oEPA
EPA 600/R-15/066 | March 2015 | www.epa.gov/research
United States
Environmental Protection
Agency
Distinguishing Intentional Releases
from Natural Occurrences and
Unintentional Releases of
anthracis:
LITERATURE SEARCH AND ANALYSIS
Office of Research and Development
National Homeland Security Research Center

-------
Disclaimer
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development, funded and managed this literature review in collaboration with the Defense
Threat Reduction Agency and the Department of Homeland Security under the
Battelle/Chemical, Biological, Radiological, and Nuclear Defense Information and Analysis
Center, Contract No. SP0700-00-D-3180, Delivery Order 0603, and Technical Area Tasks 794
and CB-11-0232. It has been reviewed by the Agency but does not necessarily reflect the
Agency's views. Reference herein to any specific commercial product, process, or service by
trade name, trademark, manufacturer or otherwise doesn not constitute or imply its endorsement,
recommendation, sale, or favoring by the EPA.
Questions concerning this document or its application should be addressed to:
Tonya Nichols
U.S. Environmental Protection Agency
National Homeland Security Research Center
Ronald Reagan Bldg., MC 8801RR
1200 Pennsylvania Ave., NW
Washington, D.C. 20460
202-564-2338
Nichol s.tonva@EPA. gov
Erin Silvestri
U.S. Environmental Protection Agency
National Homeland Security Research Center
26 W. Martin Luther King Drive, MS NG16
Cincinnati, Ohio 45268
513-569-7619
Silvestri.Erin@epa.gov
ii

-------
Table of Contents
Disclaimer ii
List of Tables	iv
List of Figures	iv
Acronyms and Abbreviations	v
Acknowledgments	vi
Executive Summary	vii
1	Introduction	1
1.1	Purpose	2
1.2	Methods	2
2	Bacillus anthracis in the Environment and Human Risk	3
2.1	Overview of B. anthracis	3
2.2	B. anthracis Persistence	3
2.2.1	B. anthracis Persistence Associated with Microenvironments	4
2.2.2	Presence of Endemic B. anthracis in the United States	5
2.3	Overview of the Classic B. anthracis Lifecycle	6
2.3.1	Initial Host Acquisition	7
2.3.2	Carcass, Sporulation, Spores, and Vegetative Survival at Carcass Sites	8
2.4	Conditions Characteristic of Outbreak Initiation	9
2.4.1	Spore Transport and Case Multiplication	9
2.4.2	Temporal Characteristics and Termination of Natural Outbreaks	12
2.5	History of Naturally Occurring Anthrax Outbreaks in Animals in the United States	13
2.6	Historical Incidence of Unintentional Human Anthrax	14
2.6.1	Unintentional Occupational Exposure in the United States Associated with
Contaminated Animal Products	16
2.6.2	Unintentional Occupational Exposure Associated with Laboratory Exposure and
Legacy Bioweapon Sites	17
2.6.3	Other Sources of Unintentional Human Cases in the United States	18
2.7	Vaccination Efficacy	19
3	Model assessing Bacillus anthracis Natural Outbreaks, Unintentional Releases or Intentional
Releases	21
3.1	Screening for the Likely Cause of a Bacillus anthracis Occurrence	23
3.2	Screening Category I: Unexpected genetic strain?	25
3.3	Screening Category II: Anomaly in Vaccine Efficacy?	27
3.4	Screening Category III: Site Anomalies Observed?	29
3.5	Screening Category IV: Anomalies in Epidemiology?	30
iii

-------
3.6	Screening Approach for Location-Specific Anomalies and Epidemiology	32
3.7	Conclusions	37
4	Implications for Clean-Up of Occurrences of B. anthracis and Identified Challenges	38
5	References	41
List of Tables
Table 1. Representative Human Anthrax Cases from Unintentional Exposures in the United States	15
Table 2. Strains of B. anthracis associated with livestock and wildlife outbreaks in the United States as
adapted from Kenefic et al. [86]	26
List of Figures
Figure 1. Summary of Bacillus anthracis lifecycle adapted from Schuch et al. [23]	7
Figure 2. Screening categories for rapidly evaluating likelihood that a B. anthracis occurrence in the
United States is intentional	25
Figure 3. Key to location-specific screening for intentional occurrences	34
Figure 4. Screening for intentional occurrences in an agricultural location OR occurrences in natural
locations (wildlife)	35
Figure 5. Screening for intentional occurrences in a laboratory location	36
Figure 6. Screening for intentional occurrences in processing plant or animal transportation locations.... 36
Figure 7. Screening for intentional occurrences in locations where animal products are in use	37
iv

-------
Acronyms and Abbreviations
AFCS
Advanced Facer Cancellar System
AVA
Anthrax vaccine absorbed
BDS
Biohazard Detection System
CDC
Centers for Disease Control and Prevention
CFU
colony forming units
ENM
ecological niche model
EPA
U.S. Environmental Protection Agency
FBI
Federal Bureau of Investigation
g
gram
mL
milliliter
MLVA
Multilocus variable tandem repeat analysis
NHSRC
National Homeland Security Research Center
PCR
polymerase chain reaction
SNR
Single nucleotide repeats
USD A
United States Department of Agriculture
USPS
United States Postal Service
WNA
Western North America
v

-------
Acknowledgments
The following individuals and organizations are acknowledged for their contributions and/or
review of this report:
U.S. Environmental Protection Agency
Charlena Bowling
Deborah McKean
Tonya Nichols
Eric Rhodes
Frank Schaefer, III
Erin Silvestri
Battelle, Contractor for the U.S. Environmental Protection Agency
Spatial Epidemiology and Ecology Research Laboratory, Department of Geography and
the Emerging Pathogens Institute, University of Florida
Jason K. Blackburn
Hadeco, LLC
Ted Hadfield
School of the Coast and Environment, Louisiana State University, Baton Rouge, LA
Martin Hugh-Jones
vi

-------
Executive Summary
The purpose of this report was to: (1) survey the scientific literature to determine the current state
of the science regarding the presence of Bacillus anthracis in the environment and outbreaks of
anthrax; (2) identify characteristics that would enable a screening of information about outbreaks
to rapidly assess whether an intentional release was a likely cause (in United States settings); and
(3) identify gaps in risk-related knowledge associated with B. anthracis events in the United
States. Being able to identify whether an event was natural (e.g., wildlife or livestock exposure to
a carcass site), unintentional (e.g., human exposure to a naturally contaminated animal hide), or
intentional (e.g., release of B. anthracis by an individual or group to deliberately cause harm)
could provide insight into the type of response that might be necessary following a
contamination event. The screening approach is not intended to be a tool for performing a
criminal investigation or for extensive public health assessment, although the information and
logic may inform both endeavors.
A review of literature was used to interpret the exposure aspects of the risk of human anthrax
associated with the environmental presence of B. anthracis. Until about 1950, the source of
human anthrax in the United States was primarily from occupational exposures to B. anthracis
spores from contaminated animal products. Outbreaks of human anthrax in the United States
declined significantly during the first half of the 20th century due to improved industrial hygiene,
the closing of domestic animal hair processing mills and tanneries removing the risk to U.S.
workers, and effective livestock vaccines. Today, in the United States, a majority of anthrax
cases remain cutaneous in nature, resulting from handling contaminated animal products or
contact with livestock in a narrowing range of enzootic areas.
B. anthracis is widely distributed across the central United States and the Mississippi Delta.
Human infection risk, while low, is most likely in enzootic areas of West Texas, North and South
Dakota, Minnesota, and Montana. Because of gaps in knowledge of B. anthracis lifecycle,
dormancy, loss/gain of virulence plasmids, and human susceptibility, the circumstances under
which human exposure to B. anthracis occurs and results in disease remains uncertain. An
outbreak of anthrax or the detection of Bacillus anthracis in an unexpected environment can
trigger local, state, or federal response, particularly when there is the potential for human
exposure. Response to the event and subsequent risk management depends in part on whether the
occurrence of B. anthracis is natural, unintentional, or intentional.
The screening approach presented here is designed to help the U.S. Environmental Protection
Agency rapidly and systematically determine the likelihood that a detection of or exposure to B.
anthracis is due to an intentional release. A limited number of parameters need to be considered
in this screening process. Given ready access to the necessary data, an initial evaluation can be
completed quickly, except for identifying the strain of the B. anthracis, which requires about 48
to 96 hours using current technologies. Use of the proposed systematic assessment of the
likelihood that an event was intentional might help indicate what type of response might be
needed.
vii

-------
1 Introduction
The U.S. Environmental Protection Agency (EPA) assists in the federal environmental response
to chemical, biological, and radiological releases. EPA is the lead federal agency for the
remediation of areas contaminated with these agents. EPA's responsibilities include threat
assessment, hazard identification, detection and reduction, environmental monitoring, and
planning and implementing site decontamination and remediation activities. Adequate and valid
knowledge is key to being prepared to effectively and efficiently execute these responsibilities.
The National Homeland Security Research Center (NHSRC), within EPA's Office of Research
and Development, provides knowledge to contain and mitigate contamination and to
decontaminate indoor and outdoor environments.
B. anthracis, the causative agent for anthrax, has been recognized as an important biothreat agent
for various reasons. The potential use of Bacillus anthracis as a biological warfare agent has
been evaluated by various nations including the United States, and has been found to meet key
factors making a biological pathogen or toxin suitable for a large-scale bio-warfare or terrorist
attack [1], As in a chemical attack, only individuals directly exposed to the released agent will be
affected because anthrax is not contagious. Unlike contagious biological agents, B. anthracis can
produce a controlled and targeted impact. In addition, B. anthracis spores are stable and can be
prepared and stored for long periods without losing viability. Inhalation anthrax, resulting from
an aerosolized release of spores, can be deadly. The Amerithrax attacks of 2001, in which spores
of Bacillus anthracis were sent by mail to targeted individuals, resulted in sickness and death.
For these reasons, anthrax attacks are viewed as an important biological threat.
In many areas of the world, including specific areas in the United States, anthrax is endemic. In
these areas outbreaks occur in animals and humans. Continued reporting of human anthrax
throughout the world, but especially in areas with increasing human incidence [2], coupled with
the potential use of B. anthracis spores as a biological weapon, leads to a need to understand
human infection risk. Human health risk associated with the presence of B. anthracis in the
environment in the United States, whether as background levels or as residual levels after an act
of bioterrorism [3, 4], depends on a variety of environmental and biological factors (e.g., soil
conditions or the pathogen's virulence). Section 2 of this report presents a literature review of the
baseline human health risk associated with B. anthracis in endemic areas. Section 2 also explores
the unintentional exposures for people who could have occupational exposure to B. anthracis and
for people who handle, process, or eat contaminated animal products.
EPA's role in response to a natural or unintentional event compared to an intentional release
would be different. Intentional releases are intended to cause deliberate harm and, when an
intentional release occurs, a crime scene is created. In a crime scene, additional agencies must
respond and different protocols must be employed in processing the scene. Section 3 presents a
screening approach to systematically evaluate whether an occurrence, defined in this report as
detection of B. anthracis in the environment or an outbreak of anthrax in human or animal
populations, is the result of a natural outbreak, an unintentional release, or an intentional release.
Differences that could be used to distinguish natural and unintentional releases from intentional
releases of B. anthracis or outbreaks of anthrax were organized into a key that could be used to
rapidly (in less than eight hours, given available data) screen for intentional occurrences.
1

-------
I.I Purpose
The purpose of this report was threefold: (1) survey the scientific literature on the presence of B.
anthracis in the environment and outbreaks of anthrax; (2) identify characteristics of an
occurrence that would enable a rapid assessment of whether an intentional release was a likely
cause (in United States settings); and (3) determine what gaps exist in risk-related knowledge
associated with detection of B. anthracis in the environment or outbreaks of anthrax in the
United States.
1.2 Methods
Information about B. anthracis from reports, peer-reviewed journal articles, books, and
government publications was collected in this literature review. Relevant journal articles and
peer-reviewed reports were initially identified using PubMed and Google Scholar. Additional
articles were also added to the search as recommended by experts in the topic areas. Data
describing the historical geographic distribution of B. anthracis in the environment or human
cases of anthrax were summarized along with the emerging understanding of the lifecycle of B.
anthracis.
2

-------
2 Bacillus anthracis in the Environment and Human Risk
2.1	Overview of B. anthracis
B. anthracis is a Gram-positive bacteria responsible for anthrax [5, 6], The name derives from
the Greek word for coal in recognition of the black, dead skin characteristic of the cutaneous
form of the disease [7], This rod-shaped, facultative anaerobe forms chains of boxcar-shaped
cells. Two plasmids, pXOl and pX02, are required for B. anthracis virulence. Three proteins
encoded by pXOl produce the anthrax toxin. Protein necessary for the capsule are produced by
pX02. The antiphagocytic properties of the capsule are important for virulence [8, 9], The strain
of B. anthracis, and presence or absence of pXOl and pX02 plasmids, provide valuable
virulence information. The presence/absence of plasmids largely determine the virulence of B.
anthracis-pXOl (coding for a tripartite toxin) and pX02 (coding for the capsule) for the strains
[10], Seventy-nine B. anthracis strains can be distinguished using amplified length
polymorphism, corresponded to specific geographic origins [10],
2.2	B. anthracis Persistence
The vegetative form of B. anthracis does not compete well with other microorganisms in nature
[11, 12] and therefore forms spores in response to adverse environmental conditions [5], The
spores enable viable B. anthracis to survive for decades under favorable conditions [13],
Generally, spores can resist prolonged desiccation, fluctuations in soil pH, temperature, pressure,
and ultraviolet and ionizing radiation [14], In pond water, B. anthracis has been reported to
survive 18 years [15], It survives in moist or dry soil for months to years (3 months to 36
months) [14-16],
Our understanding of the factors that affect persistence of B. anthracis in the environment is
evolving. As early as 1956, the persistence of B. anthracis in the environment was found to
depend on a neutral or alkaline pH, sufficient calcium, and adequate moisture [17], Further
studies have supported these findings and added high organic matter as an additional
characteristic of soils supporting the persistence of B. anthracis spores [13, 14, 18, 19], B.
anthracis persistence has been observed in these specific soil and environmental conditions in
the United States, Russia, Central Asia, South America, and South Africa [14], Poorly drained
depressions in the land can accumulate organic material and minerals from surrounding
"inhospitable" land to create patches in which B. anthracis may survive [14, 18], Generally, it
has been hypothesized that B. anthracis does not persist in calcium-poor soils, shale or
sandstone, or well-drained fields. Anthrax outbreaks are unlikely in areas with acidic soils; B.
anthracis prefers soils with a pH greater than 6 [14, 18, 20],
A debate has existed as to whether B. anthracis persists only as spores, or whether germination
of the spores and subsequent vegetative growth might account for persistence. "Incubator areas,"
areas of vegetative growth of the B. anthracis with subsequent sporulation, were hypothesized by
Van Ness [18] to be calcareous/alkaline depressions, rich in organic deposits, with water
standing for periods sufficient to kill grass; anthrax also arises in dry streams or
calcareous/alkaline hillside seep areas. Incubator areas were hypothesized to form only when
rain is sporadic or during dry periods. In an alternative hypothesis, the depressions and seeps
referenced by Van Ness [18] as "incubator areas" were referenced by Dragon and Rennie [13] as
3

-------
"storage areas" for spores. In their hypothesis, rather than reproducing, spores may be
transported, on organic matter, to low-lying storage areas, thereby concentrating the spores.
Drying of the pools would further concentrate the spores in the soil storage areas [13],
2.2.1 B. anthracis Persistence Associated with Microenvironments
Members of the Bacillus genus are primarily saprophytic, living in soil [21], In contrast, B.
anthracis has been considered an obligate pathogen, unable to reproduce outside of an infected
host in nature [22] due to loss of saprophytic capabilities attributed to a single nonsense mutation
inactivating a transcription regulator gene [23], Recent evidence suggests that conditions exist in
which reproduction can occur outside of a diseased host. While low levels of soil-borne B.
anthracis may persist, B. anthracis likely depends on mammalian hosts for significant replication
and persistence. Recent studies suggest the organism may have a dynamic life cycle in the
rhizosphere and soil biome [21, 23], B. anthracis spores, present at low levels, can germinate,
multiply, and persist in the rhizosphere when nutrients are available and predation pressures are
not too great [21], Under laboratory conditions, B. anthracis spores were shown to germinate and
multiply in the rhizosphere of grass (Festuca arundinaceae) for up to seven weeks, but not in
soil without plants [21], However, new evidence from outdoor soil experiments with wild type
strains in Etosha National Park, Namibia suggest that B. anthracis spores significantly promote
grass seed germination. While the germinating grass did not appear to enhance multiplication or
increase persistence of B. anthracis, such a mechanism would attract host ingestion by
encouraging foraging, further supporting the importance of mammals in focal pathogen
persistence [24],
Hypothetically, biofilms might provide another mechanism by which vegetative B. anthracis
could persist in soil. Under laboratory conditions, B. anthracis was shown to form vegetative
biofilms that sporulate under low nutrient conditions [25], B. anthracis reproduces in biofilms
present in soil [23, 25],
Dey et al. [26] performed laboratory experiments to determine whether B. anthracis would
germinate and reproduce in amoebas that would be found in the low-lying areas characteristic of
hypothesized "incubator areas". They demonstrated that B. anthracis spores inside of amoebas
(Acanthamoeba castellanii) do germinate and reproduce. Further, they demonstrated that the
pXOl plasmid was necessary for intracellular germination and growth in amoebas, but pX02
was not. Based on these results they hypothesized a role for amoebas in the persistence of B.
anthracis in "incubator areas" [26], B. anthracis lacking pX02 or both pXOl and pX02 is
observed in the field, with avirulent isolates occurring at spore-contaminated sites within five to
eight years [14], The presence of avirulent B. anthracis supports the hypothesis that
environmental persistence may arise from incubator areas (assumes microbial reproduction
outside of an infected host) [18] rather than spore concentrator areas (assumes no reproduction
occurs) [13], Whether avirulent B. anthracis is likely to acquire virulence plasmids in natural
settings is not known. Without the pX02 plasmid B. anthracis has a low pathogenicity and is
therefore unlikely to be able to infect another animal [22],
Beyond the laboratory, B. anthracis-likt bacteria were found in earthworm gut in nature [23] and
B. anthracis multiplied in the intestinal tract of earthworms (Eisenia fetida) [23, 27], Earthworm
4

