EPA/600/R-12/596 | September 2012 | www.epa.gov/ord
United States
Environmental Protection
Agency
             On the Use of Bacillus
             thuringiensis as a Surrogate
             for Bacillus anthracis  in
             Aerosol Research

             TECHNICAL REPORT
Office of Research and Development
National Homeland Security Research Center

-------
Technical Report
On the Use of Bacillus thuringiensis
as a Surrogate for Bacillus anthracis
in Aerosol Research
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

RESEARCH TRIANGLE PARK, NORTH CAROLINA 27711
                   11

-------
                             Table of Contents
List of Tables	iv
List of Figures	iv
Disclaimer	v
Acronyms and Abbreviations	vi
Acknowledgments	vii
Executive Summary	viii
1.0   Introduction	1
2.0   Materials and Methods	2
  2.1   Sources of Secondary Data	2
     2.1.1    Source Selection Rationale	2
  2.2   Sources of Primary Data	2
3.0   Quality Assurance/Quality Control	3
  3.1   Quality Requirements	3
  3.2   Procedures for Determining Quality	3
  3.3   Primary Data Sources	3
4.0   Results and Discussion	5
  4.1   Background and Taxonomy	5
  4.2   Bt andBa  Spore Physical Properties	5
     4.2.1    Size	5
     4.2.2   Surface Morphology	7
     4.2.3    Hydrophobicity	7
     4.2.4   Density	7
  4.3   Effects of Irradiation	7
     4.3.1    Primary Data on Irradiation Effects	8
5.0   Summary	12
6.0   References	13
                                         in

-------
                                List of Tables

Table 4-1. Size Comparison of Hydrated Bacillus Spores (Carrera et al.; 2007)16	9


                                List of Figures

Figure 1. SEM images of Bt kurstaki strain spores and crystal proteins (spores indicated by black
arrows, crystal proteins by white arrows)	10
Figure 2. SEM images of viable (non-irradiated) Ba S (A) and Btk (D) at 2 jim	10
Figure 3. SEM images of irradiated Ba S (A) and Bt 9727 (B) (mislabeled as Btk} at 2 |im	11
Figure 4. SEM images of washed irradiated liquid spore preparations for Ba S (A) and Bt 9727
(B) (mislabeled as Btk).  The white arrow in (A) points to a faintly evident collapsed spore	11
                                         IV

-------
                                   Disclaimer

The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development's National Homeland Security Research Center, funded, directed and managed this
work. This report has been peer and administratively reviewed and has been approved for
publication as an EPA document. Note that approval does not signify that the contents
necessarily reflect the views of the agency.
Mention of trade names or commercial products does not constitute endorsement or
recommendation for use of a specific product.
This report was generated using some references (secondary data) that could not be evaluated for
accuracy, precision, representativeness, completeness, or comparability and, therefore, no
assurance can be made that the data extracted from these publications meet EPA's stringent
Quality Assurance requirements.
Questions concerning this document or its application should be addressed to:
        M. Worth Calfee, Ph.D.
        Decontamination and Consequence Management Division
        National Homeland Security Research Center
        Office of Research and Development
        U.S. Environmental Protection Agency
        Mail Code E343-06
        Research Triangle Park, NC 27711
        (919)541-7600
        calfee.worth@epa.gov

-------
                    Acronyms and Abbreviations
AFM
Ba
Ba$
Bg
Bs
Bt
Btk
BSL
CDC
DNA
EBI
ECBC
EPA
kGy
LLNL
LANL
MGB
NCAUR
NHSRC
PCR
QA
QC
SEM
USAMRIID
Atomic Force Microscope
Bacillus anthracis
Bacillus anthracis var Sterne
Bacillus atrophaeus
Bacillus subtilis
Bacillus thuringiensis
Bacillus thuringiensis var kurstaki
Biological Safety Level
Centers for Disease Control and Prevention
Deoxyribonucleic acid
Electron Beam Irradiation
Edgewood Chemical Biological Center
Environmental Protection Agency
kiloGray
Lawrence Livermore National Laboratory
Los Alamos National Laboratory
Microbial Genomic and Bioprocessing Research Unit
National Center for Agricultural Utilization Research
National Homeland Security Research Center
Polymerase  Chain Reaction
Quality Assurance
Quality Control
Scanning Electron Microscope
U.S. Army Research Institute of Infectious Diseases
                                        VI

-------
                            Acknowledgments
Contributions of the following individuals and organizations to the development of this
document are gratefully acknowledged:
Post-Graduate Research Contractor
Jenia A. Tufts, M.S, ESE, for performing the literature review and authoring this document.
United States Environmental Protection Agency (EPA)
M. Worth Calfee, Ph.D. (US EPA/ORD/NHSRC, Decontamination and Consequence
Management Division) for technical reviews and mentorship.
Dugway Proving Grounds
Angelo J. Madonna, Ph.D. (Chief, Microbiology Branch, West Desert Test Center, U.S. Army
Test & Evaluation Command) for providing Scanning Electron Microscope (SEM) images of
crystalliferous Bt.
Alion Science and Technology Corporation
SEM images and the Polymerase Chain Reaction (PCR) analysis of irradiated and non-irradiated
spore samples were completed under U.S. EPA contract #EPD10070 with Alion. This support
provided by Alion and their subcontractor, RTI International, is gratefully acknowledged.
Additionally, the authors would like to thank the peer reviewers for their significant
contributions. Specifically, the efforts of Gene Rice, Vipin Rastogi and Timothy Dean are
recognized.
                                        vn

