FRANCISELLA TULARENSIS - 100 YEARS: TULAREMIA RESEARCH IN FORMER CZECHOSLOVAKIA AND IN THE CZECH REPUBLIC

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Mil. Med. Sci. Lett. (Voj. Zdrav. Listy) 2012, vol. 81(2), p. 46-55
ISSN 0372-7025
DOI: 10.31482/mmsl.2012.006
REVIEW ARTICLE
FRANCISELLA TULARENSIS - 100 YEARS: TULAREMIA
RESEARCH IN FORMER CZECHOSLOVAKIA AND IN THE
CZECH REPUBLIC
Ales Macela1, Jiri Stulik1, Zuzana Krocova1, Michal Kroca2and Klara Kubelkova3
1Institute of Molecular Pathology, Faculty of Military Health Sciences, University of Defence, Hradec Kralove,
Czech Republic
2Center of Biological Defence, Central Military Institute of Health, Techonin, Czech Republic
3Center of Advanced Studies, Faculty of Military Health Sciences, University of Defence, Hradec Kralove, Czech
Republic
Received 3th February 2012.
Revised 15th May 2012.
Published 8th June 2012.
Summary
The history of national tularemia research started in 1936 when the first outbreak was recognized in
south-east Moravia. Since then in average about one hundred cases have been recorded annually. As
tularemia was endemic in former Czechoslovakia, three research groups which concentrated on this disease
were formed during decades. The first two groups have worked from sixties and were associated with Jiri
Libich (Prague) and Darina Gurycova (Bratislava). The third group which concentrated on the research of
natural foci started during late seventies in Valtice (Zdenek Hubalek). The experimental research was, and
still is, mainly associated with military research, recently with the Proteomic Center (Faculty of Military
Health Sciences, University of Defence) in Hradec Kralove. This center opens molecular approaches to the
analysis of Francisella tularensis microbes on one side and the studies on mutual host-pathogen interaction
on the other side. One of the significant aims of the research is searching for the new typing and diagnostic
markers of Francisella tularensis for the military and medical practice. Thus, scientists from former
Czechoslovakia and the Czech Republic contributed significantly to current knowledge on Francisella
pathogenesis and their results were highly appraised by international scientific community.
The authors would like to dedicate this review to Jiri Libich, M.D., a leading researcher on
tularemia in former Czechoslovakia.
Key words: Francisella tularensis; tularemia; former Czechoslovakia; Czech Republic
University of Defence, Faculty of Military
Health Sciences, Center of Advanced Studies,
Třebešská 1575, 500 01 Hradec Králové,
Czech Republic
kubelkova@pmfhk.cz
+420 973255203
+420 495513018
ABBREVIATIONS
Δ, deletion;
AAA+, ATPases Associated with diverse cellular Ac-
tivities;
ATP, adenosine-5´-triphosphate;
dsbA, disulfide bonded protein A;

clpB, caseinolytic peptidase B;
