Chapter 108
Francisella
Anna-Lena Johansson1, Laila Noppa1, Emelie Näslund Salomonsson1 and Åke Forsberg2
1
FOI Swedish Defense Research Agency, Umeå, Sweden, 2Umeå University, Umeå, Sweden
INTRODUCTION
Francisella tularensis is a non-motile Gram-negative coc-
cobacillus and a facultative intracellular pathogen that
causes the zoonotic infection known as tularaemia.
F. tularensis is associated with a wider range of hosts
than most other zoonotic pathogens. The organism has
been found in more than 300 species, from large mammals
and vertebrates to invertebrates, arthropods and amoebas
[1]. In the environment F. tularensis has been isolated
from water, soil, and various wild animals, especially rab-
bits, hares and rodents. Outbreaks of disease in humans
often parallel outbreaks of tularaemia in wild animals that
also often correlates to peaks of rodent populations [2,3].
The most important mammalian species involved in
human infection differ between the continents. In North
America voles, mice, squirrels, muskrats and beavers are
the most important, while in Europe and Asia lagomorphs
(hares and rabbits) and voles are known hosts. Although
most frequently found to be infected, lagomorphs and
rodents are unlikely to be reservoirs for F. tularensis,
since infection often leads to acute disease in these ani-
mals. Infectious transfer to humans and other mammals
also occurs through aerosolization or direct contact with
infected material or carcasses. Another important route of
transfer between mammalian hosts involves blood-feeding
arthropods (insects) such as ticks, mosquitoes and biting
flies [4]. The most important vector species also differ
between the continents. In North America, ticks and bit-
ing flies are the most common vectors, in central Europe,
ticks are the main vector while in Northern Europe and in
Russia, the bacterium can also be transmitted by mosqui-
toes. The bacterium also persists for long periods in
watercourses, possibly in association with protozoa [5,6].
F. tularensis subspecies tularensis (also denoted
type A) is one of the most infectious bacteria known,
since inhalation of as few as 10 15 organisms is suffi-
cient to initiate infection in humans [7,8].
Molecular Medical Microbiology. DOI: http://dx.doi.org/10.1016/B978-0-12-397169-2.00108-6
© 2015 Elsevier Ltd. All rights reserved.
Due to its high infectivity, ease of aerosol dissemina-
tion, and ability to cause severe illness and death,
F. tularensis has been categorized as a potential bioter-
rorism agent. It is currently classified as a Tier 1 select
agent (formerly called category A select agent) which
specifies ‘a select agent with a documented risk of caus-
ing a high consequence event’ by the United States
Centers for Disease Control and Prevention (CDC) [9].
Indeed, subsp. tularensis was developed for use as a bio-
logical weapon by several countries including Japan, the
USA and the Soviet Union during World War II and the
Cold War [10,11]. The USA alone reported a 450 kg
stockpile of dry-powder F. tularensis when it scrapped
its bioweapons programme in 1973. The anthrax letters
in the aftermath of the 9/11 events in 2001 increased the
concern about biothreat agents in general and resulted in
new initiatives to promote preparedness and research in
the area of biodefence [12]. This increased funding has
had a major impact on tularaemia research over the past
decade [12].
TAXONOMY, EPIDEMIOLOGY AND
ECOLOGY
The genus Francisella comprises at least four recognized
species; F. tularensis, F. hispaniensis, F. philomiragia
and F. noatunensis (Table 108.1). In contrast to F. tular-
ensis, F. philomiragia is rarely associated with human
disease and has only been isolated from patients who
were nearly drowned [13 15]. F. noatunensis is closely
related to F. philomiragia and seems to be the only mem-
ber within the genus able to cause disease in fish
[16 20]. Challenge studies indicate that this pathogen
transmits the disease (francisellosis) directly between fish
[21 23]. Francisellosis in fish has during recent years
been diagnosed all over the world [18]. In the Atlantic
Ocean, both wild and farmed cod have been diagnosed
1991
1992 PART | 17 Animal and Ectoparasitic Source Infections

TABLE 108.1 Recognized Francisella Species, Virulence and Distribution

Species Subspecies Type Virulence Distribution/Region

F. tularensis tularensis A.I A Very high North America Midwest, California,
and Massachusetts

tularensis A.II A Moderate North America California,

Less virulent to humans than mountain states
Type B isolates

holarctica B High Throughout Northern hemisphere

mediasiatica High Central Asia

novicida Low, (immuno-suppressed Broad

individuals)

F. hispaniensis ?

F. philomiragia Low (immuno-suppressed Central Asia

individuals)

F. noatunensis noatunensis High, solely a fish pathogen Broad/Atlantic Ocean

orientalis High, solely a fish pathogen Broad

and the mortality rate appears to be high [20,21]. The risk
for humans to get infected from the fish and shellfish
pathogenic Francisella species is considered very low
[18]. In addition, F. hispaniensis was recently accepted as
a separate species that includes several strains previously
denoted as novicida-like isolates [24 26].
F. tularensis is subdivided into four subspecies; tular-
ensis (also denoted type A), holarctica (also denoted type
B), mediasiatica and novicida which are highly similar at
genomic level [27] but differ significantly in virulence
and geographical distribution.
Subspecies tularensis (endemic in North America) and
holarctica (predominantly found throughout the Northern
Hemisphere but recently also isolated in Australia [28])
are the most prevalent and are responsible for the majority
of human cases [29,30]. F. tularensis is the most virulent
among the subspecies with a mortality rate of up to 30%
if untreated, while F. holarctica causes a milder form of
the disease that is rarely fatal. F. mediasiatica is found
within a geographically limited niche, i.e., in central Asia,
and exhibits even lower virulence and only rarely causes
human infections [31]. Subsp. novicida has only been iso-
lated from immune-compromised patients. With the rap-
idly increasing number of genome sequences available
the knowledge on the diversity within the genus
Francisella has recently expanded substantially. This has
opened up a discussion on the boundaries between taxo-
nomic units within the genus. One example concerns
F. novicida that previously was considered as a separate
species but now has been ranked as a subspecies [32,33].
F. tularensis subsp. tularensis has been further divided
into two genetically distinct subpopulations (clades), the
highly diverse A.I group and the less diverse A.II group
[34]. These subgroups also differ in clinical severity since
A.I isolates are more virulent to humans than A.II isolates
which appear to be even less virulent to humans than
subsp. holarctica (type B) isolates [35,36]. The A.I and
A.II groups show distinct geographic distribution which
matches well with geographic ranges of known tularaemia
hosts and vectors, and abiotic components, suggesting
that the two groups occupy separate ecological niches
[34]. F. tularensis subsp. holarctica can also be further
divided into clades but these are genetically more homo-
genic. This is interesting since subsp. holarctica is distrib-
uted in a larger geographic area, throughout North
America and the whole of Eurasia with some reports also
from Australia [28]. In Northern Europe and in Russia,
subsp. holarctica is associated with blood-feeding mos-
quitoes, in association with watercourses, while in central
Europe it is more closely associated with ticks.
Tularaemia ecology is still only partially understood but
the current view describes two ecological niches, one
mainly terrestrial, i.e., tularensis, and one more connected
to aquatic environments, i.e., holarctica.
The increased availability of completely sequenced
Francisella genomes has revealed insights into evolution-
ary processes within the genus and several studies have
identified new variants of Francisella from soil and water
[26,37 39], as well as Francisella-like endosymbionts
with high similarity to F. tularensis (Fig. 108.1) [40,41].
 Chapter | 108 Francisella 1993

F. tularensis subsp. holarctica

F. tularensis subsp. mediasiatica

F. tularensis subsp. tularensis

Clade 1
F. tularensis subsp. novicida

F. hispaniensis

Wolbachia persica

F. noatunensis subsp. orientalis

F. philomiragia Clade 2

F. noatunensis subsp. noatunensis

Fangia hongkongensis

Piscirickettsia salmonis

5000

FIGURE 108.1 An overview of the whole-genome phylogeny of the genus Francisella and two genetic relatives, Fangia hongkongensis and
Piscirickettsia salmonis, based on single nucleotide polymorphism in the core genome. Figure is slightly modified from [40]. The Francisella genus is
divided into two parts, clade 1 (F. tularensis and F. novicida) and clade 2 (F. noatunensis and F. philomiragia) occupying both terrestrial and aquatic/
marine habitats, respectively. The phylogeny data show that pathogenic species in each clade (mammals and fish) have emerged independently by
similar evolutionary paths of host adaptation and that the ancestral Francisella species originated in a marine habitat [40].

Thus, members of the Francisella genus can be found in
and appear to be associated with a wide range of different
ecological niches, from specialized endosymbionts and
pathogens with different host ranges, to those that appear
to be free-living. The increased knowledge about impor-
tant sequence differences between environmental species
and F. tularensis also allows for the design of highly spe-
cific F. tularensis detection assays with improved ability
to discriminate between strains. This is particularly
important since current methods frequently falsely detect
F. tularensis in environmental samples [40,42]. The
genome information can also be used for diagnostic
purposes and epidemiological studies as well as tracing
specific strains when deliberate release is suspected in
bio-forensic analysis and studies.
TULARAEMIA
Tularaemia, also called rabbit fever, hare fever, lemming
fever, or deerfly fever, is a zoonotic infection disease
where humans could be infected through contact with
infected animals or via flies, ticks or mosquitoes. The dis-
ease onsets after an incubation period of 1 21 days
(usually between 3 5 days) [43]. The clinical symptoms
and severity depend on (i) the type of F. tularensis that
causes the infection, (ii) the infection dose, (iii) the route
of infection and (iv) the immune status of the recipient.
So far there are no confirmed reports of transfer of the
disease from human to human, even if a theoretical risk
exists due to the occurrence of F. tularensis in respiratory
fluids [44].
Clinically relevant species of Francisella that cause
human infections are F. tularensis subspecies tularensis
(type A), holarctica (type B), and mediasiatica, and in
addition immune-compromised individuals could be sus-
ceptible to F. novicida and F. philomiragia infections
[13,15,45]. If untreated, type A infections have mortality
rates of 30 60% [45], while type B infections are much
less severe and mortality following type B infections is
rare [10].
Symptoms
Diagnosis of tularaemia is relatively uncomplicated in
areas where the disease is known to occur and cases
can be expected. It is far more demanding when it
1994 PART | 17 Animal and Ectoparasitic Source Infections

TABLE 108.2 Different Types of Tularaemia

Type of Tularaemia Typical Symptoms

Ulceroglandular Skin ulcer(s) and swollen tender glands

Glandular Swollen glands without a skin ulcer

Oculoglandular Painful swollen and red eyes. Swollen cervical lymphnodes

Oropharyngeal and gastrointestinal Sore throat, stomach pain, vomiting, diarrhoea and occasionally bowel ulceration.
Swollen cervical lymphnodes