-------
                             Executive Summary
This project supports the U.S. Environmental Protection Agency (EPA), through its National
Homeland Security Research Center (NHSRC), by providing relevant information pertinent to
the selection of a Bacillus anthracis spore surrogate for use in aerosol and reaerosolization
testing.
The primary focus of this effort is to investigate the physical properties of spores of B. anthracis
(Bd) and B. thuringiensis (Bf) that impact their movement in air and interaction with surfaces,
including size, shape, density, surface morphology, structure and hydrophobicity. Also
compared are pathogenicity, genetic relatedness, and the impact of irradiation on the physical
properties of both spore species.
Many physical features of Bt and Ba have been found to be similar and, while Bt is considered
non-pathogenic, Bt is in the same family as Ba. When prepared similarly, both microorganisms
share a similar cylindrical pellet shape, an aerodynamic diameter of approximately 1 |im (in the
respirable size range), and have higher relative hydrophobicities than other species. While spore
size, morphology, and other physical properties can vary among strains of the same species, the
variations can be due to sporulation conditions and may therefore be controllable. All Bt spores
may therefore not be representative of all Ba spores. Characterization of candidate surrogate
spores prior to experimental use is therefore critical to confirm that the characteristics of the
surrogate are as closely similar as possible to the characteristics of the pathogenic agent.
Both anecdotal and direct evidence of spore structural damage caused by electron beam and
gamma irradiation contraindicates the use of irradiated spores in aerosol testing as the structural
damage has unknown effects on the aerosol properties of these particles.  Based on the results
presented here, irradiated spores do not appear to be a good surrogate to predict the behavior of
non-irradiated spores, and irradiated spores should not be used for studies to investigate the
reaerosolization potential of spores.
Based on this review and comparisons of the physical properties of Bt and Ba,  the use of Bt as a
surrogate for Ba in aerosol testing appears to be well supported. Comparative  studies should be
performed to test the hypothesis that the two species will behave similarly when suspended in a
gas (as an aerosol).
                                           Vlll

-------
1.0  Introduction
The Category A biological agent Bacillus
anthracis (Bd) has the potential to produce mass
casualties and its spores are highly persistent in
the environment.1 The environmental and public
health impacts of reaerosolization of Ba
following an outdoor release in an urban
environment are not well characterized. There
are currently insufficient data in the literature to
adequately quantify, predict, or model the risks
associated with an outdoor release. Research to
address these gaps is an immediate need.  Before
research can begin, however, a surrogate must be
identified that is an appropriate model for Ba in
aerosol and reaerosolization testing that could be
released in the environment without concerns of
pathogenicity.  Bacillus thuringiensis (Bt) has
been discussed in the literature as an appropriate
                               r\
surrogate for Ba in aerosol testing and is
currently used in wide area outdoor spraying as
an insecticide.3  The purpose of this document is
to summarize the  similarities and differences
between Bt and Ba in the context of
reaerosolization, discuss the suitability of Bt as a
surrogate for Ba and factors that may impact use
of Bt in field testing.
Based on a review of the available literature and
comparisons of the physical properties of Bt and
Ba, the use of Bt as a surrogate for Ba appears to
                 r\
be well supported. A recent paper by Greenberg
et al.2 examined several Bacillus species as
potential surrogates for Ba in aerosol and water
testing.  The authors comprehensively reviewed
the literature for historical Ba surrogates and
compared the properties of those surrogates to
select the one that would most closely mimic Ba
during aerosol and water testing.
The properties examined by the authors included
the physical properties that impact the movement
of spores in air and water, including size, shape,
density, surface morphology, structure and
hydrophobicity. Also compared were
pathogenicity and genetic relatedness to Ba, a
potential indicator of similar characteristics,2 and
survivability of spores exposed to extreme
conditions.
Of the potential surrogates considered (which
included B. atrophaeus, B. cereus, B. subtilis, B.
thuringiensis, B. anthracis Sterne (Ba S),
B. megaterium, B. mycoides, and Geobacillus
spp.), Bt was identified as the most appropriate
for aerosol and water testing.  The authors based
this selection on the lack  of pathogenicity, the
high degree  of genetic relatedness, and, most
importantly, on the many physical similarities
between Bt and Ba.
Drawing on the literature, the following sections
outline the similarities between Ba and Bt,
justifying the selection of Bt as a surrogate for Ba
in aerosol and reaerosolization testing.  The key
physical parameters compared are size, surface
morphology, hydrophobicity and density. These
parameters are considered critical in influencing
the behavior of an aerosol, and of a particle
interacting with a surface.