-------
abundance and diversity and B. anthracis persistence require similar conditions: both are
enhanced by slightly alkaline soils with high calcium and high organic matter [27], It may be
speculated that the presence of earthworms is a critical factor for B. anthracis persistence at
locations where outbreaks have occurred but no spores have been detected in soil samples.
Bacteriophage infection of B anthracis may create lysogens (the integration of phage nucleic
acid into the bacterial chromosome) to restore functionality necessary to survive and replicate in
earthworms, the rhizosphere, or in soil [23], Such lysogens were observed in nature [23],
Lysogeny with phages (from soil, fern rhizosphere, and earthworm gut) was found to inhibit
sporulation, favor biofilm formation, and support persistence in soil and anoxic earthworm gut
by activating B. anthracis genes [27], The phages responsible for the lysogens are diagnostic for
B. anthracis and were shown to be specific for B. anthracis-Wke bacteria (B. anthracis Sterne and
some related strains of B. cereus), but not B. thuringiensis, B. megaterium, B. mycoides, B.
pumilus, B. subtilis, B. brevis, Sporosarcina ureae, or Brevibacillus laterosporus [27],
Whether these, or other, alternative vegetative pathways in the B. anthracis lifecycle occur in
nature, enabling persistence between outbreaks, remains in debate. The specificity of the
bacteriophages for B. anthracis, their presence in the natural environment, the observed
characteristics of the lysogenic B. anthracis, and the detection of avirulent B. anthracis in field
samples support the hypothesis that vegetative reproduction may occur within the soil
environment supporting B. anthracis between outbreaks. However, without continuing sporadic
outbreaks the disease over time will disappear, suggesting that soil support of B. anthracis
survival is limited.
2.2.2 Presence of Endemic B. anthracis in the United States
Ecological niche models (ENM) can be used to broadly define the geographic range of Bacillus
anthracis and target surveillance efforts [28, 29], ENM have been used to predict the potential
geographic distribution of B. anthracis in the continental United States using confirmed outbreak
data from livestock and wildlife between 1955 and 2005 [30], Additional models described
potential shifts in the distribution under climate change, suggesting possible reduced suitable
habitat for pathogen survival in southern Texas and expanding habitat in the northern states [31],
Further ENM experiments were performed to refine predictions by modeling the dominant
lineage (Western North America - WNA or Al.a [32]) in the contiguous United States [33],
Broadly, such models define the geographic potential for pathogen persistence [34], Coupling
spatial statistics of outbreaks with ENM outputs allows for the identification of high risk
transmission areas (where the statistically significant clusters occur) and areas where passive
surveillance should increase (where niche models predict persistence in under-investigated areas)
[34],	Much of the recent spatial modeling of anthrax, including ENM experiments, has relied on
mortality data to understand the disease, which likely underestimates the extent of the disease
[35],	More recently, it has been suggested that serological surveillance could delineate additional
anthrax enzootic zones [36] by including species with high survival rates.
Recent soil samples were collected along a transect from Manitoba to New Mexico and Texas
and analyzed using polymerase chain reaction (PCR) technology to look for persistent B.
anthracis [37], PCR analysis was used to look for B. anthracis within the range of persistence
that has been identified by the ENM [30], A BA-SF primer (rpoB sequence) [38] was used to
5

-------
distinguish B. anthracis from other Bacillus species, including B. cereus. B. anthracis was
present in samples collected at five of 104 sample sites tested (one site each in Manitoba,
Minnesota, South Dakota, Colorado, and Nebraska) [37], The transect generally paralleled the
eastern edge of the geographic range for B. anthracis as determined by Blackburn et al. [30] and
showed B. anthracis was occasionally present. Whether the detected B. anthracis was virulent
(and a risk to human and livestock health) was not assessed.
Louisiana and Mississippi are areas with soil and other characteristics such that ecological niche
models do not predict B. anthracis would be likely to persist [30], yet there has been a history of
natural outbreaks of anthrax in the Mississippi River delta [17], B. anthracis was found in soil
samples collected in New Orleans after hurricane Katrina in 2005. The samples were analyzed
by PCR using the BA-SF primer specific for B. anthracis [37], The PCR-positive samples were
cultured and confirmed to be B. anthracis. Virulence markers (pXOl and pX02 plasmids) were
evaluated using PCR. B. anthracis were found in 26% of the samples tested. All five B.
anthracis-positive post-flood samples from New Orleans were positive for pXOl, but only one
sample had both the pXOl and pX02 plasmids needed for virulence [37], Two years later (2007)
no samples from New Orleans were positive for B. anthracis [37], While the general
environmental conditions in the Mississippi delta would be expected to inhibit B. anthracis
persistence, sporadic outbreaks and detection of B. anthracis occurred there. One potential
explanation was provided by Van Ness [18], who reported that in a study of the 1959 Pearl River
Mississippi outbreak, areas high in calcium were found to be required for anthrax to occur. Thus
hospitable microenvironments for B. anthracis may exist within an otherwise unlikely area for
persistence, resulting in recurrent anthrax in the Mississippi delta. But, the non-reappearance of
disease in this area suggests that these later outbreaks may have been due to contaminated feed,
not persistent soil contamination.
2.3 Overview of the Classic B. anthracis Lifecycle
The classic B. anthracis lifecycle is summarized in Figure 1. A necessary condition for anthrax
outbreaks is exposure of a susceptible animal to B. anthracis with both pXOl and pX02
plasmids (required for virulence). In locations with recurrent outbreaks, the presence of B.
anthracis is generally attributed to persistence in the environment and the presence at novel
locations may arise from the transport of spores to the location. Given the potential for
persistence of B. anthracis spores at a location, outbreaks tend to occur in certain seasons and
under specific environmental conditions. When the specified conditions exist, an initial case or
cases might occur. With a resulting death, release of blood containing B. anthracis and
subsequent sporulation might occur at the carcass site. Other animals could then be exposed to B.
anthracis spores at or near the carcass, or through mechanisms that transport B. anthracis,
resulting in exposure of other animals and multiplication of cases. Over time, conditions change,
resulting in the termination of the outbreak.
6

-------
Vegetative Form	Spore form
germination
	
host disease
and death
exposure
of animal
host to
spores
release of
an thrax with
fluids from
carcass
sporu/ation
SOIL OR NATURAL
ENVIRONMENTAL
RESERVOIR(S)
ANIMAL HOST
SOIL
Figure 1. Summary of Bacillus anthracis lifecycle adapted from Schuch et al. [23].
2.3.1 Initial Host Acquisition
Anthrax can infect a wide variety of livestock, wildlife, and, rarely, birds (see [39] and [36] for
extensive lists of taxa). Susceptible animals are mostly grazing mammals, but include both
grazing and non-grazing mammals. Susceptible animals include cattle, horses, sheep, pigs, dogs,
bison, elk, white-tailed deer, goats, mink, and numerous other mammals. Ruminants appear to be
most susceptible [40], Among livestock, cattle generally have higher case and mortality rates
than other herbivores. Swine and carnivores are relatively resistant to lethality resulting from
anthrax infections [40-43], However, a wide range of carnivores do contract anthrax [39, 44],
Serologic data show almost 90% of carnivores (e.g., lions and hyenas) in some areas have
survived anthrax infection, whereas no zebras survived anthrax in the same areas [41] based on
serology data. It is notable though that zebras had high seroprevalence rates elsewhere in Africa
[45], Note that in addition to surviving anthrax, high antibody titers might result from sub-
clinical anthrax or toxin-induced stimulation of antibody production [36], In the case of zebras in
Namibia, titers fluctuated in animals captured during the anthrax season and recaptured in the
non-season [45], In birds, there are only a handful of reports of anthrax mortality resulting from
natural or oral exposures including a few reports of ostriches on ostrich farms [39], of a few
captive birds in zoological gardens [39], and of a single vulture among many feeding on
carcasses in natural outbreak [46],
There is a high level of uncertainty as to the factors triggering the initial case or cases of an
epizootic, whether changes in environment or behavior increase host exposure, changes in
stressors increasing host susceptibility, or changes in environmental concentrations of B.
anthracis [47], It should not be overlooked that anthrax is typically a summer disease, a time of
heat stress that will negatively impact the innate resistance of host species [48], thereby allowing
low exposure levels to become infective. An outbreak may arise from one such stressed animal
that has been grazing contaminated soil or that has been harboring a latent infection. Generally
the index case (or early cases) occurs at or near a former carcass site [49], Several theories exist
7

-------
as to the exact route of exposure. The spores may be ingested with grazing and there may be
seasonal and behavioral changes that result in greater ingestion of soil containing spores [47] or
ingestion of grass at carcass sites locally [24], Contributing to the initial case in an outbreak,
ingesting grit and thorns could cause lesions in the digestive tract providing unimpeded entry of
B. anthracis into the blood or lymph system allowing escalation to bacteremia [40], Beyer and
Turnbull [40], citing Fox et al. [50], states the lesion theory is not consistent with empirical data.
Beyer and Turnbull [40] stated inhalation of spores while grazing over dry contaminated soils
could lead to inhalation anthrax, though there is a lack of empirical evidence. Ground
disturbance of an old carcass site could lead to an outbreak [19] from inhalation or ingestion.
More research is needed to test these infection hypotheses.
2.3.2 Carcass, Sporulation, Spores, and Vegetative Survival at Carcass Sites
B. anthracis is present at high levels, a million to a billion CFU per milliliter (mL) in the blood
of animals when they die of anthrax [51], High carbon dioxide concentrations found in
decomposing carcasses reduce sporulation. B. anthracis in carcasses appears to die within four or
fewer days under conditions supporting anaerobic digestion [Minett [52] cited in [14]] and in
competition with other microorganisms [13], Opening of the carcass by scavenging birds or other
mammals, allows body fluids to be drained from the carcass and causes dispersal of vegetative
cells into the surrounding environment [14, 19, 52, 53], Introduction of the vegetative cells to
nutrient-poor environments induces sporulation [13], Initiation and speed of sporulation depend
on the temperature and relative humidity, at least in the spore microenvironment [14, 40, 46], A
temperature of 39 °C with high relative humidity (100%) is ideal for sporulation to occur. In
Namibia, sporulation in soil was reported to occur at temperatures above 25 °C [51], At a
temperature of 18 °C sporulation is inhibited; the vegetative form of B. anthracis dies [14], In
locations and/or conditions not conducive to sporulation, spilled blood with high B. anthracis
CFU/mL may result in few or no spores in soil; only a small proportion of the B. anthracis
successfully sporulate [51], Vegetative B. anthracis survival outside of a host has been
considered to be poor (<24 h) [54] although recent evidence (discussed in Section 2.2.1) suggests
that vegetative persistence in microenvironments might be possible.
Carcass sites are the most likely locations to find B. anthracis spores. Spores may persist in soil
at some locations for years [51, 55], Spore titers vary greatly in the area around a carcass site and
between anthrax carcass sites [51, 53], In some environmental conditions, B. anthracis spore
levels at carcass sites tend to remain high for years (104 - 106 colony forming units [CFU]/gram
[g] soil) [56], At other carcass sites, B. anthracis is observed to quickly disappear from heavily
contaminated soil, becoming undetectable in soil or bone marrow from buried carcasses within a
year, whether analyzed by culture or by polymerase chain reaction [56], In other locations spore
persistence at carcass sites appears to be transient [51], More recently, investigations in Texas
recovered viable B. anthracis with both plasmids (pXOl and pX02) from soils around a carcass
12 months after an outbreak and that same genotype was recovered from a fresh deer carcass
[55], In Montana, viable spores were recovered from a bull bison skull 27 months after the
animal died during an epizootic (Blackburn, unpublished data). In a study of enzootic areas in
Namibia, Africa [51], few soil or water samples not associated with a carcass site were positive
for B. anthracis and the number of spores, where detected, was low: 3.3% of water samples (1
spore/mL) and 3.0% of soils samples (8 - 80 spores/g). Sampling 106 carcass sites, 65% of the
samples had 1 to 10,000 spores/g; 10% had >10,000 spores/g, and 4% had >1 million spores/g.
8

-------
In another field study in Kenya [57], B. anthracis spores were found in 43% of ash samples from
burned carcasses, but not in soil samples within three meters of the carcass or samples from
ephemeral pools and dry river bed channels. In contrast to the Kenya study, B. anthracis spore
concentrations were highest within two meters of old or recent carcasses and specifically in
locations where the soil was saturated by body fluids from the carcass [53], The cause of this
difference is unknown, but may arise from differences in ecosystems or environmental
conditions. Multiplication is not observed at carcass sites [51],
2.4 Conditions Characteristic of Outbreak Initiation
Broadly, recurrent outbreaks are thought to be associated with areas with frequent host-
pathogen-soil-host transmission. Anthrax outbreaks in animals in the United States are generally
limited to specific geography (discussed above), geology/soils, and seasons [19], Van Ness [18]
hypothesized livestock grazing on soils with pH above 6.0 and ambient temperature above 15.5
°C are in favorable conditions for pathogen exposure.
Under conditions conducive to an outbreak, multiple independent outbreaks may occur [58-60],
For example, in 2004 in Italy there were two outbreaks, 23 kilometers (km) apart, each caused by
a different B. anthracis strain, suggesting the outbreaks were independent [59], In the northern
hemisphere, anthrax is primarily a summertime disease, with cases occurring after May [61],
Generally, environmental conditions leading to acute outbreaks involve a wet period (typically)
followed by an extended drought or hot, dry conditions (see [14] for an in-depth review). In
Texas, Blackburn and Goodin [61] quantified spring green-up conditions, showing statistically
significant differences in years with epizootics. Using satellite measures of vegetation greenness,
the study confirmed that epizootic years greened up earlier and more intensely than years with
just sporadic cases. Vegetation indices suggested that short-term green-up events precede large
epizootics after prolonged (several week or month) dry periods, supporting that rain events in
otherwise dry times may lead to explosive outbreaks. A study from southern Canada suggested
that large rain events preceded cattle outbreaks [62], Similar conclusions were reported from
Australia following an acute regional epizootic in Victoria in 1997 [63],
2.4.1 Spore Transport and Case Multiplication
Under conditions supporting sporulation, the index case(s) may provide heavy spore loads
resulting in infection of other animals leading to cluster(s) of cases [34, 58], Spores from the
index case infect other animals when they ingest blood containing B. anthracis or ingest
vegetation contaminated with spores [49], B. anthracis spores may be transported from a carcass
site by animals or insects, expanding the area in which anthrax is observed. Spore transport
vehicles and mechanisms include movement of infected animals, insects, and predators; physical
transport of parts of the carcass; transfer of spores to feathers and feet; and spore passage through
the digestive tract and deposition into the environment at remote locations in vomit and feces.
Spores may also be transported from the carcass site in runoff and by wind [13, 40, 51],
Insects may play a key role in the spread of anthrax and transmission of spores to animals or to
vegetation subsequently eaten by animals. Delay in disposal of carcasses (two to four days) may
provide time for insects and especially blow flies to feed on the animals and then serve as vectors
of transmission [55], Peaks in fly populations (biting or non-biting) are associated with
9

-------
seasonality of anthrax and suggest a role for flies in case multiplication [64, 65], Laboratory
experiments using houseflies (Musca domestica) fed even briefly on anthrax infected blood
subsequently deposited B. anthracis in fly spots (feces and vomitus). The spores germinated and
reproduced in the fly gut, raising the titer from less than 10,000 per spot at two hours to about
25,000 - 40,000 per spot at 10 hours. The observed B. anthracis populations in fly spots later
crashed between 10 hours and 12 hours and declined to about zero per spot 24 hours after
feeding [66], In Africa, fly spots from blowflies feeding on carcasses deposited on browse near
the carcass resulted in high levels of B. anthracis on leaves. Browsing of leaves may cause new
cases, but is likely limited to within several days of an animal death [55], Additionally, the
species of flies, and their physiology, may play an important role in pathogen survival [67] and
much work is needed to more fully understand the role of insects [55, 64],
Heavy rains preceding droughts in large anthrax epidemics (noted previously) may provide
conditions resulting in the production of large numbers of flies. Large populations of
hemophagic ("biting") flies (e.g., Stomoxys calcitrans) are hypothesized to cause the broad
spread (about 5-10 km) of an anthrax epizootic [39], although to date, published evidence is
lacking to confirm this. Available evidence suggests that biting flies may locally amplify risk
within an outbreak [64], A human case of cutaneous anthrax occurred after a gadfly bite
contemporaneous with an outbreak that had resulted in the death of sheep. The infected man had
been in a pasture that was used by the herd of sheep in which the outbreak occurred [68],
However, he had had no contact with sick animals or carcasses. DNA analysis showed no
differences between the strain that killed the sheep and the strain that infected the man. This case
supports the hypothesis that there is risk of human anthrax associated with bites by contaminated
flies. Historical work in Russia suggested that biting fly risk was associated with shrub land
environments and less so with open grasslands [69] but it should be noted that female tabanids
lay their eggs in running water, which can be scarce in steppes. The enzootic zone defined by
Blackburn et al. [64] meets this shrubland definition. Ticks and mosquitoes may also ingest the
bacteria [14], While tick transmission would be limited to host-jumping species, transmission via
mosquitoes would be dependent on the density and numbers of mosquitos [14], Animals newly
infected by ticks and mosquitos, however might travel considerable distance. Both explanations
for the transmission and geographic spread of anthrax need further investigation.
Exposure of anthrax carcasses to carnivores may be a key factor in the transmission and
continuation of outbreaks in Africa, Argentina, and Canada [51, 54, 70], Carnivore and avian
scavengers are less susceptible to anthrax than herbivores. Both may deposit spores in the
vicinity of the opened carcass [53] and transport spores long distances, releasing them in feces
[13, 41], In Namibia, >50% of fecal samples from scavengers near carcasses were positive for
anthrax spores. The spore density in the fecal material was highly variable, reaching a high of
20,000 CFU/g of fecal material [51], Avian scavengers, like vultures [46, 71], and predators, like
hawks [70], may disseminate B. anthracis spores via fecal material and via contaminated feet
and feathers. While a transmission role for B. anthracis by African vultures was proposed, a
study of vultures in southern Africa did not result in the detection of B. anthracis spores and
suggests a limited (or no) role in the spread of anthrax [46], Field investigations from west Texas
were unable to recover B. anthracis from feces, but did recover spores from feathers and water
troughs frequented by vultures feeding on carcasses (Blackburn, unpublished data) supporting
early hypotheses on water contamination in Africa [72], It should be remembered that vegetative
10

-------
cells will not survive passage through the scavenger gastrointestinal tract. It is when they have
consumed spores, e.g., from an already opened carcass a few days old, that they can deposit
spores in their feces.
Spores in soil resist movement in spite of high wind and rain events. High levels may remain at
carcass sites for years [Canada [53]; Etosha National Park, Namibia [56]]. Wind movement of
spores from carcass sites may occur, with concentrations of 7.1 x 10"3 CFU/liter air measured at
18 meters from the center of the site [56], These low levels would result in few microbes being
inhaled, well below the normal infective dose number, and windborne spores from carcass sites
are therefore believed to have little impact on the spread of anthrax [56], Increased entrainment
of dust (enhanced by increased animal contact with soil during rutting aggression) has been
suggested as a potential cause of increased cases of anthrax through aerosolization of spores
[13], It should be noted that large soil particles will not penetrate far into the lung because of
particle size and particles capable of causing anthrax may be of low density in the atmosphere, as
was observed in Namibia [5, 56], Rutting causality is questionable because anthrax outbreaks in
wood bison in Canada [13] and deer in Texas [49] begin before rutting season (in northern
hemisphere summer).
While the importance of flooding in anthrax epizootics is recognized, the transport role in
geographic dispersion is uncertain. In some cases, flooding and natural drainage are assumed to
disperse spores over large geographic areas [58, 63], Alternatively, multiple independent
outbreaks over large geographic areas may be due to a large area of optimal conditions in which
B. anthracis persisted before the flood event [59], Floods and high runoff could wash soil and
spores to depressions ("concentrator sites") [13] and onto seasonally flooded water meadows.
Rain may also splash spores onto grass [47], Water may transport spores to locations of initial
cases, but the subsequent dispersion pattern around carcass sites is most consistent with
dispersion by scavengers via movement of scavenged body parts and spores in fecal material
[53] or by insects [64], Summarizing the strongest evidence for spore dispersion mechanisms
found in published reports points toward (1) herbivores ingesting spores near carcasses and
traveling some distance before dying (creating a new carcass site); (2) dispersion by insects,
through their biting, in their feces and vomitus; and (3) predator transport in their feces and by
physical dispersion of parts of the carcass.
Humans can transmit spores and enable case multiplication of livestock cases by using
contaminated bone meal in animal feeds and by administering animal vaccinations containing
virulent organisms [73, 74], However, in the United States, these two causes of anthrax in
animals are no longer observed because of requirements for treating imported bone meal and
improved vaccines. Strains of B. anthracis observed in outbreaks in the United States include
enzootic strains and imported strains. Industrial importation of animal products resulted in the
introduction of characteristic 'industrial' genotypes into the United States, which resulted in
cases of anthrax. These imported strains resulted in a small number of human cases. Some of the
imported strains become ecologically established [32, 75],
11