Pneumonic Dry cough and sharp chest pain

Typhoidal (septicaemic) Fevers, chills, muscle pain, and lack of energy. Absence of an ulcer or swollen glands

occurs in areas where tularaemia has rarely been

reported since the symptoms are shared with several
other diseases with flu-like symptoms including sudden
fever, headache and swollen lymph nodes and fatigue.
Respiratory symptoms, like cough and a sore throat,
can occur even if the infection was not acquired by
inhalation. The type of tularaemia depends on the route
of infection, where the most common are ulceroglandu-
lar, glandular and pneumonic, but oropharyngeal, ocu-
loglandular, gastrointestinal and typhoidal forms also
occur (Table 108.2) [43].
Ulceroglandular tularaemia that appears to be the most
common type is usually vector-transmitted, in Central
Europe and the USA predominantly by ticks [4,10,46]
and in Northern Europe mainly by mosquitos [10,47]. A
person with a skin wound could also acquire ulcerogland-
ular tularaemia as a result of handling infected animals
[10,48]. The clinical symptoms may vary but commonly
there is a local cutaneous papule or a granuloma at the
inoculation site, eventually followed by an abscess and an
eschar after a few days. Several days after the inoculation,
typical symptoms include fever and flu-like signs,
together with one or more lymph nodes in the proximity
of the papule becoming swollen. In more severe cases the
lymph node could eventually burst [11]. Symptoms
from glandular tularaemia are the same as during ulcero- FIGURE 108.2 Swollen lymph node in a patient suffering from oculo-
glandular tularaemia but without any skin infection. glandular tularaemia. With permission from: Dr Henrik Eliasson, Örebro
Oculoglandular tularaemia could be transmitted via direct University Hospital, Sweden.
infection/contamination of the eye. The symptoms are
often conjunctivitis followed by a cervical lymphadeno-
pathy (Fig. 108.2). The symptoms of gastrointestinal tularaemia are typically
After ingestion of infected food or water oropharyn- vomiting and diarrhoea, but in the more severe cases also
geal and gastrointestinal tularaemia could occur depend- an ulceration of the bowel [45]. For pneumonic tularaemia
ing on the focus of infection. Oropharyngeal tularaemia is the symptoms are typically flu-like with headache and
the most common presentation in Turkey and in Eastern high fever, and a mild, dry cough that occur at later stages.
European countries [49 53]. The clinical findings may There is a discrepancy between primary pneumonia and
vary but typical symptoms are a painful sore throat, swollen secondary pneumonia, where the former is a result of inha-
neck, cervical lymphadenopathy and tonsillopharyngitis. lation and secondary pneumonia could be found after a

haematogenic spread from another type of tularaemia.
[43,54]. Typhoidal tularaemia is a generic term used to
describe systemic illness in the absence of other symptoms
indicating another specific type of tularaemia [10].
Furthermore, in rare cases F. novicida, F. philomiragia
and F. noatunensis could cause bacteraemia after near-
drowning accidents in salt water, where the typical
symptoms are pneumonia [13,15,18,55].
Diagnosis
Several methods for diagnosis of tularaemia have been
developed. Different methods are used depending on the
time point after onset of the disease, the equipment avail-
able and the need for exact identification of the
Francisella subspecies that has caused the disease. If
pneumonic tularaemia is suspected, the pneumonia might
be verified using X-ray. Culturing could be performed
from ulcers, sputum, pharyngeal washing, and occasion-
ally in blood [10], however culturing could be difficult
and many clinical laboratories do not have the ability to
handle this pathogen since there is a demand of a bio-
safety 3 level laboratory when handling both type A and
type B strains [56].
Useful tools for diagnosis are agglutination tests,
ELISA and Western blotting. The main disadvantage of
using serological-based assays is the late onset of
detectable antibody levels that usually appear about 2
weeks after infection [57,58]. In order to measure the
prevalence of antibodies, whole killed cells, outer mem-
brane or LPS could be used as antigens [57,59]. The high-
est level of antibodies could be detected in blood 4 7
weeks after the onset of the disease, and detectable levels
have also been found to persist even 31 years after the ini-
tial infection [60,61]. Therefore identification of antibo-
dies against F. tularensis may represent a previous
infection of the disease as well as an acute illness.
Immunological analysis could be performed on serum,
urine, sputum or on tissue biopsies [10,62] However, due
to the delay of antibody response after onset of the dis-
ease, the optional method for rapid and early diagnosis of
tularaemia is a PCR-based approach [63,64].
Therapy
Historically, treatment of tularaemia has been based on
streptomycin or gentamicin treatment, but streptomycin is
not commonly used today due to its potential toxicity and
is therefore less available in several countries [10,65 68].
All F. tularensis species are naturally resistant to beta-
lactam antibiotics [69], but so far development of resis-
tance against any other antibiotics has not been reported.
Today ciprofloxacin and doxycycline represent the first
therapeutic choices for treatment of tularaemia, but in
Chapter | 108 Francisella 1995
more severe cases aminoglycosides like gentamicin are a
good alternative or complement [65,70]. The levels of
protection afforded by antibiotics are highly dependent
upon timely administration following infection [71 73].
PATHOGENIC LIFESTYLE OF FRANCISELLA
The very low infectious dose suggest that Francisella has
evolved to become a highly successful pathogen and cen-
tral to the pathogenic lifestyle is the ability to replicate
intracellularly and also circumvent or evade the different
stages of immune response by the host. This includes
strategies to infect via very different routes and means of
transmission by different vectors like ticks, mosquitoes
and flies or via inhalation, ingestion or small wounds.
Irrespective of the route of entry the pathogen can rap-
idly disseminate from the initial site of infection, prolifer-
ate and spread to different organs in the infected host.
Various strategies have evolved to mitigate the initial
extracellular innate immune response. Francisella can
enter various host cells like macrophages, dendritic cells,
polymorphonuclear neutrophils (PMNs), endothelial and
alveolar epithelial cells [11,74,75]. Once inside cells the
survival and replication absolutely rely on the ability to
escape from the initial phagosome. This is also a central
virulence strategy that involves a number of genes
encoded on the Francisella pathogenicity island (FPI).
The subsequent growth in the host cell cytosol also
requires successful tampering with the intracellular innate
defence mechanisms as well as a metabolic adaption to
growth inside the host cell.
In line with the pathogenic lifestyle is that cell-
mediated immunity is very important for development of
protective immunity against subsequent infection. Still, sub-
stantial antibody responses are generated in humans for
instance against LPS during human F. tularensis infections
[76 78]. This is not surprising since F. tularensis has also
been shown to exist in an extracellular phase in mice [79].
However, due to the mainly intracellular nature of
F. tularensis infections, the cell-mediated immune response
is thought to be the most important for protective immunity
against tularaemia [80].
The available genome sequences of several strains of
the different F. tularensis subspecies have shown that the
overall similarity at genomic level is very high and this
also includes the more or less non-pathogenic subsp. novi-
cida. Even though there are distinct differences even
between subgroups (clades) within each subsp. it is still
far from clear how the genetic differences translate into
the differences seen in virulence to humans [81,82]. The
vast majority of genes involved in immune evasion and/or
virulence are common for the subspecies.
While the ability to infect and replicate inside host
cells is the single most important property for successful
1996 PART | 17 Animal and Ectoparasitic Source Infections
infection and virulence in Francisella, the extracellular
phase of the infection is clearly also of relevance. It is not
entirely clear during what stages of infection Francisella
is extracellular, but it could be assumed to occur both
early after the entry into the host as well as at later stages
when the infection could be more or less systemic.
REGULATION OF GENE EXPRESSION
DURING HOST INFECTION
Bacteria have evolved the capability to sense environmen-
tal changes and adapt to these changes by altering expres-
sion of different enzymes, proteins and toxins. In many
bacterial species, the regulatory systems are well known
and a broad range of regulatory genes have been
identified. Many of these regulators are so-called two-
component systems (TCS), which are typically composed
of a membrane-localized sensor, that senses the environ-
ment, and a transcriptional response regulator located in
the cytoplasm [83]. In human virulent F. tularensis spe-
cies there is no typical complete TCS, and also only one
alternative sigma factor, RpoH, is identified [84,85].
In general, transcriptional regulators identified in
F. tularensis are relatively few compared to other bacte-
rial species, but on the other hand it seems that these
regulators control transcription of a broad range of genes.
The best-characterized transcriptional regulators in
F. tularensis are; MglA, SspA, FevR, PmrA and MigR.
All these regulators are conserved between F. tularensis
subspecies, and their regulatory function has been experi-
mentally established in more than one subspecies.
Common to these regulators is that they are involved in
regulation of FPI gene expression, but some of them have
been found to regulate several additional genes.
MlgA was one of the first identified regulatory genes
in F. tularensis [86]. MglA is a homologue to SspA of
Escherichia coli where it is a transcriptional factor that
responds to nutrient limitation and regulation during star-
vation. In F. tularensis MglA is up-regulated during
infection in macrophages and an mlgA mutant (novicida)
is unable to grow in macrophages and amoeba, and also
attenuated in mice [86 88]. An additional SspA homo-
logue is identified and these two homologues, SspA and
MglA, interact and associate with RNA polymerase to
regulate transcription [89]. It is suggested that the MglA/
SspA complex responds to environmental stress and is
therefore essential for bacterial survival and growth in
tough environments [90]. FevR (pigR) is another regula-
tory protein that acts in parallel with MglA/SspA and has
been found to promote expression of genes essential for
survival. This transcriptional up-regulation is a result of
the ability to sense the nutritional status of the cell via
ppGpp (guanosine tetraphosphate) a molecule produced
by bacteria in response to nutritional starvation [89,91].
MigR regulates gene expression of the FPI and also posi-
tively regulates transcription of fevR. It is still unclear if
the regulation is direct or indirect, due to the absence of a
DNA binding domain in MigR. In addition Mig R, as
well as FevR, was found to be involved in blockade of
neutrophil NADPH oxidase activity, a feature that is an
important strategy of F. tularensis to evade neutrophil
killing [92]. PmrA is another response regulator of a large
group of genes and has also been found to be essential for
intracellular survival and virulence in mice [93,94].
Today, it is unclear how expression of several other
virulence genes outside the FPI is regulated, but it seems
clear that environmental conditions, like increased tem-
perature and intracellular growth, are factors that initiate
induction of gene expression [95,96].
In most organisms, small non-coding RNAs (sRNAs)
have a regulatory function based on pairing with comple-
mentary mRNA [97]. This base pairing is often facilitated
by Hfq, an RNA chaperone. The hfq homologue identified
in F. tularensis was, as in many other bacteria, found to
be important for growth and resistance to different stress
conditions [98]. To date a few sRNA are identified in
F. tularensis, with variable impact on growth, intracellu-
lar survival and virulence [99,100].
Taken together the regulatory network in F. tularensis
is unique compared to many other bacteria and focuses
on assuring timely expression of key genes during the
infection cycle where the key events are rapid escape
from the phagosome and evasion of host innate defence
mechanisms.
EXTRACELLULAR PHASE OF INFECTION
Spread from Initial Infection Site
With a very low infection dose, even via vector-borne
transmission, one of the initial challenges for Francisella
is to survive the extracellular immune response and to
spread from the initial infection site. Francisella encodes
gene clusters for functional type IV pili (Tfp) and there is
some evidence that these adhesive structures may be
important for the spread from the initial site of infection.
Pilin mutants in type B strains were found to require high-
er inoculum to be able to spread and cause systemic infec-
tion [101,102], while the pilin was dispensable for
intracellular replication. The attenuated vaccine strain
LVS (type B) also lacks one of the pilin genes [103].
However, it is less clear if Tfp has a major role in the vir-
ulence of the most virulent subsp. tularensis (type A) as
different mutants in the pilin genes have been found to
be only slightly attenuated or of similar virulence as
the wild-type strains when evaluated in mouse infection
models [104,105].