F. tularensis, Francisella tularensis;
F. tularensis strain 15, Francisella tularensis subsp.
holarctica strain 15;
hsp, heat shock protein;
igl, intracellular growth locus;
LVS, live vaccine strain;
MAPK, Mitogen-Activated Protein kinase;
MHC, major histocompatibility complex;
NK, natural killer;
subsp., subspecies
INTRODUCTION
Background on Francisella tularensis
Francisella tularensis (F. tularensis) - a highly
virulent, nonsporulating, pleomorphic, facultative in-
tracellular, Gram-negative coccobacillus is capable
of causing a zoonotic disease called tularemia in a
large number of mammals. The first description of
this disease was probably made on September 19,
1907 by Ancil Martin, an ophthalmologist of
Phoenix, the territory of Arizona, in his letter ad-
dressed to F. G. Novy, a professor of bacteriology at
the University of Michigan. Martin stated that under
observation and treatment he had five cases of an in-
fection caused by the skinning and dressing of wild
jack rabbits (reviewed by [1]). The first published de-
scription of the disease may be found in the article
by R. A. Pearse who reported six atypical cases of
fever caused by a deer-fly bite. He called this disease
a "deer-fly fever" [2]. However, it is generally ac-
cepted that the history of tularemia started in 1911
when this illness was discovered in ground squirrels
in Tulare County, California by G. W. McCoy from
the US Plague Laboratory in San Francisco [3]. Sub-
sequently, the bacillus, identified as the causative
agent of tularemia, was isolated and named Bac-
terium tularense [4]. The first human case of tu-
laremia, which was confirmed bacteriologically, was
reported by Wherry and Lamb when they isolated the
bacteria from a conjunctival ulcer [5]. The man who
contributed most to our knowledge of tularemia as a
separate clinical entity was Edward Francis. Francis
studied the "deer-fly fever" in Utah and recognized
its identical traits with the illness from Tulare County
and named the infection tularemia [6]. At the same
time, the attention was attracted to the acute febrile
disease transmitted to man by wild rabbits of the
Abukuma Mountains in the eastern part of
Fukushima prefecture in Japan. The illness was
known as “yato-byo” (the hare illness) or, according
to the discoverer, as Ohara´s illness [7]. Edward
Francis and Dunlop Moore finally concluded that
Ohara's disease and tularemia were identical on the
basis of the exchanged clinical sample analysis in
1926 [8]. F. tularensis was recognized as the
causative agent of “water rat-trappers’ disease”, an
illness acquired by trappers who skinned water-rats
for their pelts [9, 10]. Soon after that, tularemia was
also reported in Norway (1929), Canada (1930),
Sweden (1931) and Austria (1935) [11]. Several other
cases of tularemia were then reported from more than
15 countries in North America, Asia, and Europe dur-
ing the third decade of the twentieth century.
Taxonomy of Francisella tularensis
Tularemia is a zoonotic disease appearing in the
entire north hemisphere. F. tularensis, as the etiolog-
ical agent of, tularemia comprised of four subspecies:
tularensis (Type A), holarctica (Type B), mediasiat-
ica, and novicida. Genotyping methods have demon-
strated that both subspecies, Type A and Type B, can
be further divided into subpopulations. The sub-
species differ in their pathogenicity and in their geo-
graphic distribution. F. tularensis subsp. tularensis
Type A (in North America), is further divided into dis-
tinct Type A1 (east) and Type A2 (west), resp., differs
according to clinical severity [12, 13], F. tularensis
subsp. holarctica Type B (in North America, Europe
and Asia), and F. tularensis subsp. mediasiatica (dom-
inating in Central Asia). The taxonomy of F. novicida
is still a matter of a debate. An attempt of Huber et al.
[14] to classify F. novicida as the fourth subspecies of
F. tularensis was refused by others [15].
Tularemia outbreaks in former Czechoslovakia
and in the Czech Republic
The first outbreak of tularemia in former Czecho-
slovakia was recognized in the area of south-east
Moravia in 1936-1937. More than 400 cases were di-
agnosed during this outbreak. All affected people
were in contact with hares [16]. After the Second
World War, the outbreaks were mainly associated
with the campaign in sugar factories. From that time,
it was recognized that tularemia is endemic in the ter-
ritory of Moravia as well as Bohemia. The number
of reported cases is about 100 per year with some ex-
ceptions (Figure 1). The majority of cases usually ap-
pears during a hunting season. The outbreak of
water-born tularemia was also registered in the area
near the town of Plzen during the onset of the new
millennium [17]
Kubelkova et al.: Tularemia research
47

48
Figure 1. The amount of reported cases of tularemia in Czech Republic during the last 16 years.
