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1 Introduction
The history of research on tularemia as well as that of
its causative agent’s taxonomic classification goes back
more than 100 years. The origin of the tularemia story is
generally dated to the year 1911, when George W. McCoy,
Director of the US Public Health Service Plague Laboratory,
undertook bacteriological investigations of bubonic
plague in ground squirrels and reported a plague-like
disease of rodents in California [1]. In parallel, physician
R. A. Pearse reported in the journal Northwest Medicine
(March 1911) six cases of fever caused by the bites of
deerflies and named this “deerfly fever” [2]. One year later,
McCoy and Chapin successfully isolated a novel organism
which was named Bacterium tularense (B. tularense) after
Tulare County in central California, the site of the original
discovery [3]. Edward Francis studied deerfly fever in Utah
during the 1920s. Because of the pathological changes of
the disease observed in animals and humans and due to
isolation of the bacterium from human blood, he renamed
the disease tularemia [4]. Almost concurrently, Hachiro
Ohara was studying wild hare disease (Yato-byo) in Japan
and recognized that disease’s similarity to tularemia.
His observation was confirmed by Francis, who isolated
B. tularense from specimens he received from Ohara [5].
Soon thereafter, tularemia was recognized in the USSR,
Norway, Canada, Sweden, and Austria [6–8]. Thus, the
names deerfly fever, rabbit fever, Pahvant Valley plague,
lemming fever, Yato-byo, Ohara’s disease, water-rat
trapper’s disease, hare meat poisoning, and probably also
other historical names are synonyms for tularemia.
From a historical point of view, tularemia was probably
first described on September 19, 1907. At the Annual
Meeting of the Medical Library Association, Portland,
Oregon, on June 25, 1940, Dr. William Levin cited a letter
of one Dr. Ancil Martin which had been sent to F. G. Novy
at the University of Michigan. In this letter, Martin had
stated that he had under observation and treatment five
cases of an infection caused by the skinning and dressing
of wild rabbits [9]. For the sake of completeness, it should
be noted that there is a hypothesis that the biblical plague
Abstract: Tularemia is a debilitating febrile and potentially
fatal zoonotic disease of humans and other vertebrates
caused by the Gram-negative bacterium Francisella
tularensis. The natural reservoirs are small rodents,
hares, and possibly amoebas in water. The etiological
agent, Francisella tularensis, is a non-spore forming,
encapsulated, facultative intracellular bacterium, a
member of the γ-Proteobacteria class of Gram-negative
bacteria. Francisella tularensisis capable of invading and
replicating within phagocytic as well as non-phagocytic
cells and modulate inflammatory response. Infection
by the pulmonary, dermal, or oral routes, respectively,
results in pneumonic, ulceroglandular, or oropharyngeal
tularemia. The highest mortality rates are associated
with the pneumonic form of this disease. All members of
Francisella tularensis species cause more or less severe
disease Due to their abilities to be transmitted to humans
via multiple routes and to be disseminated via biological
aerosol that can cause the disease after inhalation of even
an extremely low infectious dose, Francisella tularensis
has been classified as a Category A bioterrorism agent. The
current standard of care for tularemia is treatment with
antibiotics, as this therapy is highly effective if used soon
after infection, although it is not, however, absolutely
effective in all cases.
Keywords: Tularemia; Francisella tularensis; intracellular
bacterium; bioterrorism
DOI 10.1515/biol-2015-0013
Received March 17, 2014; accepted October 28, 2014
Review Article Open Access
© 2015 Kubelkova K., Macela A., licensee De Gruyter Open.
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License.
Kubelkova K.*, Macela A.
Putting the Jigsaw Together - A Brief Insight Into
the Tularemia
*Corresponding author: Kubelkova K.: Department of Molecular Pa-
thology and Biology, Faculty of Military Health Sciences, University
of Defense, 1575 Trebesska, 500 01 Hradec Kralove, Czech Republic,
E-mail: klara.kubelkova@unob.cz
Macela A.: Department of Molecular Pathology and Biology, Faculty
of Military Health Sciences, University of Defense, 1575 Trebesska,
500 01 Hradec Kralove, Czech Republic
196 Kubelkova K., Macela A.
of the Philistines and a virulent epidemic, similar to the
bubonic plague or typhus, in Ancient Egypt (around 1715
BC) may have been tularemia [10,11].
The original name and taxonomy of the etiological
agent of tularemia changed several times over the years. In
the literature can be found the name Pasteurella tularensis
and in older literature the name Brucella tularensis (due to
serological cross-reactions with the Brucella sp. antigens).
Finally, to honor the achievements of Edward Francis
in the field of tularemia research, the bacterium was
renamed to Francisella tularensis (F. tularensis) [12,13].
2 The etiological agent - Fran-
cisella tularensis
F. tularensis is a highly virulent, non-sporulating,
pleomorphic, facultative intracellular, Gram-negative
coccobacillus that is capable of causing the zoonotic
disease tularemia in a large number of mammals. F.
tularensis strains grow slowly in CO2 supplemented air
and almost all strains of this fastidious organism have
specific requirements for iron and cysteine or cystine.
After 24 hours of incubation, small, white and gray,
smooth or shiny-surfaced colonies can be determined on
an appropriate solid media.
Francisella is the only genus within the family
Francisellaceae of the γ-subclass of Proteobacteria [14].
The Francisellaceae family is distinguished by a unique set
of phenotypic characteristics such as their morphology,
a capability for degrading only a limited number of
carbohydrates, a growth requirement for cysteine, and a
unique fatty acid composition [15]. The taxonomy of the
genus Francisella is still subject to debate. A recent study
of the phylogenetic relationship of all known Francisella
species divided the genus Francisella into two genetic
clades. One is represented by F. tularensis, F. novicida, F.
hispaniensis, and the close neighbor Wolbachia persica;
the second by F. philomiragia and the fish pathogen F.
noatunensis [16]. The majority of publications divide the
species F. tularensis into four closely related subspecies
that are highly conserved in their genomic content but
differ in their virulence, biochemistry, and epidemiology:
F. tularensis subsp. tularensis (also known as Type A),
which is endemic in North America, while in Europe
there is known only one isolate that possibly represents a
laboratory escape [17]; F. tularensis subsp. holarctica (also
known as Type B), widespread throughout the Northern
Hemisphere; F. tularensis subsp. mediasiatica, endemic
in Central Asia; and F. tularensis subsp. novicida. Most
human cases of tularemia are caused by the Type A and
Type B strains, with Type A strains being significantly
more virulent. Type A strains are now newly divided into
3 genotypes (clades) A1a, A1b, and A2, all of which have
been shown to be epidemically important [18,19]. Strains
of F. tularensis subsp. tularensis are considered the most
virulent for humans, with an infectious dose of less than
10 colony forming units (CFU). The lethality of Type A
strains is up to 24% in untreated cases depending on
the Type A genotype [19]. F. tularensis subsp. holarctica
also causes debilitating diseases, albeit with a milder
course. The fatality rate barely reaches 1%. F. tularensis
subsp. mediasiatica rarely causes human illness and is
less virulent than F. tularensis subsp. holarctica [20]. F.
tularensis subsp. novicida is an opportunistic pathogen
for humans, and it is significantly less pathogenic than
the other subspecies. It can cause the disease mainly in
immunocompromised people [21,22]. Fully virulent strains
must be handled in labs under containment meeting the
requirements of Biosafety Level 3 [23]. The Centers for
Disease Control and Prevention classify F. tularensis as a
Category A bioterrorism agent¹.
