\STUDY SPECIFIC
DATA\FY06\FY06-078_TUL03\TUL-03 data files\Aeromist
nebulizer\Hospitak_nebulizer_ima
ge
F-8
Sparging Generator
n/a
\\Saturn\absl3\Agent and Study
Specific Data and Miscellaneous
Documents\STUDY SPECIFIC
DATA\FY06\FY06-078_TUL03\TUL-03 data files\Sparging
Generator\Sparging_generator_im
age
F-9
Effect of time, temperature, and media on
F. tularensis LVS viability/culturability
n/a
\\Saturn\absl3\Agent and Study
Specific Data \STUDY SPECIFIC
DATA\FY06-078_TUL-03\LVS
Growth\characterization\Stability\
Effect of time and temp on LVS
recovery_18April07.xls
F-10
Effect of delivered pressure to the
Aeromist Nebulizer on F. tularensis LVS
viability/culturability
n/a
\\Saturn\absl3\Agent and Study
Specific Data \STUDY SPECIFIC
DATA\FY06-078_TUL-03\TUL03 data files\FY06-078_TUL03_Cumulative MS3 data.xls
[Worksheet entitled “Aeromist
Pressures”]
F-11
Effect of delivered pressure to the
Aeromist Nebulizer on F. tularensis
SCHU S4 viability/culturability
n/a
\\Saturn\absl3\Agent and Study
Specific Data \STUDY SPECIFIC
DATA\FY06-078_TUL-03\TUL03 data files\FY06-078_TUL-
Page 50 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Version: 2.0
Page 51 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Table/
Figure1
Title
Notebook
Location2
(Notebook #
and page
numbers)
Accepted Date:4/23/09
Electronic Location2 (Full
Path & File Name)
03_Cumulative MS3 data.xls
[Worksheet entitled “Aeromist
Pressures”]
F-12
Effect of aerosol generation using the
Hospitak Nebulizer on F. tularensis
SCHU S4 viability and culturability
n/a
\Saturn\absl3\Agent and Study
Specific Data \STUDY SPECIFIC
DATA\FY06-078_TUL-03\TUL03 data files\FY06-078_TUL03_Cumulative MS3 data.xls
[Worksheet entitled “Hospitak
Pressures”]
F-13a
and b
Effect of delivered pressure to the
Collison Nebulizer on F. tularensis LVS
and SCHU S4 viability/culturability
n/a
\\Saturn\absl3\Agent and Study
Specific Data \STUDY SPECIFIC
DATA\FY06-078_TUL-03\TUL03 data files\FY06-078_TUL03_Cumulative MS3 data.xls
[Worksheet entitled “Collison
Pressures”]
F-14
Cumulative spray factor versus aerosol
concentration data using frozen F.
tularensis LVS and the Aeromist,
Collison, Micropump and Sparging
Nebulizers
n/a
\\Saturn\absl3\Agent and Study
Specific Data \STUDY SPECIFIC
DATA\FY06-078_TUL-03\TUL03 data files\FY06-078_TUL03_Cumulative MS3 data.xls
[Worksheet entitled “Summary
Graphs”]
Page 51 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Version: 2.0
Page 52 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Table/
Figure1
Title
Notebook
Location2
(Notebook #
and page
numbers)
Accepted Date:4/23/09
Electronic Location2 (Full
Path & File Name)
F-15
Cumulative spray factor versus aerosol
concentration data using fresh F.
tularensis LVS and the Aeromist,
Collison, and Micropump Nebulizers
n/a
\\Saturn\absl3\Agent and Study
Specific Data \STUDY SPECIFIC
DATA\FY06-078_TUL-03\TUL03 data files\FY06-078_TUL03_Cumulative MS3 data.xls
[Worksheet entitled “Summary
Graphs”]
F-16
Cumulative spray factor versus aerosol
concentration data using frozen F.