The existence of tularemia outbreaks became the
basis of the intensive research of this disease in the
Czech Republic. Altogether three independent
research groups started to study this relatively new
illness in former Czechoslovakia. One group came
from the Medical Faculty of Comenius University in
Bratislava. This group is associated with the name of
Darina Guricova. She published the first isolation of
F. tularensis subsp. tularensis Type A in Europe,
which was a unique report about the presence of Type
A in Europe [18]. Second group was established at
the branch of the Institute of Landscape Ecology of
the Academy of Sciences in the town Valtice. The
studies of this group were concentrated on the
analyses of vectors in active enzootic foci (floodplain
meadow and forest ecosystem) located mainly in
South Moravia [19-21]. The third group started their
research at the Military Institute of Hygiene,
Epidemiology and Microbiology in Prague during
the late fifties. This group was engaged solely in the
experimental research, which was motivated by the
political situation. The dominant person of this group
was Jiri Libich, M.D., the head of the Bacteriological
Division of the Military Institute of Hygiene,
Epidemiology and Microbiology in Techonin. A
transformed form of this research group has been
working successfully till now and can be currently
found at the Faculty of Military Health Sciences,
University of Defence, in Hradec Kralove.
Overview of the former Czechoslovak and Czech
tularemia research
Initially it was important to prepare the culture
media for the cultivation of Francisellae [22] and
to develop a treatment strategy for tularemia based
on existing antibiotics [23, 24]. Simultaneously, an
experimental model of inhalational tularemia was
constructed for the study of pathogenesis and
virulence of individual subspecies of F. tularensis
based on several animal species. The laboratory
experiments were enabled by the development of
the aerosol technology and exposure procedure
named Single Dose Exposure 400 (SINEX 400 –
for scheme see Figure 2). A great advantage of this
technique was the ability to accurately calculate an
inhalation and a deposition dose of microbes in
lungs of experimental animals. The calculation was
based on several aerosol parameters selected for the
experiment (e.g. concentration of dispersed
particles, stability of the aerosol, bacterial
population density in particles, parameters of the
aerosol chamber such as volume of the chamber,
air flow in the chamber, relative humidity,
temperature or the parameters of an experimental
animal used for the experiments). A mathematical
model of inhalational tularemia was developed
based on theoretical and experimental data, and
consequently was confirmed experimentally using
Kubelkova et al.: Tularemia research

49
the SINEX 400 technique [25-27]. It was
demonstrated that a number of granulomas is
strictly dependent on a number of deposited
microbes in lungs of infected animals, on a kinetics
of microbe dissemination dependent on the
infection dose after aerosol challenge, and on
generation time of both an attenuated F. tularensis
subsp. holarctica strain 15 (F. tularensis strain 15)
and a fully virulent F. tularensis subsp. holarctica
strain 130 in murine peritoneal macrophages.
After this initial period, tularemia research had
moved on to a study of a host immune response to
experimental F. tularensis infection. The conditions
for the best effectiveness of immunization against
virulent strains of F. tularensis were studied during
the eighties of the last century. The challenging of
immunized animals with the virulent strains of
subsp. holarctica Type B and tularensis Type A led
to the observation that a protective effect of
inhalational immunization is significantly better than
a subcutaneous one. Furthermore, it was also
demonstrated that the induction of protective
immunity is not an exclusive property of a live
vaccine. Some degree of protection was also
obtained when a proliferation of a live vaccine was
limited by an administration of antibiotics
(streptomycin, kanamycin) in immunized animals.
Similar protective effect was observed after an
immunization with attenuated F. tularensis strain 15
autolysate. It was also documented that an intensity
of immune reaction and a spectrum of activated
immune mechanisms were dependent on a genetic
background of mice, on a route of infection, and
partially on a dose of infection. A production of
specific antibodies, an activation of macrophages, a
blast transformation of lymphocytes, and a
production of several cytokines were used for
monitoring an immune response [28-30]. It was also
proved that an infection with F. tularensis leads to
an activation of “Natural” killer (NK) cells during
early stages of infection briefly after its discovery
[31]. The NK cell activation was accompanied by a
production of regulatory interferon gamma cytokine
[32]. Transfer experiments also confirmed an MHC
class II restriction of a protective immune response
induction [33]. A phenomenon of an early
protective response to virulent F. tularensis Type A
occurring between 24 and 48 h after immunization
was published by Karen Elkins and co-workers
later in 1997 [34].