3 Intracellular lifestyle
To establish infection, F. tularensis, as an intracellular
pathogen, needs to enter cells, find a target place to
survive, and then grow inside host cells. The ability of F.
tularensis to survive and multiply intracellularly has been
well described in both professional and non-professional
phagocytes in in vitro and in vivo models [24–26]. The
molecular mechanisms used by F. tularensis to mediate its
uptake into the host cell are mostly unknown, however. In
general, serum-opsonized F. tularensis can enter host cells
using a process dependent on the presence of complement
factor C3 in the serum and complement receptors on
the surface of the host cell, which are engaged in the
formation of pseudopod loops in the host cell’s surface
membrane [27]. Under this condition, class A scavenger
receptors, lung surfactant protein A, nucleolin, as well
as the Fcγ receptors are involved, to various degrees, in
the internalization of F. tularensis by mammalian cells
[28–30]. In the non-opsonic uptake of F. tularensis by
macrophages, a significant role is played by the mannose
receptor [28] and possibly other cell surface receptors
that have not yet been defined. The mode of entry may
influence the fate of Francisella inside the host cell by
triggering different signaling pathways that control the
1 See the website: http://emergency.cdc.gov/agent/agentlist-catego-
ry.asp
Tularemia 197
expression of intracellular defense mechanisms and, in
parallel, influence the survival of intracellularly localized
bacteria [31].
Inside the host cell, F. tularensis resides in an initial
vacuolar compartment along the general endocytic
degradative pathway, recently termed the Francisella-
containing phagosome (FCP). The FCP sequentially
acquires early and late endosomal markers, such as
EEA-1, Lamp-1, and Rab-7, but not the marker cathepsin
D, which is an indicator of phagosome–lysosome fusion
[32,33]. The next step in the intracellular trafficking of the
bacterium consists in active FCP membrane disruption
followed by escape into the cytosol, where it then
replicates [32–34]. It is still under debate whether the
FCP is acidified before disruption of the membrane by F.
tularensis. There are experimental data demonstrating
the progressive acidification of the vacuole by acquiring
vacuolar ATPase before phagosomal disruption [35,36], as
well as contradictory data that exclude FCP acidification
[32,37,38]. Whether this discrepancy is due to different
experimental conditions or different infectious agents is
not yet clear. Phagosomal escape is followed by extensive
cytosolic replication and, finally, the programmed cell
death of the host macrophage [39,40]. During later intervals
of infection, some F. tularensis have been observed in the
multi-membrane vacuolar compartment of the endocytic
pathway that has the characteristics of an autophagosome
[41,42]. Still unclear, however, are the reason why F.
tularensis reenters the membranous compartment and the
consequences for the further dissemination of infection
and induction of immune response (the scheme of whole
process see Fig. 1). If the re-entering of Francisellae into
Fig. 1: Intracellular trafficking of F. tularensis after uptake by different macrophage receptors: Francisella uses multiple mechanisms to
evade host defence. Francisella can be recognized by multiple macrophage receptors such as FcgR, complement receptor 3 (CR3), scaven-
ger receptor A (SRA), mannose receptor (MR) and surface-exposed nucleolin (SE-N). Francisella blocks the NADPH oxidase and detoxifies
reactive oxygen species (ROS). Francisella does also signal through TLR2 and may activate intracellular TLR9, but it does not signal through
TLR4. Upon entry, Francisella-containing phagosome (FCP) matures into early endosome characterized by the early endosomal antigen 1
(EEA1) and Rab5 GTPAse (Rab5). The maturation progress into the late endosome characterized by the late endosomal markers Rab7 GTPase
(Rab7), lysosomal-associated membrane protein 1 a 2 (LAMP1, LAMP2) together with proton ATPase pump. Subsequently, Francisella multi-
plies in cytosol within 24h after infection with engagement of a selected signaling pathway. Following replication, re-entering the endoso-
mal-lysosomal pathway through the autophagosome-like vacuole characterized with LAMP1/2, microtubule-associated protein light chain 3
(LC3) and major histocompatibility complex classII (MHCII) can be observed. Francisella induces host cell death (apoptosis or necrosis) with
subsequent robust infection of other cells.
198 Kubelkova K., Macela A.
multilamelar compartment is induced by Francisella itself
or if it is the result of defence mechanism orientation to
eliminate intracellular threat is still under debate.
During its history, F. tularensis has developed
molecular tools to avoid the intracellular defense
mechanisms of the host cell. More than 300 genes
considered to be virulence factors have been identified so
far. Among these are genes involved in adhesion to host
cells [43]; genes associated with capsule biosynthesis
contributing to serum resistance; and genes, including
those from the Francisella pathogenicity island coding
probably of the type VI secretion system, enabling the
“neutralization” of intraphagosomal milieu [44–47],
escape into the cytosol, and proliferation inside the
host cell [48–50]. F. tularensis is also able to delay cell
death to increase its survival and replication through
activation of Ras by the SOS2/GrB2/PKCα/PKCβI
quaternary complex, which stimulates cell survival
through the downregulation of caspase-3 activation
[51]. Moreover, Francisella spp. actively manipulate the
timing of autophagy onset, interferon signaling, Toll-
like receptor signaling, and phagocytosis [52,53], thus
manipulating one of the early cell defense mechanisms
against infection [54]. Equally important for Francisella is
the adaptation to the nutritional limitations inside a host
cell [55] Despite numerous studies dedicated to virulence
factors, F. tularensis virulence in its complexity has not yet
been sufficiently elucidated [56]. Some important factors
involved in a process of Francisella phagosomal escape
and in intracellular growth are listed in Table 1.
Altogether, the ability of intracellular pathogens to
evade clearance inside host cells and disseminate to other
areas of the body is essential to the pathogen’s virulence
and pathogenicity. It remains unclear, however, as to how
Francisella spp. actually kills their hosts. Deactivation of
immune cells, uncontrolled cytokine response, and toxin
production has all been implicated as mechanisms by
which bacterial pathogens induce death during systemic
infection. For obligatory and facultative intracellular
pathogens, factors essential for their in vitro and/or in vivo
survival and growth should be considered as virulence
determinants. Nevertheless, inasmuch as each intracellular
pathogen has developed its own strategy for survival inside
macrophages, the application of knowledge from one
pathogen to another should be made very carefully.
Francisellae are able to evade defence mechanisms of
the host. Then, there is still a question if the host is able to
clear the infection in full or if there are some Francisellae,
inside unactivated macrophages, that are able, under the
specific conditions of the host, to reactivate infectious
process. The clear evidence is still missing.