tularensis SCHU S4 and the Aeromist
and Collison Nebulizers
n/a
\\Saturn\absl3\Agent and Study
Specific Data \STUDY SPECIFIC
DATA\FY06-078_TUL-03\TUL03 data files\FY06-078_TUL03_Cumulative MS3 data.xls
[Worksheet entitled “Summary
Graphs”]
F-17
Cumulative spray factor versus aerosol
concentration data using fresh F.
tularensis SCHU S4 and the Aeromist ,
Hospitak, and Collison Nebulizers
n/a
\\Saturn\absl3\Agent and Study
Specific Data STUDY SPECIFIC
DATA\FY06-078_TUL-03\TUL03 data files\FY06-078_TUL03_Cumulative MS3 data.xls
[Worksheet entitled “Summary
Graphs”]
F-18
Growth of F tularensis LVS on CHAB
and BCGA solid media
n/a
\\Saturn\absl3\Agent and Study
Specific Data \STUDY SPECIFIC
DATA\FY06\FY06-078_TUL03\LVS Growth
characterization\48h on
CHAB.jpeg and \48h on
BCGA.jpeg
Page 52 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Version: 2.0
Page 53 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
6.2
Accepted Date:4/23/09
Appendix 2: Quality Assessment of Milestone Completion and Report
Assessment Criteria for Milestone Completion
Evaluation of Milestone Completion Report
Yes No N/A Comment
Has the Milestone Completion Report format been used and all
sections completed, including Milestone Summary, Milestone
Objectives, Methods Reagents & SOPs, Salient Original Data
Results Interpretation & Quality Control, Deliverables
Completed, and Appendices?
Does the Milestone Summary include the milestone’s goals,
milestone results, an overall interpretation of the milestone’s
data and conclusion?
Do Methods, Critical Reagents and SOPs include summarized
methods and details necessary to re-perform critical
experiments? A list of critical reagents? The completed table of
SOPs?
Are salient negative and positive original data included in the
Milestone Completion Report?
Has the Deliverables Table been completed?
X
Have the Appendices been completed?
Are the specific original data associations with experiments
clearly annotated in the “Salient Technical Data” section of the
Milestone Completion report?
X
X
Evaluation of Data included
Are the salient original data and results included in an
organized, easily interpretable format?
Is the rationale included?
Do tables and figures have legends and original data location
annotations?
X
X
UNM accepted SOPs 1, 2,
3, 4 4/23/09
X
X
The deliverables are the
MSCR and an SOP for
aerosolization; UNM
accepted SOPs 1, 2, 3, 4
and the MS3 MSCR on
4/23/09
Many of the data location
cited are identical and yet
the experiments show
different data. LBERI
added the worksheet name
in the file reference for
clarity
Yes No N/A Comment
X
X
X
Many of the data location
cited are identical and yet
the experiments show
different data. LBERI
Page 53 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 54 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Assessment Criteria for Milestone Completion
added the worksheet name
in the file reference for
clarity
Is the data interpretation clear?
Is the data storage location listed in Appendix 1 sufficient for
data retrieval in the future? (E.g. notebook numbers and pages,
electronic file locations including directory paths and file
names). Are prior data locations cross-referenced to final data
locations?
X
X
Is the data backed up electronically or in hardcopy notebooks?
X
Is the data storage location secured either in a locked fireproof
cabinet for hardcopy or on a server protected by firewall?
X
Has the data quality been assessed? How many replicates and
how reproducible is the data? Has statistical analysis been
performed on the data? What quality control has been utilized
by the subcontractor during the data generation and
assessment?
X
LBERI indicates all data
is stored electronically.
Per Trevor Brasel, Data
files are backed-up daily
and are located on a
secure server. Access is
limited to the data files by
management.
If a protein or peptide has been synthesized, how has the
protein or peptide sequence been verified? What percentage of
the sequences has been randomly verified?
X
If a genetic mutant has been made, how has the mutation been
verified e.g. DNA sequencing, PCR sequence verification?
How stable is the mutation? How has the impact of the genetic
mutation on the bacterial growth been assessed? What is the
sensitivity of the assay?
X
If an aerosol delivery system has been tested, how reproducible
is the delivery to the animal? Have sufficient animal numbers
been tested to determine reproducibility?