Subversion of Extracellular Host Defences
For a successful intracellular pathogen, uptake can be
regarded as a major evasion mechanism to avoid extracel-
lular host defences. Still, it is clear that the ability to resist
killing mechanisms and inflammatory responses is impor-
tant also for Francisella (Figure 108.3). Activation of the
complement is a major defence mechanism where host
recognition of a pathogen via antibodies or lections leads
to activation with host signalling molecules that will pro-
mote inflammation and recruit phagocytes and other
immune cells to the site of infection. In addition, comple-
ment activation also leads to activation of killing mechan-
isms [106]. Francisella avoids activation of complement
by binding to host factors like plasminogen/plasmin and
factor H and thereby prevents activation of both the kill-
ing mechanisms and a strong inflammatory response. This
strategy also leads to complement-factor-mediated uptake
by phagocytic cells that is a better alternative to activate
other powerful host defences [107,108]. The O-antigen
oligosaccharide, that is part of LPS and present on the
surface, is critical for the complement-inhibiting activity
and O-antigen mutants are also attenuated in animal
infection models [109].
FIGURE 108.3 Francisella is a successful intracellular pathogen and
in line with this has mechanisms to subvert host defences at different
stages of the infection. For details, see the respective paragraph.
1. Subversion of innate extracellular host defences, like complement
and antimicrobial peptides
2. Multiple receptors mediate uptake and also affects the level of host
innate defences inside the phagosome
3. Survival in the hostile host phagosome interference with ROS
production and ROS detoxification
4. Mechanism to rapidly escape the phagosome mediated by FPI
encoded T6SS-like mechanism
5. Intracellular replication without eliciting a strong inflammatory host
response avoiding TLR and NLR recognition
6. Uptake into autophagosomes after successful intracellular cytosolic
replication role in human infection somewhat unclear
Chapter | 108 Francisella 1997
Another important host defence mechanism is the
cationic antimicrobial peptides defensins and cathelici-
dins [110]. These positively charged antimicrobial pep-
tides are designed to disrupt the negatively charged
bacterial membrane and can be found both extracellu-
larly on mucosal surfaces and within immune cells like
macrophages and neutrophils. The host defensins and
cathelicidins appear to have a limited role in defence
against Francisella. There are two apparently efficient
mechanisms by which the pathogens avoid these antimi-
crobials. One intriguing mechanism is to lower the nega-
tive surface charge by modifying the lipid A moiety of
LPS. Lipid A of Francisella lacks two negatively
charged phosphate groups that prevents the binding of
the positively charged defensins [111]. The second
mechanism whereby Francisella can avoid these antimi-
crobial peptides is efflux systems like the AcrAB/TolC
efflux platform that promotes the active efflux of toxic
compounds and thereby preventing their antimicrobial
action [112].
INTRACELLULAR PHASE OF INFECTION
Uptake/entry and Survival in the Phagosome
Francisella readily enter different host cells but macro-
phages appear to be the primary cell where it replicates
in vivo. Francisella is also very capable of replicating
inside phagocytes so uptake can also be seen as a strategy
to evade the extracellular innate host defence
(Figure 108.3). Uptake can be promoted via interaction
with different receptors, and binding triggers uptake via
membrane protrusions formed around the bacterium
called pseudopod loops [113]. The uptake appears to
occur via a pseudopod loop mechanism irrespective of
what receptor Francisella binds to and if the bacterium is
opsonized or not. While the overall mechanism appears to
be the same, the efficacy of uptake as well as intracellular
replication differs depending on the host cell receptor
involved. Serum or antibody opsonized bacteria are
mainly taken up via CR3 and FcγR-receptors. Uptake of
opsonized bacteria is about tenfold more efficient than for
non-opsonized bacteria [114]. This could be an advantage
for an intracellular pathogen, but this route of uptake
comes at the price of a slower replication rate and delayed
escape from the phagosome. Even though Francisella is
fully capable of surviving in the hostile phagosomal envi-
ronment, escape from the phagosome is a direct require-
ment for successful infection of macrophages and in vivo
infection of animal hosts. It seems like the time
Francisella spends in the phagosome also depends on the
mode of uptake. Opsonized bacteria on average spend a
longer time within the Francisella-containing phagosome
(FCP). There are several lines of host defence that
1998 PART | 17 Animal and Ectoparasitic Source Infections
Francisella has to deal with once internalized and con-
tained within FCP.
Acidification of the phagosome is one mechanism
used by the host that hampers replication of many bacte-
rial pathogens. At the same time there are many patho-
gens that have specific strategies to handle and survive in
the acidic environment. The role of acidification for
Francisella is not entirely clear but very intriguing. While
there does not appear to be any direct link or requirement
of acidification for phagosomal escape, uptake of non-
opsonized bacteria is followed by transient acidification
15 30 minutes after uptake and rapid phagosomal escape
around 1 hour after infection [115,116]. Uptake mediated
via opsonization occurs with little or no acidification but
in this case phagosomal escape is delayed in comparison
with uptake without serum or antibody-mediated uptake
[113,117]. The significance of the transient acidification
after non-opsonized uptake is strengthened by the fact
that agents that block acidification also delay escape
while the acidification-blocking agents have no effect on
escape after uptake of opsonized bacteria. Overall, this
illustrates that Francisella have a broad array of mechan-
isms to deal with host defence at different stages of
infection.
Francisella must also handle the reactive oxygen
species (ROS) that are produced within the phagosome
in response to infection (Figure 108.3). ROS molecules
are produced by a membrane-localized enzyme system,
the NADPH oxidase, which produces highly toxic
superoxide anions from oxygen [118]. In response to
phagocytosis of a pathogen, the different subunits of the
enzyme assemble at the membrane to form the active
NADPH oxidase that then starts to produce ROS.
Francisella species can interfere with ROS production
on different levels. One mechanism is to block the
assembly of NADPH oxidase components. Four acid
phosphatases (AcpA, AcpB, AcpC and Hap) are
required for the ability to inhibit NADPH oxidase
assembly and/or block ROS production by already-
assembled complexes [119]. Francisella species encode
at least six different acid phosphatases and three of
those are truncated and may not be fully functional in
subspecies tularensis (type A) strains. Their role in viru-
lence and intercellular replication is not entirely clear
but inactivation of four acid phosphatase genes in a type
A strain had a significant impact on intracellular repli-
cation as well as on virulence [120]. Even if Francisella
is able to suppress activation of the NADPH oxidase,
low levels of ROS are still produced. Francisella
encodes proteins that can directly detoxify ROS like cat-
alase [121 123].
In addition, a third class of proteins with similarity
to Ohr proteins that mediate resistance to organic hydro-
peroxides created during the interaction of ROS with
lipids of the bacterial cell membrane have been identi-
fied [124]. Again, this verifies that Francisella has
overlapping mechanisms also to subvert the NADPH
oxidase and ROS, a key host defence against intracellu-
lar pathogen.
Phagosomal Escape
As discussed above Francisella is successful in surviv-
ing the innate host defence mechanisms prior to uptake
as well as inside the FCP. At the same time it is clear
that until this point it is mainly about surviving, there is
little replication during these initial phases of infection.
Therefore, the last step, phagosomal escape whereby
the bacterium is released from the toxic phagosome
and reaches the cytosol, is an absolutely essential
step. Mutants unable to escape the FCP are highly
attenuated and essentially avirulent (Figure 108.3). The
timing of escape, as mentioned above, largely depends
on the route of Francisella uptake where non-opsonized
Francisella can escape as early as within 1 h after entry
while opsonized Francisella have been estimated to
escape between 2 4 h after entry into the FCP
[114,125].
The mechanism of escape is not known; no
membrane-disrupting or pore-forming protein directly
mediating the escape has been identified. Still it is very
clear that most of the genes encoded by the Francisella
pathogenicity island (FPI) are absolutely required. The
FPI which is duplicated in all subspecies except subspe-
cies novicida encodes a putative type VI secretion system
(T6SS) and these genes are essential for the intracellular
lifestyle as well as for pathogenesis of Francisella
[126 128].
Of the 17 FPI proteins only three, IglA, IglB and
DotU, share significant homology to typical T6SS core
components [128] and no homologue of ClpV, the impor-
tant AAA 1 ATPase and putative energizer of T6SSs, has
been identified. A potential VgrG homologue has been
identified in the FPI but the Walker A box commonly
present in IcmF homologues is missing from the F. tular-
ensis PdpB gene. These differences suggest that the
Francisella FPI system is clearly different compared to
other T6SS. Even if these observations raise the question
whether the FPI actually encodes a T6SS it is still clear
that some proteins encoded by FPI are secreted and possi-
bly also reach the host cell cytosol from the bacteria
residing within the FCP and that this is related to phago-
somal escape. Somehow FPI-encoded proteins are
secreted and can promote or induce disruption of the
phagosomal membrane. More work is needed to elucidate
the molecular mechanisms of this essential virulence
mechanism in Francisella pathogenesis.