4 Epidemiology
Tularemia is a zoonotic infection caused by F. tularensis
that occurs endemically in most countries of the Northern
Hemisphere. It is widespread throughout the Old World
and North America [79]. On the Eurasian continent,
tularemia is traditionally reported from Scandinavian
countries, particularly from Sweden [80,81]; from
countries of Central Europe, including Germany [82,83],
Austria [84,85], the Czech Republic and Slovakia [84,86];
from countries of the former Soviet Union [87–89]; from
Central Asia [90–92], including Mongolia [93]; and
from the Japanese islands [94,95]. Tularemia has been
occurring in Turkey [96–98] and Spain [99,100] since
the 1990s. Several dozen cases have been reported from
Italy [101,102] and France [103]. The British Islands seem
to be free from the disease. It is important to note that in
Europe, Asia, and Japan the frequency of tularemia cases
has generally grown in situations when the socioeconomic
and environmental conditions of the population have
been disrupted. During the Second World War, epidemics
comprising from 10,000 to 100,000 cases each year and
large outbreaks of waterborne tularemia were recorded
in Eastern European countries [104–106]. An increased
frequency of tularemia cases during World War II was
also registered in Japan [107]. More recently, 327 cases
of tularemia were reported during the postwar period in
Kosovo (1999–2000) [108].
In the New World, the disease, or the presence of F.
tularensis in wildlife, is reported from Canada, the USA,
and Mexico. Tularemia has occurred in all Canadian
jurisdictions except the Yukon and Nunavut [109]. In the
USA, human cases have been reported from all states
except Hawaii [110,111]. An isolated small endemic area
is Martha’s Vineyard in Massachusetts, with diverse
Francisella spp. in an environment where human cases
of pneumonic tularemia occurred during recent decades
[112–114]. A serological survey of wildlife carried out
during 1988 and 1989 in Mexico documented the exposure
of two animal species to F. tularensis [115].
While it is generally known that there are zones
where tularemia has been occurring for decades, the
overall ecology of F. tularensis is not well understood, and
particularly the transmission cycle, ecological requirements
of the different subspecies, and true natural reservoir
hosts [116,117]. More than 250 species of mammals, birds,
amphibians, invertebrates, and protozoans have already
been identified as hosts for F. tularensis, which complicates
understanding the transmission cycle. In general, human
cases of tularemia are most often associated with exposure
to lagomorphs, rodents, and blood-feeding arthropods; the
Tularemia 199
Table 1: Some important factors involved in a process of a Francisella phagosomal escape and in intracellular growth
Protein/gene Name of protein Role/functionFT strainHost cellsRef.
AcpA Acid phosphatase PE F. novicida U THP-/BMMs []
IG F. novicida U THP-/BMMs []
CarA Carbamoyl-phosphate synthase small chain PE LVS BMMs []
IG LVS BMMs []
CarB Carbamoyl-phosphate synthase large chain IG LVS BMMs []
DsbA DSBA-like thioredoxin domain protein PE SchuS
FSC
J
BMMs
[]
[]
IG SchuS
SchuS
LVS
HepG
J
J
[]
[]
[]
DsbB Disulfide bond formation protein PE SchuS HepG []
FevR/PigR Francisella effector of virulence regulation/
Uncharacterized protein
PE LVS BMMs []
IG F. novicida U
LVS
BMMs
BMMs
[]
[]
IglA Intracellular growth locus A PE LVS J []
IG F. novicida U
LVS
J
J
[]
[]
IglB Intracellular growth locus B PE LVS J []
IG LVS J []
IglC Intracellular growth locus C PE LV S
F. novicida U
J
U/hMDMs
[]
[]
IG LVS
F. novicida U
J
U/hMDMs
[]
[]
IglD Intracellular growth locus D PE LVS J []
IG LVS
F. novicida U
J
U/hMDMs
[]
[]
IglG Intracellular growth locus G PE LV S J []
IglH Intracellular growth locus H PE FSC BMMs []
IG FSC BMMs []
IglI Intracellular growth locus I PE LVS
F. novicida U
J
BMMs
[]
[]
IG F. novicida U J []
MglA Macrophage growth locus, subunit A PE F. novicida U
F. novicida U
LVS
J
U/hMDMs
JA.
[]
[]
[]
IG F. novicida U
F. novicida U
LVS
J
U/hMDMs
JA.
[]
[]
[]
MglB Macrophage growth locus, subunit B IG F. novicida U J []
MigR Macrophage intracellular growth regulator PE LVS BMMs []
IG LVS BMMs []
LVS dMDMs []
PdpA Pathogenicity deteminant protein pdpA PE F. novicida U BMMs/J []
IG F. novicida U BMMs/J []
PdpB Pathogenicity deteminant protein pdpB IG F. novicida U BMMs/J []
PmrA Orphan response regulator IG F. novicida U THP-/J []
PurA Adenylosuccinate synthetase IG F. novicida U J []
table modified from [264]
1 PE – phagosomal escape; IG – intracellular growth
2 FT – Francisella tularensis; LVS - F. tularensis subsp. holarctica live vaccine strain; SchuS4 – F. tularensis subsp. tularensis SchuS4;
FSC200 - F. tularensis subsp. holarctica FSC200
3 THP-1 - humanmonocyticcell linederived from anacute monocytic leukemiapatient; BMMs - bone marrow-derived macrophages;
J774 - murine macrophagescell line; HepG2 - human hepatocellular liver carcinomacellline; U937 - human leukemic monocyte lym-
phomacellline; hMDMs - human monocyte-derived macrophage
200 Kubelkova K., Macela A.
inhalation of contaminated dust particles; or the drinking
of contaminated water. Two cycles, terrestrial and aquatic,
have been described for the disease caused by F. tularensis.
Hares and rabbits are prototypical hosts for the terrestrial
cycle while ticks, mites, and biting flies represent arthropod
vectors. Beavers, muskrats, and voles are mammalian
hosts that can contaminate water within the aquatic
cycle [118,119]. The aquatic cycle is associated with rivers,
streams, and flooded landscapes, and it can be promoted
by the persistence of the bacterium within protozoans
[116,120–122].
Outbreaks of tularemia in humans are typically
associated with outbreaks of tularemia in animal
populations. Thus, rural populations, and especially those
individuals who spend some time in endemic areas, such
as farmers, hunters, forest workers, and tourists, are most
at risk of tularemia [123–125]. For example, two outbreaks of
pneumonic tularemia on Martha’s Vineyard, Massachusetts,
were associated with the use of lawn mowers or brush cutters
while people were working around the houses [112,126].
Some animal species such as skunks and raccoons moving
within the environment of this island are seropositive and
may constitute the source of infecting agent [127]. Moreover,
the ability of F. tularensis subsp. tularensis to survive in
salt-influenced soil or moisture on this island has been
already documented and this probably contributed to the
epidemiological situation in this territory [128]. Another
example of pneumonic tularemia outbreaks associated with
farming can be seen in outbreaks during work campaigns at
sugar factories within the former Czechoslovakia in the 1950s
and 1960s [129]. Based on previously accepted techniques,
heaps of sugar beets were being washed using jets of water
that obliterated the corpses of Francisella-infected small
rodents inside the heap and subsequently created an aerosol
that was inhaled by the workers.