-------
2.4.2 Temporal Characteristics and Termination of Natural Outbreaks.
During an epizootic, new cases may occur for weeks or months following the initial outbreak
[19, 49, 58, 63, 76], In natural outbreaks impacting multiple farms in the United States, the
maximum number of newly affected premises is normally identified in weeks 3 to 5 after the
initial case, and all affected premises are identified within 9 weeks. The typical number of
livestock deaths per premise is five, with cattle mortality about 6% and horse morality about
17% [19], In recent U.S. wildlife outbreaks in Texas, deer mortality rates have varied from ~1-
7% [64], with some ranches reporting a near 100% loss [55], The disease is present in all years,
but in the sporadic years mortality is ten or fewer deer [64],
Mortality in wildlife may result in higher death rates when wildlife densities are high [14],
though this has not been quantified or modeled. In a major outbreak in wood bison, —10% of a
herd of 1,800 died in a single outbreak. By the following year, 93% of surviving adults had
developed high antibody titers with sub-clinical anthrax, survival of the infection resulting in
immunity, or absorbing toxin components that stimulate antibody formation (see [36]). High
percentages of carnivores in an enzootic area may exhibit elevated titers to B. anthracis
indicating sub-clinical anthrax or survival [41, 77]; other animals, e.g., zebras, appear to have
much lower survival rates in the Serengeti ecosystem [41],
In natural settings, outbreaks can continue until conditions change, such as beginning of seasonal
rains [14] or cold weather [13], For example, it has been posited in South Africa, Tanzania, and
West Texas, that the end of outbreaks is coincident with seasonal rain [14], Such weather may
remove spores deposited by insects from vegetation by washing B. anthracis from the leaves
[55], In the latter case, field evidence suggests this may be in fewer than 21 days [55], Outbreaks
in wood bison in northern Canada end with cold weather [13], Cold weather termination of an
outbreak may be due to unfavorable conditions for sporulation, change in animal behavior,
movement of the animal to an area free of spores, loss of fly populations, or other causes.
Outbreaks may be ended or mitigated by active management actions such as quarantine, insect
control, soaking carcasses in 10% formaldehyde to discourage scavengers and disinfect surface
of the carcass, removing and/or incinerating carcasses, use of antibiotics to stop incubating
infections, and vaccination of animals in the area outbreaks [59, 60, 76, 78-80], If vaccination is
adequately administered, lower mortality rates have been reported during outbreaks [19, 79],
Delayed germination of spores can result in anthrax cases after an antibiotic regimen has been
completed [81],
The rapid removal of carcasses reduces the likelihood of a widespread epidemic by reducing the
transport and transmission potential of insects [66], reduces soil contamination and reduces
scavenger dispersal of spores [76], Burning the carcass or using quicklime, or agricultural lime,
to cover the carcass [57, 82] may not be effective or effectiveness may not have been
determined [82], Because the persistence of B. anthracis spores is enhanced in some instances by
alkaline conditions with high levels of calcium, lime may support persistence of viable spores.
More recently, a critical review has suggested that lime be discontinued as a decontaminant and
considered a spore promoter [83],
12

-------
2.5 History of Naturally Occurring Anthrax Outbreaks in Animals in the United States
Anthrax, or anthrax-like, outbreaks have been reported in the historical record for thousands of
years [84] and have been reported in livestock and wildlife nearly worldwide [85], Anthrax has a
long history in the United States dating back at least 200 years [32], although it might have
arrived on the continent prior to European exploration [86], While the initial introduction to the
U.S. is and will remain unclear until more genomic studies are performed, the presence of
multiple divergent genetic lineages, as defined by single nucleotide polymorphisms (SNPs),
confirms multiple introductions over time [32, 87], Anthrax epizootics continue to occur in the
United States in livestock and wildlife [14], with climatic drivers associated with severity (less
green and drier summers found during epizootic years) [61], Generally, animal outbreaks were
associated with historic livestock pastures or farms. In particular, many of these outbreaks
occurred at known historical outbreak sites [19], There may be years or decades between
outbreaks at a given location [79], However, it is likely that livestock and wildlife outbreak
reports underestimate the number of cases due to biases in search efforts [35], difficulties in
finding carcasses [61], and lack of diagnosis and underreporting [85, 88], Recent serological
reports from Africa [41, 45] and Ukraine [89] further indicate frequent, regular infection. In the
latter example of Bagamian et al. [89], serology confirmed infection in wild boar in the absence
of livestock or other wildlife reporting. In Namibia, wildlife outbreaks occur annually in Etosha
National Park [47], Across these studies, there is evidence for frequent transmission events.
Clearly, viable B. anthracis populations persist in areas of prior outbreaks and, under favorable
conditions, initiate sporadic or seasonal outbreaks in susceptible livestock or wildlife hosts [13,
30],
Prior to 1950, anthrax in animal populations was reported regularly in several areas of the United
States, including parts of California; Texas to Missouri west of the Mississippi River; along the
borders between North and South Dakota, Minnesota, Iowa, and Nebraska; along the New
York/Pennsylvania and New York/New Jersey borders; and the northeastern border of New York
[73], Historical enzootic sites along the New York border were near transportation terminals
where livestock densities were high. Historically, cattle were driven from central Ohio to
Dunkirk in southwest New York. There they were loaded on trains to Buffalo [90], and Canada,
and shipped via the Black River Canal. Both the New York and Erie Railroad [91] and the Black
River Canal [92] connected with the Erie Canal, which carried livestock to New York City.
Areas near Dunkirk, New York, the Black River Canal at Carthage, and New York City were
historically considered enzootic [73],
From 1945 through 1950, anthrax outbreaks were widespread, with reports in 32 states, of which
16 had no prior reported cases. The outbreaks were primarily attributed to animal feed containing
bone meal contaminated with B. anthracis [73]; in some cases infection was attributed to live
(attenuated) vaccinations [93], The epizootiology of outbreaks due to bone meal and vaccines
was different from endemic outbreaks in several ways [73]: 1) cases occurred in new states
where anthrax was not considered enzootic; 2) outbreaks associated with bone meal or
vaccinations occurred in every season including winter months; 3) outbreaks occurred across
many states; and 4) cases were predominantly or solely in animals eating the contaminated feed
or receiving vaccine. Anthrax following vaccinations with killed or weakened bacteria exhibited
an incubation period of 3 to 120 days and swelling occurred around the injection site [73],
13

-------
The geography of anthrax in the United States contracted through the 1960s continued to
decrease in area as efficacious animal vaccine and restrictions on international bone meal were
introduced [94], The current enzootic areas are south and west Texas [14, 64] and the Dakotas
region [95, 96], Whether viable and virulent B. anthracis spores survive at, and could be released
by disturbance of, old carcass sites in the northeastern states is unknown. Outbreaks in wildlife
[97] and livestock [98] in Mississippi suggest there might be localized areas of enzootic anthrax
in the southwestern Mississippi delta.
From 2000 to 2005, outbreaks were confirmed in North Dakota, South Dakota, Minnesota,
Oklahoma, Texas, Nevada, Louisiana, and California [82, 94, 99], Since 2006, livestock and
wildlife outbreaks have been reported from Texas [55, 64] and Montana [14, 42], and livestock
outbreaks have occurred in the Dakotas, Minnesota [95], and Colorado [100], Outbreaks from
the past decade have been concentrated in areas defined as having soil conditions for pathogen
survival [17, 20], With the exception of the 2008 epizootic in plains bison {Bison bison), Rocky
Mountain elk (Cervus elaphus nelsoni), and white-tailed deer [42], outbreaks in Montana have
only been reported from eastern Montana in the last century [42], Evidence of confirmed
bacteremia in bison from western Montana in 2010 confirms the area may be a re-emerging zone
[42], Large and periodic outbreaks in wildlife species in Texas [55] and Montana [42] suggest
that wildlife surveillance is critical to fully defining the extent of ongoing anthrax risk in the
United States. Livestock outbreaks have occurred at legacy sites, for example, at a burial site for
infected hides at a leather manufacturer; at locations 2.5 km downstream of a contaminated
facility; and in plaster with horsehair as a binder [101],
2.6 Historical Incidence of Unintentional Human Anthrax
Humans are susceptible to four anthrax infection types from four routes of exposure: cutaneous
anthrax, the most common form of the disease, by direct contact with spores in open lesions or
insect bites; gastrointestinal anthrax, by ingesting contaminated meat or possibly drinking
contaminated water; inhalation anthrax, by breathing in spores [22]; and, recently identified,
injection anthrax by injection of contaminated heroin [102, 103], The range of incubation periods
in humans depends on the route of exposure. For cutaneous anthrax, the incubation range is 1 to
12 days; for gastrointestinal anthrax, the range is 1 to 7 days; and for inhalational anthrax the
incubation period is 4 to 45 days, consistent with prolonged spore dormancy within the lung
[104],
Typically 20,000 to 100,000 human anthrax cases occur annually [105], with 95% of cases being
cutaneous [106], Most human cases occur in developing countries and are associated with
infected animal slaughter or contaminated meat [107], Despite recent increases in human anthrax
incidence in developing countries such as Georgia [2] and Bangladesh [108], unintentional cases
in the United States are rare [96], Environmental locations in the United States where human
exposures to B. anthracis may occur are not ubiquitous, but are discrete locations where spores
were transported or persist.
For human cases of anthrax to occur: 1) B. anthracis, most likely spores, must be produced at, or
transported to, the site of the outbreak; 2) virulent B. anthracis must persist, as spores or through
vegetative growth, at the site until a host is infected; 3) conditions must exist supporting
14

-------
exposure and infection of animals or of the direct exposure of humans; 4) if humans are not
directly infected from the persistent B. cmthracis at the site (unusual), B. cmthracis must be
transferred to humans from the infected animals, animal products, or fomites (from transfer), or
via aerosolization, e.g., through soil disturbance. Several sources of unintentional exposure are
summarized in Table 1 and discussed in the sections below.
Table 1. Representative Human Anthrax Cases from Unintentional Exposures in the
United States.
Location, Year
Source of Exposure
Number of Cases and
Route of Exposure
Reference(s)
PA, MA, NJ, NY, DE 1919 - 1925
Leather industry (Tannery)
Not reported
Smyth etal. [109]
PA and other states, 1919-1925
Wool
Not reported
Smyth etal. [109]
PA and other states, 1919-1925
Hair and brush
Not reported
Smyth etal. [109]
United States
Animal contact
Not reported
Smyth etal. [109]
NY and other states, 1919 -1925
Shaving brush
Not reported
Smyth etal. [109]
Philadelphia, PA 1929 - 1942
Goat skins
Not reported
Smyth etal. [110]
Philadelphia, PA 1929 - 1942
Goat hair
Not reported
Smyth etal. [110]
Philadelphia, PA 1939, 1942
Horse hair
Not reported
Smyth etal. [110]
Philadelphia, PA 1929 - 1942
Wool
Not reported
Smyth etal. [110]
Philadelphia, PA 1929 - 1942
Wool and hair
Not reported
Smyth etal. [110]
Philadelphia, PA 1941
Fur pelts
Not reported
Smyth etal. [110]
DE, MA, NJ, NY, PA, and other
states 1939-1943
Hides and skins
Not reported
Smyth etal. [110]
NJ, NY, PA, and other states,
1939-1943
Wool and hair
Not reported
Smyth etal. [110]
U.S., 1939-1943
Shaving brush, fur
coat/collars, others
Not reported
Smyth etal. [110]
CA, LA, SD, TX, and other states,
1939-1943
Agricultural
Not reported
Smyth etal. [110]
Northeastern States (PA, NY, NJ,
MA, CT, NH), 1945-1951
Mostly industrial exposure
372 cases
Steele and Helvig [73]
FL, 1951
Exposure to skinning dead
cow
5 cases
Steele and Helvig [73]


5 cases of inhalation

Manchester, NH, 1957
Goat hair
anthrax
4 cases of cutaneous,
anthrax
Cohen and Whalen [111]
Manchester, NH, 1957
Across street from goat
hair processing mill
1 case inhalation anthrax
Cohen and Whalen [111]
U.S., 1957
Workers at hide or hair
mills
4 cases of inhalation
anthrax
Cohen and Whalen [111]
U.S., 1948-1957
Near tannery
3 cases of inhalation
anthrax
Cohen and Whalen [111]
Four mills in Northeastern U.S.,
1955 - 1959
Goat hair processing
21 cases cutaneous
5 cases inhalation
Brachman et al. [112]
U.S., 1961
Secretary in goat hair
processing mill
1 case of inhalation
anthrax
Bales et al. [113]
15

-------
Location, Year
Source of Exposure
Number of Cases and
Route of Exposure
Reference(s)
U.S., 1966
Truck driver who unloaded
goat hair
1 case cutaneous anthrax
Bales et al. [113]
U.S., 1957- 1971
Veterinarians who
necropsied infected
animals
6 cases of cutaneous
anthrax
Bales et al. [113]
OH, 1964
Pipe insulator goat hair
exposure
1 case
Bales et al. [113]
CA, 1976
Weaver with goat hair
exposure
1 case
Bales et al. [113]

Livestock; infected animal
carcass

Centers for Disease
ND, 2000
1 case cutaneous anthrax
Control and Prevention
(CDC) [114]
TX, 2002
Laboratory worker
handling Bacillus
cmthracis
1 case cutaneous anthrax
Page et al.[l 15]
NY, 2006
Made drum heads from
1 case inhalation anthrax
CDC [116]
CT, 2007
African animal hides
ProMED Mail [117]
NH, 2009
Exposed to drumming on
"African" drum skin
1 case gastrointestinal
anthrax
Adalja [118]
2.6.1 Unintentional Occupational Exposure in the United States Associated with Contaminated
Animal Products
Virtually all human cases of anthrax in the United States, other than those due to intentional
releases, are cutaneous and are typically associated with the handling and preparing of
contaminated animals, processing carcasses, or handling contaminated animal products (i.e.,
meat, skins/hides, hair, bones, other components of infected animals, products made from
contaminated animal parts such as goat hair insulation, imported yarn, knitted sweater, goat hair
from horse saddle pads, goatskin handicrafts, bone meal, and drum heads made from
contaminated goat hides) [40, 113, 119-121], Anthrax cases in agricultural settings in the United
States typically involve ranchers, veterinarians, or others exposed to diseased animals from direct
animal contact during handling, slaughter, butchering, necropsy, ingestion of contaminated meat,
or disposal [113], Trade in infected animals and animal products can transport B. cmthrcicis over
long distances [40], Transport of crops in contaminated animal hair sacks, with contaminated
hides, or in contaminated ship hulls or containers has resulted in cross-contamination [122, 123]
cited in Turnbull et al. [5]). Likewise, bone meal associated with outbreaks has been shown to be
contaminated with B. cmthracis [73, 74], With the introduction of anthrax vaccines for livestock
and encouragement of their use, by 1945 human cases of anthrax attributed to contact with
infected animals had declined [124],
In the U.S. there have been 32 documented inhalational anthrax cases between 1900 and 2005.
Except for the cases associated with letters from a terrorist containing B. cmthrcicis spores (2001)
and four cases with no known connection to contamination (1923 - 1947), all human cases of
inhalation anthrax were associated with animal hide processing, proximity to animal hide
processing, or proximity to animal hair processing [73, 111, 113], Anthrax occasionally, but
rarely, occurred in persons who did not directly handle the contaminated animal parts, such as a
16

-------
secretary in the main office of a goat-hair processing mill, delivery personnel, and other persons
living or working near the contaminated animal processing facility [113], During a North
Carolina outbreak, one textile worker's home was positive for B. anthracis out of four textile
workers' homes sampled [113], Routine occupational exposure (daily inhalation of 600 particles
containing B. anthracis spores) of unvaccinated workers rarely resulted in diagnosed cases of
inhalation anthrax. Where a cluster of cases of occupational anthrax was observed, such as in a
Manchester, New Hampshire processing plant in 1957 (five cases of inhalation anthrax and four
cases of cutaneous anthrax), an unusually high level of B. anthracis spores was assumed to be
present on the goat hair in use and introduced into the air [111],
Eating meat contaminated with B. anthracis was reported to present a low risk factor in the U.S.
[113], but has been an important source in the former Soviet Union [107, 125], While a claim of
"low risk" is supported by only one confirmed case of human gastrointestinal anthrax in the
United States [118], the risk of gastrointestinal anthrax may be higher than cutaneous anthrax in
rural areas of the world where anthrax is enzootic and where one is culturally more likely to eat
undercooked meat from ill animals [80, 126], A Thai outbreak of anthrax associated with an
outbreak in cattle and water buffalo resulted in both gastrointestinal anthrax and cutaneous
anthrax [126], Contaminated water has been suspected as a source of infection, but there is no
evidence to support the conjecture [113],
2.6.2 Unintentional Occupational Exposure Associated with I .aboratory Exposure and I .egacy
Bioweapon Sites
In addition to accidental exposure to contaminated animal produces, humans have received
accidental exposure to spores at historic bioweapon test sites or after release from a laboratory
[e.g., Sverdlovsk outbreak from facility release [104]]. Most, if not all, publicly available
information on human exposure after a release of B. anthracis spores from biological warfare
facilities comes from an accidental release at Sverdlovsk, Union of Soviet Socialist Republics
(now Ekaterinburg, Russia) [127], The release resulted from an accidental venting of spores from
the bioweapons facility into the outside air where it was dispersed by the wind. Downwind of the
facility, at least 68 people, age 24 or older, died from anthrax [104], The characteristics of the
outbreak cases were mapped and the following was observed:
•	Human cases (at least 90%) were people who resided or worked within a narrow zone 4
km long extending from the assumed site of the release and consistent with the prevailing
wind direction on the date of release (determined to be April 2, 1979)
•	Livestock anthrax cases were on the same vector to a distance of 67 km [128]
•	Human cases began 2 to 3 days after the release; the last fatal case occurred after an
incubation period of 45 days
•	Animal cases preceded human cases [104]
•	No anthrax cases were known to occur in the region after 1979 [104],
Legacy sites represent a potential source for the introduction of B. anthracis spores into the
environment. Spores buried at legacy biological weapon production sites may persist. The U.S.
biological warfare program included sites in Maryland (Ft. Detrick/Edgewood Arsenal),
Mississippi (Horn Island), Indiana (Vigo Ordnance Plant), and Utah (Dugway Proving Ground),
and, for animal diseases, New York (Plum Island). Although B. anthracis was used at some of
17