Evasion of Host Recognition Outside Cells
and Inside the Phagosome
The host defence includes an array of receptor molecules
(pathogen recognition receptors (PRRs)) specialized in
detecting different conserved microbial components known
as PAMPs (pathogen-associated molecular patterns) [129].
The receptors that recognize these microbial components
can be found in the extracellular space and inside the phago-
some are denoted Toll-like receptors (TLRs) and these can
trigger phagocytosis and inflammatory signalling pathways.
The TLR signalling activates transcription factors like NF-
ĸB and IRF3 that in turn induce expression of inflammatory
cytokines and type I interferons that activate innate and
adaptive immune cells [130]. The different TLRs recognize
different bacterial molecules; TLR2 is specific for bacterial
lipoproteins (BLPs) and peptidoglycan (PGN) [131]; TLR4
for lipopolysaccharide (LPS) structures from Gram-negative
bacteria; TLR5 for flagellin and TLR9 recognizes bacterial
CpG DNA [130].
As discussed above most of Francisella’s successful
strategy as a pathogen relies on the ability to avoid recog-
nition and induce a strong host inflammatory response in
the host. A significant part of this strategy is linked to
successful evasion and/or suppression of inflammatory
signalling mediated by TLRs. For E. coli and many other
Gram-negative bacteria TLR4 is a primary sensor of
infection. In contrast to many other pathogens Francisella
LPS does not activate an efficient TLR4 response. One
important difference is that Francisella actively removes
or modifies LPS structures that induce a strong TLR4
response [132]. The importance of these modifications
has been verified by showing that mutation of the gene
that removes an additional 4phosphate group present in
lipid A in other bacteria that induce TLR4 responses also
resulted in rapid clearance in an in vivo infection model
[111].
Similar to TLR4, TLR5, another important host sensor
of infection, is also not activated during infection, as
Francisella does not encode flagellin [133].
Even though there is evidence supporting that
Francisella DNA is recognized by the TLR system,
TLR9 seems to be of limited importance for the in vivo
host response to Francisella [134]. It is possible that
Francisella avoids TLR9 activation by its resistance to
host antimicrobial attacks both before uptake and within
the phagosome and thereby prevents release of bacterial
DNA. Here it is likely that the rapid escape of Francisella
from the phagosome is critical for minimizing bacterial
damage or killing and subsequent DNA release to prevent
activation of TLR9 (see also above).
Studies conducted so far indicate that TLR2 is the pri-
mary TLR involved in the host response to Francisella
infection [135]. So far lipoproteins have been identified
Chapter | 108 Francisella 1999
as the primary inducers of the TLR2 response. Even if
only a few lipoproteins have been directly identified as
TLR2 agonists [136] it is likely that the interplay between
lipoproteins and TLR2 signalling is of major importance
as Francisella encodes many lipoproteins. There is also
substantial evidence that TLR2 is required for the early
inflammatory response to Francisella infection, both from
in vitro infected macrophages, as well as in vivo
Francisella infections, where TLR2 has been shown to be
required for control of infection [135,137].
Francisella has also evolved ways to deal with the
induced TLR2-dependent signalling. It also seems to be
the case that infection of macrophages with Francisella
has been shown to be blocked from TLR2 and TLR4 acti-
vation in response to E. coli lipoproteins and LPS and
infection with F. tularensis has also been shown to reduce
TLR2 expression [138,139]. Further, the ability of the bac-
teria to resist damage by antimicrobials and minimize the
release of BLPs is another indirect but efficient way of
evading TLR2 signalling. An even more sophisticated
mechanism to avoid TLR2 induction is to selectively use
the CRISPR/Cas to degrade the mRNA coding for a lipo-
protein known to induce TLR2 signalling [140]. CRISPR/
Cas (clustered regularly interspaced palindromic repeats/
CRISPR-associated) systems are bacterial defence systems
against foreign nucleic acids derive, that use an array of
small CRISPR RNAs (crRNAs) with repetitive sequences
flanking unique spacers to recognize their targets and then
the Cas proteins to mediate degradation of target mRNA.
This intricate mechanism is nicely illustrated and supported
by in vivo experiments showing that strains with a non-
functional cas gene were highly attenuated as a result of
inability to block lipoprotein expression and that lipopro-
tein mutants rescued virulence [140].
All this shows that TLR2 signalling is a major battle-
field between host and pathogen that is very decisive for
the outcome of Francisella infections where the host uses
this pathway as a major innate defence mechanism and
where the pathogen has multitudes of mechanisms, more
or less sophisticated to subvert and deal with TLR2
signalling.
Immune Subversion at the Level of
Intracellular Replication
After escape from the phagosome Francisella reaches the
host cell cytosol thus the pathogen has overcome TLRs
and harsh phagosomal defences. Even though this could
be seen as a major achievement, replication in the cytosol
is also faced with a number of challenges. Until this point
bacterial replication has normally been limited, but in the
cytosol replicating to high numbers also increases the risk
of triggering an immune response. Bacterial replication
2000 PART | 17 Animal and Ectoparasitic Source Infections
could result in release of PAMPs that in turn could be rec-
ognized by cytosolic PRRs (Figure 108.3). The host cell
cytosol also has numerous PRRs capable of recognizing a
large number of bacterial products and elicits a host
response to eliminate the invaders. The Nod-like receptor
(NLR) family responds to a diverse set of PAMPs includ-
ing PGN (Nod1 and Nod2), flagellin (NLRC4, NAIP5
and NAIP6) [141] and damage induced by pore-forming
toxins (NLRP3) [142]. The role of Nods during
Francisella infection is less well established than TLRs,
but it is possible that they recognize and respond to PGN.
The presence of less pathogenic subspecies novicida in
the cytosol is sensed in a type I interferon (IFN)-depen-
dent manner that activates the absent melanoma (AIM2)-
containing inflammasome [143]. The AIM2 complex acti-
vation leads to induction of secretion of proinflammatory
cytokines IL1ß and pro-IL18 and programmed inflamma-
tory cell death by pyroptosis. This will recruit and activate
other immune cells that can promote bacterial clearance
[144]. Thereby the bacteria released into the extracellular
environment can be taken up by other cells like neutrophils
that under these inducing conditions are not permissive for
replication [145]. The activation of the AIM2 inflamma-
some is potentiated by TLR2 signalling and very important
for eliminating Francisella from its intracellular prolifera-
tion niche inside macrophages [146,147]. AIM2 has been
shown to recognize double-stranded DNA and at least for
subspecies novicida the AIM2 activation has been found to
be linked to release of bacterial DNA in the cytosol [147].
It is important to bear in mind that these studies have
been performed in murine systems and the less virulent
subspecies novicida. In addition, macrophages infected
with the highly virulent subspecies tularensis secrete only
low levels of the inflammasome dependent cytokine
interleukin (IL)-18 and dendritic cells infected with
F. tularensis fails to efficiently activate the inflamma-
some [148]. Still, the findings strongly support that TLR2
and IFN signalling are the two major host defence path-
ways in controlling Francisella infections and both con-
tribute to inflammasome activation. It is likely that the
higher virulence of type A strains, at least in part, is the
result of having additional ways to limit or prevent
inflammasome activation.
It is not clear how Francisella after extensive replica-
tion exits from infected host cells and disseminates
throughout the host without evoking inflammation. These
infected macrophages could serve as Trojan horses, traf-
ficking the bacteria to promote systemic spread.
Xenophagy is another host strategy to capture intracel-
lular microbes within a double-membrane vacuole, the
autophagosome, for delivery and degradation in the lyso-
somal compartment [149]. After long periods of cytosolic
replication (.18 hours), some of the intracellular
Francisella become enclosed and reside inside
autophagosomes [150] (Figure 108.3). It is not clear
whether this is a host-induced response to control infec-
tion or a mechanism induced by the pathogen to promote
infection. F. tularensis modulates the expression of
autophagy-related genes that could indicate that
Francisella subverts this host defence system [151]. An
important thing to bear in mind, with regard to the
significance of autophagy, is that it has only been
observed in murine macrophages and has not yet been
observed in human macrophages [152,153].
The extensive and rapid replication in the cytosol of
host cells that occurs without induction of an inflamma-
tory response is a major hallmark of the successful and
essential immune subversion employed by F. tularensis.
Many of the molecular determinants and mechanisms that
promote the evasion of cytosolic defences remain to be
identified.
Nutritional Requirements and Defences
An intracellular pathogen must be well adapted for intra-
cellular replication and enough nutrients must be
available and possible for Francisella to acquire, to
sustain high growth. The host on the other hand has
developed mechanisms that limit the availability of spe-
cific nutrients, thereby establishing defence against patho-
gens at the nutritional level and the battle for nutrients is
another critical aspect of host pathogen interactions.
Several specific metabolic pathways critical to
Francisella replication in the host, such as the biosynthe-
sis of purines and pyrimidines, gluconeogenesis and gly-
colysis have been identified [154]. The host also actively
limits some nutrients during infection and for these the
pathogen needs to develop strategies to directly access
these nutrients.
Iron is necessary for a variety of enzymatic functions
in bacteria and is a vital component of various redox reac-
tions that take place during growth. Within the host, most
of the total iron is found in the intracellular space [155].
The iron outside host cells is associated with iron storage
molecules that bind iron with very high affinity like
haem, lactoferrin and transferrin. In principle Francisella
in part overcomes the barrier to iron acquisition when it
enters the host cell, where the iron content is significantly
higher. Also within host cells, iron-containing redox
enzymes and iron-storage proteins sequester iron and
therefore iron acquisition for the pathogen is still very dif-
ficult [156,157]. In addition, the host also sequesters iron
away in response to infection [158]. In response to the
challenges from the host, Francisella utilizes a
rhizoferrin-like siderophore [159]. Siderophores are small
molecules that bind iron with very high affinity and can
compete for iron from the host iron-sequestering com-
pounds [160]. The Francisella genes that encode the iron-