Significant vectors of tularemia in the countryside
are blood-sucking arthropods, including ticks, flies, and
mosquitos. In the United States four tick vectors consisting
of Amblyomma americanum, Ixodes scapularis [130,131],
and Dermacentor variabilis in the southeastern and
south–central states and D. andersoni in the west were
identified to be the most important for the transmission
of tularemia to humans. All developmental stages, larva,
nymph, as well as imago, can carry the disease-causing
agent. Larvae of D. variabilis in the US as well as larvae of
Ixodes ricinus in Europe have been shown able to acquire,
maintain, and transstadially transmit F. tularensis [132–
134]. The percentage of ticks infected with F. tularensis
in endemic areas is nevertheless relatively low. Over a
period of 3 years, out of 4,246 D. variabilis ticks assayed at
Martha’s Vineyard, only 0.7% were positive in the specific
PCR assay for F. tularensis [135]. Similarly, studies carried
out in Central Europe demonstrated a minimal infection
rate of around 2.0% for D. reticulatus and less than 0.5%
for Ixodes ricinus [136–138]. Transmission, especially
by the deerfly, Chrysops discalis, and by the Tabanidae
(horseflies), has been documented in western regions of
the USA. While in the western USA both biting flies and
ticks are considered important vectors, in the eastern USA
only ticks seem to be significant vectors [139].
continuedTable 1: Some important factors involved in a process of a Francisella phagosomal escape and in intracellular growth
Protein/gene Name of protein Role/functionFT strainHost cellsRef.
PurF Amidophosphoribosyltransferase IG F. novicida U J []
PurMCD Phosphoribosylformylglycinamidine cyclo-
ligase/Fusion protein PurC/PurD
IG LVS J []
LVS PEC []
PyrB Aspartate carbamoyltransferase IG LVS BMMs []
VgrG VgrG protein PE F. novicida U BMMs []
IG F. novicida U J []
FTT Uncharacterized protein IG SchuS BMMs []
FTT Uncharacterized protein PE SchuS BMMs []
IG SchuS BMMs []
FTT Hypothetical membrane protein PE SchuS BMMs []
IG SchuS BMMs []
table modified from [264]
1 PE – phagosomal escape; IG – intracellular growth
2 FT – Francisella tularensis; LVS - F. tularensis subsp. holarctica live vaccine strain; SchuS4 – F. tularensis subsp. tularensis SchuS4; FSC200
- F. tularensis subsp. holarctica FSC200
3 THP-1 - humanmonocyticcell linederived from anacute monocytic leukemiapatient; BMMs - bone marrow-derived macrophages; J774 -
murine macrophagescell line; HepG2 - human hepatocellular liver carcinomacellline; U937 - human leukemic monocyte lymphomacellline;
hMDMs - human monocyte-derived macrophage
Tularemia 201
In northern European countries, such as Sweden,
Finland, and the northern part of Russia, mosquitoes
are the dominant vector transmitting tularemia to
humans [139,140]. By contrast, mosquitoes in Central
Europe probably do not carry F. tularensis in natural foci
of tularemia; contact with infected animals, ingestion
of contaminated food or water, along with possible
infection caused by tick vectors are the dominant modes
of transmission in this region [129,138]. The seasonality
of reported tularemia cases corresponds well with these
transmission modes.
5 The disease – signs and
symptoms
Signs and symptoms of tularemia in wild animals are not
well documented and unfortunately are based mostly
on postmortem examinations of carcasses. Some data
can be found describing naturally infected animals
such as rabbits, hares, cats, and prairie dogs [141–144].
The majority of information on tularemia symptoms in
animals has originated from laboratory experiments using
mice, rabbits, guinea pigs, and monkeys as animal models
of natural infection. In these cases, however, most studies
utilized the F. tularensis live vaccine strain or F. novicida,
which in murine models induce symptoms similar to the
infection in humans caused by wild virulent strains [145].
The clinical manifestations of tularemia depend on the
route of infection and the susceptibility of any particular
animal species to tularemia.
Similarly to other bacterial zoonoses, tularemia is
transmitted to humans by direct contact with infected
animals, tissues or fluids from infectious animals or
by bites from infected arthropod vectors. Inhalation of
aerosol or ingestion of contaminated food and water
are other sources of infection. Thus, the gateways for
F. tularensis into the body include the skin, mucosal
membranes, lungs, and gastrointestinal tract. In general,
infection is characterized by common symptoms as are
fever, sweats, headache, body ache, nausea, vomiting,
and diarrhea. Pulse-temperature dissociation is seen in
less than half of the patients. The incubation time, which
delineates the period of delay between infection and the
outbreak of symptoms, varies around 6 days. Moreover,
the delay between the onset of symptoms and the seeking
of treatment ranges around 7 days [146,147]. Disease onset
is abrupt, usually within 3 to 5 days, but it can be as rapid
as 1 day or as prolonged as 14 days post-exposure.
According to a study by Dr. Francis, tularemia
was delineated into four major clinical manifestations
(ulceroglandular, glandular, oculoglandular, and
typhoidal) while there also exist additional manifestations,
including oropharyngeal, gastrointestinal, pneumonic,
and other rare forms [148]. The most common form
of human tularemia is the ulceroglandular form that
results from contact with infectious material or from
vector-borne transmission. Typically, a papule develops
into a pustule surrounded by a zone of inflammation
at the site of infection, subsequently manifesting by
enlargement of the regional lymph node. An ulcer can
persist for months. A similar form is taken by glandular
tularemia, which is characterized by similar symptoms
but without appearance of the primary lesion in the form
of an ulcer. During the incubation period (typically 3 to
6 days), bacteria disseminate from the site of infection
via lymphatic vessels to the regional lymph node [149].
The enlargement of the draining lymph nodes often
resembles the buboes of bubonic plague. Finally, the
bacteria disseminate to such other tissues as the spleen,
liver, lung, or peritoneum. It is likely the bacteria are
carried there by phagocytes of the bloodstream, although
bacteremia occurs transiently and relatively early after
infection. The mortality rate for this form of tularemia is
less than 3% [150]. The oculoglandular form occurs as
a result of infection though the eye by touching the eye
with a contaminated finger or by the ingress of infected
dust particles into the eye. The conjunctiva is the primary
site of infection. The appearance of ulcers and nodules
on the conjunctiva is a characteristic feature. Without
treatment, the bacteria disseminate to the draining
lymph node and to other organs. This form is not so
frequent and according to data in the older literature
comprises less than 1% of all human cases of tularemia
[151,152]. Typhoidal tularemia, an acute form of the
disease caused mainly by F. tularensis subsp. tularensis,
is characterized by various clinical symptoms typical for
septicemia, without the formation of a primary lesion or
lymphadenopathy. Occasionally, the patients are delirious
and this stage may be followed by shock. Nevertheless,
clinical classification and acceptance of a typhoidal
designation is generally applied only for cases when no
route of infection is diagnosed. Typhoidal tularemia has
a substantial mortality rate of 30–60% without antibiotic
treatment [153–155]. Outbreaks of pneumonic tularemia
resulting from the inhalation of infected aerosols are
commonly associated with activities which may aerosolize
F. tularensis from environmental and animal reservoirs
[149]. This is probably the most acute form of the disease.
A substantial complication is the problem of diagnosis,
because the clinical and roentgenological picture is not
specific for tularemia.Respiratory tularemia may present
202 Kubelkova K., Macela A.
symptoms of pneumonia, including a cough, chest pain,
increased respiratory rate, and high fever, as well as
other unspecific symptoms such as nausea and vomiting.
Pneumonia may occur as a primary manifestation of the
respiratory form, but secondary pneumonia may also
appear as a complication in any form of tularemia. This is
a consequence of the bacteria’s dissemination throughout
the body, including the lungs.
Why is the pneumonic tularemia the most acute
and the most severe form of the disease? The answer to
this question was searched using different experimental
models.