-------
the sites, including field testing, (e.g., Dugway Proving Ground), at other sites B. anthracis was
not present (e.g., Vigo Ordnance Plant was never operational for B. anthracis production) [129],
The risk of human exposure to B. anthracis in the environment at a legacy site would depend on
many factors including (but not limited to) access to historic weapons sites, the strain B.
anthracis established in the environment, and whether conditions favor persistence of the spores.
Deliberate releases of B. anthracis spores, such as documented on Gruinard Island, Scotland,
present a different release and dispersal scenario compared to Sverdlovsk. Small bombs
containing B. anthracis spores were repeatedly detonated from a gantry over-ground for a
prolonged period, resulting in a spore pattern in the residual soil that was highly directional and
assumed to reflect the prevailing wind at the time of release. After 40 years, spores were detected
more than 50 meters from the point of detonations. Spore concentration in the soil did not show a
consistent pattern of decrease with distance; pockets of spores of relatively high concentration
were observed beyond areas of lower concentration [130], The soil at Gruinard Island is a
sandstone base overlaid with acidic peat bog topsoil [131], conditions in which B. anthracis
spores would not be expected to persist [14, 19], The reason that the spores remained viable has
not been reported in the literature and may not be known. Of note, no spores were found in the
area in which sheep had died of anthrax after deliberately being exposed to spores dispersed
from the bomb [132], Thus, the soil and environmental conditions did not appear to support
persistence of the "natural" spores released from the dead sheep. Persistence of spores intended
and prepared as weapons might be different from natural spores, but the original spore-release
concentration was very dense.
Occupational anthrax infections in diagnostic and research laboratories in the United States have
historically been cutaneous in nature [133], There continues to be a risk of accidental laboratory-
related exposure, primarily for laboratory workers, such as by receiving putative non-viable B.
anthracis samples containing viable B. anthracis or by discarding animal bedding from infected
laboratory animals as solid waste rather than as infectious waste [134], Such exposure occurred
without any laboratory workers becoming ill, although most of the exposed laboratory workers
were treated with antibiotics once the live B. anthracis was detected [134], Centers for Disease
Control and Prevention (CDC) confirmed that B. anthracis samples believed to be non-viable
were shipped to laboratories that subsequently determined that the samples contained viable B.
anthracis. A recent transfer of B. anthracis within the CDC was inadvertently transported
between agency laboratories without confirmation that the spores were completely inactivated
[135],
2.6.3 Other Sources of Unintentional Human Cases in the United States
Insect bites (hemophagic flies, mosquitoes, or others) were implicated in the transmission of
anthrax in Texas, South Africa, Zimbabwe, and India [14, 65], In laboratory studies, the potential
for such transmissions was demonstrated [65], In a recent human case in Italy, a hematophagous
fly bite was identified as the most likely source of infection [68], In that report, a sheepherder
several kilometers from an anthrax-confirmed sheep death presented with a cutaneous lesion at a
fly bite on his arm. Molecular sub-typing linked the herder's strain with that isolated from the
dead sheep. No other animal cases were found and the insect bite was the most plausible
explanation for infection. That report, coupled with the spatial relationship between biting flies
18

-------
and deer cases reported in Texas [64], support the need for more empirical work on the role of
biting insects in transmission.
While not observed in the United States, intravenous drug users in Scotland, Norway, and
Germany contracted anthrax; most cases [15] were in Scotland [102, 103], Speculation attributes
these cases to have resulted from the transport of heroin in contaminated animal skins [102, 103]
or to heroin being cut with contaminated bone meal [118],
2.7 Vaccination Efficacy
Vaccination of mammals with live attenuated Sterne strain has been successful in livestock since
its introduction in the 1930s [136, 137], However, cases of anthrax in vaccinated animals may
occur [138] for a variety of reasons and has recently been reported in a bison in Montana [42],
Residual virulence due to unpredictable attenuation occasionally results in outbreaks [73, 139],
Vaccinations may be effective in animals for only about six months to a year. A multiple dose
regimen is required to ensure effectiveness following an outbreak. Administering vaccine and
antibiotics concurrently may prevent development of immunity [79],
While regular vaccination of animals may be effective at preventing outbreaks when used
consistently, but because of the high persistence of B. anthracis, outbreaks can recur if the
vaccinations are terminated [59, 140], even 30 years after a prior outbreak [140], Commonly an
outbreak absence after 10 years without vaccination is considered sufficient to indicate a
pragmatic cessation of risk.
Because of occasional severe reactions at the injection site, as well as animal deaths from live
vaccine, the Anthrax Vaccine Absorbed (AVA) is used for humans in the United States. The
AVA vaccine is a cell-free extract of a non-encapsulated strain prepared from an aluminum
potassium sulfate precipitation that contains Protective Antigen [136, 138], Protective antigen is
a component of the genesis of both B. anthracis toxins: lethal toxin and edema toxin [141],
Antibodies to protective antigen provide protection against anthrax. The vaccination consists of a
series of three subcutaneous injections over four weeks, injections at 6, 12, and 18 months, and
subsequent annual booster injections [138],
Vaccine efficacy data for human anthrax are limited to animal model studies and one field study
[142], On the basis of those studies, AVA vaccine is considered to be efficacious in humans and
primate models [112, 138, 139, 142, 143], Results using the rhesus monkey model indicate a
two-dose regimen is sufficient to provide protection for almost two years [143], Depending on
the strain of B. anthracis, up to 100% of vaccinated rabbits (which have a similar response to
rhesus macaques) survived after an aerosol challenge of approximately 100,000 spores, except
for one death from a Namibian isolate [142], Bacteremia was observed in 0% - 80% of the
vaccinated rabbits, depending on the isolate. The two most vaccine-resistant isolates (ASIL
K7978/Namibia and ASIL K9729/Turkey) were used to provide an aerosol challenge to
vaccinated rhesus macaques. Survival was 90% with 25% experiencing transient bacteremia
[142],
Human efficacy data is limited to a field study involving comparison of mill workers with
occupational exposure to anthrax receiving an AVA-like vaccine to those receiving a placebo (or
19

-------
neither vaccine nor placebo) [112], Brachman et al. [112] concluded the vaccine was 92.5%
effective at preventing anthrax. Brachman et al. [112] also noted there were no infections among
individuals with prior anthrax infections. Some mill workers with only two doses of the anthrax
vaccine and an occupational exposure did contract anthrax; three doses are therefore assumed to
be necessary [139],
20

-------
3 Model assessing Bacillus anthracis Natural Outbreaks, Unintentional Releases or
Intentional Releases
Reviewing the literature on anthrax outbreaks provided insight into characteristics of intentional
releases that may distinguish them from natural or unintentional occurrences. Recent studies
have provided insight for distinguishing natural from non-natural outbreaks of animal anthrax
using specific epizootiological and ecological characteristics [19] and distinguishing natural from
non-natural outbreaks of animal diseases using a broad range of characteristics [144-146], A few
of the common criteria across the studies for distinguishing natural from non-natural outbreaks
included geographic distribution of the anthrax cases [19, 144-146], season or time that the
outbreak occurred [19, 144, 145], appearance of an unknown strain [144-146], presence of an
epidemic in a specific population (animal or human) [19, 144, 145], peculiarities in the clinical
manifestation of the disease [19, 144, 145], and identification of the agent as a biological warfare
agent [144],
An outbreak of anthrax or the detection of Bacillus anthracis in an unexpected environment
could trigger local, state, and federal responses, particularly when there is the potential for
human exposure. Response to the event and subsequent risk management might depend on
whether the occurrence of B. anthracis is natural, e.g., associated with wildlife or livestock;
unintentional, e.g., exposure to a naturally contaminated animal hide or a laboratory-related
exposure; or an intentional release of B. anthracis. "Intentional release," as used herein, could
arise from a variety of scenarios in which an individual or group use a disease agent (i.e., B.
anthracis) to deliberately cause harm in which the release is no longer considered accidental or
unintended, and creates a crime scene [147], An intentional release could result from exposures
arising from criminal production, packaging, handling, or transport of B. anthracis. An
intentional release could be masked to appear as a natural or accidental event. A claim of
responsibility for an intentional release is not proof of anything other than opportunism. Factual
linkage must be demonstrated, not assumed or presumed, to establish responsibility for the
release. However more weight might be given to such a claim if it were made before the
outbreak is recognized and its scale appreciated.
The correct classification of a B. anthracis exposure incident as natural, unintentional, or
intentional in origin is needed to elicit the appropriate governmental response. This paper's
primary concern is the recognition of an intentional event involving B. anthracis in the United
States compared to naturally occurring outbreaks or unintentional releases. The following
categories have been identified to provide examples of potential causes of natural, unintentional,
and intentional occurrences that could result in anthrax cases or outbreaks:
[1] Example causes of naturally occurring animal outbreaks:
•	unvaccinated animals, or animals without timely boosters, grazing in a pasture where
previous outbreaks had occurred or infected carcasses were buried, especially in relation
to ground disturbance (ditch clearing, pipe laying, repeated leveling, excavated tannery
waste);
•	sporadic outbreaks in enzootic areas of west and south Texas, and central and eastern
Dakotas, Montana, Saskatchewan, Manitoba;
21

-------
•	hay made in such a pasture contaminated with grave soil, resulting in distant cases in
herds where the purchased hay is consumed;
•	displacement of contaminated grave soil by floodwater and deposition on downstream
water meadows;
•	riverbed dredging downstream of an old tannery and deposition on meadows;
•	animals fed mineral/salt supplements contaminated with spores from bone meals;
•	pigs and dogs with access to carcasses;
•	biting-fly infected animal(s) near unreported case;
•	animals browsing blow fly-contaminated scrub/bush leaves in proximity to a carcass.
•	heavy spring rains and dry summer resulting in excess tabanid biting-fly hatching with
access to moribund or dead animals which could transport infection to neighboring or
distant ranches [14];
•	multi-herd access to a large contaminated batch of commercial feed, minerals or salt;
•	feeding captive carnivores meat and bones from a purchased infected carcass;
•	sporadic cases in wildlife (i.e., long history in and around to the Wood Bison National
Park, Northwest Territories, Canada, and in the exotic wildlife and deer ranches of west
Texas and additional reemergence in elk and bison in western Montana).
[2]	Examples of unintentional occurrences:
•	skinning and butchering, handling of sun-dried hides, or careless disposal an infected
carcass;
•	contaminated drum heads;
•	processing or exposure to contaminated horse hair in old plaster, wool or goat hair
('Bradford Disease');
•	buying, receiving, or eating contaminated meat;
•	addict injecting contaminated heroin;
•	bitten by a tabanid (biting) fly with contaminated mouthparts;
•	laboratory exposure;
•	inadequate hand washing after handing contaminated objects or wearing contaminated
clothes;
•	immunocompromised individual in a contaminated environment;
•	inexplicable singular cases with no known cause, e.g., tourist hospitalized in Minnesota
with anthrax infection with unusual strain from unidentified exposure; serological studies
will presumably reveal more such incidents.
[3]	Examples of intentional occurrences:
•	white powder (hoax) letters to an abortion clinic, mixed with ricin and sent to prison
officials, judges, and prosecutors in a recent instance or mailing of contaminated letters
[148];
•	targeting a much loved or valuable animal, e.g., racehorse, or the devastation of a herd,
with a high financial cost;
•	using B. anthracis with the intent to cause fear in a targeted community, irrespective of
the number of victims, whether structured or chaotic.
22

-------
3.1 Screening for the Likely Cause of a Bacillus anthracis Occurrence
This section describes an approach for rapidly screening whether detection of the bacterium or
outbreak of the disease is likely an intentional release. While much of what is described here
may also apply to Canada and Western Europe, it is too restricted for use in regions where the
disease is poorly controlled.
The presence of B. anthracis in the environment can be identified in a variety of ways: culture or
molecular assay (i.e. PCR) of environmental samples; diagnosis of anthrax through the animal
health systems [19, 144]; and BioWatch1 Actionable Result (detection via PCR) [149],
Confirmation of the presence of B. anthracis may lag two to three days or more from the time of
an exposure to B. anthracis spores (by humans, animals, or air sampler) and their
isolation/identification. Some of these methods to identify B. anthracis are based on culture
while others use more subjective evaluations including observation or direct testing of samples
with molecular assays. Direct testing of environmental samples is risky because of the ever-
present possibility of multiple organisms contributing to positive responses in the molecular
assays. Both immunological assays for B. anthracis spores and PCR assays for pXOl and pX02
genes have resulted in the misidentification of Bacillus species other than anthracis as B.
anthracis [150], Consequently, use of culture for B. anthracis detection remains important for
confirmation of positive molecular tests from direct sample testing [150], Diagnosis of anthrax
through the health care and public health systems or syndromic surveillance detecting an
increase in anthrax-like symptoms [149] might also indicate that an exposure might have
occurred. The recovery of B. anthracis spores from a place is not proof of clinical exposure, only
of a potential for exposure. Genotyping of the isolates from the environmental location and the
clinical samples, and then demonstrating they are identical, confirms the exposure.
A screening approach, including four categories for analysis (shown in Figure 2), is proposed to
rapidly and systematically distinguish whether, in the United States, a surveillance detection of
B. anthracis or clinical diagnosis of anthrax is due to a natural, unintentional, or intentional
occurrence. Statements shown in italics are summary statements of information that, collectively,
might discriminate intentional from natural or unintentional occurrences (screening statements).
These are supplemented by keys leading to indicators suggesting an event may be intentional.
The screening approach considers whether:
• In a natural occurrence, observed B. anthracis strain(s) is/are consistent with those
previously recovered at the site or area, or is/are consistent with a likely source of spores
[10, 86], Normal isolates in a natural occurrence will show no evidence of sophisticated
spore modification, e.g., microencapsulation for environmental protection and for
rendering spores invisible to biosensors; of multiple antibiotic resistance; or of having
been genetically modified to remain fully virulent in spite of prior vaccination.
1 Department of Homeland Security BioWatch System is a program for detection of biological warfare
agents in the air via air filters in major U.S. cities.
23

-------
•	An intentional occurrence is indicated when immunized livestock and/or people are
contracting anthrax at a higher than expected incidence [21, 73, 79, 112, 139, 142, 143,
151], However, immunity is naturally short and in livestock it can be minimal if they
were vaccinated while being treated with antibiotics.
•	A natural or unintentional occurrence is indicated when the location of the occurrence is
at the site of a previous outbreak or along a cattle trail in a region with calciferous
alkaline soils, or near a biological laboratory, near a tannery or other animal processing
facility, or where a potentially contaminated animal product is or was in use [18, 40, 113]
•	Natural outbreaks in animals often coincide with the time of year for grazing or browsing
livestock or wildlife (late spring, summer, or early fall) [14, 19, 76]; winter outbreaks are
not unknown but are common only when livestock are fed commercial feeds containing
contaminated bone meal.
•	The environmental factors are appropriate for an outbreak. For example, during a natural
outbreak, the outbreak is often preceded by heavy rains followed by drought [13, 58, 59,
63, 140]; during a hot-dry period outbreaks will follow when there is a brief shower that
stimulates growth of vegetation for grazing. Sporadic outbreaks will occur during a
drought but characteristically seldom involve secondary cases.
•	The livestock most commonly affected are cattle, both from enteric and cutaneous
disease during a natural occurence. Anthrax is also common in sheep and goats. Pigs are
semi-resistant but will be affected with contaminated fibrous feeds or from scavenging.
Horses, though they can be infected from grazing, textbook-traditional cutaneous lesions
would result from being infected by biting flies radiating out from a previous case [152],
Hyenas, wolves, and coyotes are to differing degrees resistant when scavenging
carcasses. African wild dogs (Lycaonpictus) appear more sensitive to anthrax [153],
Serological surveys confirm that domestic dogs, which are moderately resistant, can act
as sentinels when index cases are missed, though dogs do succumb to infection [19, 30,
40,41,44,51,54, 76, 79, 154],
•	Epidemiology and pattern of cases are consistent with a natural cause: the number of
human or animal cases, pattern of cases over geographic region and time (e.g., radiating
from an index case when biting flies are involved, or a number of farms more or less at
the same time when a contaminated feed product is involved and the distribution reflects
sales), and type of human infections (e.g., mostly cutaneous) [13, 14, 19, 58, 60, 76, 79,
104, 155],
•	Unintentional occurrences such as laboratory-based infections are not uncommon though
this depends on the organism: for example, brucellar infections are a constant threat.
Between 1941 and 1975, laboratories in the United States suffered 40 anthrax infections
with three deaths in 1941, 1951, and 1958, in spite of the use of laminar flow cabinets
since 1950 [156, 157], As anthrax is non-contagious, accidental laboratory infections will
be limited to a direct or indirect exposure, not infrequently from centrifugation, and will
be from a documented laboratory strain and will usually result in the cutaneous form of
the disease. Cases more often arise in those individuals working with the organism, then
from accidents, infected animals/carcasses, and contaminated discarded glassware.
Clerical and maintenance personnel are known to have been exposed [158], A cutaneous
case occurred in 2002 in a laboratory worker as a result of unknowingly handling
contaminated vials of B. anthracis when not wearing gloves [159, 160], Exposure is
usually limited but an exception is the Sterne vaccine strain, which has a reputation for
24

-------
environmental contamination. Unless the laboratory is handling specially processed dry
or microencapsulated spores, a lung infection would not be expected.
Figure 2. Screening categories for rapidly evaluating likelihood that a B. anthracis
occurrence in the United States is intentional.
3.2 Screening Category I: Unexpected genetic strain?
Bacillus anthracis is a highly clonal bacterium, typified by a high degree of genetic homogeneity
[32, 161], Genetic diversity has most often been defined using single nucleotide polymorphisms
(SNPs; [162]), multilocus variable number tandem repeat analysis (MLVA) using eight [161], 15
[32], 25 [163], or 31 [164]) markers. More rapidly evolving changes are often measured with
single nucleotide repeats (SNR; [95]). Broadly, MLVA types fit within major lineages defined
by canonical SNPs (those that identify terminal branches within a phylogenetic analysis) and
SNRs are interpreted within MLVA-types [165] progressive hierarchical resolving assays using
nucleic acids scheme). Geographically, canonical SNPs identify broad lineages, usually at the
national or regional scale. MLVA diversity appears to be related to more regional spatial scales
[32, 87], with SNRs most useful locally and within MLVA-types. SNRs are subject to
homoplasy and less useful when like SNR types are defined between MLVA-types. With this in
mind, it is reasonable to expect the genetic diversity during an outbreak in an enzootic region to
be consistent with that observed in previous outbreaks at that location or neighboring locations
within the region. Multiple MLVA and SNR types have been observed during a natural outbreak

Unexpected
Strain
Anomalies in
Epidemiology and
Pattern Cases
Anomaly in
Vaccine
Efficacy
Site-Specific
Anomalies

Unexplained Location
Unexpected Time of Year
Unexpected Season
Unusual Host
Unexplained Number of
Cases
Unexplained Pattern of
Cases over Geographic
Region and Time
Unexplained Type of
Infection
25

-------
when environmental conditions are favorable suggesting multiple historical introductions [59],
However, the genetic diversity observed will be dependent on the geographic scale of the
outbreak, with larger numbers of farms or wildlife populations equating to a potential for higher
diversity. Within-farm diversity is often quite limited and more likely detected with SNRs once
MLVA types are established. However, unpublished observations by Hugh-Jones indicate that
when the strains recovered are from an area historically exposed to contaminated feed or salt, or
from a recent distribution of a contaminated feed, there might be interfarm variance, differences
between animals within a herd, and even sometimes multiple strains recovered from a singular
animal.
Phylogenetically, B. cmthrcicis is divided into five lineages (A-E following Lista et al. [163] or
A-D and Ap following Maho et al. [166]). Many available isolates in the global strain collections
represent three major lineages, designated A branch (A.Br.), B branch (B.Br.), or C branch
(C.Br.) based on canonical SNPs and their respective sub-lineages or sub-groups based on
MLVA types. Genetic analyses of B. cmthracis samples from a variety of North American
sources associated with livestock and wildlife outbreaks, confirmed the presence of a single
strain (i.e., A.Br. Western North America [WNA]) in 91% (352 WNA of 387 strains analyzed) of
SNP samples tested [86], although sample selection in that paper under-represented diversity of
Ames and Sterne-like strains from west and South Texas [32, 55, 87], As shown in Table 2, the
167 samples analyzed by Kenefic et al. [86] showed that most were from western states where
anthrax is enzootic (Colorado [CO], Minnesota [MN], Montana [MT], North Dakota [ND], New
Mexico [NM], Nevada [NV], and South Dakota [SD]) and were exclusively A.Br.WNA (using
canonical SNPs from Van Ert et al. [32]).
Table 2. Strains of B. anthracis associated with livestock and wildlife outbreaks in the
United States as adapted from Kenefic et al. [86]	
State
Observed Strains
No. of
Samples
State
Observed Strains
No. of
Samples
CA
B.Br.OO 1/002
2
OK
A.Br.Aust94
1
CO
A.Br.WNA 1
SD
A.Br.WNA
53
FL
A.Br.Ames 1
TX
A.Br.001/002
3
IA
ABr.WNA 1
TX
A.Br.Vollum
1
LA
C.Br.A1055 1
TX
A.Br.WNA
4
MD
A.Br.Vollum 1
UT
A.Br.WNA
2
MD
ABr.WNA 1
WT
A.Br.WNA
1
MN
ABr.WNA
23
WY
C.Br.A1055
1
MS
A.Br.Vollum 1
WY
A.Br.WNA
2
MT
ABr.WNA 1
USA*
A.Br.001/002
1
NC
ABr.WNA 1
USA*
A.Br.Aust94
1
ND
ABr.WNA
26
USA*
A.Br.003/004
1
NM
ABr.WNA
1
USA*
A.Br.Vollum
2
NV
ABr.WNA
5
USA*
A.Br.WNA
28
Source: Kenefic, L.J. et al., (2009). Pre-Columbian origins for North American anthrax. PLoS
One, 4 (3): e4813.
*No state specified. WNA, Western North America. No specific years for outbreaks given.
26