sequestering system are fslABCDE (also termed
figABCDE). These genes encode the proteins that provide
production as well as export of the Francisella sidero-
phore [159,161]. FslE is not involved in siderophore pro-
duction but is instead required for siderophore binding
and/or import [161,162]. Francisella appears to lack the
highly conserved siderophore receptor and importer genes
tonB, exbB and exbD genes found in many bacteria [163].
Instead there are two fslE orthologues present in the
genome that may promote siderophore import that are
denoted fupAB and in the type B vaccine strain LVS a
deletion event has resulted in these two genes being fused
to a single protein. This deletion event is also linked to
significant virulence attenuation indicating the importance
of this gene for siderophore import [162,164,165]. It is
clear that siderophore production and iron acquisition pro-
teins are vital for Francisella to maintain iron homeosta-
sis and to survive and sustain replication in the host. The
importance of fslABC, fupAB and feoB, a ferrous iron
importer [159,166] have each been verified by in vivo vir-
ulence screening or by direct mutagenesis identified in
in vivo virulence screens in macrophages or in animal
models [167,168].
The host also limits the intracellular concentration of
the amino acid tryptophan in response to infection [169].
Francisella overcomes this nutritional host defence by its
ability to synthesize tryptophan de novo [170] and strains
with mutations in genes involved in tryptophan biosynthe-
sis are also significantly attenuated in vivo [171,172] con-
firming the importance of tryptophan prototrophy for
Francisella pathogenesis. Francisella has several muta-
tions in genes encoding enzymes required for several bio-
synthetic pathways [154,173] and as a consequence it is
auxotrophic for 13 amino acids, i.e. Francisella can not
de novo synthesize these amino acids. The only amino
acids that Francisella can synthesize de novo are alanine,
glutamate, glutamine, asparagine, glycine, phenylalanine
and tryptophan [154,163,173]. Therefore, the ability to
acquire these essential amino acids within the host is also
essential for pathogenesis.
EVASION OF DEVELOPMENT OF
ADAPTIVE IMMUNITY
As discussed above F. tularensis efficiently evades early
immune responses and can also modulate the activity of
important innate immune cells. Macrophages and den-
dritic cells have a very important role as antigen-
presenting cells (APCs) and the ability of these cells to
present microbial antigens and activate T cells is a key
link between the innate and adaptive immune systems. As
discussed above Francisella uses several different
mechanisms to subvert macrophage defences like entry
Chapter | 108 Francisella 2001
via non-activating receptors, escape of phagosomal killing
and modulation of cytosolic defence pathways too.
One important observation is that if macrophages are
activated by the pro-inflammatory cytokine IFN-gamma,
they become highly capable of preventing its replication
in the cytosol [174,175]. IFN-gamma that is primarily
produced by NK cells and dendritic cells during infec-
tion regulates more than 200 genes. Many of these are
involved in enhancing nitric oxide production, inducing
autophagy, and promoting enhanced major histocompati-
bility complex (MHC) class I and II antigen presentation
[176,177]. All these pathways could enhance clearance
of Francisella and promote host survival. In line with
this lack of IFN-gamma in vivo leads to increased suscepti-
bility of mice to Francisella infection [175,178]. F. tular-
ensis also induces production of immunomodulatory lipid
prostaglandin E2 (PGE2) in macrophages [179]. PGE2
lowers IFN-gamma production from T cells and it also
induces the expression of a host factor that directs
ubiquitin-dependent lysosomal degradation of MHC class
II molecules, thereby significantly lowering the number of
MHC class II on the surface of the macrophages [180].
Thereby Francisella also affects the induction of adaptive
immune responses by limiting antigen presentation through
MHC class II.
Dendritic cells produce cytokines that control T-cell
activation and the immune responses that they then pro-
mote in induction of cell-mediated immunity. F. tularen-
sis can suppresses proinflammatory cytokine production
from dendritic cells [181] and one of these, IL-12, is
important for the development of IFN-gamma-producing
T cells [182].
F. tularensis also subverts dendritic cell function to
make them produce anti-inflammatory cytokines like
transforming growth factor beta (TGF-β) and IL-10
[179,181], which also contributes to the lack of an early
inflammatory response as IL-10 inhibits macrophage pro-
liferation and proinflammatory cytokine production [183].
This can also lead to reduced MHC class II expression
and further suppression of the adaptive immune response
[184] and they can also promote development of regula-
tory T cells that in turn can suppress the inflammatory
activity of other T cells [185].
The molecular mechanisms of the F. tularensis-medi-
ated redirection of APCs towards an anti-inflammatory
response are not known but inducing immune tolerance
through the induction of IL-10 or TGF-β is a strategy also
used by important pathogens such as Yersinia pestis,
Coxiella burnetii and Chlamydia pneumoniae [186].
Cell-mediated immunity is regarded as essential for
controlling intracellular bacterial infection. It is appar-
ent that Francisella encodes multiple strategies to early
modulate APCs that can interfere with activation of the
adaptive immune system. This provides an environment
2002 PART | 17 Animal and Ectoparasitic Source Infections
that promotes replication in subsequent phases of
infection by interfering with the activation of CD8 T
cells, which could kill infected host cells harbouring
Francisella.
INFECTION MODELS
Unlike some intracellular pathogens, F. tularensis natu-
rally infects a wide variety of mammals, including
humans and rodents, via multiple routes including the
respiratory and gastrointestinal tracts, the skin and the
conjunctiva [1]. An advantage with this is that there are
many different natural infections and infection routes that
can be used in research to study naturally occurring infec-
tion. Importantly, there are many significant differences
when hosts and routes for infections are used.
The small animal models used; mouse, guinea pig,
rabbit and hamster, also differ in sensitivity. The viru-
lence of the attenuated live vaccine strain, LVS (subsp.
holarctica) in the most commonly used animal model,
mice, has been shown to be dependent on the route of
delivery, while the subsp. tularensis strain SCHU S4 is
fully virulent regardless of the route of infection. To date,
none of the small animal models described in the litera-
ture behaves as the human host with respect to their sus-
ceptibility to different subspecies and strains like
SCHU S4 (type A) or LVS (attenuated type B). The fact
that the vaccine strain LVS is still virulent in mice makes
this model limited for evaluation of virulence properties
for the human infection [187 189]. Further, F. novicida,
which is essentially avirulent in humans, is more virulent
in the mouse infection model compared to LVS (type B)
[190,191]. Because of these differences and limitations
there is a need to develop and use other animal models.
Non-human primates are important and highly relevant
for vaccine and protection studies. The LVS vaccine
strain provides protection in monkeys in a similar way as
in humans [192,193]. Still, the model is not ideal as mon-
keys are more sensitive to infections with type B strains
compared to humans [194]. A rat model has also been
used but is less well established. Rats are less susceptible
to LVS but instead they appear to be more resistant to
SCHU S4 (type A) that may limit their use [195].
Arthropod vectors are important transmission routes
for tularaemia, and the fruitfly Drosophila melanogaster
has recently been included in the list of possible in vivo
models of Francisella infections. F. tularensis can spread
throughout the tissues of the fly but it is not yet known
how Francisella survives the efficient arthropod immune
response. This model might be valuable as it can enable
studies of bacterial survival and virulence in an arthropod
host and also provide insight into vector-borne transmis-
sion [196].
TULARAEMIA VACCINE DEVELOPMENT
One of the long-term goals with tularaemia research has
been to develop a vaccine against the disease. Currently,
there is no licensed vaccine for general use against tular-
aemia, but vaccine development has become a renewed
priority since the anthrax letters in the aftermath of the
9/11 events in 2001. Vaccine efforts against tularaemia
started in the 1930s and since then two different types of
tularaemia vaccine have been studied in humans. The
Foshay vaccine, in honour of the inventor, Dr. Lee
Foshay, consisted of chemically killed (phenol-killed)
F. tularensis and was effective at reducing the incidence
of laboratory-acquired tularaemia cases from 100% to
30% in the 1950s (Box 108.1) [198]. However, this vac-
cine provided only minimal protection against aerosol
challenge with type A Francisella. At the same time a
live vaccine strain (LVS) derived from type B
Francisella (F. tularensis subsp. holarctica) was used to
immunize millions of individuals in the former Soviet
Union. Samples of the Russian vaccine strain which was
attenuated by several in vitro passages were given to the
United States as part of a formal scientific exchange pro-
gramme. However, tularaemia in the former Soviet
Union was exclusively caused by type B subsp. holarcti-
ca strains and therefore the efficacy of the Russian vac-
cine against type A, subsp. tularensis was unknown.
During the 1950s and 1960s, the LVS vaccine was fur-
ther characterized and after several passages in labora-
tory media and through mice, the strain was finally
designated LVS [199].
This vaccine provided better protection against aerosol
challenge with type A Francisella than the Foshay-
vaccine and has ever since been used to vaccinate at-risk
staff (laboratory and military personnel) in some coun-
tries. The vaccine confers a long-lasting humoral and cell-
mediated immunity that is effective for up to 25 years
[200]. This is in accordance with the protective immunity
obtained after a natural infection [201 203].
Box 108.1 Laboratory-Acquired Tularaemia
Tularaemia is caused by F. tularensis and due to the very
low infection dose and risk for aerosolization during han-
dling of bacterial cultures and plates, there is a relatively
high frequency of accidental infection of laboratory person-
nel exposed to F. tularensis. One illustration of this is when
12 microbiology laboratory personnel were exposed to
F. tularensis, and received prophylactic antibiotics [197].
To avoid this type of accident it is very important that labo-
ratory personnel are well trained and informed about the
high risk for aerosol exposure and that appropriate protec-
tive measures are implemented routinely.