One of the options can be a spectrum of cells in the
lung that are infected by Francisellae. Lung macrophages
and dendritic cells, as the mobile phagocytes and
effective APCs, but also lung endothelial cells and
structural alveolar type II epithelial cells all are targets of
F. tularensis LVS as well as F. tularensis Schu S4 invasion.
The promiscuity of Francisellae in relation to the cell types
that are infected in the lung can have a substantial impact
on the course of infection through modulated functions
of infected cell [156]. There are evidences that pulmonary
macrophages and DCs infected with virulent Francisellae
produce a significant amount of immunosuppressive
cytokine TGF-β [157] that, through the feedback loop,
can differentially modulate secretory and phagocytic
functions of the lung APCs [158,159], can promote the
development and activation of tolerogenic DCs. Produced
IL-10 and TGF-β stimulate proliferation of T(regs) that
may restrain Th1-type of protective immune response
[160]. Moreover, migrating DC may serve as an effective
carrier of F. tularensis during the early stages of infection
and can play a role in pathogen dissemination [161,162].
Infected endothelial cells may attract polymorphonuclear
leukocytes to transmigrate across the endothelium, but,
concurrently, downregulate responsiveness of the PMN to
subsequent activation [163]. Invasion of F. tularensis into
and proliferation within nonphagocytic lung epithelial
cells [164,165] may modulate their multiple biological
functions, some of them are associated with immune
responsiveness of the host [166].
Thus, collectively with our own results demonstrating
questionable function of neutrophils and B cells in the
early stages of inhalation infection with F. tularensis
subsp. holarctica strain 15L and 130 [129,167], all the
effects on secretory and functional profile of infected cells
within lung may resulted into poorly controlled protective
response and severity of pneumonic tularemia.
Important differences can be observed during the
course of infection by the Type A versus Type B subspecies
[15]. Tularemia infection associated with Type A shows
a high mortality rate for infected hosts in general. In
humans the onset of Type A infection is often sudden and
is characterized by chills, high fever, dyspnea, cough,
pharyngitis, chest pain, headache, profuse sweating,
and general weakness. If this is the case, the patient’s
condition is extremely severe, symptoms and signs may
mimic those of typhoid fever, and the changes are highly
variable [168–170]. Infections induced by Type B differ
from infections caused by the Type A subspecies. Type
B infected patients in general suffered high fever, chest
pains, and flu-like symptoms [171].
To summarize, the clinical forms of tularemia can
be divided into various syndromes that complicate
diagnosis. The incubation period is usually 3 to 5 days
after inoculation. Clinical manifestation of illness begins
with the rapid onset of fever, chills, headache, malaise,
fatigue, and myalgia. Some patients suffer from coughing,
nausea, and vomiting. Other findings may include skin
ulcers, sore throat, pleural effusion, primary or secondary
pneumonia, acute respiratory distress syndrome, and
pericarditis. General signs and symptoms of different
clinical types of tularemia are included in Table 2.
6 Host immune response
Immunity against F. tularensis has been studied for
decades, but unanswered questions remain. Some kinds
of bacteria have evolved mechanisms for survival inside
host cells, and Francisella spp. is among these. These
pose complications for the immune system and its ability
effectively to respond. The immune system is classically
divided into innate and adaptive branches, and the innate
branch is the most evolutionarily conserved part of the
host defense. A substantial part of innate responses are
based on such phagocytic cells as macrophages and
neutrophils that represent the cellular component while
the complement system constitutes the most important
humoral component of the innate system. Just macrophages
are considered to be primary host cells for F. tularensis,
but several other cell types within the organism, such as
neutrophils, dendritic cells, hepatocytes, and alveolar
epithelial cells, also serve as host cells for Francisella
spp. [172,173]. Recently, B cells also have been shown to
be infected by F. tularensis [174]. The complement system
is the second major key of the innate immune response,
and it plays an essential role in defense against foreign
pathogens. Generally, the complement system’s crucial
activity is in inducing immune responses via the optimal
contact of target antigens with macrophages, dendritic
cells, and both T and B cells. However, the process of
Tularemia 203
eliminating Francisella spp. from the body – similar to that
for other intracellular pathogens – is controlled primarily
by adaptive immunity and depends on the function of T
cell subsets which finalize the expression of protective
immunity [175,176]. Regarding intracellular bacteria,
T-cell-mediated immune responses are paramount for the
control of both primary and secondary infections. The
crucial role of T cells in both the control and eradication
of F. tularensis has been predominantly demonstrated in
experiments carried out on experimental animals [145].
In humans, an immunospecific T cell response can be
demonstrated after the first 2 weeks from disease onset
[21]. Notwithstanding this fact, during the initial stages
of tularemia the immune response is almost entirely
independent of T cells. Meanwhile, the final resolution
and clearance of bacteria from the cells and tissues is
completely dependent on αβ T cells which need to be
activated [175,176]. Furthermore, in contrast to the well-
known role of CD4+ and CD8+ T cell subpopulations in the
immune response against F. tularensis, the role of other T
cell subpopulations is not well understood. For example,
it seems likely that CD4−CD8− double negative T cells play
Table 2: Common clinical signs and manifestation of various type of tularemia
Characteristics Means of spread Portal of entry
Ulceroglandular type Skin papule followed by persistent ulcer
Enlargement of regional lymph node
Chronic granulomatous inflammation
Fever
Vector-borne transmission
Direct contact
Indirect contact (tools)
Skin
Glandular
type
Tender lymphadenopathy
(usually axillary/epitrochlear)
Absence of visible skin lesions
Fever
Vector-borne transmission
Direct contact
Indirect contact (tools)
Unknown
(probably skin)
Oropharyngeal
type
Severe pharyngitis
Tonsillitis
Regional neck lymphadentis
Cervical adenitis
Persistent fever
Ingest of contamined food/water Oropharyngeal mucosa
Oculoglandular
type
Unilateral conjunctivis
Swelling of eyelids
Photophobia
Mucopurulent discharge
Enlargement of regional lymph node
Parinaud´s syndrome
Touch eye with contamined fingers
Infective dust
Conjunctiva
Typhoidal
type
Myalgia
Headache
Fever of unknown origin
Skin or mucous membrane lesions
Lymph node enlargement
Unknown
(probably oral/ respiratory)
Oropharyngeal mucosa
Respiratory tract
Pneumonic
/respiratory
Type A
Sudden onset of symptoms
Pneumonia
Bronchopneumonia
Chest pain
Dry or productive cough
Dyspnoea
Fever
Profuse sweating
Mental deterioration
Septicemia
Erythema nodosum
Inhaling contaminated dust
Laboratory-acquired infection
Respiratory tract
Pneumonic
/respiratory
Type B
Hilar adenopathy
Pneumonic infiltration
Erythema nodosum
Pneumonia (rarely)
Inhaling contaminated dust
Laboratory-acquired infection
Respiratory tract
204 Kubelkova K., Macela A.
a substantial role during pulmonary F. tularensis infection
by producing IL-17A and IFN-γ cytokines that additively
contribute to the control of the infection [177,178].
Considerable attention has also been devoted to
understanding effector mechanisms provided by the γδ T
cells. Although these cells appear to play a minimal role
during primary infection of mice, they seem to have a
larger role in the case of F. tularensis infection in humans,
where their numbers remain elevated for as long as one
year after infection [179,180]. Their contribution to the
protective response is still rather unclear.
