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Abstract. Brucella has traditionally been considered a
biological weapon. It was the subject of extensive offen-
sive research in the past, and still belongs to category B
pathogens on most lists. Its propensity for airborne trans-
mission and induction of chronic debilitating disease re-
quiring combined antibiotic regimens for treatment, its
abundance around the world and its vague clinical char-
acteristics defying rapid clinical diagnosis are some of
the characteristics that apply to the pathogen’s weapons
potential. Yet minimal mortality, availability of treatment
options, protracted inoculation period and the emergence
of new, more virulent potential weapons means that its in-
clusion among agents of bioterrorism is nowadays mainly
of historical significance. Nevertheless, in the interest of
literacy and of avoiding panic, physicians and the pub-
lic both should be aware of the most common zoonosis
Keywords. Brucella, brucellosis, bioterrorism.
Brucellosis is an ancient disease. It remains the most
common anthropozoonosis worldwide, inducing an of-
ten chronic, often incapacitating disease with low mor-
tality. Its significance as a potential agent of bioterror-
ism was acknowledged early, and the pathogen remains
on the category B biodefense research list of both the
Centers for Disease Control and Prevention (CDC) 
and the National Institute of Allergy and Infectious
Diseases (NIAID) . It is also invariably included on
the non-stratified lists of potential biological weapons
of other organizations, such as the World Health Orga-
nization (WHO), the North Atlantic Treaty Organiza-
tion (NATO), and the Biological and Toxin Weapons
Convention (BTWC). Certain epidemiological, mi-
crobiological and clinical parameters of the pathogen
render it an attractive agent for malicious use. We will
analyze these parameters, along with other aspects that
emerge following a hypothetical deliberate release of
Brucella belongs to the genus of a-proteobacteria and
consists of seven species: B. melitensis, B. abortus, B. suis
and B. canis are known to induce human disease, while
B. neotomae and B. ovis are not virulent to humans. B.
pinnipediae and B. cetaceae are marine species pathogens
discovered recently and provisionally named  that may
also be human pathogens . The genome of B. melitensis
, B. abortus  and B. suis  has been fully decoded,
and extended active research on the significance of vari-
ous proteins expressed by the bacterium will probably al-
low for better understanding of the unique pathogenetic
processes involved in human brucellosis . Brucellosis
is principally a zoonosis, a common cause of abortions
in sheep and goats (B. melitensis), cows (B. abortus) and
pigs (B. suis). B. canis is a canine pathogen, B. ovis is
also a sheep pathogen, and B. neotomae is found in ro-
dents. Furthermore, though other species, including wild-
life, can serve as the reservoir of Brucella, although the
prevailing subtypes in wildlife may be of minimal hu-
man importance , as in France. That is not the case in
United States though, where B. abortus-infected bisons
were recently slaughtered in certain Midwestern states,
Brucella as a biological weapon
*, P. Panagopoulou
, L. Christou
and N. Akritidis
Institute for Continuing Medical Education of Ioannina (Greece)
Internal Medicine Department, University Hospital of Ioannina, Velissariou 15-19, 45221, Ioannina (Greece),
Fax: +30 26510 49045, e-mail: firstname.lastname@example.org
Internal Medicine Department, General Hospital ‘G. Hatzikosta’ of Ioannina (Greece)
Online first ❚❚❚
* Corresponding author.
Cell. Mol. Life Sci.
© Birkhäuser Verlag, Basel, 2006
Cellular and Molecular Life Sciences
2 G. Pappas et al. Brucellosis and bioterrorism
raising concerns about the cost effectiveness and political
correctness of the whole procedure . Brucella does
not form spores, but it is still significantly environmen-
Brucellosis is an ancient disease. It induced disease in a
significant number of inhabitants of Pompei , and is
possibly the cause, in a bizarre form of ‘divine bioterror-
ism’, of the fifth plague of Egypt, which decimated Egyp-
tian cattle. Yet the disease is also described as incapaci-
tating equine species, and others have suggested that the
fifth plague was in fact anthrax (the sixth plague, which
is a more accurately describes human and animal anthrax
disease, is suggested by some to be smallpox). At the end
of the 19
century, brucellosis was prevalent among Brit-
ish troops stationed in Malta. And although it is tempting
to assume that locals deliberately infected troops by offer-
ing raw goat milk, we know that raw milk was considered
an excellent tool for strengthening patients supposedly
suffering from typhoid. Sir David Bruce, a British Army
officer, was the first to isolate the organism and along
with his coworkers subsequently managed to trace the
epidemiology back to goat milk. He developed the first
serum agglutination test for to diagnose brucellosis .