LVS is administered by scarification, and induces a
high level of protection against type B infections, while
protection against type A is more limited [199]. LVS vac-
cination by aerosol, i.e. administration directly into the
lungs, improved the efficacy against respiratory challenge
but also elicited serious side effects [204]. However,
although the LVS vaccine has been used as prophylaxis in
a large number of people over the last few decades, it has
not yet been licensed for general use. Major obstacles for
approval are concerns regarding its undefined attenuation,
mechanism of protection and reversion frequency [203].
Recent studies using the mouse infection model have
revealed that the virulence attenuation of LVS is the
result of two gene-deletion events, one affecting a pilin
gene and the other a protein mediating iron uptake [103].
The total number of clinical type A cases (type A
restricted to North America) is low but due to renewed
concerns regarding the threat of bioterrorism, there has
been an increased interest in licensing a tularaemia vac-
cine for general use. Therefore, the goal has been to
develop a Francisella vaccine that can also provide pro-
tection from an aerosol challenge, i.e., the pneumonic
form of the disease, in the event of a deliberate release in
a biological attack.
New experimental vaccines, including novel formula-
tions of LVS, live (and defined) attenuated type A strains,
killed whole cell, and subunit vaccine candidates are
currently developed and under evaluation [12]. Subunit-
based vaccines would be preferable from a safety
perspective but have proven not to confer high levels of
protection. This is also the experience from other intracel-
lular bacterial pathogens. The most common strategy for
the on-going vaccine development is to find ways to
attenuate type A strains to be both safe and provide high
levels of protection [205].
A vaccine must elicit both innate immunity and immu-
nological memory, i.e. persist long enough to develop a
strong adaptive immunity. Live attenuated vaccines would
be able to elicit both and confer protection.
CONCLUSIONS
Francisella tularensis, the causative agent of tularaemia,
is a zoonotic intracellular pathogen that can be found in a
very large number of species ranging from large mam-
mals and vertebrates to invertebrates, arthropods and
amoebas. Disease in humans often occurs in parallel with
tularaemia in wild animals.
Human infection can occur through aerosolization,
direct contact with infected animals and via arthropod
vectors like ticks, mosquitos and biting flies.
F. tularensis subspecies tularensis (type A) is one of
the most infectious bacteria known and inhalation of as
Chapter | 108 Francisella 2003
few as ten organisms can be sufficient to establish an
infection in humans that if untreated could be fatal.
The success of F. tularensis as a pathogen lies in its
unique ability to adapt a lifestyle as an intracellular patho-
gen and to very timely subvert host defences both at
extracellular and intracellular levels. Key events in the
infection process are the ability to rapidly escape the pha-
gosome after uptake by phagocytic cells and the ability to
replicate to high levels inside the host cells without evok-
ing a host inflammatory response.
REFERENCES
[1] Keim P, Johansson A, Wagner DM. Molecular epidemiology,
evolution, and ecology of Francisella. Ann N Y Acad Sci
2007;1105:30 66.
[2] Morner T. The ecology of tularaemia. Rev Sci Tech
1992;11:1123 30.
[3] Tarnvik A, Sandstrom G, Sjostedt A. Epidemiological analysis of
tularemia in Sweden 1931 1993. FEMS Immunol Med Microbiol
1996;13:201 4.
[4] Petersen JM, Mead PS, Schriefer ME. Francisella tularensis: an
arthropod-borne pathogen. Vet Res 2009;40:7.
[5] Berdal BP, Mehl R, Meidell NK, Lorentzen-Styr AM, Scheel O.
Field investigations of tularemia in Norway. FEMS Immunol Med
Microbiol 1996;13:191 5.
[6] Abd H, Johansson T, Golovliov I, Sandstrom G, Forsman M.
Survival and growth of Francisella tularensis in Acanthamoeba
castellanii. Appl Environ Microbiol 2003;69:600 6.
[7] Saslaw S, Eigelsbach HT, Wilson HE, Prior JA, Carhart S.
Tularemia vaccine study. I. Intracutaneous challenge. Arch Intern
Med 1961;107:689 701.
[8] Saslaw S, Eigelsbach HT, Prior JA, Wilson HE, Carhart S.
Tularemia vaccine study. II. Respiratory challenge. Arch Intern
Med 1961;107:702 14.
[9] Darling RG, Catlett CL, Huebner KD, Jarrett DG. Threats in bio-
terrorism. I: CDC category A agents. Emerg Med Clin North Am
2002;20:273 309.
[10] Dennis DT, Inglesby TV, Henderson DA, Bartlett JG, Ascher MS,
Eitzen E, Working Group on Civilian, B., et al. Tularemia as a
biological weapon: medical and public health management.
JAMA 2001;285:2763 73.
[11] Oyston PC, Sjostedt A, Titball RW. Tularaemia: bioterrorism
defence renews interest in Francisella tularensis. Nat Rev
Microbiol 2004;2:967 78.
[12] Conlan JW. Tularemia vaccines: recent developments and remain-
ing hurdles. Future Microbiol 2011;6:391 405.
[13] Hollis DG, Weaver RE, Steigerwalt AG, Wenger JD, Moss CW,
Brenner DJ. Francisella philomiragia comb. nov. (formerly
Yersinia philomiragia) and Francisella tularensis biogroup novici-
da (formerly Francisella novicida) associated with human disease.
J Clin Microbiol 1989;27:1601 8.
[14] Mailman TL, Schmidt MH. Francisella philomiragia adenitis and
pulmonary nodules in a child with chronic granulomatous disease.
Can J Infect Dis Med Microbiol 2005;16:245 8.
[15] Wenger JD, Hollis DG, Weaver RE, Baker CN, Brown GR,
Brenner DJ, et al. Infection caused by Francisella philomiragia
2004 PART | 17 Animal and Ectoparasitic Source Infections
(formerly Yersinia philomiragia). A newly recognized human
pathogen. Ann Intern Med 1989;110:888 92.
[16] Brevik OJ, Ottem KF, Nylund A. Multiple-locus, variable number
of tandem repeat analysis (MLVA) of the fish-pathogen
Francisella noatunensis. BMC Vet Res 2011;7:5.
[17] Ottem KF, Nylund A, Karlsbakk E, Friis-Moller A, Kamaishi T.
Elevation of Francisella philomiragia subsp. noatunensis
Mikalsen et al. (2007) to Francisella noatunensis comb. nov. [syn.
Francisella piscicida Ottem et al. (2008) syn. nov.] and character-
ization of Francisella noatunensis subsp. orientalis subsp. nov.,
two important fish pathogens. J Appl Microbiol
2009;106:1231 43.
[18] Birkbeck TH, Feist SW, Verner-Jeffreys DW. Francisella infec-
tions in fish and shellfish. J Fish Dis 2011;34:173 87.
[19] Colquhoun DJ, Duodu S. Francisella infections in farmed and
wild aquatic organisms. Vet Res 2011;42:47.
[20] Olsen AB, Mikalsen J, Rode M, Alfjorden A, Hoel E, Straum-Lie
K, et al. A novel systemic granulomatous inflammatory disease in
farmed Atlantic cod, Gadus morhua L., associated with a bacte-
rium belonging to the genus Francisella. J Fish Dis
2006;29:307 11.
[21] Nylund A, Ottem KF, Watanabe K, Karlsbakk E, Krossoy B.
Francisella sp. (Family Francisellaceae) causing mortality in
Norwegian cod (Gadus morhua) farming. Arch Microbiol
2006;185:383 92.
[22] Ostland VE, Stannard JA, Creek JJ, Hedrick RP, Ferguson HW,
Carlberg JM, et al. Aquatic Francisella-like bacterium associ-
ated with mortality of intensively cultured hybrid striped
bass Morone chrysops x M. saxatilis. Dis Aquat Organ
2006;72:135 45.
[23] Kamaishi T, Miwa S, Goto E, Matsuyama T, Oseko N. Mass mor-
tality of giant abalone Haliotis gigantea caused by a Francisella
sp. bacterium. Dis Aquat Organ 2010;89:145 54.
[24] Escudero R, Elia M, Saez-Nieto JA, Menendez V, Toledo A,
Royo G, et al. A possible novel Francisella genomic species iso-
lated from blood and urine of a patient with severe illness. Clin
Microbiol Infect 2010;16:1026 30.
[25] Whipp MJ, Davis JM, Lum G, de Boer J, Zhou Y, Bearden SW,
et al. Characterization of a novicida-like subspecies of Francisella
tularensis isolated in Australia. J Med Microbiol
2003;52:839 42.
[26] Kugeler KJ, Mead PS, McGowan KL, Burnham JM, Hogarty MD,
Ruchelli E, et al. Isolation and characterization of a novel
Francisella sp. from human cerebrospinal fluid and blood. J Clin
Microbiol 2008;46:2428 31.
[27] Johansson A, Farlow J, Larsson P, Dukerich M, Chambers E,
Byström M, et al. Worldwide genetic relationships among
Francisella tularensis isolates determined by multiple-locus vari-
able-number tandem repeat analysis. J Bacteriol 2004;186:
5808 18.
[28] Jackson J, McGregor A, Cooley L, Ng J, Brown M, Ong CW,
et al. Francisella tularensis Subspecies holarctica, Tasmania,
Australia, 2011. Emerg Infect Dis 2012;18:1484 6.
[29] Olsufjev NG, Meshcheryakova IS. Infraspecific taxonomy of tula-
remia agent Francisella tularensis McCoy et Chapin. J Hyg
Epidemiol Microbiol Immunol 1982;26:291 9.
[30] Ellis J, Oyston PC, Green M, Titball RW. Tularemia. Clin
Microbiol Rev 2002;15:631 46.
[31] Sandstrom G, Sjostedt A, Forsman M, Pavlovich NV, Mishankin
BN. Characterization and classification of strains of Francisella
tularensis isolated in the central Asian focus of the Soviet Union
and in Japan. J Clin Microbiol 1992;30:172 5.
[32] Johansson A, Celli J, Conlan W, Elkins KL, Forsman M, Keim
PS, et al. Objections to the transfer of Francisella novicida to the
subspecies rank of Francisella tularensis. Int J Syst Evol
Microbiol 2010;60:1717 8; author reply 1718 20.
[33] Busse HJ, Huber B, Anda P, Escudero R, Scholz HC, Seibold E,
et al. Objections to the transfer of Francisella novicida to the sub-
species rank of Francisella tularensis - response to Johansson
et al. Int J Syst Evol Microbiol 2010;60:1718 20.
[34] Farlow J, Wagner DM, Dukerich M, Stanley M, Chu M, Kubota
K, et al. Francisella tularensis in the United States. Emerg Infect
Dis. 2005;11:1835 41.
[35] Staples JE, Kubota KA, Chalcraft LG, Mead PS, Petersen JM.
Epidemiologic and molecular analysis of human tularemia, United
States, 1964 2004. Emerg Infect Dis 2006;12:1113 8.
[36] Kugeler KJ, Mead PS, Janusz AM, Staples JE, Kubota KA,
Chalcraft LG, et al. Molecular epidemiology of Francisella tular-
ensis in the United States. Clin Infect Dis 2009;48:863 70.
[37] Barns SM, Grow CC, Okinaka RT, Keim P, Kuske CR. Detection
of diverse new Francisella-like bacteria in environmental samples.
Appl Environ Microbiol 2005;71:5494 500.
[38] Petersen JM, Carlson J, Yockey B, Pillai S, Kuske C, Garbalena
G, et al. Direct isolation of Francisella spp. from environmental
samples. Lett Appl Microbiol 2009;48:663 7.
[39] Schrallhammer M, Schweikert M, Vallesi A, Verni F, Petroni G.
Detection of a novel subspecies of Francisella noatunensis as
endosymbiont of the ciliate Euplotes raikovi. Microb Ecol
2011;61:455 64.
[40] Sjodin A, Svensson K, Ohrman C, Ahlinder J, Lindgren P, Duodu
S, et al. Genome characterisation of the genus Francisella reveals
insight into similar evolutionary paths in pathogens of mammals
and fish. BMC Genomics 2012;13:268.
[41] Scoles GA. Phylogenetic analysis of the Francisella-like endo-
symbionts of Dermacentor ticks. J Med Entomol
2004;41:277 86.
[42] Kugeler KJ, Gurfield N, Creek JG, Mahoney KS, Versage JL,
Petersen JM. Discrimination between Francisella tularensis and
Francisella-like endosymbionts when screening ticks by PCR.
Appl Environ Microbiol 2005;71:7594 7.
[43] Tarnvik A, Berglund L. Tularemia. Eur Respir J 2003;21:361 73.
[44] Jones RM, Nicas M, Hubbard A, Sylvester MD, Reingold A. The
infectious dose of Francisella tularensis (Tularemia). Appl
Biosafety 2005;10:227 39.
[45] Ellis J, Oyston PCF, Green M, Titball RW. Tularemia. Clin
Microbiol Rev 2002;15:631 46.
[46] Goethert HK, Telford 3rd SR. Differential mortality of dog tick
vectors due to infection by diverse Francisella tularensis tularen-
sis genotypes. Vector Borne Zoonotic Dis 2011;11:1263 8.
[47] Li J, Ryder C, Mandal M, Ahmed F, Azadi P, Snyder DS, et al.
Attenuation and protective efficacy of an O-antigen-deficient mutant
of Francisella tularensis LVS. Microbiology 2007;153:3141 53.
[48] Francis E. Tularemia. JAMA 1925;84:7.
[49] Helvaci S, Gedikoglu S, Akalin H, Oral HB. Tularemia in Bursa,
Turkey: 205 cases in ten years. Eur J Epidemiol 2000;16:
271 6.