Figure 2. The scheme of an aerosol technique constructed and named by Jiri Libich as Single Dose Exposure 400 (SINEX 400).
An aerosol chamber of 400 liter volume and a pneumatic nebulizer constructed for creation of monodisperse aerosol with a
mean diameter of particles 3.5 um were used for an inhalation model of infection. Three impingers were put into the aerosol
chamber during nebulization of 1ml bacterial suspension per minute. One impinger contains a simple cultivation medium for
aerosolized bacteria. The constant flow of air was drowned through the impinger for an indicated time (axis x). A number of
microbes in the aerosol was determined by plating the impinger liquid on a solid thioglycolate-glucose-blood-agar plate. An
amount of bacteria in the aerosol was expressed as C/Cmax (axis y) calculated from the impinger liquid.
Kubelkova et al.: Tularemia research

50
* Mice were s.c. infected with F. tularensis 15 infection in a dose of 1.05 x 102live microbes, 72h after irradiation.
Table 1. A survival of C3H/Cbi/Bom mice irradiated by 60Co in a dose of 4 Gy. The resistance to F. tularensis infection is totally
abrogated using gamma irradiation (60Co) higher than 3 Gy. A significant rapid depression in a specific T lymphocyte count is
observed after irradiation (3 – 4 Gy), and their nadir is reached 36 to 48 h after an irradiation event. The duration of this decrease
correlates with a radiation dose, while the recovery begins 10 to 15 days after a dose of 3 – 4 Gy.
* Mice were s.c. infected with F. tularensis 15 infection in a dose of 1.05 x 102live microbes, 72h after irradiation.
** Mice were infected at the indicated day after irradiation using gamma irradiation (60Co).
Table 2. A survival of C3H/Cbi/Bom mice irradiated by 60Co in a dose of 4 Gy. A natural recovery of resistance to infection
started during a second week after irradiation. A significant rapid depression in a specific T lymphocyte count is observed after
irradiation (3 – 4 Gy), and their nadir is reached 36 to 48 h after an irradiation event. The duration of this decrease correlates
with a radiation dose, while the recovery begins 10 to 15 days after a dose of 3 – 4 Gy.
A separate set of experiments was performed
using 60Co gamma irradiation of mice as a model of
immunocompromised animals. It was demonstrated
that gamma irradiation of mice greater than 3 Gy
totally abrogated resistance to an infection induced
by F. tularensis strain 15 (Table 1). Minimum two
weeks were required to reach full natural recovery
from this deep decline of resistance (Table 2). It
means that a live vaccine immunization of
generally immunocompromised individuals is
practically impossible. An immunization with
killed microbes or microbial protein extracts lack
a sufficient protective effect. Thus, one of the
possibilities how to protect irradiated individuals
is a passive transfer of immunity. Both immune
cells and antibodies are effective in a passive
transfer of protective immunity to naive recipient
(Table 3). This knowledge can conclude that
specific antibodies provide some degree of
protection probably mediated by an antibody
dependent cell mediated bactericidal activity,
originally published by Lovell at al. 1979 [35, 36].
Later, the same conclusion was published by
Stephan Stenmark in 2003 [37].