-------
2.0  Materials and Methods
2.1  Sources of Secondary Data
Sources of data for this literature review were
unclassified peer reviewed journal articles,
conference proceedings and textbooks found by
searching citation databases including the EPA
Desktop Library, PubMed, Google Scholar, and
books.  Articles included research, reviews and
epidemiological studies.  Due to the dearth of
published data on the physical properties of these
spore species, the search was not limited by the
age of the data. However, books cited were
published within the last 7 years or are the most
recent edition of an established source.
2.1.1   Source Selection Rationale
Peer-reviewed sources are generally accepted as
reliable in the scientific community because the
study methods and results have been verified by
independent reviewers.  Important sources were
defined as those crucial to answering research
questions pertaining to the physical properties of
the target spore species because they pertain to
aerosol testing as well as pathogenicity and
genetics. Five general assessment factors were
considered in the evaluation of scientific and
technical information, as outlined in the EPA
General Assessment Factors for Evaluating the
Quality of Scientific and Technical Information
(EPA/100/B-03/001):
    •   Soundness:  The extent to which the
       scientific and technical procedures,
       measures, methods, or models employed
       to generate the information was
       reasonable for, and consistent with, the
       intended application.
    •   Applicability and Utility: The extent to
       which the information was relevant for
       the intended use.
    •   Clarity and Completeness: The degree of
       clarity and completeness with which the
       data, assumptions, methods, QA, and
       analyses employed to generate the
       information are documented.
    •   Uncertainty and Variability: The extent
       to which variability and uncertainty
       (quantitative and qualitative) related to
       results, procedures,  measures, methods,
       or models are evaluated and
       characterized.
    •   Evaluation and Review:  The extent of
       independent verification,  validation, and
       peer review of the information or of the
       procedures, measures, methods, or
       models.
2.2  Sources of Primary  Data
To determine what effects gamma irradiation
may have on exposed spores, EPA compared
samples of irradiated Bt 9727 and Ba S with
viable preparations of Bt kurstaki {Bt k) and Ba S
using polymerase chain reaction  (PCR) and
scanning electron microscopy (SEM).  The
irradiated samples were inactivated using an
exposure dose of 44 ± 4 kiloGray (kGy).
PCR is a standard microbiological technique to
identify microorganisms through the isolation,
amplification and analysis of DNA in a sample.
SEM is a widely used microscopy technique that
allows viewing and imaging of a sample on a
submicron scale.

-------
   3.0     Quality Assurance/Quality Control
Quality assurance (QA)/quality control (QC)
procedures were performed in accordance with
the EPA QA program. QA/QC procedures are
summarized below.

3.1  Quality Requirements

A criteria based approach was followed to select
the data and information to develop this report.
Although the  quality of the data used cannot be
quantified or qualified based on the source
selection and  assessment process, we have some
certainty that  the information is credible.

Data collected were from peer-reviewed journals,
conference proceedings and textbooks, all of
which were critically assessed through peer
critique, typically by experts in the field, prior to
publication. This peer review process usually
ensures a high level of quality in the reported
data.

3.2  Procedures for Determining Quality

Peer-reviewed publications were identified by
reviewing the editorial statements of the journal.
Research articles presenting data were
considered to be of sufficient quality if the data
were produced using accepted analytical methods
and techniques.  Accepted analytical methods are
previously documented, commonly utilized, and
recognized laboratory techniques, procedures,
measures,  methods, and models employed to
generate information. Information on quality
parameters considered for the literature review
includes the following:
   •   Source:  The characteristics of the source
       were considered as an indicator of
       quality.  Publications that were peer-
       reviewed and published in a reputable
       journal were generally regarded as
       reliable,  although, because the quality of
       peer-reviewed articles can vary greatly,
       care was taken to assess each article
       carefully as outlined in Section 2.2.
    •   Experimental Design: Articles that
       contained details of experimental design
       (quality control samples, statistically
       valid conclusions) indicating that
       appropriate scientific methodology was
       employed were regarded as high quality.
    •   Data Quality: The quality of data was
       assessed by examining the types and
       numbers of QC samples used, the
       consistency of results and other statistical
       analyses of data. Since most peer-
       reviewed articles do not include all
       QA/QC information and data generated
       during a study, a review of such
       information and data was not possible.
       Instead, sources were assessed for
       evidence of the use of controls, replicates
       and other QC samples as applicable to the
       study and the evaluation of variability
       and uncertainty in the data.
    •   Data Presentation: Concise, well-
       substantiated articles were sought and
       regarded as high quality. Poorly
       presented information (disorganized,
       rambling, and poorly supported by factual
       information) was viewed with extreme
       caution.
    •   Data Interpretation:  The authors'
       interpretation and conclusions were
       reviewed for scientific soundness.
       Results were reviewed to ensure they
       supported the conclusions presented, and
       other published sources substantiating the
       conclusions were sought out.

3.3  Primary Data Sources

The SEM images shown in Figure 1 were
obtained from Dugway Proving Ground rather
than the literature.  The PCR data and SEM
images discussed in Section 4.2.1 and shown in
Figures 2-4 were collected by EPA during efforts
to characterize spore preparations that had the
potential for use in aerosol testing in the planning
process.

-------
All of the data were collected using established
methods. QC samples for PCR analysis include
positive, negative and internal controls. Positive
controls ensure that the test is sufficiently
sensitive and have an expected positive result for
the target DNA sequence. Negative controls
ensure aseptic techniques and are expected to
contain no target DNA.  Internal controls are
present in all samples, including control samples,
to ensure that the method has the expected
sensitivity and that PCR inhibitors did not
negatively bias the results.
QA/QC procedures for SEM include detector
calibration, detector beam alignment and the
periodic measurement of a known standard.