-------
What is clear from the North American phylogenetic analyses is that the A.Br.WNA group is
well established from Canada into Texas, matching its wide distribution throughout the world
[32, 154, 163], Other genetically-divergent strains of B. anthracis found in the United States are
assumed to have originated from handling, processing, and using imported animal hides, hair,
wool, bone meal, and other animal parts contaminated with B. anthracis spores indicative of
their regions of origin [32], Where justified by suspicion of an intentional release, additional
genetic testing of a strain (A.Br.WNA for example) may be useful to compare the suspect strain
to the local strain(s) at a higher level of genetic detail [167], Over time the reassessment of
archived reference strains have shown instructive minor differences. For example, SNR diversity
has been used to differentiate B. anthracis strains within large multi-property epizootics in Italy
with related minor strains of the singular major epidemic strain taking advantage of the epidemic
to infect a few spatially related herds [59],
B. anthracis Ames (A.Br.Ames) has been broadly used in biodefense research [32] as a
challenge strain. The organism used in the 2001 Amerithrax attacks was a laboratory strain of
Ames [168], The natural distribution of the classic Ames strain is limited in the U.S. and
localized to an area west of Uvalde, Texas; it was originally recovered from a single cow carcass
in 1981 in south Texas [32, 86, 169], Ames-like strains have also been reported from Kazakhstan
and Kyrgyzstan [154] and across Inner Mongolia in China [87], and would have reached the U.S.
in imported contaminated goat hair or hides sometime in the latter 20th century. Presence of an
Ames-like strain in any location except western Texas (defined as the Enzootic Zone by
Blackburn et al. [64]) and southern Texas, within Jim Hogg County, could indicate human-
cultured spores and (in the absence of an accidental laboratory exposure or release, or import of
contaminated animal products from southwest Texas) could signal a possible intentional release.
Additional genetic testing of an A.Br.WNA strain may be useful to compare the suspect strain to
the local strain(s) at a higher level of genetic detail [167], Following Lista et al. [163], a high
number of markers, such as MLVA-25, should be considered.
The stage in the analysis considers whether there is a likely source for the exotic or rare strain
that has not been intentionally released. Genetic data are required on the identified background
strains, strains in use in nearby laboratories, or strains endemic to the origin of potentially
contaminated animal products at that location. The presence of an unexplained genetic isolate of
B. anthracis is a sufficient anomaly to be subject to intentional release suspicion.
3.3 Screening Category II: Anomaly in Vaccine Efficacy?
Vaccination of mammals with live Sterne strain (lacks the pX02 plasmid) has been successful in
livestock since its introduction in the 1930s [136, 137], The Sterne vaccine is currently the most
widely used anthrax vaccine and is generally safe and effective for most animals [137, 139],
Reduced doses are needed when vaccinating llamas and goats because of their susceptibility to
the low pathogenicity of Sterne. Animals acquire immunity from a single dose in 7-10 days and
protection lasts about 9-12 months [136], When used to protect animals during an outbreak,
deaths from incubating infections are observed to decline substantially by the tenth day after
vaccination [60], Anthrax cases among effectively vaccinated livestock or individuals are of
concern if only because it reflects exposure, but it can also indicate inadequate vaccine
protection. For as yet unknown reasons this is a constant problem with certain Sterne livestock
vaccines [170],There are limited studies on the efficacy of the human vaccines [139], Also
27

-------
livestock deaths are sometimes seen in purchased unvaccinated stock added to a vaccinated herd
grazing a pasture with a history of previous anthrax cases.
Cases of anthrax in vaccinated animals may occur for a variety of reasons. Residual virulence
due to unpredictable attenuation in the Pasteur vaccine resulted in a 3% mortality in vaccinated
animals, which is why it was abandoned [73, 139], Anthrax cases appearing soon after
vaccination of animals could be due to a lethal incubating infection prior to the onset of
protection. If due to the vaccine, the strain can be isolated and confirmed as the vaccine strain
(e.g., Sterne). Vaccinations are effective in animals for six months to a year. During an epidemic,
a multiple dose regime may be required to ensure effectiveness especially with high value stock.
Administering vaccine and antibiotics concurrently may prevent the development of immunity
[79], However some have argued that as spore germination is not simultaneous, sufficient spores
will germinate late after the antibiotic blood titer has fallen to an ineffective level and thus an
adequate immunity will follow. Although it is better to wait seven to ten days after prophylactic
antibiotic treatment before vaccinating, working a herd twice carries both an added stress cost to
the livestock and financial cost to the owner.
A cell-free antigen vaccination method is used for humans in the United States [139, 170], AVA
efficacy was tested against several strains of B. anthracis in animal models. For certain isolates,
AVA was highly protective in both rhesus monkeys and rabbits, but only variably effective in
guinea pigs [142], The only placebo-controlled study of the cell-free human anthrax vaccine
evaluated effectiveness among workers in four textile mills who experienced chronic
occupational exposure to B. anthracis spores on contaminated goat hair. The study reported
92.5% effectiveness with a lower 95% confidence limit of 65% [112], The duration of immunity
from the two-inoculation human vaccine is believed to be 1-2 years [136, 171], with a three-
inoculation series protecting up to four years [139], A five-dose series of a cell-free vaccine
(BioThrax™ Anthrax Vaccine Adsorbed, Emergent BioSolutions, Rockville, MD) over 18
months is currently the human vaccination approach approved by the U. S. Food and Drug
Administration [172], According to the BioThrax™ product insert, incomplete vaccination
results in a decline in protective antigen antibodies thereby lowering protection.
Effectively vaccinated animals are those that received the vaccine within the past six months and
were not receiving antibiotics at the time of the vaccination or the weeks thereafter. Effectively
vaccinated humans are those who are within the expected period of immunity for the series of
vaccine they have received. Anthrax in effectively vaccinated animals or humans during the
expected period of protection (minimally six months) should be considered suspect. Given the
high efficacy of vaccines, anthrax occurring among a significant number of effectively
vaccinated animals may indicate the presence of a particularly virulent strain of B. anthracis in
the environment, or a deliberate release of spores of unknown origin for which existing vaccines
may not be effective. Intentional releases should be suspected in outbreaks among effectively
vaccinated animals by an unexpected strain, e.g., genetically modified B. anthracis [173].
Anthrax cases in vaccinated humans, especially those without known health compromises,
should be considered an indication of possible intentional contamination. However as present
human vaccines require a discipline of multiple injections stretched over time, incomplete series
are always a possibility.
28

-------
3.4 Screening Category III: Site Anomalies Observed?
Normal anthrax outbreaks are not ubiquitous, but occur at discrete locations where B. anthracis
was transported or persists due to an alkaline soil with a high calcium content. Locations where
anthrax outbreaks may occur include those near:
•	Historic outbreak sites or carcass burial sites, and downstream (as in water) from such
sites - downwind risk is limited to <20 meters with high wind speeds or mechanical soil
disturbance, and minimal risk because of large particle sizes [56];
•	Historic cattle trails or wildlife (herbivore) grazing habitats within the historical range of
anthrax;
•	Historic or current industrial animal processing sites (e.g., slaughter houses, tanneries,
wool processing, hair processing, fur processing, feed manufacture); nearby locations
where spores are carried by wind or washing waters, or where waste has been buried;
•	Biodefense laboratories, culture collections, testing ranges, or historic biological
laboratories; off-site handling of contaminated laboratory clothing can result in
reaerosolization, which can pose a hazard [174];
•	Laboratories with United States Department of Agriculture (USDA)/CDC approval for
research on virulent B. anthracis
Recently there have been two singular human pneumonic anthrax cases, neither from an
identified exposure. Fortunately, both survived. Their exposures were probably accidental and
not purposeful. One case in the United States is an amateur rock collector while on holiday and
traveling [96], The other is a vehicle maintenance mechanic in the United Kingdom armed forces
and previously vaccinated [175], Over time and random serum testing others may be identified
with no known exposure.
In addition, human anthrax may occur during the transport of contaminated animal products, e.g.,
containers, vehicles, and storage areas; or the use of such animal products, e.g., drum heads,
bone meals, and horse hair plaster binding. Human cases of anthrax arising from exposure to
contaminated animal products or accidental laboratory release can occur at any time of the year
and in any weather. The key factor is exposure to B. anthracis spores from such contaminated
sources by contact, ingestion, or inhalation. Exposures have been associated with disturbance of
legacy carcass sites [5], playing drums made from contaminated skins [118], and disturbing pipe
insulation made with contaminated goat hair [113],
In contrast, human cases arising from exposure to sick livestock or wildlife are most likely to
occur in the United States during drought conditions following a wet spring [14], Such
conditions can also trigger high biting fly activity, which may expand the size of an epizootic
[55] and lead to subsequent human cases, particularly those in close contact with animals, like
herders [68], However, regional drought/rain conditions may not trigger region-wide outbreaks.
Recently, Blackburn and Goodin [61] illustrated that epizootics are associated with localized
changes in vegetation greenness (a proxy for moisture) that appeared to promote anthrax in one
29

-------
area of a region and not another. In the United States, such natural outbreaks, as well as singular
sporadic cases, most often occur during the late spring through early fall, and end with the onset
of cold weather [14, 61], Human cases of anthrax arising from contact with wildlife or livestock
during the winter months or cold weather in enzootic regions should be considered a possible
intentional occurrence when other explanations for the initial wildlife or livestock exposure have
been ruled out, e.g., livestock consumption of contaminated feed. Wintertime livestock anthrax is
not usually expected in the United States, though cases among grazing animals are known to
have occurred.
Anthrax can and does infect a wide variety of livestock, wildlife, and rheas and ostriches [19, 22,
40, 58, 140], In the United States, the typical livestock hosts for anthrax are grazing animals,
primarily cattle [19], followed by plains bison [14], Other susceptible animals include wood
bison, white-tailed deer, and antelope, kudu, and lions in exotic animal ranches (common in
Texas and elsewhere), and horses, sheep, pigs, dogs, goats, and mink [19, 39, 40, 44, 60, 70, 79,
140], Cattle generally have more cases and higher mortality rates than other domestic herbivores;
mortality rates in white-tailed deer can reach 100% and though only 10% of wood bison will die,
another 70% may serologically reveal exposure [36], However, the presence of anthrax illness in
large numbers of animals thought to be resistant to anthrax illness (i.e., require extremely high
doses to contract illness) may suggest an intentional release-related exposure. For example, dogs
and pigs are moderately resistant to anthrax [176], Thus an outbreak among dogs in the United
States might indicate that affected dogs were exposed to high levels of aerosolized B. anthracis
spores (as was noted in Sverdlovsk in 1979 [104]), or had scavenged on undocumented carcasses
[41], or had been fed contaminated meat (as recently reported in Ukraine [44]). Efforts should be
made to determine the sources of dog infections. Thus, in the United States, canine exposure in a
farmyard in an enzootic area is not unexpected but, in an urban area, canine exposure is notable.
Laboratory based infections with B. anthracis do occur even when care is taken. Unless a
specially processed strain is involved that would have increased its aerosol potential, e.g.,
microencapsulation, it is usually just a minor cutaneous infection. But centrifuge accidents, e.g.,
broken vials, can and do result in aerosol exposure as well as laboratory contamination. Workers
must be aware that outer gloves used within a laminar flow biosafety cabinet should be removed
prior to removing hands from the cabinet and outer gloves replaced before touching any other
surface outside of the cabinet. However, a review of select agent theft, loss and release reports in
the U.S. between 2004 and 2010 revealed 727 such reports of which 12% were losses (primarily
due to sample mislabeling or misplacement within the lab) and 88% were release reports - or one
to nine reports per 1,000 worker years - of which only 11 resulted in laboratory acquired
infections, none of which were with B. anthracis; ten were other bacterial infections and one a
fungal agent. Thus, laboratory infections with select agents appear to be rare and, therefore, of
consequence when observed, but unrecognized releases are by definition unrecorded [177], That
should not stop investigations to look for releases, for example, through routine blind swabs of
surfaces likely to get contaminated.
3.5 Screening Category IV: Anomalies in Epidemiology?
Human cases of anthrax in the United States are rare [114], Most anthrax cases in the United
States (other than those arising from the 2001 letters) are cutaneous, traditionally from skinning
an infected carcass. However, a few pneumonic cases have been associated with contaminated-
30

-------
hide drum heads. When wool and hide mills were common, up to 60 years ago, processing
imported contaminated wool, hair, and hides, pneumonic anthrax posed a constant threat but they
have been shut down. Two cases with no known exposure have occurred in the United States and
United Kingdom (see Section 3.4) and the source of their infections remains unknown. With
prompt treatment, cutaneous anthrax is rarely fatal; without treatment, the fatality rate for
cutaneous anthrax ranges between 10% -20% and is virtually certain if systemic symptoms are
present [114], Only one case of gastrointestinal anthrax has been confirmed recently in the
United States and it was associated with contaminated skins used on drums rather than eating
contaminated meat [118, 178], This was in a community hall that had witnessed some 30 years
of public drumming events. In 2000 six clinical cases were suspected following the consumption
of some contaminated meat but not confirmed because of prior antibiotic treatment [179], While
family and community-based outbreaks of cutaneous and gastroenteric anthrax occur in Central
Asia and Africa due to the slaughter and butchering of sick animals and the scavenging of meat
from dead animals, such events are not seen in the United States and Canada - the Minnesota
incident was an exception. Recently injection anthrax has been seen multiple times in the United
Kingdom and Europe from the use of contaminated heroin, and has resulted in high death rates
[180], While many rural physicians are aware of the risk, especially in North and South Dakota,
Texas, and the Canadian prairie provinces, urban doctors are less likely to recognize the disease
until it is severe or at autopsy.
The immunity to anthrax, whether from a natural exposure or from vaccination, is short-lived
[36], Therefore any unvaccinated person demonstrating antibodies has been exposed sometime in
the previous six months; the same applies to a previously vaccinated person with a higher than
expected titer. Not all animals die from anthrax and thus animals exposed during an outbreak or
epidemic, with or without clinical disease, may demonstrate antibody titers [36, 45, 89], This can
also be in the absence of known coincident outbreaks. For example, as part of the annual
brucellosis surveillance, repeated random bison bleedings at the MacKenzie Bison Sanctuary
over many years demonstrated sporadic protective antigen antibody positive animals even
though the routine aerial surveillance for anthrax carcasses had found no cases at the time (Betty
Golsteyn-Thomas, CFIA - ADRI Lethbridge, personal communication, April 11, 2014). This
represents a silent or paradoxical epidemiology but note should be taken when anthrax occurs in
the absence of any logical normal explanation, e.g., an urban office worker distant from any
hypothetical natural exposure.
Most cases of human anthrax occur within seven days of exposure [151], The incubation period
range for cutaneous anthrax is from one to 12 days; for gastrointestinal anthrax one to seven
days; and for inhalational anthrax one to 45 days, consistent with prolonged spore dormancy
within the lung [40, 104, 151, 181-183], While inhalation anthrax incubation is generally
reported as about one to five days, the incubation period is dose-dependent, with a lower dose
corresponding to a longer incubation period [184], Long latency periods, e.g., 30 days, have been
occasionally observed in humans [104] and over months in animals [14, 185], However, a
continued high level of human cases beyond day seven of an initial outbreak may indicate
possible on-going intentional releases and exposures. Given the rarity of anthrax cases in the
United States today, if multiple human cases cannot be attributed to a common source of
exposure such as a contaminated animal, carcass site, laboratory, or a contaminated animal
product, an intentional contamination should be suspected. Cases of human inhalation anthrax
31

-------
are extremely rare and typically occur as individual cases. Multiple cases of inhalation anthrax
are likely to be intentional or associated with an unintentional laboratory release.
As a result of the 2001 contaminated letters incident, the United States Postal Service® (USPS)
set up a Biohazard Detection System (BDS) to provide mail security without disrupting the flow
of mail from individuals and small organizations. The BDS assumes that all bulk mail from
commercial mailers is safe. The BDS is attached to a specific piece of automated postal
processing equipment, Advanced Facer Canceller System (AFCS) machines, which are used to
scan mail for their proper postage. As the mail is processed by the AFCS, air samples are
continually collected by the BDS and analyzed for the presence of B. anthracis spores. A
positive test signal stops the mail processing, triggers alarms, and informs emergency
responders. But absent a positive signal the air sampling does not affect the mail flow. The
unique characteristic of the AFCS machines is that they exclusively process collection mail put
into street collection boxes, from homes, small businesses, and individuals. Other equipment,
such as Delivery Bar Code Sorters are used for bulk mailings from large mailers. BDS was not
the only security system to appear after the anthrax letters. The USPS introduced a suite of
services known as 'Intelligent Mail' to confront bioterrorism. It is an assemblage of interlinked
technologies, including bar codes, scanning equipment, and software that generate, store, and
manipulate real-time data from the postal network. The Intelligent Mail barcodes contain unique
identifier information indicating mailer and recipient, routing details, and service type. The real-
time processing and distribution data generated can be used to isolate cross-contaminated mail
and reroute other mailings away from cross-contamination sites. This wealth of Intelligent Mail
data would aid investigators in retrospectively tracing the movement of contaminated mail
through the system [186], Assuming it is as efficient and reliable as claimed, the postal system
not only identifies individual pieces of contaminated mail, but provides a holistic view of the
event, whether it is unintentional or intentional, as it evolves.
3.6 Screening Approach for Location-Specific Anomalies and Epidemiology
There are a number of significant singular events which alone indicate a possible intentional
event in the United States and are worthy of further investigation:
[1]	multiple unrelated cases of anthrax in humans, especially inhalational anthrax;
[2]	an outbreak in livestock east of the Mississippi;
[3]	a human outbreak that does not involve drums or an obvious occupational exposure;
[4]	an outbreak of inhalational anthrax of no obvious cause;
[5]	simultaneous or near-simultaneous outbreaks in multiple locations (multi-foci) without an
obvious common cause;
[6]	cases in individual animals or persons who had been fully vaccinated within 6 months with a
recognized valid vaccine;
[7]	isolation of multidrug-resistant strains of B. anthracis;
[8]	an incident that involves envelope(s)/packages containing loose dry B. anthracis spores;
[9]	the recovery of loose spores in an atypical location (e.g., as in purposefully contaminated
animal feed, in HVAC filters, on urban surfaces) or in a vial, outside of a laboratory, especially
with evidence of sophisticated spore modification, e.g., microencapsulation;
[10]	the recovery of an unexplained genotype or of genetically manipulated spores.
32