By the beginning of World War II, the medical and veteri-
nary aspects of the disease had been extensively outlined,
and brucellosis emerged as an attractive candidate in the
still premature biowarfare industry. The attractiveness of
Brucella was based on certain combat parameters of the
era: an agent that could caused a protracted incapacitat-
ing disease with minimal mortality would mean that most
of the enemy’s troops would be sidelined by illness, and
a significant percentage of non-infected army members
would be needed to care for them (this percentage would
be higher and implicated for a longer period than the one
needed for dealing with dead bodies, had a more lethal
pathogen been used). Practically every major national
program for offensive biological weapon development
dealt with Brucella. Brucella was one of the agents with
which Japan experimented in the infamous 731 Manchu-
ria Unit before and during World War II. In the United
States, B. suis was the first agent weaponized in 1952,
and extended field testing with B. suis-filled bombs
took place thereafter . Soon though, other, more po-
tent weapons were targeted. In the former Soviet Union,
Brucella was one of the agents developed for offensive
purposes by Biopreparat, the extensive Soviet biological
weapons program. Ken Alibek, a former deputy director
who relocated in United States in 1992 stated that un-
treatable, antibiotic-resistant forms had been developed,
the agent was weaponized both in dry and liquid forms,
production capability ranged at the level of 100 tons of
bacteria and the means to deliver the pathogen had been
extremely sophisticated. As with other agents developed
by the Soviet Union, extended field testing was per-
formed on the island of Vozroshdeniye, in the midst of
the Aral Sea . Despite its historical significance and
attractiveness in the era of traditional combat situations,
by the end of the 20
century interest in Brucella gradu-
ally waned: it is characteristic that Alibek states that Bru-
cella was dropped from the Soviet program in favour of
Burkholderia pseudomallei, which was considered more
potent. Still, as with other aspects of the Biopreparat pro-
gram, questions about the subsequent whereabouts of the
resistant strains developed remain.
Brucellosis, particularly caused by B. melitensis, remains
the commonest zoonotic disease worldwide, and more-
over seems to be relocating and re-emerging in recent
years . Middle Eastern countries as Syria, Iran and
Iraq Figure prominently on a list of endemic countries,
but new foci that have emerged include all the former
communist Asian republics, such as Kazakhstan, Kyr-
gizstan and especially Mongolia. The situation is slowly
improving in the European Union, although the disease
is still endemic in Greece, Spain, Portugal and southern
Italy. International travel and the importation of exotic
food from endemic areas account for a limited number
of cases reported annually from brucellosis-free industri-
alized countries. The same stands for the United States,
where most cases appear in states neighboring Mexico, in
patients with Hispanic origin and related to importation
of infected dairy products from the still endemic Mexico
. North Africa remains an endemic area, while the
situation in sub-Saharan Africa cannot be adequately
evaluated; furthermore, other infectious disease-related
priorities exist in these countries. Three important bio-
terrorism-related aspects emerge from the current global
disease status. First, Brucella can be easily obtained
practically anywhere in the world, in contrast with agents
such as smallpox, and thus its use as a biological weapon
could be kick-started rather easily. In that vein one has
to question the rationale behind widely circulated reports
about Iraq obtaining Brucella strains from a US firm at
the end of the 1980s: brucellosis was already endemic in
Iraq during that period, so strains could easily be isolated
from naturally occurring human cases. Second, one has
to note that certain endemic areas coincide with areas
where active foreign army operations have been evolv-
ing; thus, a naturally occurring case in a US soldier in
Iraq could, at least initially, raise concerns about pos-
sible deliberate release. In this context, brucellosis was
related to the development of Gulf War Syndrome (see
following sections). Containment of naturally occurring
Cell. Mol. Life Sci. Multi-author Review Article 3
disease in these areas does not seem feasible at present,
since the disease is related to overall socioeconomic sta-
tus and political factors. The emergence of brucellosis
in Kosovo and Bosnia-Herzegovina in recent years after
lengthy political unrest and extended military operations
and the mechanisms of disease trafficking in the Balkans
is a typical example . The third important aspect is
that of awareness: brucellosis being a rare disease in the
developed world, many physicians and infectious disease
specialists are not familiar with its characteristics, lead-
ing to delayed diagnoses or false alarms . More on
this follows in upcoming sections.