-------
    4.0     Results and Discussion
4.1  Background and Taxonomy
Bt is widely distributed in nature and is
commonly used as an insecticide in the
management of mosquitoes, moths and black
flies.3 Like Ba, Bt is a gram-positive spore-
forming Bacillus that is in the B. cereus group.4
Bt is genetically similar to Ba, with Bt 9727, a
strain that has shown wound infectivity and
virulence in immunocompromised mice,5
demonstrating the highest homology.6 There
have also been some isolated reports of infection
caused by strains ofBt,5'7 and a recent mouse
study indicated that repeated low-dose aerosol
exposures can cause sub-chronic lung
inflammation in mice.8 However, while
exposure to Bt may cause skin and eye irritation,9
based on its use in the field and laboratory
studies,9 Bt is considered a non-pathogenic
Biosafety Level-1 (BSL-1) organism.
4.2  /fraud Ba Spore Physical Properties
The key physical parameters compared are size,
density, surface morphology, and
hydrophobicity. These parameters are
considered critical in influencing the behavior of
an aerosol, and how a particle interacts with a
surface. Identifying a surrogate spore in the
same size range as Ba is essential for aerosol and
reaerosolization studies because particle size is
the most important factor in the behavior of
aerosols, and all aerosol properties are dependent
upon this parameter.10 Particle density is a key
parameter because it impacts aerodynamic
diameter, particle settling velocity and inertial
properties.10 Surface morphology plays an
important role in particle adhesion because the
primary adhesive forces (van der Waals,
electrostatic, and capillary) are influenced by
particle surface features.10 For example, surface
roughness will determine how many points of
contact a particle makes with a surface, and
particle elasticity will play a part in particle
deformation onto a surface and an increase in the
adhesive force.10  As with morphology,
hydrophobicity influences particle-surface
interactions, as has been demonstrated
experimentally with hydrophobic B. cereus
spores which adhere more strongly to
hydrophobic than to hydrophilic surfaces.11'12
On a micro level, surface features such as
morphology and hydrophobicity may help
predict the likelihood that a spore will adhere to a
particular  surface type.  However, after the first
layer of spores has settled, the spore-on-spore
adhesive properties are  stronger than the
adhesive forces between the spores and the
surface they initially landed on, and a layer of
particles will be detached more easily as larger
agglomerates than as single particles from
surfaces.10
4.2.1   Size
Both Bt and Ba single spores are in the same size
range and  have similar aspect ratios and
diameters.13"16 In a recent study comparing the
physical dimensions of seven hydrated strains of
Ba spores  to seven other hydrated spores of
Bacillus species,16 B. subtilis (Bs) 1031 (source
laboratory not given) and B.atrophaeus (Bg)
ATCC B-385 spores were found have  smaller
dimensions than all Ba strains compared, while
B. cereus (Be)  ATCC 10702 and Bt 4055
(Microbial Genomic and Bioprocessing Research
Unit, NCAUR, Peoria, H,  USA) spores were
found to have the dimensions most similar to the
Ba species compared. All spores were prepared
under similar conditions using the same media to
ensure comparability. The Bs and Bg strains
tested were smaller in length  and diameter than
the Ba strains.  However, the Bt strain tested was
closer in both aspects with an average  of 1.42 x
0.83 jim.  The  average length and width of the
seven Ba strains measured were 1.42 x 0.83 jim,
although there was variation between strains.

-------
There were more between-strain variations in the
length of Ba spores (range of 1.23 to 1.67 jim)
than in the diameters (range 0.81 to 0.86 jim).
Table 4-1 outlines the results obtained by Carrera
etal.
16
In another study13 where more variation in length
than width was also seen, the authors prepared Bt
israelemis (ATCC 35646) andBg ATCC 9372
spores using both a plate-wash method and a
liquid sporulation approach. For Bt, the resulting
average length for plate-grown dried spores was
2.17 jim with an absolute deviation (D) of 0.18
|im and an average width of 0.937 jim (D =
0.049 |im). For solution-grown dried Bt, the
average length was 2.00 jim (D = 0.16  jim) and
the average width was 0.872 jim (D = 0.047 jim).
For Bg, plate-grown dried spore average length
was 1.68 |im (D = 0.13 jim) and width  was 0.647
|im (D = 0.028 jim); solution-grown dried Bg
spores averaged 1.79 jim (D = 0.19 jim) and
width 0.686 |im (D = 0.027 |im).  These results
indicate that there are differences in the size of
spores as determined by the two methods.13 The
authors also noted differences of up to a factor of
2 X in length and 1.5 X in width between the
smallest and largest spores measured in each
population.  These wide size distributions in
unique spore populations will have significant
impact on environmental fate and transport
models.13
Both Ba and Bt have been described as oval,17
cylindrical18 and ellipsoid16'18in shape,  depending
on the viewer.  Carrera et al. (2007)16 used the
shape of an ellipsoid to calculate the volume of a
spore. However, this calculation may
underestimate the volume as the actual  shape of
these spores is more like a cylindrical pellet.
However, to calculate the volume of a cylindrical
pellet one needs the radius at each end. In the
absence of radius data, the equation for the
equivalent diameter for a cylindrical pellet was
used to estimate the volume of a spore, and this
value was used to estimate the aerodynamic
                                               diameters. Equivalent diameter was calculated
                                               by
                                                    d =
0.7/J
9 \
*)\
                                                                                       (1)
                                                                                               19
                                               where h is the hydrated length (|im), and dis the
                                               hydrated diameter (|im).
                                               Using the physical dimensional measurements in
                                               Carrera et al. (2007)16 and the calculated average
                                               equivalent diameter of each spore species, the
                                               average aerodynamic size for the hydrated Ba
                                               species was calculated by
                                               d,. =
\PpCcde

  PoX
(2)
                                                                                          10
                                               where de is the equivalent diameter calculated
                                               above; pp is the density of the particle as reported
                                                                    90
                                               in Carrera et al. (2008);  Cc is the Cunningham
                                               slip correction factor as calculated by equation
                                               3.22 in Hinds (1999);10 p0 is standard particle
                                               density (1  g/cm3); and %is the dynamic shape
                                                                                 91
                                               factor, as calculated by Sturm (2011)  for the
                                               estimation of non-spherical particle transport in
                                               the human respiratory tract.