Irradiation Infection* Survival % MTD (days)
-+45/49 91.8 8.5
1 Gy + 18/20 90.0 9.0
2 Gy + 8/20 40.0 9.8
3 Gy + 0/20 0.0 8.8
4 Gy + 0/20 0.0 7.8
5Gy + 0/20 0.0 6.4
5 Gy - 20/20 100.0 -
Infection* after irradiation** (days) Survival % MTD (days)
1 0/8 0.0 8.0
3 0/8 0.0 7.1
7 0/8 0.0 7.0
14 7/8 87.5 7.0
21 8/8 100.0 -
28 7/8 87.5 7.0
42 8/8 100.0 -
56 8/8 100.0 -
Un-irradiated 15/16 93.7 8.0
Un-infected 16/16 100.0 -
Kubelkova et al.: Tularemia research

51
At the turn of the millennium, brand-new
tularemia research stimuli were discovered on a
molecular level due to the development of
advanced proteomic analyses of both pathogen and
infected host cells. A proteomic technology based
on combining various gel electrophoresis
procedures, a Western blot technique, and mass
spectrometry identification approaches were used
for analyzing F. tularensis immunoreactive proteins
[38-42], for identifying unique typing markers of
Francisella subspecies [43-45], and for studying
host-pathogen interaction at a molecular level.
These studies were ranging from a subcellular
proteome of bacterial membranes [46-48] to
bacterial secreted proteins important for early
stages of a host-pathogen interaction [49]. Highly
sophisticated approaches of quantitative shotgun
proteomics were then applied to a protein profiling
study of bacteria exposed to stress conditions in
vitro. Such conditions were able to immitate
prevailing conditions inside host cells in vivo [51-
54] alongside with the identification of several
proteins whose expression was changed. Among
them there were proteins encoded by an igl operon,
a Hsp100 chaperone ClpB with its assumed
function in reactivation of aggregated proteins
under in vivo stress conditions, and an ORF
FTL_0200 encoding a protein of putative AAA+
ATPase of a MoxR subfamily. All these proteins
seem to be indispensable for the resistance to stress
conditions and are substantial factors controlling a
virulence and a pathogenicity of F. tularensis.
Further, the comparative shotgun proteome
analyses of F. tularensis subtypes revealed several
promising candidate proteins for constructing a new
type of attenuated live vaccines. Deletion mutants
were prepared for some of the identified proteins
and two of them, FSC200ΔdsbA and FSC200ΔiglH,
were successfully tested for their attenuation and
their immunogenicity. Regarding the DsbA deletion
mutant, a molecular mechanism of its attenuation
was studied comparing protein patterns of the
original wild and deletion mutant strains. Several
proteins accumulating in a membrane of the mutant
strain were found and some of them were later
identified as important factors of F. tularensis
* Mice were s.c. infected with F. tularensis 15 infection in a dose of 1.05 x 102live microbes, 72h after irradiation.
** Passive transfer of cells or sera was realized 2 h before infecting irradiated mice. A treatment of irradiated mice by a
commercial USSR preparation Tularin (antigenic material for skin tests used frequently in the past as a prototype of dead
vaccine) was realized 2 h after irradiation.
Table 3. A survival of C3H/Cbi/Bom mice irradiated by 60Co in a dose of 4 Gy, passively protected by transfer of cells, sera
against F. tularensis 15 or Tularin.
Irradiation Infection* Treatment** Survival % MTD
4 Gy --8/8 100.0 -
4 Gy + Tularin 0/8 0.0 8.2
4 Gy + Naïve spleen cells 2/22 9.1 7.9
4 Gy + Naïve thymus cells 1/10 10.0 7.8
4 Gy + Immune spleen cells 22/22 100.0 -
4 Gy + Immune spleen cells + NMS + C 10/10 100.0 -
4 Gy + Immune spleen cells + anti Thy1.2 Ab + C 10/10 100.0 -
4 Gy + Ultrasound destroyed immune spleen cells 2/10 20.0 8.9
4 Gy + Mouse serum naïve 0/10 0.0 8.6
4 Gy + Immune serum (3rd day - live vaccine) 3/7 42.8 8.7
4 Gy + Immune serum (7th day - live vaccine) 4/5 80.0 7.0
4 Gy + Immune serum (11thday - live vaccine) 10/10 100.0 -
4 Gy + Immune serum (21st day - live vaccine) 10/10 100.0 -
4 Gy + Immune serum (56th day - live vaccine) 7/7 100.0 -
4 Gy + Immune serum (21stday - heat inactivated vaccine) 7/7 100.0 -
-+ - 20/22 90.9 7.5
Kubelkova et al.: Tularemia research

virulence [55, 56]. Current proteomic experiments
are focused on structural characterization of F.
tularensis proteins, especially on identifying
bacterial membrane glycoproteins [50].