[50] Kantardjiev T, Ivanov I, Velinov T, Padeshki P, Popov B, Nenova
R, et al. Tularemia outbreak, Bulgaria, 1997 2005. Emerg Infect
Dis 2006;12:678 80.
[51] Meric M, Sayan M, Dundar D, Willke A. Tularaemia outbreaks in
Sakarya, Turkey: case-control and environmental studies.
Singapore Med J 2010;51:655 9.
[52] Tarnvik A, Priebe HS, Grunow R. Tularaemia in Europe: an epi-
demiological overview. Scand J Infect Dis 2004;36:350 5.
[53] Willke A, Meric M, Grunow R, Sayan M, Finke EJ, Splettstosser
W, et al. An outbreak of oropharyngeal tularaemia linked to natu-
ral spring water. J Med Microbiol 2009;58:112 6.
[54] Matyas BT, Nieder HS, Telford 3rd SR. Pneumonic tularemia on
Martha’s Vineyard: clinical, epidemiologic, and ecological charac-
teristics. Ann N Y Acad Sci 2007;1105:351 77.
[55] Brett M, Doppalapudi A, Respicio-Kingry LB, Myers D, Husband
B, Pollard K, et al. Francisella novicida bacteremia after a near-
drowning accident. J Clin Microbiol 2012;50:2826 9.
[56] Titball RW, Sjostedt A, Pavelka Jr. MS, Nano FE. Biosafety and
selectable markers. Ann N Y Acad Sci 2007;1105:405 17.
[57] Bevanger L, Maeland JA, Naess AI. Agglutinins and antibodies to
Francisella tularensis outer membrane antigens in the early diag-
nosis of disease during an outbreak of tularemia. J Clin Microbiol
1988;26:433 7.
[58] Brown SL, McKinney FT, Klein GC, Jones WL. Evaluation of a
safranin-O-stained antigen microagglutination test for Francisella
tularensis antibodies. J Clin Microbiol 1980;11:146 8.
[59] Grunow R, Splettstoesser W, McDonald S, Otterbein C, O’Brien
T, Morgan C, et al. Detection of Francisella tularensis in biologi-
cal specimens using a capture enzyme-linked immunosorbent
assay, an immunochromatographic handheld assay, and a PCR.
Clin Diagn Lab Immunol 2000;7:86 90.
[60] Koskela P, Salminen A. Humoral immunity against Francisella
tularensis after natural infection. J Clin Microbiol 1985;22:973 9.
[61] Sato T, Fujita H, Ohara Y, Homma M. Microagglutination test for
early and specific serodiagnosis of tularemia. J Clin Microbiol
1990;28:2372 4.
[62] Tarnvik A, Lofgren S, Ohlund L, Sandstrom G. Detection of anti-
gen in urine of a patient with tularemia. Eur J Clin Microbiol
1987;6:318 9.
[63] Sjöstedt A, Eriksson U, Berglund L, Tarnvik A. Detection of
Francisella tularensis in ulcers of patients with tularemia by PCR.
J Clin Microbiol 1997;35:1045 8.
[64] Johansson A, Ibrahim A, Göransson I, Eriksson U, Gurycova D,
Clarridge 3rd JE, et al. Evaluation of PCR-based methods
for discrimination of Francisella species and subspecies and
development of a specific PCR that distinguishes the two major
subspecies of Francisella tularensis. J Clin Microbiol 2000;38:
4180 5.
[65] Eliasson H, Broman T, Forsman M, Back E. Tularemia: current
epidemiology and disease management. Infect Dis Clin North Am
2006;20:289 311, ix.
[66] Francis E. Streptomycin in treatment of tularemia. Trans Assoc
Am Physicians 1947;60:181 6.
[67] Maurin M, Mersali NF, Raoult D. Bactericidal activities of anti-
biotics against intracellular Francisella tularensis. Antimicrob
Agents Chemother 2000;44:3428 31.
[68] Sanford JP. Landmark perspective: Tularemia. JAMA 1983;
250:3225 6.
Chapter | 108 Francisella 2005
[69] Baker CN, Hollis DG, Thornsberry C. Antimicrobial susceptibility
testing of Francisella tularensis with a modified Mueller-Hinton
broth. J Clin Microbiol 1985;22:212 5.
[70] Johansson A, Berglund L, Gothefors L, Sjostedt A, Tarnvik A.
Ciprofloxacin for treatment of tularemia in children. Pediatr Infect
Dis J 2000;19:449 53.
[71] Piercy T, Steward J, Lever MS, Brooks TJ. In vivo efficacy of
fluoroquinolones against systemic tularaemia infection in mice. J
Antimicrob Chemother 2005;56:1069 73.
[72] Mills E, Gold R, Thipphawong J, Barreto L, Guasparini R,
Meekison W, et al. Safety and immunogenicity of a combined five-
component pertussis-diphtheria-tetanus-inactivated poliomyelitis-
Haemophilus B conjugate vaccine administered to infants at two,
four and six months of age. Vaccine 1998;16:576 85.
[73] Steward J, Piercy T, Lever MS, Simpson AJ, Brooks TJ.
Treatment of murine pneumonic Francisella tularensis infection
with gatifloxacin, moxifloxacin or ciprofloxacin. Int J Antimicrob
Agents 2006;27:439 43.
[74] McCaffrey RL, Allen LA. Francisella tularensis LVS evades kill-
ing by human neutrophils via inhibition of the respiratory burst
and phagosome escape. J Leukoc Biol 2006;80:1224 30.
[75] Hall JD, Craven RR, Fuller JR, Pickles RJ, Kawula TH. Francisella
tularensis replicates within alveolar type II epithelial cells in vitro
and in vivo following inhalation. Infect Immun 2007;75:1034 9.
[76] Sundaresh S, Randall A, Unal B, Petersen JM, Belisle JT, Hartley
MG, et al. From protein microarrays to diagnostic antigen discov-
ery: a study of the pathogen Francisella tularensis. Bioinformatics
2007;23:i508 18.
[77] Cole LE, Elkins KL, Michalek SM, Qureshi N, Eaton LJ,
Rallabhandi P, et al. Immunologic consequences of Francisella
tularensis live vaccine strain infection: role of the innate immune
response in infection and immunity. J Immunol 2006;176:6888 99.
[78] Koskela P, Herva E. Cell-mediated and humoral immunity
induced by a live Francisella tularensis vaccine. Infect Immun
1982;36:983 9.
[79] Forestal CA, Malik M, Catlett SV, Savitt AG, Benach JL, Sellati
TJ, et al. Francisella tularensis has a significant extracellular
phase in infected mice. J Infect Dis 2007;196:134 7.
[80] Sjostedt A, Sandstrom G, Tarnvik A, Jaurin B. Molecular cloning
and expression of a T-cell stimulating membrane protein of
Francisella tularensis. Microb Pathog 1989;6:403 14.
[81] Molins CR, Carlson JK, Coombs J, Petersen JM. Identification of
Francisella tularensis subsp. tularensis A1 and A2 infections by
real-time polymerase chain reaction. Diagn Microbiol Infect Dis
2009;64:6 12.
[82] Champion MD, Zeng Q, Nix EB, Nano FE, Keim P, Kodira CD,
et al. Comparative genomic characterization of Francisella tular-
ensis strains belonging to low and high virulence subspecies.
PLoS Pathog 2009;5:e1000459.
[83] Beier D, Gross R. Regulation of bacterial virulence by two-
component systems. Curr Opin Microbiol 2006;9:143 52.
[84] Bell BL, Mohapatra NP, Gunn JS. Regulation of virulence gene
transcripts by the Francisella novicida orphan response regulator
PmrA: role of phosphorylation and evidence of MglA/SspA inter-
action. Infect Immun 2010;78:2189 98.
[85] Hrstka R, Stulik J, Vojtesek B. The role of MAPK signal path-
ways during Francisella tularensis LVS infection-induced apopto-
sis in murine macrophages. Microbes Infect 2005;4:4.
2006 PART | 17 Animal and Ectoparasitic Source Infections
[86] Baron GS, Nano FE. MglA and MglB are required for the intra-
macrophage growth of Francisella novicida. Mol Microbiol
1998;29:247 59.
[87] Bonquist L, Lindgren H, Golovliov I, Guina T, Sjostedt A. MglA
and Igl proteins contribute to the modulation of Francisella
tularensis live vaccine strain-containing phagosomes in murine
macrophages. Infect Immun 2008;76:3502 10.
[88] Lauriano CM, Barker JR, Yoon SS, Nano FE, Arulanandam BP,
Hassett DJ, et al. MglA regulates transcription of virulence factors
necessary for Francisella tularensis intraamoebae and intrama-
crophage survival. Proc Natl Acad Sci USA 2004;101:4246 9.
[89] Charity JC, Costante-Hamm MM, Balon EL, Boyd DH, Rubin EJ,
Dove SL. Twin RNA polymerase-associated proteins control viru-
lence gene expression in Francisella tularensis. PLoS Pathog
2007;3:e84.
[90] Guina T, Radulovic D, Bahrami AJ, Bolton DL, Rohmer L,
Jones-Isaac KA, et al. MglA regulates Francisella tularensis
subsp. novicida (Francisella novicida) response to starvation and
oxidative stress. J Bacteriol 2007;189:6580 6.
[91] Brotcke A, Monack DM. Identification of fevR, a novel regulator
of virulence gene expression in Francisella novicida. Infect
Immun 2008;76:3473 80.
[92] Buchan BW, McCaffrey RL, Lindemann SR, Allen LA, Jones
BD. Identification of migR, a regulatory element of the
Francisella tularensis live vaccine strain iglABCD virulence
operon required for normal replication and trafficking in macro-
phages. Infect Immun 2009;77:2517 29.
[93] Mohapatra NP, Soni S, Bell BL, Warren R, Ernst RK, Muszynski
A, et al. Identification of an orphan response regulator required
for the virulence of Francisella spp. and transcription of pathoge-
nicity island genes. Infect Immun 2007;75:3305 14.
[94] Sammons-Jackson WL, McClelland K, Manch-Citron JN,
Metzger DW, Bakshi CS, Garcia E, et al. Generation and charac-
terization of an attenuated mutant in a response regulator gene of
Francisella tularensis live vaccine strain (LVS). DNA Cell Biol
2008;27:387 403.
[95] Horzempa J, Carlson Jr. PE, O’Dee DM, Shanks RM, Nau GJ.
Global transcriptional response to mammalian temperature pro-
vides new insight into Francisella tularensis pathogenesis. BMC
Microbiol 2008;8:172.
[96] Wehrly TD, Chong A, Virtaneva K, Sturdevant DE, Child R,
Edwards JA, et al. Intracellular biology and virulence determi-
nants of Francisella tularensis revealed by transcriptional profil-
ing inside macrophages. Cell Microbiol 2009;11:1128 50.
[97] Waters LS, Storz G. Regulatory RNAs in bacteria. Cell
2009;136:615 28.
[98] Janovska S, Pavkova I, Hubalek M, Lenco J, Macela A, Stulik J.
Identification of immunoreactive antigens in membrane proteins
enriched fraction from Francisella tularensis LVS. Immunol Lett
2007;108:151 9.
[99] Postic G, Dubail I, Frapy E, Dupuis M, Dieppedale J, Charbit A,
et al. Identification of a novel small RNA modulating
Francisella tularensis pathogenicity. PLoS One 2012;7:e41999.
[100] Postic G, Frapy E, Dupuis M, Dubail I, Livny J, Charbit A, et al.
Identification of small RNAs in Francisella tularensis. BMC
Genomics 2010;11:625.
[101] Forslund AL, Kuoppa K, Svensson K, Salomonsson E,
Johansson A, Bystrom M, et al. Direct repeat-mediated deletion
of a type IV pilin gene results in major virulence attenuation of
Francisella tularensis. Mol Microbiol 2006;59:1818 30.
[102] Salomonsson EN, Forslund AL, Forsberg A. Type IV Pili in
Francisella - A Virulence Trait in an Intracellular Pathogen.
Front Microbiol 2011;2:29.
[103] Salomonsson E, Kuoppa K, Forslund AL, Zingmark C,
Golovliov I, Sjostedt A, et al. Reintroduction of two deleted viru-
lence loci restores full virulence to the live vaccine strain of
Francisella tularensis. Infect Immun 2009;77:3424 31.
[104] Forslund AL, Salomonsson EN, Golovliov I, Kuoppa K, Michell
S, Titball R, et al. The type IV pilin, PilA, is required for full vir-
ulence of Francisella tularensis subspecies tularensis. BMC
Microbiol 2010;10:227.
[105] Ark NM, Mann BJ. Impact of Francisella tularensis pilin homo-
logs on pilus formation and virulence. Microb Pathog
2011;51:110 20.
[106] Rus H, Cudrici C, Niculescu F. The role of the complement sys-
tem in innate immunity. Immunol Res 2005;33:103 12.
[107] Ben Nasr A, Klimpel GR. Subversion of complement activation at
the bacterial surface promotes serum resistance and opsonophago-
cytosis of Francisella tularensis. J Leukoc Biol 2008;84:77 85.
[108] Clinton SR, Bina JE, Hatch TP, Whitt MA, Miller MA. Binding
and activation of host plasminogen on the surface of Francisella
tularensis. BMC Microbiol 2010;10:76.
[109] Clay CD, Soni S, Gunn JS, Schlesinger LS. Evasion of
complement-mediated lysis and complement C3 deposition are
regulated by Francisella tularensis lipopolysaccharide O antigen.
J Immunol 2008;181:5568 78.
[110] Cederlund A, Gudmundsson GH, Agerberth B. Antimicrobial
peptides important in innate immunity. FEBS J 2011;278:
3942 51.
[111] Wang X, Ribeiro AA, Guan Z, Abraham SN, Raetz CR.
Attenuated virulence of a Francisella mutant lacking the lipid A
4’-phosphatase. Proc Natl Acad Sci U S A 2007;104:4136 41.
[112] Bina XR, Lavine CL, Miller MA, Bina JE. The AcrAB RND
efflux system from the live vaccine strain of Francisella tularen-
sis is a multiple drug efflux system that is required for virulence
in mice. FEMS Microbiol Lett 2008;279:226 33.
[113] Clemens DL, Lee BY, Horwitz MA. Francisella tularensis enters
macrophages via a novel process involving pseudopod loops.
Infect Immun 2005;73:5892 902.
[114] Geier H, Celli J. Phagocytic receptors dictate phagosomal escape
and intracellular proliferation of Francisella tularensis. Infect
Immun 2011;79:2204 14.
[115] Santic M, Asare R, Skrobonja I, Jones S, Abu Kwaik Y.
Acquisition of the vacuolar ATPase proton pump and phagosome
acidification are essential for escape of Francisella tularensis
into the macrophage cytosol. Infect Immun 2008;76:
2671 7.
[116] Chong A, Wehrly TD, Nair V, Fischer ER, Barker JR, Klose KE,
et al. The early phagosomal stage of Francisella tularensis deter-
mines optimal phagosomal escape and Francisella pathogenicity
island protein expression. Infect Immun 2008;76:5488 99.
[117] Clemens DL, Lee BY, Horwitz MA. Francisella tularensis pha-
gosomal escape does not require acidification of the phagosome.
Infect Immun 2009;77:1757 73.
[118] Nauseef WM. Assembly of the phagocyte NADPH oxidase.
Histochem Cell Biol 2004;122:277 91.