                                               Using the above-cited equation, the average
                                               aerodynamic size for the hydrated Ba spores is
                                               0.95 |im (ranging between 0.90 and 1.01 |im)
                                               and 0.91 |im for Bt4055.  The estimated
                                               aerodynamic diameters forBgB-385 andBs
                                               1031 are estimated to be 0.68 jim and 0.50 jim,
                                               respectively.
                                               Dried Bt spores expand by 4% under high
                                               humidity conditions and hydrated spores contract
                                               at low humidity,13 so the size range will vary
                                               depending upon environmental conditions.22
                                               This type of size  variation is also true for Bg,
                                               which was shown to shrink by as much as 12%
                                               from a hydrated state  when air-dried,13 and may
                                               also be true for Ba,  because it is a close relative.
                                               However, there are  no confirmation studies

-------
currently in the literature addressing this property
forBa.
4.2.2  Surface Morphology
Numerous atomic force microscopy (AFM)
image studies of several Bacillus species are
                        1 ^ 9^ 9R
available in the literature, '  "   detailing three-
dimensional views of the surface architecture of
different Bacillus spore species, size
distributions, changes in spore size due to
different hydration states, and changes in
ultrastructure due to different spore treatments or
preparations. However, because spore surface
properties are impacted by their preparation,
previously published AFM studies may not be
useful in planning large-scale field
reaerosolization studies.  New microscopy
images (AFM or SEM) should be obtained as
part  of surrogate spore characterization prior to
field testing.

4.2.2.1  Crystal-Producing^ Strains
One factor not considered by Greenberg et al.2 in
their discussion of Ba surrogate selection is the
production of crystal proteins in some Bt strains
that  are not present in Ba. While it is possible
these crystals adhere to Bt spores, impacting their
aerodynamic and other properties, the effect of
these crystals, shown in Figure 1, on
reaerosolization is currently unknown because it
this has not yet been studied. Accordingly, until
more is known about the impact of the crystals
on the movement or adhesion of Bt spores,
acrystalliferous Bt strains should be favored as
Ba surrogates.
4.2.3  Hydrophobicity
Hydrophobicity studies with hexadecane and
other hydrophobic solvents, conducted to
understand Ba and Bt spore behavior in the
aqueous phase better, have shown that both Bt
and Ba spores can bind to hydrophobic
solvents.29"31 However, the degree of
hydrophobicity varies both between species and
within strains.29  While these results indicate that
33
both Ba and Bt spores have potentially similar
hydrophobic properties in aqueous media,
whether this similarity is applicable to spore
resuspension under ambient conditions is
uncertain. Wshile these studies are not
determinative for the degree of hydrophobicity of
each species, they do indicate that both Ba and Bt
have higher relative hydrophobicities than other
Bacillus species.  Hydrophobicity for Bt and Ba
spores may be linked to the presence and makeup
of their exosporia.30'32
4.2.4  Density
When prepared in the same manner, the wet
spore density and volume of some Bt species are
in the same range as some strains of Ba16'20 The
average wet and  dry densities for seven strains of
Ba were determined to be 1.17 g/cm3 and 1.42
g/cm3, respectively.16 In the same study,  the
average wet density of Bt was measured as 1.17
g/m3 and the dry density was not determined.
4.3   Effects of  Irradiation
Irradiation is one of several methods used to
inactivate virulent Ba strains to prevent anthrax
infection resulting from occupational exposure34
and to sterilize contaminated samples and
equipment from  release sites.35  The use of
irradiated Ba surrogate spores has been
suggested to eliminate concerns of pathogenic!ty,
to facilitate approval for outdoor surrogate
releases,  and to better compare the behavior of
inactivated Ba to inactivated surrogates in
laboratory studies. For these reasons, the effects
of irradiation on  spore properties should be
understood.  A recent study35 on the effects of
electron beam irradiation (EBI) on Bg spores in
solution revealed that EBI of up to 20 kGy (2
megarads) resulted in structural damage, DNA
fragmentation, reduction in spore size, and other
effects. The changes seen in the EB irradiated
spores was dose-dependent, with increasing
damage seen at higher doses.35 While there are
currently no published studies or images of the
physical or structural effects of gamma