-------
Figures 3-7 present keys incorporating both location-specific characteristics and epidemiology
for evaluating the likelihood that a B. anthracis occurrence is intentional. The keys are not
strictly dichotomous; rather, they constitute a systematic path for gathering indications that an
occurrence is (or is not) intentional. Therefore, in some cases a statement to "suspect intentional
activity" is followed by a loop back into the key to answer additional questions that may lead to
additional indicators of an intentional occurrence. As described following the keys, cumulative
indicators of suspicion lead to an overall conclusion as to whether suspicion of an intentional
release is warranted. Only in specific cases, e.g., presence of spores displaying sophisticated
spore modification, will a single indicator lead to a strong conclusion that an intentional release
is likely.
The quick screen is biased toward an assumption of an intentional release. There are exceptions
to the logic that is used. For example, long latency of B. anthracis could cause an outbreak in an
unexpected season. Further, individual cases of anthrax sometimes occur with no suspicion of an
intentional act, but without a source of exposure ever being found.
Questions in the key in Figure 3 point to the location-appropriate key (in Figures 4 through 7). If
the location of an occurrence falls outside of the logical locations where B. anthracis might be
expected, the occurrence should be interpreted as a potential intentional release.
Using the keys above in the sequence shown, the following interpretation is proposed. A single
response of "assumed to be an intentional exposure" suggests a high likelihood of an intentional
release incident. Two or more questions in the keys yielding "suspect intentional exposure"
outcomes likewise suggest a high likelihood of an intentional incident. A single "suspect
intentional exposure" coupled with other answers suggesting a deliberate act as a possible
explanation requires further investigation beyond the screening approach presented here to
distinguish whether the occurrence is likely an intentional release. The screening approach
provides a rapid indication of whether the occurrence is likely intentional. It is not intended for
making decisions regarding whether the act was criminal in nature or to be used for public health
decisions.
33

-------
Did the infected person have contact with cattle,
sheep, goats or wildlife OR was this occurrence near a
prior anthrax site, or along a historic cattle drive trail?
Yes

No
Does the infected person work in a facility where B.
anthracis is grown or stored OR is this occurrence near
a laboratory or defense location currently or
historically using B. anthracis?
Yes
I
No
Go to
Clrr. A
Go to

Yes

Was this occurrence near an existing or historic site of
Go to
v Figure 6 >
an animal transportation or processing facility?
W
1 No


Were animal products (hair, hides, wool, bone meal) in
use near the occurrence?
No
i r
Yes
Unless there is another suspected source of natural
or unintentional exposure, this outbreak is assumed
to be an intentional exposure.
Go to
"7
Figure 3. Key to location-specific screening for intentional occurrences.
34

-------
Did the infected person have contact with cattle, sheep, goats or
wildlife OR was this occurrence near a prior anthrax site, or along
a historic cattle drive trail?		.—
Yes
No
Yes
No
Yes
Yes
Yes
No
Yes
No
Suspect intentional
exposure
Consistent with
natural outbreak
Suspect intentional
exposure via
contaminated food
Suspect intentional
exposure
Suspect intentional
exposure, but
check Figure 6
Consistent with
natural outbreak
Assume intentional exposure.
Multiple instances or broad distribution of human cases?
Were there few or no human cases of cutaneous anthrax?
Human inhalation anthrax (possibly with other types)?
Human gastrointestinal cases (possibly in addition to
cutaneous)?
Flood followed by drought, radiating pattern of multiple cases
from index case(s) (first carcasses not immediately disposed)?
Animal outbreak occurred during usual anthrax season (late
spring to late summer)?
Herbivores infected (similar species to prior outbreaks), one or
few cases OR large number of cases if infected carcass not
disposed promptly?
Figure 4. Screening for intentional occurrences in an agricultural location OR occurrences
in natural locations (wildlife).
35

-------
Does the infected person work in a facility where B. anthracis is
grown or stored OR is this occurrence near a laboratory or
defense location currently or historically
using B. anthracis?
Yes
One or few human cases consistent with occupational
exposure?
No
Geographic distribution of cases (inhalation and cutaneous)
consistent with prevailing wind from laboratory at assumed
time of release?
.No
Suspected intentional exposure.
Figure 5. Screening for intentional occurrences in a laboratory location.
Occurrence was near an existing or historic site of an animal
transportation or processing facility?
Single human case?
No
Yes
Likely
unintentional
exposure
Multiple human cases: suspect intentional exposure
Figure 6. Screening for intentional occurrences in processing plant or animal
transportation locations.
Potential
unintentional or
intentional release
Likely unintentional
exposure or a
deliberate homicide
or suicide
36

-------
Sporadic, singular human cases?
Likely unintentional
exposure
Likely unintentional
exposure linked to
contaminated drugs.
Intentional exposure
possible
Exposure linked to injection of illegal drugs?
Suspect intentional exposure
Were animal products in use near the occurrence, e.g.,
African drum, animal feed containing bone meal, animal
skins for heroin transport, use of heroin cut with bone
meal?
Livestock/wildlife feed intentionally contaminated?
No
Likely unintentional
exposure
Suspect intentional exposure
Figure 7. Screening for intentional occurrences in locations where animal products are in
use.
3.7 Conclusions
Compared to a natural outbreak or epidemic, an intentional anthrax spore release will occur in
the wrong place, at the wrong time, in the wrong group, or with the wrong strain; if from a
massive aerosol, there may be multiple cases. Ignoring the repetitive and imitative talcum-
powder attacks on abortion clinics, such an attack with anthrax spores is, at the moment, more a
matter of theory than reality but ricin has appeared in letters [187], The footprint for an
intentional attack is unpredictable. While the great majority of anthrax related events might be
naturally occurring, understanding of the nature of the occurrence might be confused by our still
evolving knowledge of the di sease, the agent, and its epidemiology. The screening approach
presented here is designed to help rapidly and systematically determine the likelihood that a
37

-------
detection or exposure is intentional. A limited number of parameters need to be considered in
this screening process and, given ready access to necessary data, an initial evaluation can be
completed quickly, except for identifying the strain of the B. anthracis, which will require about
48-96 hours using current technologies.
The culture collections associated with anthrax sites in the United States are limited. It is
possible, even likely, over the coming years new and unexpected strains of B. anthracis will be
discovered in natural environments that support spore survival. This will not reflect an
intentional release event, just the discovery of new pre-existing strains in the environment.
Following an intentional release, recovery of the organism might be possible from multiple
surfaces in the exposed zone; in ranch land it might be recoverable from trees, flowers, grass,
soil, and fruits from trees; in urban locations, from windowsills, air condition filters, exposed
granite surfaces, benches, automobiles, and so on. Typically, B. anthracis is difficult to isolate
from environmental surfaces so having multisource samples all yielding B. anthracis in the
absence of carcasses points to an intentional release.
4 Implications for Clean-Up of Occurrences of B. anthracis and Identified Gaps
Roles and actions in the response to an occurrence of biological threat agent depends both on the
nature of an occurrence and the risks associated with an occurrence. It is important to know
whether an occurrence of biological threat agent is natural, unintentional, or an intentional
human act. The risk to humans (or animals) of a biological threat agent detected in the
environment needs to be understood to inform response and decontamination decisions. Risk is a
function of dose and exposure. If the detected biological threat agent was deliberately released
there might be associated factors that increase the risk, e.g., high concentration of spores for
exposure, and/or sophisticated spore modifications to enhance aerosolization and retention in the
lungs.
Among biological threat agents, B. anthracis has unique characteristics that make it attractive for
deliberate use: under simple storage conditions the spores remain viable and virulent for decades;
the disease is not contagious (not transmitted person to person) thereby limiting spread beyond
the target; the spores can be prepared in a form that is readily aerosolized; and inhalation anthrax
in humans has a high mortality rate. For these reasons, significant effort has been focused to both
understand human exposure to B. anthracis, as well as to understand the risks associated with
exposures. Identifying factors that trigger timely and appropriate responses could be challenging.
A survey of scientific literature was conducted to determine the current state of the science
regarding the presence of B. anthracis and corresponding natural or unintentional outbreaks of
anthrax. Appropriate response, as well as both the actual and perceived human health risk, is
different when a release of B. anthracis is deliberate rather than natural or unintentional. During
the literature review of natural and unintentional occurrences of anthrax, a variety of
characteristics became apparent that would enable a screening of information regarding anthrax
outbreaks to rapidly assess whether the occurrence was intentional. The screening would inform
the evaluation as to whether an intentional release had occurred. The screening approach is not
intended to be a tool for performing the criminal investigation or for the extensive public health
assessment, although the information and logic may inform both endeavors.
38

-------
Knowledge related to exposure from occurrences of B. anthracis were identified. It is important
to understand under what circumstances human exposure to B. anthracis occurs and how B.
anthracis may attenuate or persist after a deliberate release into the environment. Questions
include:
•	Why do dormancy periods occur between anthrax outbreaks at a given location and what
synergies occur to break dormancy?
•	What causes and what prevents natural attenuation of virulent B. anthracis in the
environment?
•	What are the natural reproductive cycles? What is the role (if any) of lysogeny?
•	How can detection of virulent B. anthracis in the environment be improved?
•	How susceptible are human populations to anthrax? What factors influence
susceptibility? How can susceptibility data be incorporated into human dose-response
relationships?
•	How does the background level of virulent or avirulent B. anthracis in the environment
(and other factors) determine the likelihood of sporadic outbreaks?
•	What is the role of seasonal environmental conditions in the severity of epizootics and
subsequent risk to humans?
•	What is the role of mechanical vectors, and associated spatial and ecological factors, in
transmission risk to both animals and humans during outbreaks?
•	What is the likelihood and particle size distribution of reaerosolized B. anthracis spores
in the natural environment?
To rapidly and accurately evaluate whether an occurrence in an intentional release, necessary
data must be obtained quickly, including:
•	data from CDC/USD A and the Department of Defense on strains of B. anthracis in use or
historically used at laboratories in the region;
•	list of historic outbreak sites and of carcass burial and burn locations in the region;
•	environmental background levels of B. anthracis typically found at the burial and burn
locations;
•	determination of the B. anthracis strains present.
To reduce uncertainty associated with the proposed rapid evaluation, key knowledge and
technology gaps need to be filled. For example, accurate knowledge of the locations of anthrax
outbreaks in the last two decades is available. However, locations of outbreaks during the 1950s
and 1960s and the locations where the carcasses were buried that might be contributing to
outbreaks today are known with less certainty. Information on the strains of B. anthracis in use
in laboratories may not be readily accessible. Although most reliable laboratories use MLVA24
typing system, SNP assays and SNRs, there is no standard, and some use just the MLVA8 typing
system. Fortunately the MLVA8 is included in the MLVA25 thereby identifying the same clade
and subclade but missing the finer detail. The result is that a backup genomic identification
sometimes has to be organized to more fully characterize the B. anthracis strains present at an
occurrence. Ensuring that positive diagnostic cultures are not destroyed but promptly shared with
the CDC strain archive would retain valuable information.
39

-------
When non-industrial cases of human anthrax occur in the United States, they are usually
cutaneous [5], The locations of such outbreaks typically correspond to endemic areas from North
Dakota to Texas. Outside of these areas anthrax is rarely seen in the United States in this century.
Occasionally anthrax is associated with contaminated animal products, typically imported, such
as animal-hide drumheads. Even more rare are cases of human inhalation anthrax in the United
States and, in some cases, no source of exposure is ever identified. While all human anthrax
cases are a cause for concern, natural and unintentional occurrences are generally addressed
through routine USDA and CDC sampling procedures. When the outbreak results in significant
environmental contamination, EPA might assist in cleanup, requiring appropriate and timely
response to an intentional incidents. Use of the proposed systematic assessment of the likelihood
that an event was intentional, might help indicate what type of response might be needed.
40

-------
5 References
1.	Eitzen, E.M.J. (1997). Medical aspects of chemical and biological warfare, ed. F.R.
Sidell, E.T. Takafuji, and D.R. Franz. Washington, D.C.: Borden Institute, Walter Reed
Army Medical Center.
2.	Kracalik, I., L. Malania, N. Tsertsvadze, J. Manvelyan, L. Bakanidze, P. Imnadze, S.
Tsanava, and J.K. Blackburn. (2014). Human cutaneous anthrax, Georgia 2010-2012.
Emerg Infect Dis, 20(2): 261-4.
3.	Fennelly, K.P., A.L. Davidow, S.L. Miller, N. Connell, and J.J. Ellner. (2004). Airborne
infection with Bacillus anthracis—ixom mills to mail. Emerg Infect Dis, 10(6): 996-1002.
4.	Price, P.N., M.D. Sohn, K.S. Lacommare, and J. A. McWilliams. (2009). Framework for
evaluating anthrax risk in buildings. Environ Sci Technol, 43(6): 1783-7.
5.	Turnbull, P.C., ed. (2008). Anthrax in Humans and Animals. 4th ed., World Health
Organization: Geneva.
6.	Koch, R. (1876). The etiology of anthrax, based on the life history of Bacillus anthracis.
Beitrage zur Biologie der Pflanzen, 1(2): 277-310.
7.	Turnbull, P.C. and S.V. Shadomy. (2011). From ancient times to the 19th century, in
Bacillus anthracis and Anthrax, N.H. Bergman, Editor., Wiley-Blackwell: New York. p.
1-16.
8.	Rail, J.M. and T.M. Koehler. (2011). Bacillus anthracis virulence gene regulation, in
Bacillus anthracis and Anthrax, N.H. Bergman, Editor., John Wiley and Sons, Inc.:
Hoboken, N.J. p. 157-178.
9.	Coker, P.R., K.L. Smith, P.F. Fellows, G. Rybachuck, K.G. Kousoulas, and M.E. Hugh-
Jones. (2003). Bacillus anthracis virulence in Guinea pigs vaccinated with anthrax
vaccine adsorbed is linked to plasmid quantities and clonality. J Clin Microbiol, 41(3):
1212-8.
10.	Keim, P., A. Kalif, J. Schupp, K. Hill, S.E. Travis, K. Richmond, D.M. Adair, M. Hugh-
Jones, C.R. Kuske, and P. Jackson. (1997). Molecular evolution and diversity in Bacillus
anthracis as detected by amplified fragment length polymorphism markers. JBacteriol,
179(3): 818-24.
11.	Dragon, D.C., R.P. Rennie, and B.T. Elkin. (2001). Detection of anthrax spores in
endemic regions of northern Canada. JApplMicrobiol, 91(3): 435-41.
12.	Minett, F.C. and M.R. Dhanda. (1941). Multiplication of B. anthacis and CI. chauvoei in
soil and water. Indian J Vet Sciences, 11: 308-328.
13.	Dragon, D.C. and R.P. Rennie. (1995). The ecology of anthrax spores: tough but not
invincible. Can Vet J, 36(5): 295-301.
14.	Hugh-Jones, M. and J. Blackburn. (2009). The ecology of Bacillus anthracis. Mol
Aspects Med, 30(6): 356-67.
15.	Sinclair, R., S.A. Boone, D. Greenberg, P. Keim, and C.P. Gerba. (2008). Persistence of
category A select agents in the environment. Appl Environ Microbiol, 74(3): 555-63.
16.	Baxter, R.G. (1977). Anthrax in the dairy herd. JS Afr Vet Assoc, 48(4): 293-5.
17.	Van Ness, G. and C. Stein. (1956). Soils of the United States favorable for anthrax. J Am
Vet Med Assoc, 128: 7.
18.	Van Ness, G.B. (1971). Ecology of anthrax. Science, 172(3990): 1303-7.
19.	Johnson, R. (2007). Differentiation of naturally occurring from non-naturally occurring
epizootics of anthrax in livestock populations: USD A-APHIS.
41

-------
20.	Van Ness, G. (1967). Geologic implications of anthrax. The Geological Society of
America Special Paper 90: 61-64.
21.	Saile, E. and T.M. Koehler. (2006). Bacillus anthracis multiplication, persistence, and
genetic exchange in the rhizosphere of grass plants. Appl Environ Microbiol, 72(5): 3168-
74.
22.	Turnbull, P., R. Bohm, M. Hugh-Jones, and J. Melling. (2008). Guidelines for the
Surveillance and Control of Anthrax in Humans and Animals. Fourth Edition: 2019.
23.	Schuch, R. and V.A. Fischetti. (2009). The secret life of the anthrax agent Bacillus
anthracis: bacteriophage-mediated ecological adaptations. PLoS One, 4(8): e6532.
24.	Ganz, H.H., W.C. Turner, E.L. Brodie, M. Kusters, Y. Shi, H. Sibanda, T. Torok, and
W.M. Getz. (2014). Interactions between Bacillus anthracis and plants may promote
anthrax transmission. PLoSNegl Trop Dis, 8(6): e2903.
25.	Lee, K., J.W. Costerton, J. Ravel, R.K. Auerbach, D.M. Wagner, P. Keim, and J.G. Leid.
(2007). Phenotypic and functional characterization of Bacillus anthracis biofilms.
Microbiol, 153(Pt6): 1693-701.
26.	Dey, R., P.S. Hoffman, and I.J. Glomski. (2012). Germination and amplification of
anthrax spores by soil-dwelling amoebas. Appl Environ Microbiol, 78(22): 8075-81.
27.	Schuch, R., A.J. Pelzek, S. Kan, and V.A. Fischetti. (2010). Prevalence of Bacillus
anthracis-lxke organisms and bacteriophages in the intestinal tract of the earthworm
Eisenia fetida. Appl Environ Microbiol, 76(7): 2286-94.
28.	Mullins, J., L. Lukhnova, A. Aikimbayev, Y. Pazilov, M. Van Ert, and J.K. Blackburn.
(2011). Ecological niche modelling of the Bacillus anthracis Al.a sub-lineage in
Kazakhstan. BMC Ecol, 11: 32.
29.	Collier, F.A., S.L. Elliot, and R.J. Ellis. (2005). Spatial variation in Bacillus
thuringiensis/cereus populations within the phyllosphere of broad-leaved dock (Rumex
obtusifolius) and surrounding habitats. FEMS Microbiol Ecol, 54(3): 417-425.
30.	Blackburn, J.K., K.M. McNyset, A. Curtis, and M.E. Hugh-Jones. (2007). Modeling the
geographic distribution of Bacillus anthracis, the causative agent of anthrax disease, for
the contiguous United States using predictive ecologic niche modeling. Am J Trop Med
Hyg, 77(6): 1103-10.
31.	Blackburn, J.K. (2010). Integrating geographic information systems and ecological niche
modeling into disease ecology: A case study of Bacillus anthracis in the United States
and Mexico, in Emerging and Endemic Pathogens: Advances in Surveillance, Detection,
and Identification, K.P. O'Connell, E.W. Skowronski, A. Sulakvelidze, and L. Bakanidze,
Editors., Springer, p. 59-88.
32.	Van Ert, M.N., W.R. Easterday, L.Y. Huynh, R.T. Okinaka, M.E. Hugh-Jones, J. Ravel,
S.R. Zanecki, T. Pearson, T.S. Simonson, and J.M. U'Ren. (2007). Global genetic
population structure of Bacillus anthracis. PLoS One May 2(5): e461.
33.	Mullins, J.C., G. Garofolo, M. Van Ert, A. Fasanella, L. Lukhnova, M.E. Hugh-Jones,
and J.K. Blackburn. (2013). Ecological niche modeling of Bacillus anthracis on three
continents: Evidence for genetic-ecological divergence? PLoS One, 8(8): e72451.
34.	Kracalik, I.T., J.K. Blackburn, L. Lukhnova, Y. Pazilov, M.E. Hugh-Jones, and A.
Aikimbayev. (2012). Analysing the spatial patterns of livestock anthrax in Kazakhstan in
relation to environmental factors: a comparison of local (Gi*) and morphology cluster
statistics. GeospatHealth, 7(1): 111-26.
42