In recent decades, research on anti-infection
immunity has been focused on understanding the
effects of important cytokines and chemokines during
tularemia infection. Studies on murine models have
clearly demonstrated that, as early as the initial stage of
infection, cells of the innate immune system can produce
IFN-γ that activates mononuclear phagocytes and thus
controls the retardation of bacterial replication. It is
known that CD 4+ T helper (Th) 1 cells produce interleukin
2 and IFN-γ and mediate macrophage activation. Th2
cells, on the other hand, are able to produce interleukin
4 and interleukin 5 and provide B cell help. Macrophages
secrete TNF-α and IL-12 that stimulate natural killer (NK)
cells to produce IFN-γ as well [181,182]. Thus one major
mechanism of cytokine-mediated early host response is
operated through the activation of immunocompetent
cells, namely NK cells and T cells. Both activated cell
types produce IFN-γ and TNF-α that subsequently activate
mononuclear phagocytes to escalate their bactericidal
effect and eliminate bacteria from cells and tissues.
Other studies have also described the major role of
IL-12 and IL-23. Both IL-12 and IL-23 have been found able
to positively regulate IFN-γ production despite the fact
that IL-23 still has an unidentified role in the clearance
of bacteria during intradermal sublethal F. tularensis
LVS infection [183,184, 265]. Moreover, IL-23 is also an
important contributor to promotion of Th17 response that
is critical for host immunity to type A F. tularensis infection
during primary immune response but not required during
secondary immune response [185,186].
In contrast, Th2 cell cytokines production has not
been studied in similar detail as have Th1 cytokines.
This is despite the fact that data have been accumulated
confirming the role of B cells that, in classic model need
the Th2 assistance. Moreover, in looking for the role of
Th-2 cytokines one comes upon conflicting data from
experiments utilizing the vaccine and the virulent F.
tularensis strains. Recent studies have demonstrated, for
example, that IL-6, one of the Th-2 cytokines, is essential
for primary resistance to F. tularensis LVS [187], but it fails
to exert any effect on the progression of virulent strain
infections [188].
Protective immunity against F.
tularensis infection is usually attributed to
an effective T cell response. Indeed, there is evidence
that B cells are necessary for mice to develop fully
protective immunity to primary and secondary LVS
infections [189,190]. Nevertheless, the role of antibodies in
protecting against intracellular pathogens remains poorly
understood. In general, antibodies can offer only a minimal
protective advantage during intracellular infection due to
the fact that pathogens can be sheltered from antibodies
inside the cells. F. tularensis has a significant extracellular
phase in the host, however, and that makes it vulnerable
to humoral immune responses [191].
While many laboratories have demonstrated that
serum antibodies are mainly directed against F. tularensis
lipopolysaccharide, serum antibodies with reactivity to
bacterial proteins have also been detected. Among these
are antibodies oriented against some outer membrane
proteins such as FopA, OmpA [192,193], and Tul4 [194];
against other intracellular proteins such as GroEL, KatG
[192], and DnaK; and against several putative virulence
markers such as nucleoside diphosphate kinase, isocitrate
dehydrogenase, the RNA-binding protein Hfq, and the
molecular chaperone ClpB [194,195]. Thus, antibodies
can potentially contribute to the protective response by
eliminating Francisella virulence factors and, together
with the antibody-independent functions of B cells, can
demonstrate the potential of B cells to collaborate with
T cells in the induction, regulation, and expression of
protective immunity against F. tularensis infection.
The construction of B-cell-deficient mice renders
studying the role of B cells in protective immune
mechanisms against F. tularensis more feasible [190,196].
They have no B cells and no detectable antibody levels,
but a fully functional T cell compartment. Nevertheless,
today’s knowledge of B cell functions still does not allow
us to draw clear and unambiguous conclusions. It is
generally accepted, however, that only the concerted
action of both cell-mediated immunity and humoral
immunity can ensure effective protection against this
intracellular bacterial pathogen.
7 Diagnosis, detection, and labora-
tory confirmation
Diagnosis and detection of tularemia is still most
challenging point of “tularemiology”. Due to its various
acquisition routes and entrance sites, tularemia presents
Tularemia 205
different types of clinical pictures. The ulceroglandular,
oropharyngeal, glandular, pneumonic, typhoid, and
ocular forms all have characteristic symptoms that make
the diagnosis of tularemia rather difficult. Moreover,
respiratory-acquired tularemia does not have specific
signs or symptoms. In endemic areas, therefore, where
farming and hunting are risk factors for acquiring
tularemia, a patient who has been exposed to wild small
rodents, rabbits, hares, or ticks and biting flies should be
suspected of being infected by F. tularensis. Serological
confirmation of the disease is possible only until the
second week after infection, which presents another
complication in diagnosing the disease. The pneumonic
form and secondary pneumonia are associated with
abnormal chest radiographic findings. Oval opacities,
hilar adenopathy, and pleural effusions are more likely
associated with tularemia. If this is the case, then
epidemiological circumstances should be considered
because pneumonia can be the consequence of a variety
of zoonotic and environmental agents [15].
Laboratory confirmation of the tularemia diagnosis
relies mainly on serology or on any of the polymerase
chain reaction (PCR) techniques. However, cultivation
of F. tularensis requires specifically enriched media.
Recently, solid as well as liquid media have been
made commercially available (see Supplementary).
Nevertheless, some modified agglutination tests or
enzyme-linked immunosorbent assay (ELISA) show
sufficient sensitivity and specificity and still dominate
in common immunological labs [15]. Cross-reactions are
possible only with serum obtained from patients with
brucellosis or yersiniosis, but sera with a titer lower than
1:320 do not agglutinate Brucellae at all [129,197]. The
agglutination test can be followed by the Rose Bengal
plate test, which is often used as a rapid screening test
in the diagnosis of brucellosis [198], or, according to our
experience, with immunoproteomic techniques to exclude
cross-reactions [194].
Identification of tularemia antigens in clinical
or environmental specimens using ELISA or RNA
hybridization is also possible [199,200]. Recently, after
sequencing of the genomes of individual F. tularensis
subspecies, mass spectrometric identification of F.
tularensis and its typing into subspecies and even into
individual strains has become possible [201–204]. Another
possibility for precisely identifying F. tularensis isolates
consists in automated genotyping assay based on the
analysis of variable number tandem repeat (VNTR) or
multiple loci VNTR analysis (MLVA) markers [205,206].
Thus, advanced laboratory techniques have recently
come into existence that are highly sensitive and selective
while having the capacity to characterize individual
isolates of F. tularensis and predict their geographical
relationships. Some of these, however, are costly
and require specific samples preparation and skilled
laboratory personnel.
8 Prophylaxis
Research on a tularemia vaccine was initiated soon after
identification of the bacterial agent. The first attempts
to prepare a vaccine, carried out in the 1930s, were
directed toward killing F. tularensis with nitric acid. This
corpuscular vaccine was then completed by adding 0.5%
of phenol (the so-called Foshay vaccine) [207,208]. The
efficacy of this type of vaccine was subsequently tested on
humans. The Foshay vaccine sufficiently protected against
small doses of intradermal challenge with the virulent F.
tularensis Schu S4 strain, but it failed to protect against
infection by inhalation [209,210]. Similarly, a vaccine
based upon ether extract from bacteria demonstrated
marginal protection of experimental animals against
infection by virulent strains [211–213]. The protective effect
of this chemo-vaccine was tested on human volunteers,
but also with limited success [214].