-------
irradiation, there are some anecdotal
observations in the literature from researchers
studying other aspects of Bacillus spore
irradiation as well as some unpublished data
suggesting gamma irradiation can cause
structural damage to spores similar to that
described for EB irradiated spores.
A study by CDC authors34 explored the gamma
radiation dose  needed to inactivate 0.1 mL
aliquots from suspensions of live virulent spores
of eight Ba strains, including Ames.  These
authors report  a dose of 25 kGy (2.5  megarads)
to achieve a 6 log reduction (99.9999%) of
spores of a concentration of 107 CFU/mL across
all strains tested. At 15 kGy (1.5 megarads), the
authors reported >99% reduction in the
suspension aliquots tested. The authors reported
that under microscopic examination, irradiated
spores "appeared irregularly shaped" and
concluded that gamma irradiation "induces
changes in structural components." Since this
was not the focus of their study, no further
details were provided. However, the authors also
noted an increase in chromosomal DNA
detection by real-time PCR, and hypothesize that
as a result of spore structural damage caused by
irradiation, DNA is more readily  accessible in
the suspension from damaged irradiated spores
than from non-irradiated spores.  These
observations are consistent with the unpublished
EPA research,  discussed below, indicating that
irradiation causes internal structural damage and
the evacuation of the spore core.  In another
earlier study36 that examined the reaction of
irradiated and non-irradiated Ba Ames spores to
monoclonal antibodies, the researchers noted
structural damage to spores following irradiation
at a dose of 30 kGy (3 megarads). No further
information was given by the researchers.
4.3.1   Primary Data on Irradiation Effects
Analysis of DNA purification wash fractions
from irradiated Ba S preparations by gel
electrophoresis showed substantial amounts of
DNA present.  The irradiated Bt 9727 spores
appeared more intact than the irradiated Ba S,
with little to no DNA visualized on the gels from
the washes.  No amplification was seen  on the
irradiated spores, although the Bt primers should
amplify both the Bt k and the Bt 9727.  This lack
of amplification could indicate that the quality of
the extracted DNA was too poor to readily
perform PCR on due to damage  from irradiation
or that the PCR reactions require further
optimization.
Figure 2, below, shows SEM images of viable
(non-irradiated) intact spores of Bt k and Ba S in
liquid.  In contrast, Figure 3 shows SEM images
of irradiated Ba S and Bt k spores in liquid. A
thick particulate layer in all of the irradiated
samples was likely comprised of cell debris and
expended media.  All images in Figures 2 and 3
were taken of liquids under vacuum at a scale of
2.0 |im. Figure 4  contains SEM images of the
irradiated spores following centrifugation-based
washes. This figure demonstrates that damaged
spores are still present after the wash steps and
are, therefore, likely to comprise a large fraction
of the preparation.

-------
Table 4-1. Size Comparison of Hydrated Bacillus Spores (Carrera et a/.; 2007)
                                                                 16
Type
Pathogenic
anthracis
Attenuated
anthracis
Surrogates
Neighbors
Species
B. anthracis 1087 (USAMRIID)
B. anthracis 1029 (USAMRIID)
B. anthracis Amesa
B. anthracis LAI (1088) (USAMRIID)
B. anthracis Sterne (ECBC)
B. anthracis A-Sterne (ECBC)
B. anthracis Pasteur 3132 (USAMRIID)
B. atrophaeus ATCC B-385
B. subtilis 1031 (Source unknown)
B. cereus ATCC 10702
B. thuringiensis 4055 (MGB, NCAUR)
B. megaterium CDC 684
B. stearothermophilus ATCC 12980
B. sphaericus ATCC 4245
Size Comparison of Hydrated Bacillus spores (Carrera et a/.; 2007)16
Hydrated Length (u.m)
Mean
(n=100)
1.67
1.26
1.52
1.23
1.49
1.55
1.23
1.22
1.07
1.55
1.61
1.73
1.74
1.07
+
0.20
0.13
0.19
0.08
0.17
0.15
0.11
0.12
0.09
0.16
0.18
0.16
0.14
0.10
Range
Min
1.66
0.92
1.14
1.09
1.09
1.23
0.96
1.05
0.89
1.20
1.07
1.35
1.45
0.82
Max
2.17
1.65
2.27
1.35
2.13
2.05
1.47
1.63
1.52
1.99
1.99
2.18
2.04
1.47
Hydrated Diameter (u.m)
Mean
(n=100)
0.85
0.81
0.81
0.81
0.85
0.86
0.81
0.65
0.48
0.90
0.80
0.88
0.98
0.85
+
0.09
0.06
0.06
0.07
0.08
0.07
0.07
0.05
0.03
0.07
0.07
0.06
0.07
0.06
Range
Min
0.53
0.69
0.70
0.67
0.66
0.67
0.65
0.58
0.41
0.76
0.59
0.69
0.76
0.74
Max
1.11
0.95
1.00
1.03
1.09
1.06
0.96
0.86
0.67
1.14
0.96
1.01
1.16
1.00
aBiological Defense Research Division, US Navy, Washington, D.C.

-------
 Title: Bt K crystals
 Comment: Fermentation sample
Date: 09-09-2003  Time: 16:06
    Filename: 030692.TIF
Title: Bt K crystals
Comment: Fermentation sample
Date: 09-09-2003 Time: 16:20
    Filename: 030693.TIF
Figure 1. SEM images of Bt kurstaki strain spores and crystal proteins (spores indicated by black arrows, crystal proteins

by white arrows)
Figure 2. SEM images of viable (non-irradiated) Ba S (A) and Bt k (B) at 2 jim
                                                               10

-------
Figure 3. SEM images of irradiated Ba S (A) and Bt 9727 (B) (mislabeled as Bt k) at 2 jim
       ^ w  "#  j
v    *•'     '•••'•    .
 v*   •
Figure 4. SEM images of washed irradiated liquid spore preparations for Ba S (A) and Bt 9727 (B) (mislabeled as Bt k).
The white arrow in (A) points to a faintly evident collapsed spore.
                                               11