X
If UVA/psoralen treatment kills the bacteria but leaves them
metabolically active, how is killing assessed? How sensitive is
the assessment of killing? How is expression of bacterial
epitopes determined?
Do UNM and the subcontractor agree that the data supports the
scientific interpretation of the milestone?
X
X
Page 54 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 55 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Assessment Criteria for Milestone Completion
Evaluation of Deliverables, as outlined in the
Statement of Work
Yes No N/A Comment
Have Standard Operating Protocols have been written by
subcontractor, reviewed by UNM, revised by subcontractor as
requested, and accepted by UNM? The milestone completion
report will not be accepted by UNM until all the SOPs are
accepted by UNM.
X
Has the Milestone Completion Report been written by
subcontractor, reviewed by UNM, revised by subcontractor as
requested, and accepted by UNM?
Has data from the milestone been submitted by the
subcontractor, reviewed by UNM, data presentation revised by
the subcontractor as requested for clarity, and accepted by
UNM?
For deliverable reagents, have the minimum number of vials,
the minimum concentration, the minimum block size and the
minimum weight of tissue been mutually agreed by UNM and
the subcontractor?
X
X
X
Have bacterial strains and tissues been banked at the
subcontractor’s institution and backup stocks and aliquots been
received by UNM for long term storage? A minimum number
of vials of -20C /-80C bacterial stocks at specified
concentration in glycerol are stored at both institutions. A
minimum size paraffin block or minimum weight of
cryopreserved frozen tissues are stored at both institutions.
Evaluation of SOPs
Do SOPs contain standard sections e.g. purpose, list of
supplies and equipment required including vendors and model
numbers, reagent preparation, method, results expected,
description of data interpretation, criteria for accepting or
rejecting results, description of data storage location, date SOP
is in service, names of people who prepared and reviewed the
SOP?
Can an independent scientist read and understand the standard
operating procedure?
UNM accepted LBERI
SOPs # 1, 2, 3 and 4 on
4/23/09. The deliverables
include a protocol for the
optimized aerosolization.
MS3 MSCR and 4 SOP’s
accepted by UNM 4/23/09
The SOPs and MSCR are
the deliverables
X
Yes No N/A Comment
X
X
UNM accepted LBERI
SOPs # 1, 2, 3 and 4 on
4/23/09.
Page 55 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Version: 2.0
Page 56 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
6.3
6.3.1
Accepted Date:4/23/09
Appendix 3: Additional Data/Figures not included in the Text of the Milestone
Completion Report (Section 4)
Formulations for Solid Growth Media
Table A1. Formulations for Solid Growth Media (per liter)
Component
BCGA
SBCGA
CHA
BGCA
GCAT
CHAH2
CHAH10
CHAB
CA
CAH
Agar
Peptone
Lab Lemco
Peptone
Meat
extract
NaCl
L-cysteine
L-histidine
Glucose
Horse blood,
defibrinated
IsovitaleX®
Glucose
L-cysteine
HCl
Lglutamine
Adenine
Thiamine
pyrophosp
hate
Vitamin
B12
GuanineHCl
Fe(NO3)39H2O
p-aminobenzoic
acid
ThiamineHCl
12.5 g
20 g
3g
1.9 g
12.5 g
20 g
3g
1.9 g
-
-
14 g
-
-
-
-
14 g
-
14 g
-
1.1 g
1.1 g
-
-
-
-
-
-
-
-
5g
1g
1g
25 g
5g
1g
1g
25 g
-
1g
25 g
5g
25 g
-
-
-
-
-
50 mL
-
-
50 mL
50 mL
-
-
50 mL
-
-
-
-
10 mL