As already stated, Brucella was initially attractive as bio-
warfare partly due to its ability to induce chronic disease.
The pathogenesis of brucellosis is unique, and animal
models often cannot accurately reproduce events evolv-
ing during human infection. Brucella is a Gram-negative
pathogen, yet its surface lipopolysaccharide induces far
smaller immune response comparing with other Gram-
negative bacteria. Brucellae have a propensity for invad-
ing the reticuloendothelial system, practically hiding
inside macrophages and non-professional phagocytes.
In there, they reside in specialized compartments with
acidic environments, and multiply using parts of the cy-
toskeleton, without interrupting cell cycle and function
; on the contrary they are apoptosis inhibitors, thus
creating a frame for eternal survival and replication. Im-
mune response is partly muted by certain Brucella fac-
tors, inhibition of tumor necrosis factor-alpha (TNF-
being a prominent event. Cellular immune responses
predominate , although antibody production serves
as a diagnostic tool. It has been long postulated that the
outcome of the disease reflects the equilibrium developed
between the bacterium and the human immune response,
and that relapses and chronic disease should also be
viewed in this context.
Transmission to humans
One important bioterrorism-related characteristic of
Brucella is the small inoculum needed to induce human
disease, traditionally described in the levels of 10–100
microorganisms. The commonest means of Brucella
transmission to humans is by consuming unpasteurized
dairy products as milk and soft cheese. Direct contact
through skin abrasions with infected animal tissues (as in
slaughterhouse workers) is also implicated, but the most
important means of transmission in the context of bio-
terrorism importance is airborne transmission. Brucella
can be easily aerosolized, and when in air, can be easily
transmitted through the airways and induce disease, while
staying for a protracted period in this virulent form. Char-
acteristically, brucellosis is considered the commonest
laboratory-acquired infection worldwide , and thus
certain isolation and experimental procedures should be
performed in Biosafety Level 3 (BSL3), and laboratory
workers should be informed ahead of time about the di-
agnostic possibility of brucellosis in order to implement
appropriate diagnostic precautions. Its propensity for
aerosolization and easy spread is another of the impor-
tant bioterrorism-related characteristics of the bacterium.
Using Brucella as a biological weapon through the food
chain could be feasible, but would result in localized clus-
ters of cases: one would have to intervene at a post-in-
dustrial level, since pasteurization kills the pathogen. The
potential for such an approach in order to induce massive
disease is obviously low, but should also be entertained
by policy makers of the area, and is beyond the scope of
The inoculation period is relatively protracted, especially
when compared with other pathogens considered as po-
tential biological weapons, ranging 9–60 days. This alone
could be a drawback in the context of biowarfare, since
a deliberate release would not lead to a sharp outbreak
curve, but would rather induce a smooth curve of gradual
increase, and subsequent decrease over a period of 1–2
months. Thus, more time would be allowed for authori-
ties to diagnose and intervene, and less public unrest and
health authorities’ burden would be created . Since
deliberate release of a pathogen theoretically aims, at
least partly, at social disruption, Brucella is by far an un-
suitable agent. Still, certain approaches to bioterrorism
risk argue that penetration of infectious sequelae deep
in time might be more important for social disruption in
long term , and brucellosis should be re-evaluated in
this context. The possible existence of an inverse relation-
ship between microbial inoculum and inoculation period
should also be further studied.
Inapparent-to-apparent infection ratios cannot be ad-
equately calculated, owing to discrepancies in different
series. A genetic predisposition seems to exist [G. Pappas
et al., unpublished data].