The above-mentioned proteome technologies
were also used in a study of a host response to an
ongoing infection. These studies clarified that a
mutual interaction is stressful for both organisms.
Various production of a highly stress-inducible hsp72
protein, a member of the hsp70 family, has been
demonstrated in macrophages of three different
inbred strains of mice exhibiting either a resistance
or a susceptibility to an F. tularensis LVS infection.
The hsp72 was observed to be preferentially
produced and accumulated in intracellular space of a
murine peritoneal adherent cell [57]. F. tularensis
LVS induces apoptosis in a macrophage after
infecting these cells. This process requires activation
of a p42/p44 MAPK pathway and is associated with
a reduced p38 MAPK activity, indicating that
infection-induced cell death can be caused by
perturbation of these two signalling pathways [58].
A mapping of Francisella intracellular trafficking
inside macrophages discovered a complicated fate of
intracellularly localized bacteria. Bacteria of
F. tularensis strain LVS disintegrate a phagosome
early after its entry to a host cell followed by an
escape to a cytosol where it intensively proliferates.
A significant part of bacterial population merges into
an autophagosome expressing MHC class II
molecules. Subsequently, formed autophagosome
can be a source of immunogenic signal for
CD4+ T cells [59].
Macrophages are considered to be primary host
cells for F. tularensis, but several other cell types in
the immune system also serve as host cells for
tularemia infection. F. tularensis is able to infect,
“disturb”, and to activate not only cells of a
mononuclear phagocytic system but also dendritic
cells [60], epithelial cells [61] and hepatocytes [62].
Moreover, Krocova and co-workers demonstrated
that F. tularensis also infects mouse (A20) or
human (Ramos RA-1) B cell line cells as well as
murine primary spleen B cells in series of
experiments [63]. Within an infection of B cells, it
has been observed that F. tularensis FSC200
activates several caspases such as caspase 8, 9 and
3. An activation of Bid, cytochrome c, apoptosis-
inducing mitochondria factor and proapoptic Bcl-2
family member has also been determined. This
causes depolarization of a mitochondrial membrane
potential in a Ramos cell line, thus leading these cells
to apoptosis. Unlike live bacteria, killed F. tularensis
FSC200 is capable of activating caspase 3 only and
does not cause apoptosis of Ramos cells. Killed
bacteria also cause accumulation of anti-apoptotic
protein BclxLin mitochondrial membranes.
Therefore, live F. tularensis activates both caspase-
dependent pathways (receptor-mediated and
intrinsic) and caspase-independent mitochondrial
death in B cells [64].
CONCLUSION
In conclusion, former Czechoslovak and
consequently Czech scientists contributed
significantly to current knowledge on tularemia
pathogenesis and their results are highly appraised
by international scientific community. Former
Czechoslovak and Czech scientists underwent a long
way from identifying the illness at the beginning to
recent molecular analyses of a bacterial virulence
during the last 50 years. Therefore, the authors
would like to dedicate this article to a successful,
long-time work of Jiri Libich (Figure 3), which
attracted many of us to become a part of tularemia
research community.
52
Figure 3. One of the very rare photos of Jiri Libich who
was the leading researcher on tularemia in former
Czechoslovakia during the sixties and the seventies during
the last century.
Kubelkova et al.: Tularemia research

ACKNOWLEDGEMENTS
This article was supported by the grant P302/11/1631
obtained from the Czech Science Foundation.
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