It is generally accepted that cell-free extracts or lysates
of F. tularensis prepared under specific conditions contain
immunogenic substances. To look for these, attempts were
made to utilize genetically modified microorganisms that
combined the “live bases” of the modified microorganism
and an immunogenic component coded by the introduced
gene. For example, experiments with oral administration
of the Salmonella enterica serovar Typhimurium strain
expressing the F. tularensis 17 kDa membrane protein
(Tul4) showed that the 17-kDa protein mediated a limited
protective response against F. tularensis that was not as
high as the LVS-mediated protection in the mouse model
[215]. This 17-kDa protein was also incorporated into the
immunostimulatory complexes (ISCOMs), but again the
induced immunity was lower than that induced by LVS
[216].
In spite of limited success with simple subunit
vaccines, the sequencing of the F. tularensis strain
genomes will facilitate the identification of protective
antigens through bioinformatics. Moreover, it is likely
that a subunit vaccine will be composed of a number of
immunogens to provide protection against virulent strains.
New adjuvants promoting general immune response will
be needed for constructing an effective subunit vaccine.
A recent report demonstrates that a mucosal subunit
vaccine composed of the F. tularensis heat shock protein
206 Kubelkova K., Macela A.
DnaK, Tul4, and quillaja saponin derivate GPI as an
adjuvant had substantial protective effect against lethal F.
tularensis LVS infection in the murine model [217]. To date,
however, and despite intensive research on virulence
factors, immunogenic proteins, and other membrane-
associated components, decisive immunogens suitable for
constructing a subunit vaccine have not been identified.
The first attempts at the construction of a vaccine
based on attenuated strains were made in the 1930s by
Francis, Kudo, and Gotschlich, but without satisfactory
results [129,218]. In 1942, however, Elbert and Gaiskii
successfully attenuated the “Moscow” strain, obtained on
the basis of a natural isolate of F. tularensis at the Irkutsk
Anti-Plague Institute in the USSR [219,220]. Later, Elbert
and Gaiskii prepared other attenuated strains, among
which especially strain 155 and strain 15 were extensively
tested and recommended for the preparation of a
commercially safe and effective vaccine. Such a vaccine
was introduced in 1946 for mass vaccination within the
USSR [221–224].
In 1956, a vial of the Soviet commercial live vaccine
was transferred from the Gamaleia Institute in Moscow
to the US Army Medical Research Institute of Infectious
Diseases, at Fort Detrick, Maryland. The isolation of one
selected F. tularensis colony from this ampoule gave rise to
the F. tularensis Live Vaccine Strain (LVS). After protective
efficacy testing on animal models, LVS was tested for
safety and efficacy in humans and used for vaccination
of at-risk personnel [209,210,225]. Retrospective analysis
of laboratory-acquired F. tularensis infections among
civilian employees at Fort Detrick vaccinated with the
Foshay vaccine (data analyzed from the years 1950 to
1959) and LVS (from 1960 to 1969), respectively, revealed
that vaccination with the LVS was more effective than
vaccination with the chemo-vaccine. Nevertheless, LVS
could not eliminate ulceroglandular tularemia, even
though the signs and symptoms become less pronounced
[226]. Despite the undisputed protective effect of the live
vaccine strain, however, the vaccine was not licensed for
human use due to difficulties with its standardization.
To eliminate this problem researchers prepared targeted
mutants of the LVS, the holarctica strain FSC 200, and the
Schu S4 strain that are highly attenuated and are protective
against challenge with virulent strains of F. tularensis
[227–229]. Some of these may constitute a promising basis
for the construction of a new live vaccine in the future.
To complete the information on the prophylaxis of
tularemia, we should also mention attempts at passive
transfer of immunity by immune sera or antibodies
against F. tularensis. Classical experiments on animals
have already demonstrated that injection of immune
serum before virulent challenge only prolongs survival
but cannot ensure protection against even low doses of
virulent bacteria [230,231]. These results nevertheless
clearly demonstrated that immune sera contain protective
antibodies and their protective effect correlated well with
precipitating antibody content [232]. More recently, studies
on the passive transfer of immunity against tularemia in
prophylactic mode utilizing both (hyper)immune sera as
well as monoclonal antibodies have shown that specific
antibodies limited manifestation of the disease, thereby
facilitating a sterilizing T cell response to resolve the
infection [233–238]. Moreover, antibodies can also be used
in therapeutic mode, especially when given early after
infection [193,239]. In combination with the information
that immune sera can protect irradiated mice against
otherwise lethal LVS infection [240], immune sera or
monoclonal antibodies represent one of the promising
tools for immediate prophylaxis of tularemia during
threats of modern warfare [241,242]. In our view, however,
the use of antibodies in medical practice for protection
or therapy against tularemia will require much more
information than we have at the present time.
9 Treatment
Since the description of Francisella spp. as an emerging
pathogen in 2001, many molecular tools for diagnosis
have been developed to rapidly confirm tularemia-positive
patients and type the strain in order to recommend
therapeutic treatment and predict patient prognosis.
Tularemia Type A, as well as the less virulent Type B, is
often associated with various complications that involve
substantial periods of convalescence. Due to the long
incubation period of tularemia it is imperative to treat
the patient immediately after the onset of the symptoms.
However, still, only antibiotics we have at disposal and
from this reason we need new therapeutic strategies for
tularemia including the development of new antibiotics
or new ways of using existing (summarized in [243]). It
is worth recollecting, too, that while the discovery of
antibiotics had once led people to the idea that infectious
diseases would soon be eradicated, it very quickly became
clear that this notion had been only a poor assumption.
Intracellular parasitism further complicated the situation;
only xenobiotics that could cross cell membranes could
effectively exert their effect on bacteria inside the cellular
compartments.
Many studies characterizing antimicrobial
compounds effective against Francisella spp. have already
been published. The key targets of current antibacterial
Tularemia 207
treatment are the inhibition of DNA replication and
translation and inhibition of cell wall synthesis. The
aforementioned biochemical pathways are generally
essential for all bacteria to survive and cause infection.
Streptomycin became established early on as the drug
of choice against tularemia infection, and especially
tularemia meningitis [244]. But parental administration
of streptomycin is not generally preferred these days. The
aminoglycoside family of antibiotics is currently used
and is effective against most cases of tularemia, whereas
beta-lactams such as penicillin are ineffective. Prompt
treatment with streptomycin, gentamicin, doxycycline, or
ciprofloxacin is recommended because the first signs of
tularemia (due to the incubation time) typically occur 3 to 5
days after exposure. Streptomycin and gentamicin are the
preferred antibiotics for the treatment of tularemia due to
their bactericidal activity, which clears the host of bacteria
and thus significantly reduces the relapse rate [149,245].
In comparison with the murine model of tularemia, where
streptomycin has shown a better therapeutic effect,
gentamicin has become a useful alternative for parenteral
treatment of humans. Gentamycin is the antibiotic of
choice for reasons of its better tolerability. In spite of the
fact, that gentamicin has limited cell penetration, it is
still capable of successfully treating tularemia [245–247].