Brucellosis can cause practically any clinical syndrome
, and in endemic areas the tagline ‘everything is Bru-
cella until proven different’ might actually be useful. The
commonest syndrome presented is one of a flu-like ill-
ness, with fever that may be protracted (often present-
ing as fever of unknown origin ), arthralgia, myalgia,
fatigue and malodorous perspiration. The propensity for
reticuloendothelial system invasion leads often to hepato-
megaly, splenomegaly and lymphadenopathy. Uncompli-
4 G. Pappas et al. Brucellosis and bioterrorism
cated disease is readily responsive to antibiotic treatment.
Complications reported are abundant, the commonest be-
ing arthritis (either peripheral or, often, sacroiliitis) and
spondylitis , which can be debilitating and difficult to
treat . The propensity of the pathogen for granuloma
formation can lead to abscess formation in various sites.
Epididymo-orchitis , mild hepatitis, rashes and asci-
tes  are often reported. The most serious complica-
tions of the disease are neurobrucellosis, which can pres-
ent in various forms , and endocarditis, which is the
main cause of mortality (altogether very low) and often
requires surgical intervention . Respiratory complica-
tions in brucellosis are more usual than generally thought;
yet there is no connection between airborne transmission,
the probable route of transmission after deliberate release
and emergence of respiratory complications, as outlined
in a large series of cases . Laboratory characteristics
include cytopenia of varying range and severity ,
mild increases in serum aminotransferase levels and rela-
In pregnancy brucellosis is related to an increased risk of
spontaneous abortion , while in childhood the disease
is generally thought to be more benign . The relatively
few data on brucellosis in immunocompromised patients
 suggest that clinical severity is not enhanced in this
Chronic brucellosis is an entity much talked about, but
inadequately understood. By the traditionally accepted
definition, the disease is chronic when exhibiting a
course of more than 6 months. Yet chronicity can present
as frequent relapses, residual disease after treatment and
sometimes as persistent behavioral changes accompanied
by ill-defined neurological syndromes, weight loss and
fatigue, in the absence of any laboratory evidence of bru-
cellosis relapse. This syndrome is familiar to brucellosis
specialists; some argue that its nature is autoimmune. It
is a syndrome strikingly familiar to chronic fatigue syn-
drome (CFS), an equally vaguely described syndrome
developed by Gulf War veterans after the first Gulf War.
Brucellosis was endemic in the battlefield area, and fear
of deliberate exposure to Brucella aerosols from the Iraqi
army was also prominent. Thus, brucellosis was one of
the first diagnoses entertained in the approach of CFS.
No significant evidence has emerged, yet one should
remember the long-standing hypotheses on the relation-
ship between Brucella and demyelinating syndromes
. Advances in diagnostic options might help to more
clearly define and understand the exact nature of chronic
brucellosis, and decisively outline the presence or not of
any etiological relationship with CFS.
The clinical presentation of brucellosis being protean,
in the event of a deliberate release a clinical diagnosis
might not be easily achieved. Most patients would ex-
perience a constellation of symptoms also pointing to-
wards other pathogens, such as Francisella tularensis,
Coxiella burnetii and several viruses. Yet establishing of
a clinical diagnosis would be largely related to clinicians’
awareness of the disease, which in turn is influenced
by the effectiveness of educational programs on bioter-
rorism and the endemicity of the agent in the targeted
area. A recent educational US program including a mul-
tiple-choice questionnaire outlined this fact: 15% of par-
ticipating physicians wrongly diagnosed brucellosis in a
patient presenting with severe pneumonia. A co-resident
of the patient had died 4 days earlier with hemoptysis
and dyspnea , and the physicians thus attributed to
brucellosis a predominantly respiratory distress course
and the capacity for person-to-person transmission (more
worrying, though, was the fact that only 15% accurately
diagnosed pneumonic plague, which was the correct di-
agnosis). Although most imported cases in the developed,
brucellosis-free world can be accurately traced to travel
in, or importation of food from, endemic areas , this
relationship is not always evident. Even in endemic areas,
a minority of patients (14%) could not readily identify the
source of the infection . A diagnosis of brucellosis in
a patient from a non-endemic area in the absence of spe-
cific risk factors for acquisition of the disease should lead
at least to enhanced awareness for the following days, in
order to readily identify an evolving trend and respond
adequately and rapidly.