-------
    5.0     Summary

                                                    comparison, irradiated spores do not appear to be
T,,        ..   •     ..  ,  , •   ,, •  ff  . •   ,  ,  ,        a good surrogate for non-irradiated spores.
The properties investigated in this effort included         &        &                     F
the physical properties that impact the movement
of spores in air and water, including size, shape,
density, surface morphology, structure and
hydrophobicity.  Also compared were
pathogenicity, genetic relatedness, and the
impact of irradiation on the physical properties of
both spore species.  Based on this review and
comparisons of the physical properties of Bt and
Ba, the use of Bt as  a surrogate for Ba in aerosol
testing appears to be well supported.
Comparative tests should be carried out to test
the hypothesis that the two species will behave
similarly when suspended in a gas (as an
aerosol).
While there are generally many features of Bt
and Ba that are similar, spore size, morphology,
and other physical properties are variable even
between strains of the same species.  The
variations can be due to sporulation conditions,
among other factors.13'37'38 For this reason, one
cannot conclude that all Bt spores are
representative of all Ba spores.  Prior to any
experimentation, it is critical to characterize the
surrogate to be used sufficiently and confirm that
the characteristics of the surrogate are adequately
similar to the characteristics of the live agent for
the intended use.
Both anecdotal and  direct evidence suggests that
spore ultrastructure  damage occurs as a result of
irradiation.   Evidence of spore structural damage
caused by EB and gamma irradiation calls into
question the use of irradiated spores for
reaerosolization experiments as the noted
structural changes have unknown effects on the
aerosol properties of these particles.  Based on
the results of the EBI study35 and the EPA
                                                12

-------
6.0     References
1.  Inglesby T. V., Henderson D. A., Bartlett J. G., Ascher M. S., Eitzen E., Friedlander A. M., Hauer
   I, McDade J., Osterholm M. T., O'Toole T., Parker G., Perl T. M., Russell P. K., Tonat K.
   Anthrax as a biological weapon: Medical and public health management. Journal of the American
   Medical Association. 1999;281 (18): 173 5-1745
2.  Greenberg D. L., Busch J. D., Keim P., Wagner D. M. Identifying experimental surrogates for
   Bacillus anthracis spores: A review. Investigative Genetics. 2010; 1(4): 1-12.
3.  Van Cuyk S., Deshpande A., Hollander A.,Duval N., Ticknor L., Layshock J., Gallegos-Graves L.,
   Omberg K. M. Persistence of Bacillus thuringiensis subsp. kurstaki in urban environments
   following spraying.  Applied and Environmental Microbiology. 2011;77(22):7954-61.
4.  Rasko D.A., Altherr M.R., Han C.S., Ravel J.: Genomics of the Bacillus cereus group of
   organisms. FEMSMicrobiology Reviews. 2005; 29(2):303-329.
5.  Hernandez E., Ramisse F., Ducoureau J-Pierre, Cruel T.,  Cavallo J-D. Superinfection: Case report
   and experimental  evidence of pathogenicity in immunosuppressed mice. Journal of Clinical
   Microbiology. 1998;36(7):2138-2139.
6.  Radnedge L., Agron P. G., Hill K. K., Jackson P. J., Ticknor L. O., Keim P., Andersen G. L.
   Genome differences that distinguish Bacillus anthracis from Bacillus cereus and Bacillus
   thuringiensis. Applied and Environmental Microbiology.  2003;69(5):2755-2764.
7.  Samples J. R., Buettner H. Ocular infection caused by a biological insecticide.  The Journal of
   Infectious Diseases. 1983;148(3):614.
8.  Barfod K. K., Poulsen S. S., Hammer M., Larsen S. T. Sub-chronic lung inflammation after airway
   exposures to Bacillus thuringiensis biopesticides in mice. BMC Microbiology. 2010;10:233.
9.  Siegel J. P. The mammalian safety of Bacillus thuringiensis-based insecticides. Journal of
   Invertebrate Pathology. 2001; 77( 1): 13 -21.
10. Hinds, W. C. Aerosol Technology: Properties, behavior, and measurement of airborne particles;
   Wiley: New York, 1999; pp 8, 50, 53, 54 and 141-145.
11. Ronner  U., Husmark U., Henricksson A.  Adhesion of Bacillus spores in relation to
   hydrophobicity. Journal of Applied Bacteriology. 1990; 69:550-556.
12. Husmark U., Ronner U. The influence of hydrophobic, electrostatic and morphologig properties on
   the adhesion of Bacillus  spores. Biofouling. 1992; 5:335-344.
13. Plomp M., Leighton T. J., Wheeler K. E., Malkin A. J. The high-resolution architecture and
   structural dynamics of Bacillus spores. Biophysical Journal. 2005;88(l):603-8.
14. Zolock R. A, Li G., Bleckmann C., Burggraf L., Fuller D. C. Atomic force microscopy  of Bacillus
   spore surface morphology. Micron. 2006;37(4):363-369.
15. Zandomeni R. O., Fitzgibbon J. E., Carrera M., Stuebing  E., Rogers I.E., Sagripanti J-L. Spore size
   comparison between several Bacillus species. Proceedings of the 2003 Joint Service Scientific
   Conference on Chemical & Biological Defense Research, Towson, MD. DOD publication ECBC-
   SP-018, Approved for unlimited distribution. Aberdeen Proving Ground, Maryland 21010-5424,
   September.
16. Carrera  M., Zandomeni R. O., Fitzgibbon J., Sagripanti J-L. Difference between the spore sizes of
   Bacillus anthracis and other Bacillus species. Journal of Applied Microbiology. 2007; 102(2): 303-
   312.
                                           13