-------
35.	Bellan, S.E., O. Gimenez, R. Choquet, and W.M. Getz. (2013). A Hierarchical Distance
Sampling Approach to Estimating Mortality Rates from Opportunistic Carcass
Surveillance Data. Methods Ecol Evol, 4(4).
36.	Bagamian, K.H., K.A. Alexander, T.L. Hadfield, and J.K. Blackburn. (2013). Ante- and
postmortem diagnostic techniques for anthrax: rethinking pathogen exposure and the
geographic extent of the disease in wildlife. J Wildl Dis, 49(4): 786-801.
37.	Griffin, D., T. Petrosky, S. Morman, and V. Luna. (2009). A survey of the occurence of
Bacillus anthracis in North American soils over two long-range transects and within
post-KatrinaNew Orleans. Appl Geochem, 24: 1464-1471.
38.	Ko, K.S., J.M. Kim, J.W. Kim, B Y. Jung, W. Kim, I.J. Kim, and Y.H. Kook. (2003).
Identification of Bacillus anthracis by rpoB sequence analysis and multiplex PCR J Clin
Microbiol, 41(7): 2908-14.
39.	Hugh-Jones, M.E. and V.D. Vos. (2002). Anthrax and wildlife. Revue Scientifique et
Technique (International Office of Epizootics), 21(2): 359-383.
40.	Beyer, W. and P.C. Turnbull. (2009). Anthrax in animals. Mol Aspects Med, 30(6): 481 -
9.
41.	Lembo, T., K. Hampson, H. Auty, C.A. Beesley, P. Bessell, C. Packer, J. Halliday, R.
Fyumagwa, R. Hoare, E. Ernest, C. Mentzel, T. Mlengeya, K. Stamey, P.P. Wilkins, and
S. Cleaveland. (2011). Serologic surveillance of anthrax in the Serengeti ecosystem,
Tanzania, 1996-2009. EmergInfect Dis, 17(3): 387-94.
42.	Blackburn, J.K., V. Asher, S. Stokke, D.L. Hunter, and K.A. Alexander. (2014). Dances
with anthrax: wolves (Canis lupus) kill anthrax bacteremic plains bison (Bison bison
bison) in southwestern Montana. J Wildl Dis, 50(2): 393-6.
43.	Skrypnyk, V., R. Koziy, A. Skrypnyk, I. Rublenko, K. Bagamian, J. Farlow, M. Nikolich,
A. Mezhenskiy, O. Nevolko, and J. Blackburn. (2014). Anthrax in Dogs, yna CynaCCui
nayKoei po3po6KU, 1(215): 14-17.
44.	Blackburn, J.K., A. Skrypnyk, K.H. Bagamian, M.P. Nikolich, M. Bezymennyi, and V.
Skrypnyk. (2014). Anthrax in a backyard domestic dog in Ukraine: a case report. Vector
Borne Zoonotic Dis, 14(8): 615-7.
45.	Cizauskas, C.A., S.E. Bellan, W.C. Turner, R.E. Vance, and W.M. Getz. (2014).
Frequent and seasonally variable sublethal anthrax infections are accompanied by short-
lived immunity in an endemic system. JAnim Ecol.
46.	Turnbull, P., M. Diekman, J. Killian, W. Versfeld, V.D. Vos, L. Arntzen, K. Wolter, P.
Bartels, and A. Kotze. (2010). Naturally acquired antibodies to Bacillus anthracis
protective antigen in vultures of southern Africa Onderstepoort Journal of Veterinary
Research, 75: 95-102.
47.	Turner, W.C., P. Imologhome, Z. Havarua, G.P. Kaaya, J.K. Mfune, I.D. Mpofu, and
W.M. Getz. (2013). Soil ingestion, nutrition and the seasonality of anthrax in herbivores
of Etosha National Park. Ecosphere, 4: artl3.
48.	Blecha, F. and K. Kelley. (1989). Heat exposure and immune function, Chapter 3.5, in
Animal Health and Production at Extremes of Weather. World Meteorological
Organization: Geneva, Switzerland, p. 104-108.
49.	Blackburn, J.K., A. Curtis, T.L. Hadfield, B. O'Shea, M.A. Mitchell, and M.E. Hugh-
Jones. (2010). Confirmation of Bacillus anthracis from flesh-eating flies collected during
a West Texas anthrax season. J Wildl Dis, 46(3): 918-22.
43

-------
50.	Fox, M.D., J.M. Boyce, A.F. Kaufmann, J.B. Young, and H.W. Whitford. (1977). An
epizootiologic study of anthrax in Falls County, Texas. J Am Vet Med Assoc, 170(3):
327-33.
51.	Lindeque, P.M. and P.C. Turnbull. (1994). Ecology and epidemiology of anthrax in the
Etosha National Park, Namibia. Onderstepoort J Vet Res, 61(1): 71-83.
52.	Minett, F.C. (1950). Sporulation and viability of B. anthracis in relation to environmental
temperature and humidity. J Comp Pathol, 60(3): 161-76.
53.	Dragon, D.C., D.E. Bader, J. Mitchell, and N. Woollen. (2005). Natural dissemination of
Bacillus anthracis spores in northern Canada. Appl Environ Microbiol, 71(3): 1610-1615.
54.	Atlas, R.M. (2002). Responding to the threat of bioterrorism: a microbial ecology
perspective—the case of anthrax. Int Microbiol, 5(4): 161-7.
55.	Blackburn, J.K., M. Van Ert, J.C. Mullins, T.L. Hadfield, and M.E. Hugh-Jones. (2014).
The necrophagous fly anthrax transmission pathway: Empirical and genetic evidence
from wildlife epizootics. Vector Borne Zoonotic Dis, 14(8): 576-83.
56.	Turnbull, P.C., P.M. Lindeque, J. Le Roux, A.M. Bennett, and S.R. Parks. (1998).
Airborne movement of anthrax spores from carcass sites in the Etosha National Park,
Namibia. J Appl Microbiol, 84(4): 667-76.
57.	Muoria, P., P. Muruthi, W. Kariuki, B. Hassan, D. Mijele, and N. Oguge. (2007). Anthrax
outbreak among Grevy's zebra (Equus grevyi) in Samburu, Kenya. Afr J Ecol, 45: 483-
489.
58.	Epp, T., C. Argue, C. Waldner, and O. Berke. (2010). Spatial analysis of an anthrax
outbreak in Saskatchewan, 2006. Can Vet J, 51(7): 743-8.
59.	Fasanella, A., G. Garofolo, D. Galante, V. Quaranta, L. Palazzo, F. Lista, R. Adone, and
M.H. Jones. (2010). Severe anthrax outbreaks in Italy in 2004: Considerations on factors
involved in the spread of infection. New Microbiol, 33(1): 83-6.
60.	Turner, A.J., J.W. Galvin, R.J. Rubira, R.J. Condron, and T. Bradley. (1999). Experiences
with vaccination and epidemiological investigations on an anthrax outbreak in Australia
in 1997. J Appl Microbiol, 87(2): 294-7.
61.	Blackburn, J.K. and D.G. Goodin. (2013). Differentiation of springtime vegetation
indices associated with summer anthrax epizootics in west Texas, USA, deer. J Wildl Dis,
49(3): 699-703.
62.	Parkinson, R., A. Rajic, and C. Jenson. (2003). Investigation of an anthrax outbreak in
Alberta in 1999 using a geographic information system. Can Vet J, 44(4): 315-8.
63.	Turner, A.J., J.W. Galvin, R.J. Rubira, and G.T. Miller. (1999). Anthrax explodes in an
Australian summer. J Appl Microbiol, 87(2): 196-9.
64.	Blackburn, J.K., T.L. Hadfield, A. Curtis, and M.E. Hugh-Jones. (2014). Spatial and
temporal patterns of anthrax in White-Tailed Deer, Odocoileus virginianus, and
hematophagous flies in West Texas during the summertime anthrax risk period. Ann
Assoc Am Geographers, 104(5): 939-958.
65.	Turell, M.J. and G.B. Knudson. (1987). Mechanical transmission of Bacillus anthracis by
stable flies (Stomoxys calcitrans) and mosquitoes {Aedes aegypti and Aedes
taeniorhynchus). Infect Immun, 55(8): 1859-61.
66.	Fasanella, A., S. Scasciamacchia, G. Garofolo, A. Giangaspero, E. Tarsitano, andR.
Adone. (2010). Evaluation of the house fly Musca domestica as a mechanical vector for
an anthrax. PLoS One, 5(8): el2219.
44

-------
67.	von Terzi, B., P.C. Turnbull, S.E. Bellan, and W. Beyer. (2014). Failure of Sterne-and
Pasteur-like strains of Bacillus anthracis to replicate and survive in the urban bluebottle
blow fly Calliphora vicina under laboratory conditions. PLoS ONE, 9: e83860.
68.	Fasanella, A., G. Garofolo, M. Galella, P. Troiano, C. De Stefano, L. Pace, A. Aceti, L.
Serrecchia, andR. Adone. (2013). Suspect vector transmission of human cutaneous
anthrax during an animal outbreak in Southern Italy. Vector Borne Zoonotic Dis, 13(10):
769-71.
69.	Olsuf ev, N.G. and P.P. Lelep. (1935). On the importance of tabanids in the spread of
anthrax, in Parasites, Transmetteurs, Anim. Venimeux. Rec. Trav. 25e Anniv. Sci
Pavovsky 1909-34. p. 145-197.
70.	Saggese, M.D., R.P. Noseda, M.M. Uhart, S.L. Deem, H. Ferreyra, M.C. Romano, M.C.
Ferreyra-Armas, and M. Hugh-Jones. (2007). First detection of Bacillus anthracis in
feces of free-ranging raptors from central Argentina. J Wildl Dis, 43(1): 136-41.
71.	Turnbull, P.C.B., J.A. Carman, P.M. Lindeque, F. Joubert, O.J.B. Hiibschle, and G.H.
Snoeyenbos. (1989). Further progress in understanding anthrax in the Etosha National
Park. Madoqua, 16: 93-104.
72.	Pienaar, U. (1967). Epidemiology of anthrax in wild animals and the control of anthrax
epizootics in the Kruger National Park, South Africa. Federation Proceedings, 26(5):
1496-1502.
73.	Steele, J.H. and R.J. Helvig. (1953). Anthrax in the United States. Public Health Rep,
68(6): 616-23.
74.	Davies, D.G. and R.W. Harvey. (1972). Anthrax infection in bone meal from various
countries of origin. JHyg (Lond), 70(3): 455-7.
75.	Kenefic, L.J., J. Beaudry, C. Trim, L. Huynh, S. Zanecki, M. Matthews, J. Schupp, M.
Van Ert, and P. Keim. (2008). A high resolution four-locus multiplex single nucleotide
repeat (SNR) genotyping system in Bacillus anthracis. J Microbiol Methods, 73(3): 269-
72.
76.	Nishi, J.S., T.R. Ellsworth, N. Lee, D. Dewar, B.T. Elkin, and D.C. Dragon. (2007).
Cross-Canada disease report: An outbreak of anthrax (Bacillus anthracis) in free-roaming
bison in the Northwest Territories, June-July 2006. Can Vet J, 48: 37-38.
77.	Bellan, S.E., C.A. Cizauskas, J. Miyen, K. Ebersohn, M. Kusters, K.C. Prager, M. Van
Vuuren, C. Sabeta, and W.M. Getz. (2012). Black-backed jackal exposure to rabies virus,
canine distemper virus, and Bacillus anthracis in Etosha National Park, Namibia. J Wildl
Dis, 48(2): 371-81.
78.	Lewerin, S.S., M. Elvander, T. Westermark, L.N. Hartzell, A.K. Norstrom, S. Ehrs, R.
Knutsson, S. Englund, A.C. Andersson, M. Granberg, S. Backman, P. Wikstrom, and K.
Sandstedt. (2010). Anthrax outbreak in a Swedish beef cattle herd—1st case in 27 years:
Case report. Acta Vet Scand, 52: 7.
79.	Mongoh, M.N., N.W. Dyer, C.L. Stoltenow, and M.L. Khaitsa. (2008). Risk factors
associated with anthrax outbreak in animals in North Dakota, 2005: A retrospective case-
control study. Public Health Rep, 123(3): 352-9.
80.	Siamudaala, V.M., J.M. Bwalya, H.M. Munang'andu, P.G. Sinyangwe, F. Banda, A.S.
Mweene, A. Takada, and H. Kida. (2006). Ecology and epidemiology of anthrax in cattle
and humans in Zambia. Jpn J Vet Res, 54(1): 15-23.
45

-------
81.	Mongoh, M., N. Dyer, C. Stoltenow, and M.L. Khaitsa. (2008). A review of management
practices for the control of anthrax in animals: The 2005 anthrax epizootic in North
Dakota-case study. Zoonoses Public Health, 55: 279-290.
82.	Coker, P.R. (2002). Bacillus anthracis Spore Concentrations at Various Carcass Sites, in
Department of Pathobiological Sciences. Louisiana State University and Agricultural and
Mechanical College: Baton Rouge, p. 87.
83.	Himsworth, C.G. (2008). The danger of lime use in agricultural anthrax disinfection
procedures: The potential role of calcium in the preservation of anthrax spores. Can Vet
J, 49: 1208.
84.	Mikesell, P., B.E. Ivins, J.D. Ristoph, M.H. Vodkin, T.M. Dreier, and S.H. Leppia.
(1983). Plasmids, Pasteur, and Anthrax. ASM News, 19: 320-322.
85.	Hugh-Jones, M. (1999). 1996-97 Global Anthrax Report. JApplMicrobiol, 87(2): 189-
91.
86.	Kenefic, L.J., T. Pearson, R.T. Okinaka, J.M. Schupp, D.M. Wagner, A.R. Hoffmaster,
C.B. Trim, W.K. Chung, J.A. Beaudry, L. Jiang, P. Gajer, J.T. Foster, J.I. Mead, J. Ravel,
and P. Keim. (2009). Pre-Columbian origins for North American anthrax. PLoS One,
4(3): e4813.
87.	Simonson, T.S., R.T. Okinaka, B. Wang, W.R. Easterday, L. Huynh, J.M. U'Ren, M.
Dukerich, S.R. Zanecki, L.J. Kenefic, J. Beaudry, J.M. Schupp, T. Pearson, D.M.
Wagner, A. Hoffmaster, J. Ravel, and P. Keim. (2009). Bacillus anthracis in China and
its relationship to worldwide lineages. BMC Microbiol, 9: 71.
88.	Fasanella, A., D. Galante, G. Garofolo, and M.H. Jones. (2010). Anthrax undervalued
zoonosis. Vet Microbiol, 140(3-4): 318-31.
89.	Bagamian, K.H., A. Skrypnyk, Y. Rodina, M. Bezymennyi, O. Nevolko, V. Skrypnyk,
and J.K. Blackburn. (2014). Serological anthrax surveillance in wild boar (Sus scrofa) in
Ukraine. Vector Borne Zoonotic Dis, 14(8): 618-20.
90.	Brayer, G. and H. Brayer. (1952). American Cattle Trails 1540-1900. Denver, CO:
Smith-Brooks Printing Company.
91.	McNamara, W.J. Old route of the Erie Railroad. 1956; Available from:
http://www.wnvrails.net/railroads/erie/oldroute.htm.
92.	Booneville Black River Canal Museum. Black River Canal History. 2005; Available
from: http://www.blackrivercanalmuseum.com.
93.	Stein, C. and M. Stoner. (1953). Anthrax oubreaks in livestock during 1952. Vet Med, 48:
257-262.
94.	Blackburn, J.K. (2006). Evaluating the spatial ecology of anthrax in North America:
Examining epidemiological components across multiple geographic scales using a GIS-
based approach, in Department of Geography and Anthropology. Baton Rouge:
Louisiana State University.
95.	Kenefic, L.J., J. Beaudry, C. Trim, R. Daly, R. Parmar, S. Zanecki, L. Huynh, M.N. Van
Ert, D.M. Wagner, T. Graham, and P. Keim. (2008). High resolution genotyping of
Bacillus anthracis outbreak strains using four highly mutable single nucleotide repeat
markers. Lett Appl Microbiol, 46(5): 600-3.
96.	Griffith, J., D. Blaney, S. Shadomy, M. Lehman, N. Pesik, S. Tostenson, L. Delaney, R.
Tiller, A. DeVries, T. Gomez, M. Sullivan, C. Blackmore, D. Stanek, R. Lynfield, and T.
Anthrax Investigation. (2014). Investigation of inhalation anthrax case, United States.
Emerg Infect Dis, 20(2): 280-3.
46

-------
97.	Kellogg, F.E., A.K. Prestwood, and R.E. Noble. (1970). Anthrax epizootic in white-tailed
deer. J Wildl Dis, 6(4): 226-8.
98.	McGee, E.D., D.L. Fritz, J.W. Ezzell, H.L. Newcomb, R.J. Brown, and N.K. Jaax.
(1994). Anthrax in a dog. Vet Pathol, 31(4): 471-3.
99.	LSUAgCenter.com. Anthrax in Louisiana. 2014.
100.	Jones, C. (2012). Northeast Colorado veterinary officer explains recent anthrax
outbreak, in Journal Advocate, http://www.iournal-advocate.com/sterling-
local news/ci 21700418/logan-countv-anthrax-outbreak-richanne-lomkin-northeast-
eolorado-veterinarv-otYicer: Sterling, CO.
101.	Sharp, R.J. and A.G. Roberts. (2006). Anthrax: The challenges for decontamination.
Journal of Chem Technol Biotechnol, 81: 1612-1625.
102.	Ramsay, C.N., A. Stirling, J. Smith, G. Hawkins, T. Brooks, J. Hood, G. Penrice, L.M.
Browning, S. Ahmed, G.G.C. Nhs, and T. Scottish National Outbreak Control. (2010).
An outbreak of infection with Bacillus anthracis in injecting drug users in Scotland. Euro
Surveill, 15(2).
103.	Radun, D., H. Bernard, M. Altmann, I. Schoneberg, V. Bochat, U.v. Treeck, R. Rippe, R.
Grunow, M. Elschner, and W. Biederbick. (2010). Preliminary case report of fatal
anthrax in an injecting drug user in North-Rhine-Westphalia, Germany, December 2009.
Euro Surveillance: bulletin europeen sur les maladies transmissibles European
Communicable Disease Bulletin 15.
104.	Meselson, M., J. Guillemin, M. Hugh-Jones, A. Langmuir, I. Popova, A. Shelokov, and
O. Yampolskaya. (1994). The Sverdlovsk anthrax outbreak of 1979. Science, 266(5188):
1202-8.
105.	Swartz, M.N. (2001). Recognition and management of anthrax—An update. NEngl J
Med, 345(22): 1621-6.
106.	Rao, G.R., J. Padmaja, M.K. Lalitha, P.V. Rao, K.V. Gopal, H.K. Kumar, and P.
Mohanraj. (2005). An outbreak of cutaneous anthrax in a non-endemic district—
Vi sakhapatnam in Andhra Pradesh. Indian J Dermatol Venereol Leprol, 71(2): 102-5.
107.	Kracalik, I.T., L. Malania, N. Tsertsvadze, J. Manvelyan, L. Bakanidze, P. Imnadze, S.
Tsanava, and J.K. Blackburn. (2013). Evidence of local persistence of human anthrax in
the country of Georgia associated with environmental and anthropogenic factors. PLoS
Negl Trop Dis, 7(9): e2388.
108.	Chakraborty, A., S.U. Khan, M.A. Hasnat, S. Parveen, M.S. Islam, A. Mikolon, R.K.
Chakraborty, B.N. Ahmed, K. Ara, N. Haider, S.R. Zaki, A.R. Hoffmaster, M. Rahman,
S.P. Luby, and M.J. Hossain. (2012). Anthrax outbreaks in Bangladesh, 2009-2010. Am J
Trop MedHyg, 86(4): 703-10.
109.	Smyth, H.F., V.S. Cheney, S.D. Hubbard, and S.H. Osborn. (1926). Industrial Anthrax.
Am J Public Health (N Y), 16(1): 42-4.
110.	Smyth, H.F. and W.D. Higgins. (1943). Report of the Committee on Industrial Anthrax
(Check Anthrax-A Warning and a Plea) : Industrial Hygiene Section. Am J Public Health
Nations Health, 33(7): 854-7.
111.	Cohen, M. and T. Whalen. (2007). Implication of low level human exposure to respirable
B. anthracis. ApplBiosafety, 12: 109-115.
112.	Brachman, P.S., H. Gold, S.A. Plotkin, F.R. Fekety, M. Werrin, and N.R. Ingraham.
(1962). Field Evaluation of a Human Anthrax Vaccine. Am J Public Health Nations
Health, 52(4): 632-45.
47