Isolating the organism remains the gold diagnostic stan-
dard, although blood culture positivity is reported to vary
widely , and bone marrow aspiration and culture,
considered by some as extremely sensitive , remains
an invasive, painful procedure. Moreover, in the context
of an outbreak, the traditionally protracted period needed
for species culture and identification (ranging from 3
days to 6 weeks), means that other diagnostic procedures
should be sought.
Serology, in the form of various agglutination tests target-
ing surface antigens, and enzyme-linked immunosorbent
assay (ELISA), targeting other bacterial antigens ,
is extremely useful; sensitivity and specificity are well
above 85% for both approaches. Drawbacks of serum ag-
glutination tests include false-negative results of varying
etiology (delayed seroconversion, blocking antibodies,
prozone phenomenon) and cross-reaction-induced false
positive results, and the inability to serologically follow
patients up due to protracted persistence of increased an-
tibody titers. ELISA is more sensitive, and a diagnostic
procedure of choice for cerebrospinal fluid specimens in
neurobrucellosis, but evolution of antibody titers in fol-
low-up and detection of relapses are still troublesome.
Various polymerase chain reaction (PCR) assays have
been developed but clinical studies are limited .
Cell. Mol. Life Sci. Multi-author Review Article 5
Moreover- real-time PCR (rtPCR) is now emerging as an
important diagnostic tool [44, 45]. Specific PCR assays
for field detection of significant bioterror pathogens have
also been developed by the military .
rtPCR may offer a rapid (less than an hour), exquisitely
sensitive and specific diagnosis in a deliberate release
outbreak, although traditional serology might be more
suitable as a diagnostic tool in such a situation: the trend
would be rapidly recognized. (Even when taking into ac-
count false negatives, the majority of patients will sero-
convert, and even in an endemic area, high titers could
not be attributed to previous contact with the pathogen.
On the other hand, false positives would be extremely un-
likely, and should be entertained only as a possible prob-
lem in a deliberate release of F. tularensis, where a false
positive diagnosis of brucellosis might steer response in
a wrong direction). Microorganism isolation for further
characterization and recognition of any genetic modifi-
cations would of course remain paramount in the overall
Various principles apply to brucellosis treatment: the or-
ganism hides inside macrophages which requires antibi-
otics with adequately intracellular penetration. Moreover,
these antibiotics need to be active in the acidic environ-
ment where the bacteria reside. The optimal treatment
is a combination regimen, since monotherapy has been
traditionally associated with an increased percentage of
treatment failure and relapse [46, 47]. Duration of treat-
ment also matters , and 6-week regimens are asso-
ciated with an acceptable percentage of relapses. The
World Health Organization (WHO) endorses regimens
that combine doxycycline, 100 mg b.i.d., and rifampicin,
600–1200 mg daily, for 6 weeks, or doxycycline for 6
weeks and streptomycin, 15 mg/kg daily, for 2–3 weeks.
The latter combination is considered superior , but
demands parenteral administration. Gentamicin can
adequately replace streptomycin, at a dose of 5 mg/kg
for 5–7 days. Alternatives include trimethoprim-sulfa-
methoxazole in various combinations, and combinations
including ofloxacin or ciprofloxacin. Quinolone-contain-
ing regimens are generally adequate, but cost-effective-
ness and the possibility of community resistance are is-
sues to be considered . Triple or quadruple protracted
regimens should be used in serious complications, in con-
junction with invasive procedures, as indicated. Rifam-
picin and trimethoprim-sulfamethoxazole are the main-
stays of treatment in pregnancy and pediatric populations,
respectively. Future options may incorporate adjuvants
aiming at altering the acidic intracellular environment or
new antibiotics .
The development of a vaccine for brucellosis suitable for
humans would be an ideal solution to the problems of in-
adequate veterinary control of animal disease, inadequate
epidemiological study of human disease and inadequate
antibiotic treatment. The absence of such a vaccine un-
derlines the absence of interest in a common, albeit usu-
ally non-fatal, zoonosis, at least in areas with adequate
scientific and financial tools for such development and
the still incomplete knowledge about important steps of
the molecular pathogenesis of brucellosis.