Recently, new antibiotics, including tigecycline, ketolides,
and fluoroquinolones have been evaluated for treatment
of tularemia [248]. The explanation may lie in pinocytosis
that allows gentamycin to reach the cell interior or in
the effect on F. tularensis during its extracellular phase.
Oxytetracyclines or chlortetracyclines were used for
treating tularemia in the 1950s [249,250]. Because a higher
rate of recurrence has been shown during the application
of tetracyclines, however, tetracyclines are not considered
first-line therapeutics in this case [149]. Although
considered more risky, tetracyclines can be alternatively
used, in unavoinable case, for the oral treatment of
tularemia. The risk of relapse can be minimized by using a
prolonged treatment period [245,251].
At least in part, however, the choice of antibiotic
type for tularemia treatment is dependent on the clinical
manifestation of the illness, the subspecies of the
infectious agent, and the immune status of the host. For
example, chloramphenicol, which is a bacteriostatic,
penetrates into the cerebrospinal fluid relatively easily
and may be used for treating tularemia meningitis [252].
Prophylactic use of doxycycline may be useful in the early
post-exposure period. In addition to aminoglycosides,
fluoroquinolone ciprofloxacin has been shown to have
bactericidal activity against Francisella spp. both in vitro
and in animals, and it is considered to be a frontline
therapeutic that can offer new options for the treatment of
tularemia, especially for children. Moreover, quinolones
can be seamlessly used for treating both Type A and Type
B tularemia [253].
Newer and newer antibiotics are being tested for
the treatment of tularemia. A “consensus” summary of
preferred and alternative choices of antibiotic treatments
for tularemia is presented at the Centers for Disease Control
and Prevention website². Streptomycin and gentamycin are
the preferred choices for all categories of patients (adults,
children, and pregnant woman) while doxycycline,
chloramphenicol, and ciprofloxacin are alternative
choices for adults and children. Chloramphenicol is not
recommended for pregnant women. Practically the same
recommendations for tularemia treatment can be found
in the WHO Guidelines on Tularemia published in 2007³.
In addition to testing new antibiotics, attempts are
being made to use chemotherapy and immunotherapy
against tularemia. The specific enzymes that F. tularensis
needs for its survival and proliferation are the targets
of enzyme inhibitors in the form of proteins or small
molecules. These include, for example, recombinant
cystatin 9 [254] and small compounds prepared by ligand-
and structure-based drug design [255]. Currently, promising
moderate therapy against tularemia comprises the
immunosuppressive and/or immunomodulatory effects of
antimicrobial peptides, such as human cathelicidin LL-37
peptide, novel synthetic hybrids designed from cecropin
A, magainin II, granulysin peptides, or specific fly
antimicrobial peptides as are attacin, cecropin, drosocin
and drosomycin from Drosophila melanogaster [256–
259]. These positively charged antimicrobial peptides
are capable to disrupt the negatively charged bacterial
membrane and limit the proliferation of microbes. Such
antimicrobial peptides and enzyme inhibitors are effective
in cell-based in vitro and in animal in vivo systems,
display significant growth inhibition of F. tularensis or
reduce organ bacterial burden, and improve survival of
experimental mice. In the future, alternatives to antibiotic
therapy may be offered in cases of natural or intentionally
generated F. tularensis (multi)resistance.
2The conclusions of the paper by Dennis are summarized on the
Centers for Disease Control and Prevention website at: http://www.
bt.cdc.gov/agent/tularemia/tularemia-biological-weapon-abstract.
asp#4
3 See the website: http://apps.who.int/iris/handle/10665/43793
208 Kubelkova K., Macela A.
10 Decontamination/disinfection
The etiological agent of tularemia, F. tularensis subsp.
tularensis, is classified as a biological agent that poses
a military and terrorist threat due to its ability to be
weaponized. The CDC’s list of bioterrorism agents
includes this bacterium in Category A, meaning that it is
a bacterium that can “result in high mortality rates and
[has] the potential for major public health impacts, might
cause public panic and social disruption, and require[s]
special action for public health preparedness” . In the
case of a biological contamination incident, therefore,
it is imperative to limit the spreading of the agent and
to have an effective decontamination and disinfection
strategy for environmental surfaces, hospitals, and
households. Francisella spp., as non-spore forming
bacteria, exist only in vegetative form and therefore
their ability to survive outside the host is considerably
less than is that of spore-forming bacteria. Thus, the
majority of general decontamination tools can be used
for the decontamination and cleaning of Francisella-
contaminated surfaces. Generally, it can be said that
chlorine dioxide in all chemical states is the disinfecting
agent of first choice. Among other disinfectants, chlorine
dioxide was tested using a spray-based application
method on several environmental surfaces (aluminum,
carpet, concrete, glass, and wood coupons) [260], its
gaseous form was tested for the decontamination of
hospital rooms [261], and a chlorine dioxide solution
in potable water was tested for the efficacy to inactivate
bacterial threat agents [262].
Of course, there are other commercially available
preparations; their spectra and names, however, keep
changing over time. According to our experience, a very
simple preparation can be prepared as a 0.5% water
solution of peracetic acid. This has been utilized in our
labs for decades, and it can be recommended as an
alternative for decontamination in laboratory practice. One
cannot be certain that the decontamination procedures
recommended in the literature and in homeland security
leaflets will have 100% efficacy. Experimental results
have documented that decontamination efficacy is
dependent upon the ambient temperature and porosity
of the surfaces to be decontaminated. Woody surfaces
and carpets are particularly difficult to decontaminate
[260]. Moreover, the number of bacteria in the area to be
decontaminated, the solution from which the agent was
dispersed (proteinaceous coating can stabilize bacteria
4 See the website: http://www.bt.cdc.gov/agent/agentlist-category.
asp#a
against the effect of external influences including chemical
treatment), and the possible unexpected expansion of the
agent can complicate decontamination. Thus, in all cases
it is necessary to have a sensitive and selective checking
system to identify residual bacterial contamination.
11 Conclusion
The etiological agent of tularemia, F. tularensis, is an
enigmatic bacterium. During informal discussions at
conferences, it is sometimes referred to as a microbe
from another world. In some publications, it has been
characterized as a stealth pathogen [162,263]. It is a
close neighbor to the endosymbionts, and its behavior
inside the host cell resembles a search for a suitable
niche for long-term persistence. It is frequently used as
a model for the study of intracellular parasitism. Various
immune mechanisms have been shown to be important
for protection against infection, but the identification
and sequence of cellular and molecular factors (entities)
involved in creating protective immunity are still mostly
unknown. The same can be said about the determinants
of F. tularensis virulence. To date, we still lack an effective
vaccine for prophylaxis of human tularemia as well as
no satisfactory tools for therapy. In spite of its more than
100-year history of study, tularemia remains a continuing
scientific challenge for the future.
Acknowledgement: This work was supported by Long-
term Organization Development Plan 1011 from the
Ministry of Defense, Czech Republic. Prof. Macela was
also supported by Grant No. P302-11-1631 from the Czech
Science Foundation. We are grateful to English Editorial
Services for critical reading of the manuscript. We
attempted to mention all of the relevant studies and have
undoubtedly omitted some. We apologize in advance to
those authors whose work we did not cite.
Conflict of interest: Authors declare nothing to disclose.
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