-------
17. Giorno R., Bozue J., Cote C., Wenzel T., Moody K-S., Mallozzi M., Ryan M., Wang R., Zielke R.,
   Maddock J. R., Friedlander A., Welkos S., Driks A. Morphogenesis of the Bacillus anthracis
   spore. Journal of Bacteriology. 2007;189(3):691-705.
18. Logan A., Berkeley R.C.W. Identification of Bacillus strains using the API system. Journal of
   General Microbiology. 1984;130:18711882.
19. Branan, C.R. ed. Estimating equivalent diameters of solids. In: Rules of Thumb for Chemical
   Engineers. Fourth ed. Burlington, MA: Elsevier; 2005:409.
20. Carrera M., Zandomeni R. O., Sagripanti J-L. Wet and dry density of Bacillus anthracis and other
   Bacillus species. Journal of Applied Microbiology. 2008;105(l):68-77.
21. Sturm R. Modeling the deposition of bioaerosols with variable size and shape in the human
   respiratory tract - A review. Journal of Advanced Research. 201 l;In press.
22. Westphal A.J., Price P.B., Leighton T.J., Wheeler K.E. Kinetics of size changes of individual
   Bacillus thuringiensis spores in response to changes in relative humidity. Proceedings of the
   National Academy of Sciences. 2003;100(6):3461-3466.
23. Plomp M., Malkin A. J. Mapping of proteomic composition on the surfaces of Bacillus spores by
   atomic force microscopy-based immunolabeling. Langmuir. 2009;25(1):403-409.
24. Plomp M., Leighton T. J., Wheeler K. E., Pitesky M. E., Malkin A. J. Bacillus atrophaeus outer
   spore coat assembly and ultrastructure. Langmuir. 2005;21:10710-10716.
25. Plomp M., Leighton T. J., Wheeler K. E., Malkin A. J.  Architecture and high-resolution structure
   of Bacillus thuringiensis and Bacillus cereus spore coat surfaces. Langmuir. 2005;21(17):7892-
   7898.
26. Plomp M., Leighton T. J., Wheeler K. E., Hill H. D., Malkin A. J. In vitro  high-resolution
   structural dynamics of single germinating bacterial spores. Proceedings of the National Academy
   of Sciences.  104; 2007:9644-9649.
27. Malkin A., Plomp M., Leighton T. J., McPherson A., Wheeler K. E. Unraveling the architecture
   and structural dynamics of pathogens by high-resolution in vitro atomic force microscopy. In:
   Microscopy andMicroanalysis. Cambridge, United Kingdom; Cambridge University Press; 2006
   (11); 32-85.
28. Malkin, A. J.; Plomp, M., High-resolution architecture and structural dynamics of microbial and
   cellular systems: Insights from in vitro atomic force microscopy. In Scanning Probe Microscopy
   of Functional Materials. Kalinin, S. V.; Gruverman, A., Eds. Springer New York: 2011; pp 39-68.
29. Doyle R. J., Nedjat-Haiem F., Singh J. S. Hydrophobic  characteristics of Bacillus spores. Current
   Microbiology. 1984;10(6):329-332.
30. Koshikawa T., Yamazaki M., Yoshimi M., Ogawa  S., Yamada A., Watabe K., Torii M. Surface
   hydrophobicity of spores of Bacillus spp. Journal of General Microbiology. 1989;135:2717-2722.
31. Leishman O. N., Labuza T. P., Diez-Gonzalez F. Hydrophobic properties and extraction of
   Bacillus anthracis spores from liquid foods.  Food Microbiology. 2010;27:661-666.
32. Brahmbhatt T. N., Janes B. K., Stibitz E. S.,  Darnell S. C., Sanz P., Rasmussen S. B., O'Brien A.
   D. Bacillus anthracis exosporium protein BclA affects spore germination,  interaction with
   extracellular matrix proteins, and hydrophobicity. Infection and Immunity. 2007;75(11):5233-9.
33. Yul, J. R., Choi, J. Y., Li, S. M., Jin, B. R., and Je, Y. H. Bacillus thuringiensis as a specific,  safe,
   and effective tool for insect pest control.  Journal of Microbiology and Biotechnology. 2007;
   17(4);547_559.
34. Dauphin L., Newton B. R., Rasmussen M. V., Meyer R. F., Bowen M. D. Gamma irradiation can
   be used to inactivate Bacillus anthracis spores without compromising the sensitivity of diagnostic
   assays. Applied and Environmental Microbiology. 2008; 74( 14): 4427-443 3.

                                            14

-------
35. Fiester S.E., Helfmstine S.L., Redfearn J.C., Uribe R.M., Woolverton CJ. Electron beam
   irradiation dose dependency damages the Bacillus spore coat and spore membrane. International
   Journal of Microbiology. 2012; 579593.
36. Phillips P., Campbell M. Quinn R. Monoclonal antibodies against spore antigens of Bacillus
   anthracis. FEMSMicrobiology Immunology. 1988; 1(3): 169-78.
37. Baweja R. B., Zaman M. S., Mattoo A. R., Sharma K., Tripathi V., Aggarwal A., Dubey G. P.,
   Kurupati R. K., Ganguli M., Chaudhury N. K., Sen S., Das T. K., Gade W. N., Singh Y.
   Properties of Bacillus anthracis spores prepared under various environmental conditions. Archives
   of Microbiology. 2008;189(l):71-9.
38. Setlow P. Spores of Bacillus subtilis: Their resistance to and killing by radiation, heat and
   chemicals. Journal of Applied Microbiology.  2006; 101(3):514-525.
                                           15

-------
United States
Environmental Protection
Agency
PRESORTED STANDARD
 POSTAGE & FEES PAID
         EPA
   PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460

Official Business
Penalty for Private Use
$300

-------