Numerous vaccines have been tested in the past; none
of them have gained wide acceptance . An intrader-
mally administered vaccine derived from B. abortus 19
strain has been used extensively in the Asian Republics
of the former Soviet Union, causing a 5–11 fold reduc-
tion in the annually reported cases of human brucellosis.
Still, the vaccine offers limited protection of short dura-
tion and requires booster doses. Moreover, an increased
number of hypersensitivity reactions were reported, with
76% local reactions and 3–7% generalized adverse ef-
Another vaccine used in the same area, similar to the
previous but administered intramuscularly, appeared
to evoke minimal reactions and similar protection. Its
reported efficacy after 75,000 doses performed in Ka-
zhakstan reached 79%. Strains of B. abortus 84-C and
104-M have been utilized for intradermal injection or
inhalation in the former Soviet Union and in China,
respectively . The vaccines were considered effec-
tive but of high risk for serious adverse reactions. The
French experience with a vaccine utilizing a phenol-
insoluble peptidoglycan fraction of B. melitensis strain
M-15 raised questions in the past about its efficacy. The
vaccine was administered in two subcutaneous doses
and supposedly offered protection for a 2-year period
. Efficacy of other vaccines that could be consid-
ered for humans has been proven in animals: the vari-
ous preparations include a lipopolysaccharide-protein
conjugate, a purified protein antigen L7/L12, Cu-Zn
SOD, and glyceraldehyde-dehydrogenase. Theoretical
vaccine targets for the future include the RB51 strain
(although it induces minimal human disease, which is
rifampicin resistant) , purE mutants (that still have
significant residual virulence), rfbK mutants of B. meli-
tensis, Omp 19, Omp 28, and the cytoplasmic protein
Yet even if developed, the efficacy of a human vaccine in
the setting of a deliberate release outbreak would be mini-
mal. Sufficient prophylaxis in such a case would demand
pre-emptive vaccine administration, since following ex-
posure, and despite the prolonged incubation period of the
disease, antibody production would not be brisk enough.
Antibiotics would be the only option in such a case. The
use of antibiotic prophylaxis for asymptomatic persons
exposed to Brucella is an inadequately studied issue ;
6 G. Pappas et al. Brucellosis and bioterrorism
most data derive from accidental laboratory exposure. In
general, the most prudent approach would be to follow
up for seroconversion, and subsequently treat, even when
no symptoms appear. With this strategy, prophylaxis ad-
ministration would be minimized, and persons at risk of
developing brucellosis (the ones seroconverting) would
be adequately treated. Another option would be to with-
hold antibiotics in the absence of any symptoms, even
for persons exhibiting seroconversion. But the insidious
course of the disease would leave these patients at risk
of developing brucellosis for practically the rest of their
A deliberate release outbreak of brucellosis would obvi-
ously have profound environmental effects, their range
depending on characteristics of the targeted area. Areas
whose economy is largely based on animal productivity
would suffer the most, due to massive loss of livestock
and diminished trade in dairy products in the future.
Even though the overall financial burden of health ser-
vices would be lower than most other potential biological
weapons , the long-term effect on the targeted region’s
economy would be profound, more so when taking into
account environmental pollution, which has the potential
for secondary airborne community outbreaks.
The art of developing attack scenarios, and in that manner
outlining problems that might emerge in a real-life situa-
tion, has been inadequately explored. This is particularly
important since the same attack would have different out-
comes in different targeted areas, depending on numer-
ous characteristics. Even the public response would be
different, depending on awareness, and endemicity. A tra-
ditional attack scenario  projected minimal fatalities
in a Brucella attack under ‘optimal’ circumstances. Yet
when a similar scenario was transcribed in an endemic
area, the projected outcome was far less morbid .
In this latter scenario, an attack in an endemic city of
100,000 inhabitants would result in only two deaths, and
the level of social disruption would be minimal, due to
public awareness of the pathogen.
The importance of Brucella as a biological weapon may
only be historical nowadays, due to its minimal mortal-
ity and protracted inoculation period. Yet our times have
taught us that everything is possible. Educating physi-
cians and public alike, walking the fine line between in-
adequate awareness and fear of a ‘nasty bug’, might help
in limiting unnecessary interventions, in determining ar-
eas of research that need to be addressed, and in creating a
web of response that is adequate yet not overly restrictive
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