-------
113.	Bales, M.E., A.L. Dannenberg, P.S. Brachman, A.F. Kaufmann, P.C. Klatsky, and D.A.
Ashford. (2002). Epidemiologic response to anthrax outbreaks: field investigations, 1950-
2001. EmergInfectDis, 8(10): 1163-74.
114.	CDC. (2001). Human anthrax associated with an epizootic among livestock — North
Dakota, 2000. Centers fo Disease Control and Prevention. Morbidity and Mortality
Weekly Report, 50: 677-680.
115.	Page, E., K. Martinez, T. Seitz, B. Bernard, A. Tepper, R. Weyant, C. Quinn, N.
Rosenstein, B. Perkins, and T. Popovic. (2002). Public health dispatch: Update:
Cutaneous anthrax in a laboratory worker-Texas, 2002 Morbidity and Mortality Weekly
Report, 51(22): 482.
116.	CDC. (2006). Inhalation anthrax associated with dried animal hides - Pennsylvania and
New York City, 2006. Centers fo Disease Control and Prevention. Morbidity and
Mortality Weekly Report, 55: 280.
117.	ProMed Mail. (2007). Anthrax, Animal Skin - USA (Connecticut) (03).
118.	Adalja, A. A. (2010). CBN report: Unusual case of GI anthrax in New Hampshire raises
questions, in Clinicians'Biosecurity Network.
119.	Thappa, D.M., S. Dave, K. Karthikeyan, and S. Gupta. (2000). An outbreak of human
anthrax: A report of 15 cases of cutaneous anthrax. Indian J Dermatol 45: 186-191.
120.	Watson, A. and D. Keir. (1994). Information on which to base assessments of risk from
environments contaminated with anthrax spores. Epidemiol Infect, 113(3): 479-90.
121.	CDC. (2008). Cutaneous anthrax associated with drum making using goat hides from
West Africa - Connecticut, 2007. Centers fo Disease Control and Prevention. Morbidity
and Mortality Weekly Report, 57(23): 629-631.
122.	Jackson, R. (1930). The introduction into Great Britain of anthrax infection by means of
products of animal origin. J Comp Pathol Therapeutics, 43: 95-98.
123.	Stockman, S. (1911). The epizootiology of anthrax. J Comp Pathol Therapeutics, 24: 97-
108.
124.	Smyth, H.F. and W.D. Higgins. (1945). Report of the Committee on Industrial Anthrax
(Anthrax in the United States, 1939-1943) : Industrial Hygiene Section. Am J Public
Health Nations Health, 35(8): 850-8.
125.	Woods, C.W., K. Ospanov, A. Myrzabekov, M. Favorov, B. Plikaytis, and D.A. Ashford.
(2004). Risk factors for human anthrax among contacts of anthrax-infected livestock in
Kazakhstan. Am J Trop MedHyg, 71(1): 48-52.
126.	Sirisanthana, T. and A.E. Brown. (2002). Anthrax of the gastrointestinal tract. Emerg
Infect Dis, 8(7): 649-51.
127.	Wampler, R.A. and T.S. Blanton. (2001). Volume V: Anthrax at Sverdlovsk, 1979: U.S.
intelligence on the deadliest modern outbreak.
128.	Djupina, S. (1996). Features of epizootic situation on anthrax in the Sverdlovsk region in
1979, in Prognosis of epizootic situation (on the model of the anthrax epizootic process).
Russian Academy of Agricultural Sciences, Siberian Branch, Institution of the
Experimental Veterinary of Siberia and Far East, Novosibirsk, p. 119-130.
129.	Army. (1977). U.S. Army Activity in the U.S. Biological Warfare Programs
(unclassified) Volume 1. U.S. Army. FA-09-0021.
130.	Manchee, R.J., M.G. Broster, I.S. Anderson, R.M. Henstridge, and J. Melling. (1983).
Decontamination of Bacillus anthracis on Gruinard Island? Nature, 303(5914): 239-40.
48

-------
131.	Lassiter, T. and M. Conte. (2006). The history of bioterrorism, in Focus on Bioterrorism,
G. Foster, Editor., Nova Science Publishers: New York. p. 43-66.
132.	Manchee, R.J., M.G. Broster, A.J. Stagg, and S.E. Hibbs. (1994). Formaldehyde Solution
Effectively Inactivates Spores of Bacillus anthracis on the Scottish Island of Gruinard.
ApplEnviron Microbiol, 60(11): 4167-71.
133.	CDC. (2009). Section IV-Laboratory Biosafety Level Criteria. Biosafety in
Microgiological and Biomedical Laboratories (BMBL).
134.	CDC. (2005). Inadvertent laboratory exposure to Bacillus anthracis — California, 2004.
Centers for Disease Control and Prevention. Morbidity and Mortality Weekly Report, 54:
301-304.
135.	CDC. (2014). Report on the potential exposure to anthrax.
136.	Oggioni, M.R., A. Ciabattini, A.M. Cuppone, and G. Pozzi. (2003). Bacillus spores for
vaccine delivery. Vaccine, 21 Suppl 2: S96-101.
137.	Turnbull, P.C. (1991). Anthrax vaccines: past, present and future. Vaccine, 9(8): 533-9.
138.	Wright, J.G., C.P. Quinn, S. Shadomy, andN. Messonnier. (2010). Use of anthrax
vaccine in the United States. Morbid Mortal Weekly Report, 59: 1-30.
139.	Saile, E. and C.P. Quinn. (2011). Anthrax vaccines, in Bacillus anthracis andAntrhax,
N.H. Bergman, Editor., John Wiley & Sons, Inc.: Hoboken, NJ.
140.	Schild, A.L., E.S. Sallis, M.P. Soares, S.R. Ladeira, R. Schramm, A.P. Priebe, M.B.
Almeida, and F. Riet-Correa. (2006). Anthrax in cattle in southern Brazil: 1978-2006.
Pesquisa VeterinariaBrasileira, 26(243-248).
141.	Moayeri, M. and S.H. Leppla. (2011). Anthrax toxins, in Bacillus anthracis and Anthrax,
N.H. Bergman, Editor., John Wiley & Sons, Inc.: Hoboken, N.J. p. 121-156.
142.	Fellows, P.F., M.K. Linscott, B.E. Ivins, M.L. Pitt, C.A. Rossi, P.H. Gibbs, and A.M.
Friedlander. (2001). Efficacy of a human anthrax vaccine in guinea pigs, rabbits, and
rhesus macaques against challenge by Bacillus anthracis isolates of diverse geographical
origin. Vaccine, 19(23-24): 3241-7.
143.	Ivins, B., P. Fellows, M. Pitt, J. Estep, S. Welkos, P. Worsham, and A. Friedlander.
(1996). Efficacy of a standard human anthrax vaccine against Bacillus anthracis spore
challenge in rhesus monkeys. Salisbury Medical Bulletin Special Supplement, 87: 125-
126.
144.	Hugh-Jones, M. (2006). Distinguishing between natural and unnatural outbreaks of
animal diseases. Scientific and Technical Review of the Office International des
Epizooties, 25(1): 173-186.
145.	Grunow, R. and E.J. Finke. (2002). A procedure for differentiating between the
intentional release of biological warfare agents and natural outbreaks of disease: its use in
analyzing the tularemia outbreak in Kosovo in 1999 and 2000. Clin Microbiol Infect,
8(8): 510-21.
146.	National Research Council. (2008). Department of Homeland Security bioterrorism risk
assessment: A call for change. National Academies Press.
147.	FEMA. (2009). Awareness and Response to Biological Events: Participant Guide.
Version 1.2. National Center for Biomedical Research and Training, Academy of
Counter-Terrorist Education, Louisiana State University and A&M College.
148.	Nowak, J. (1982). Courier from Warsaw, in Wayne State University Press: Detroit, p.
62-63.
49

-------
149.	Institute of Medicine (IOM) and National Research Council (NRC). (2011). BioWatch
and Public Health Surveillance: Evaluating Systems for the Early Detection of Biological
Threats. Abbreviated Version Washington, D.C: The National Academies Press.
150.	Luna, V.A., D.S. King, K.K. Peak, F. Reeves, L. Heberlein-Larson, W. Veguilla, L.
Heller, K.E. Duncan, A.C. Cannons, P. Amuso, and J. Cattani. (2006). Bacillus anthracis
virulent plasmid pX02 genes found in large plasmids of two other Bacillus species. J Clin
Microbiol, 44(7): 2367-77.
151.	CDC. (2010). Use of anthrax vaccine in the United States: Recommendations of the
Advisory Committee on Immunization Practices (ACIP), 2009. Centers for Disease
Control and Prevention. Morbidity and Mortality Weekly Report, 59: 1-23.
152.	Palazzo, L., E. Carlo, G. Santagada, L. Serrecchia, A. Aceti, A. Guarino, R. Adone, and
A. Fasanella. (2012). Recent Epidemic-Like Anthrax Outbreaks in Italy: What Are the
Probable Causes? Open J Vet Med, 2(2): 74-76.
153.	Creel, S.M., N. Creel, J.A. Matovelo, M.M.A. Mtambo, E.K. Batamuzi, and J.E. Cooper.
(1995). The effects of anthrax on endangered African wild dogs (Lycaon pictus). J Zool,
236(2): 199-209.
154.	Aikembayev, A.M., L. Lukhnova, G. Temiraliyeva, T. Meka-Mechenko, Y. Pazylov, S.
Zakaryan, G. Denissov, W.R. Easterday, M.N. Van Ert, P. Keim, S.C. Francesconi, J.K.
Blackburn, M. Hugh-Jones, and T. Hadfield. (2010). Historical distribution and
molecular diversity of Bacillus anthracis, Kazakhstan. Emerg Infect Dis, 16(5): 789-96.
155.	Crouch II, J. (2002). Cooperative threat reduction program Senate Armed Services
Committee, Subcommittee on Emerging Threats, U.S. Senate.
156.	Pike, R.M. (1979). Laboratory-associated infections: Incidence, fatalities, causes, and
prevention. Ann Rev Microbiol, 33: 41-66.
157.	Pike, R.M. (1976). Laboratory-associated infections: Summary and analysis of 3921
cases. Health Lab Sci, 13(2): 105-114.
158.	Sulkin, S.E. and R.M. Pike. (1951). Survey of laboratory-acquired infections. Am J
Public Health Nations Health, 41(7): 769-81.
159.	CDC. (2002). Suspected cutaneous anthrax in a laboratory worker - Texas, 2002. Centers
for Disease Control and Prevention. Morbidity and Mortality Weekly Report, 51: 279-
281.
160.	CDC. (2002). Public Health Dispatch: Update: Cutaneous Anthrax in a Laboratory
Worker — Texas, 2002. Morbidity and Mortality Weekly Report, 51: 482.
161.	Keim, P., L.B. Price, A.M. Klevytska, K.L. Smith, J.M. Schupp, R. Okinaka, P.J.
Jackson, and M.E. Hugh-Jones. (2000). Multiple-locus variable-number tandem repeat
analysis reveals genetic relationships within Bacillus anthracis. JBacteriol, 182(10):
2928-36.
162.	Pearson, T., J.D. Busch, J. Ravel, T.D. Read, S.D. Rhoton, J.M. U'Ren, T.S. Simonson,
S.M. Kachur, R.R. Leadem, M.L. Cardon, M.N. Van Ert, L.Y. Huynh, C.M. Fraser, and
P. Keim. (2004). Phylogenetic discovery bias in Bacillus anthracis using single-
nucleotide polymorphisms from whole-genome sequencing. Proc Natl Acad Sci USA,
101(37): 13536-41.
163.	Lista, F., G. Faggioni, S. Valjevac, A. Ciammaruconi, J. Vaissaire, C. le Doujet, O.
Gorge, R. De Santis, A. Carattoli, A. Ciervo, A. Fasanella, F. Orsini, R. D'Amelio, C.
Pourcel, A. Cassone, and G. Vergnaud. (2006). Genotyping of Bacillus anthracis strains
50

-------
based on automated capillary 25-loci multiple locus variable-number tandem repeats
analysis. BMC Microbiol, 6: 33.
164.	Beyer, W. and P.C. Turnbull. (2013). Co-infection of an animal with more than one
genotype can occur in anthrax. Lett ApplMicrobiol, 57(4): 380-4.
165.	Keim, P., M.N. Van Ert, T. Pearson, A.J. Vogler, L.Y. Huynh, and D.M. Wagner. (2004).
Anthrax molecular epidemiology and forensics: using the appropriate marker for different
evolutionary scales. Infect Genet Evol, 4(3): 205-13.
166.	Maho, A., A. Rossano, H. Hachler, A. Holzer, E. Schelling, J. Zinsstag, M.H. Hassane,
B.S. Toguebaye, A.J. Akakpo, M. Van Ert, P. Keim, L. Kenefic, J. Frey, and V. Perreten.
(2006). Antibiotic susceptibility and molecular diversity of Bacillus anthracis strains in
Chad: detection of a new phylogenetic subgroup. J Clin Microbiol, 44(9): 3422-5.
167.	Reed, T. (2011). The Bacillus anthracis genome, in Bacillus anthracis and Anthrax, N.H.
Bergman, Editor., John Wiley and Sons, Inc.: Hoboken, N.J. p. 67-87.
168.	Rasko, D.A., P.L. Worsham, T.G. Abshire, S.T. Stanley, J.D. Bannan, M.R. Wilson, R.J.
Langham, R.S. Decker, L. Jiang, T.D. Read, A.M. Phillippy, S.L. Salzberg, L.J. Kenefic,
P.S. Keim, C.M. Fraser-Liggett, and J. Ravel. (2011). Bacillus anthracis comparative
genome analysis in support of the Amerithrax investigation. Proceedings of the National
Academy of Sciences of the United States of America 108(12): 5027-5032.
169.	Kenefic, L.J., T. Pearson, R.T. Okinaka, W.K. Chung, T. Max, C.P. Trim, J.A. Beaudry,
J.M. Schupp, M.N. Van Ert, C.K. Marston, K. Gutierrez, A.K. Swinford, A.R.
Hoffmaster, and P. Keim. (2008). Texas isolates closely related to Bacillus anthracis
Ames. EmergInfect Dis, 14(9): 1494-6.
170.	Ivins, B.E. and S.L. Welkos. (1988). Recent advances in the development of an
improved, human anthrax vaccine. Eur J Epidemiol, 4(1): 12-9.
171.	CDC. (2000). Use of anthrax vaccine in the United States. Centers for Disease Control
and Prevention. Morbidity and Mortality Weekly Report, 49(RR15): 1-20.
172.	U.S. Food and Drug Administration. (2012). Vaccines, blood, and biologies: Anthrax..
http://www.fda.gOv/biolo2icsbloodvaccmes/vaccmes/ucm061751.htm#. Accessed 5/6/14.
173.	Pomerantsev, A.P., N.A. Staritsin, V. Mockov Yu, and L.I. Marinin. (1997). Expression
of cereolysine AB genes in Bacillus anthracis vaccine strain ensures protection against
experimental hemolytic anthrax infection. Vaccine, 15(17-18): 1846-50.
174.	Layshock, J.A., B. Pearson, K. Crockett, M.J. Brown, S. Van Cuyk, W.B. Daniel, and
K.M. Omberg. (2012). Reaerosolization of Bacillus spp. in outdoor environments: A
review of the experimental literature. Biosecur Bioterror, 10(3): 299-303.
175.	Sykes, A., T. Brooks, M. Dusmet, A.G. Nicholson, D.M. Hansell, and R. Wilson. (2013).
Inhalational anthrax in a vaccinated soldier. Eur Respir J, 42(1): 285-7.
176.	Langston, C. (2005). Postexposure management and treatment of anthrax in dogs-
executive councils of the American Academy of Veterinary Pharmacology and
Therapeutics and the American College of Veterinary Clinical Pharmacology. AAPS J,
7(2): E272-3.
177.	Henkel, R., T. Miller, and R.S. Weyant. (2012). Monitoring select agent theft, loss and
release reports in the United States, 2004 - 2010. ApplBiosafety, 17(4): 171-180.
178.	CDC. (2010). Gastrointestinal anthrax after an animal-hide drumming event - New
Hampshire and Massachusetts, 2009.
179.	CDC. (2000). Human ingestion of Bacillus anthracis - contaminated meat - Minnesota,
August, 2000. 49(36): 813-816.
51

-------
180.	National Anthrax Oubreak Control Team. An outbreak of anthrax among drug users in
Scotland, December 2009 to December 2010. 2011; Available from:
http://www.documents.hps.scot.nhs.uk/giz/anthrax-outbreak/anthrax-outbreak-report-
2011-12.pdf.
181.	Abramova, F.A., L.M. Grinberg, O.V. Yampolskaya, and D.H. Walker. (1993).
Pathology of inhalational anthrax in 42 cases from the Sverdlovsk outbreak of 1979. Proc
Natl Acad Sci USA, 90(6): 2291-4.
182.	Coleman, M.E., B. Thran, S.S. Morse, M. Hugh-Jones, and S. Massulik. (2008).
Inhalation anthrax: dose response and risk analysis. Biosecur Bioterror, 6(2): 147-60.
183.	Glomski, I. (2011). Bacillus anthracis dissemination through hosts, in Bacillus anthracis
and Anthrax, N.H. Bergman, Editor., John Wiley & Sons, Inc: Hoboken, NJ.
184.	Wilkening, D.A. (2006). Sverdlovsk revisited: Modeling human inhalation anthrax.
Proceedings of the National Academy of Sciences 103(20): 7589-7594.
185.	Friedlander, A.M., S.L. Welkos, M.L. Pitt, J.W. Ezzell, P.L. Worsham, K.J. Rose, B.E.
Ivins, J.R. Lowe, G.B. Howe, P. Mikesell, and et al. (1993). Postexposure prophylaxis
against experimental inhalation anthrax. J Infect Dis, 167(5): 1239-43.
186.	Ellis, R. (2014). Creating a secure network: The 2001 anthrax attacks and the
transformation of postal security. Sociol Rev, 62(S1): 161-182.
187.	McLaughlin, E.C. Texas actress who sent Obama ricin sentenced to 18 year. 2014;
http://www.cnn.eom/2014/07/16/iustice/texas-ricin-actress-sentenced/index.html1.
Available from: http://www.cnn.eom/2014/07/16/iustice/texas-ricin-actress-
sentenced/index. html.
52

-------
&EPA
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

-------