1g
-
-
-
-
10 mL
1g
-
-
-
-
0.26 g
-
1g
-
-
0.26 g
-
-
-
-
0.1 g
-
-
-
-
0.1 g
-
-
-
-
0.01 g
-
-
-
-
0.01 g
-
-
-
-
1 mg
-
-
-
-
1 mg
-
-
-
-
1 mg
-
-
-
-
1 mg
-
-
-
-
3 mg
-
-
-
-
3 mg
-
-
-
-
2 mg
-
-
-
-
2 mg
-
-
-
-
0.13
mg
-
-
-
-
0.13
mg
-
-
-
-
0.03
mg
-
0.05 mg
-
-
0.03
mg
-
-
Page 56 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 57 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Table A1. Formulations for Solid Growth Media (per liter)
Component
Cysteine Heart
Agar
Beef heart,
solids from
infusion
Agar
Glucose
Proteose
peptone
NaCl
L-cysteine
Hemoglobin
solution
Nutrient agar
Agar
Pancreatic
digest of
gelatin
Beef
extract
Papaic digest
of soybean
meal
Beef heart for
infusion
Chamberlain’s
medium
BCGA
SBCGA
CHA
BGCA
GCAT
CHAH2
CHAH10
CHAB
CA
CAH
-
-
51 g
-
-
51 g
51 g
51 g
-
-
-
-
10 g
-
-
10 g
10 g
10 g
-
-
-
-
15 g
10 g
-
-
15 g
10 g
15 g
10 g
15 g
10 g
-
-
-
-
10 g
-
-
10 g
10 g
10 g
-
-
-
-
5g
1g
-
-
5g
1g
5g
1g
5g
1g
-
-
-
10 g
10 g
-
-
2g
10 g
-
-
10 g
-
-
-
850 mL
12.8 g
-
-
-
-
-
-
-
-
-
4.3 g
-
-
-
-
-
-
-
-
-
2.8 g
-
-
-
-
-
-
-
-
-
-
10 g
-
-
-
-
-
-
-
-
-
3g
-
-
-
-
-
-
-
-
-
-
-
-
-
24.2 g
24.2 g
Table A1. Formulations for solid growth media tested for optimal F. tularensis growth. As shown, solid
agar media tested throughout Milestone 3 were diverse in components. Data location:
\\Saturn\absl3\Agent and Study Specific Data and Miscellaneous Documents\STUDY SPECIFIC
DATA\FY06-078_TUL-03\TUL-03 data files\Working Group\F. tularensis media comparison.pdf
Page 57 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Version: 2.0
Page 58 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
6.3.2
Accepted Date:4/23/09
Formulations for Liquid Broth Media
Table A2. Formulations for Liquid Broth Media (per liter)
Component
MMHB
MCPH
MCPHI
CB
CBH
NaCl
-
6.25 g
6.25 g
-
-
CaCl2
0.14 g
-
-
-
-
MgCl2-6H2O
0.21 g
-
-
-
-
Mueller Hinton broth
Acid hydrolysate of
casein
Beef extract
10.5 g
-
-
-
-
8.8 g
12.5 g
12.5 g
-
-
1.5 g
-
-
-
-
0.8 g
-
-
-
-
0.006 g
-
-
-
-
2 mL
-
10 mL
-
-
Starch
Ferric pyrophosphate
IsovitaleX®
Glucose
0.2 g
-
1g
-
-
L-cysteine HCl
0.052 g
0.1 g
0.26 g
-
-
L-glutamine
0.02 g
-
0.1 g
-
-
Adenine
Thiamine
pyrophosphate
Vitamin B12
2 mg
-
0.01 g
-
-
0.2 mg
-
1 mg
-
-
0.2 mg
-
1 mg
-
-
Guanine-HCl
0.06 mg
-
3 mg
-
-
Fe(NO3)3- 9H2O
P-aminobenzoic
acid
Thiamine-HCl
0.04 mg
-
2 mg
-
-
0.026 mg
-
0.13 mg
-
-
0.0006 mg
25 mg
25 mg
-
-
Bacto Yeast extract
-
6.25 g
6.25 g
-
-
K2HPO4
-
1.4 g
1.4 g
-
-
KH2PO4
Chamberlain’s
Medium
Hemoglobin
-
3.3 g
3.3 g
-
-
-
-
-
24.2 g
24.2 g
-
-
-
-
10 g
Table A2. Formulations for solid growth media tested for optimal F. tularensis growth. As with solid
agar media, tested liquid broths were diverse in their constituents. Data location: \\Saturn\absl3\Agent
Page 58 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 59 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
and Study Specific Data and Miscellaneous Documents\STUDY SPECIFIC DATA\FY06-078_TUL03\TUL-03 data files\Working Group\F. tularensis media comparison.pdf
Comparison of Aerosol Parameters between Separate Aeromist, Collison, and
Micropump Nebulizers
Figures A1-A4 present results from testing observed when comparing separate Aeromist,
Hospitak, Collison, and Micropump nebulizers to one another. All evaluations were performed
using sterile water as the generator solution.
Aeromist
Water Output vs Pressure
27
Water Output
( L/L air)
6.3.3
25
23
Nebulizer #4
21
Nebulizer #5
19
Nebulizer #6
17
15
0
5
10
15
20
25
30
35
Pressure (psig)
Figure A1. Water output versus delivered pressure for three separate Aeromist nebulizers.
Results demonstrated little difference between the units at the tested parameters. Data location:
\\Saturn\absl3\Agent and Study Specific Data and Miscellaneous Documents\STUDY SPECIFIC
DATA\FY06-078_TUL-03\TUL-03 data files\FY06-078_TUL-03_Summary Bioaerosol
Generator Output Testing.xls
Page 59 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 60 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Collison 3-Jet Nebulizer:
Water Output vs Pressure
100
Water Output
( L/ L air)
80
Nebulizer #4
60
Nebulizer #5
40
Nebulizer #6
20
0
0
5
10
15
20
25
30
35
Pressure (psig)
Figure A2. Water output versus delivered pressure for three separate Collison 3-jet nebulizers.
Results demonstrated significant differences between the units at lower delivered pressures,
though performances were very similar at standard operating conditions (20-30 psig). Data
location: \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous Documents\STUDY
SPECIFIC DATA\FY06-078_TUL-03\TUL-03 data files\FY06-078_TUL-03_Summary
Bioaerosol Generator Output Testing.xls
Page 60 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 61 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
Aeroneb® Micropump Nebulizer:
Water Output vs Pressure
Water Output
( L/L air)
100
80
Nebulizer #1
60
Nebulizer #2
40
Nebulizer #3
20
0
0
5
10
15
20
25
Pressure (psig)
Figure A3. Water output versus delivered pressure for three separate Micropump nebulizers.
Results demonstrated significant differences between the units at the tested parameters. Data
location: \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous Documents\STUDY
SPECIFIC DATA\FY06-078_TUL-03\TUL-03 data files\FY06-078_TUL-03_Summary
Bioaerosol Generator Output Testing.xls
Page 61 of 62
Tularemia Vaccine Development Contract
Contract No. HHSN266200500040-C and ADB Contract No. N01-AI-50040
Prime Contractor: University of New Mexico
Milestone Completion Report: MS #3
Institution: LBERI
Author: Trevor Brasel
MS Start
Date:2/23/2006
MS End
Date:8/31/2008
Report Date: 9/2/2008
Accepted Date:4/23/09
Version: 2.0
Page 62 of 62
Reviewed by : Barbara Griffith, 9/26/08, 10/23/08,
1/29/09, 3/3/09, 4/22/09, 6/26/09,
12/3/09,2/17/10,2/18/10
HospitakTM Nebulizer
Flow Rate vs Water Output
Water Output
(mL/min)
0.2
0.18
0.16
0.14
0.12
0.1
4
4.5
5
5.5
6
6.5
7
Flow Rate (L/min)
Output, mL/min
Figure A4. Flow rate versus water output versus for 25 separate Hospitak nebulizers. Results
demonstrated inconsistencies between different units. Data location: \\Saturn\absl3\Agent and
Study Specific Data and Miscellaneous Documents\STUDY SPECIFIC DATA\FY06-078_TUL03\TUL-03 data files\FY06-078_TUL-03_Summary Bioaerosol Generator Output Testing.xls
Page 62 of 62