[119] Mohapatra NP, Soni S, Rajaram MV, Dang PM, Reilly TJ,
El-Benna J, et al. Francisella acid phosphatases inactivate
the NADPH oxidase in human phagocytes. J Immunol
2010;184:5141 50.
[120] Mohapatra NP, Soni S, Rajaram MV, Strandberg KL, Gunn JS.
Type A Francisella tularensis acid phosphatases contribute to
pathogenesis. PLoS One 2013;8:e56834.
[121] Lindgren H, Shen H, Zingmark C, Golovliov I, Conlan W,
Sjostedt A. Resistance of Francisella tularensis strains against
reactive nitrogen and oxygen species with special reference to
the role of KatG. Infect Immun 2007;75:1303 9.
[122] Bakshi CS, Malik M, Regan K, Melendez JA, Metzger DW,
Pavlov VM, et al. Superoxide dismutase B gene (sodB)-deficient
mutants of Francisella tularensis demonstrate hypersensitivity
to oxidative stress and attenuated virulence. J Bacteriol
2006;188:6443 8.
[123] Melillo AA, Mahawar M, Sellati TJ, Malik M, Metzger DW,
Melendez JA, et al. Identification of Francisella tularensis live
vaccine strain CuZn superoxide dismutase as critical for resis-
tance to extracellularly generated reactive oxygen species.
J Bacteriol 2009;191:6447 56.
[124] Llewellyn AC, Jones CL, Napier BA, Bina JE, Weiss DS.
Macrophage replication screen identifies a novel Francisella
hydroperoxide resistance protein involved in virulence. PLoS
One 2011;6:e24201.
[125] Golovliov I, Baranov V, Krocova Z, Kovarova H, Sjostedt A. An
attenuated strain of the facultative intracellular bacterium
Francisella tularensis can escape the phagosome of monocytic
cells. Infect Immun 2003;71:5940 50.
[126] Barker JR, Chong A, Wehrly TD, Yu JJ, Rodriguez SA, Liu J, et al.
The Francisella tularensis pathogenicity island encodes a secretion
system that is required for phagosome escape and virulence. Mol
Microbiol 2009;74:1459 70.
[127] Nano FE, Schmerk C. The Francisella pathogenicity island. Ann
N Y Acad Sci 2007;1105:122 37.
[128] Broms JE, Sjostedt A, Lavander M. The Role of the Francisella
Tularensis pathogenicity island in type VI secretion, intracellular
survival, and modulation of host cell signaling. Front Microbiol
2010;1:136.
[129] Janeway Jr. CA, Medzhitov R. Innate immune recognition. Annu
Rev Immunol 2002;20:197 216.
[130] Kawai T, Akira S. The role of pattern-recognition receptors in
innate immunity: update on Toll-like receptors. Nat Immunol
2010;11:373 84.
[131] Asong J, Wolfert MA, Maiti KK, Miller D, Boons GJ. Binding
and cellular activation studies reveal that toll-like receptor 2 can
differentially recognize peptidoglycan from Gram-positive and
Gram-negative bacteria. J Biol Chem 2009;284:8643 53.
[132] Raetz CR, Guan Z, Ingram BO, Six DA, Song F, Wang X, et al.
Discovery of new biosynthetic pathways: the lipid A story. J
Lipid Res 2009;50(Suppl):S103 8.
[133] Li H, Nookala S, Bina XR, Bina JE, Re F. Innate immune
response to Francisella tularensis is mediated by TLR2 and
caspase-1 activation. J Leukoc Biol 2006;80:766 73.
[134] Elkins KL, Rhinehart-Jones TR, Stibitz S, Conover JS, Klinman
DM. Bacterial DNA containing CpG motifs stimulates
lymphocyte-dependent protection of mice against lethal infection
with intracellular bacteria. J Immunol 1999;162:2291 8.
Chapter | 108 Francisella 2007
[135] Abplanalp AL, Morris IR, Parida BK, Teale JM, Berton MT.
TLR-dependent control of Francisella tularensis infection and
host inflammatory responses. PLoS One 2009;4:e7920.
[136] Thakran S, Li H, Lavine CL, Miller MA, Bina JE, Bina XR, et
al. Identification of Francisella tularensis lipoproteins that stim-
ulate the toll-like receptor (TLR) 2/TLR1 heterodimer. J Biol
Chem 2008;283:3751 60.
[137] Cole LE, Shirey KA, Barry E, Santiago A, Rallabhandi P, Elkins
KL, et al. Toll-like receptor 2-mediated signaling requirements
for Francisella tularensis live vaccine strain infection of murine
macrophages. Infect Immun 2007;75:4127 37.
[138] Telepnev M, Golovliov I, Grundstrom T, Tarnvik A, Sjostedt
A. Francisella tularensis inhibits Toll-like receptor-mediated
activation of intracellular signalling and secretion of TNF-
alpha and IL-1 from murine macrophages. Cell Microbiol
2003;5:41 51.
[139] Butchar JP, Cremer TJ, Clay CD, Gavrilin MA, Wewers MD,
Marsh CB, et al. Microarray analysis of human monocytes
infected with Francisella tularensis identifies new targets of host
response subversion. PLoS One 2008;3:e2924.
[140] Sampson TR, Saroj SD, Llewellyn AC, Tzeng YL, Weiss DS. A
CRISPR/Cas system mediates bacterial innate immune evasion
and virulence. Nature 2013;497:254 7.
[141] Kofoed EM, Vance RE. Innate immune recognition of bacterial
ligands by NAIPs determines inflammasome specificity. Nature
2011;477:592 5.
[142] Miao EA, Mao DP, Yudkovsky N, Bonneau R, Lorang CG,
Warren SE, et al. Innate immune detection of the type III secre-
tion apparatus through the NLRC4 inflammasome. Proc Natl
Acad Sci U S A 2010;107:3076 80.
[143] Jones JW, Kayagaki N, Broz P, Henry T, Newton K, O’Rourke
K, et al. Absent in melanoma 2 is required for innate immune
recognition of Francisella tularensis. Proc Natl Acad Sci USA
2010;107:9771 6.
[144] Fink SL, Cookson BT. Apoptosis, pyroptosis, and necrosis:
mechanistic description of dead and dying eukaryotic cells.
Infect Immun 2005;73:1907 16.
[145] Sjostedt A, Conlan JW, North RJ. Neutrophils are critical for
host defense against primary infection with the facultative intra-
cellular bacterium Francisella tularensis in mice and participate
in defense against reinfection. Infect Immun 1994;62:2779 83.
[146] Fernandes-Alnemri T, Yu JW, Juliana C, Solorzano L, Kang S,
Wu J, et al. The AIM2 inflammasome is critical for innate
immunity to Francisella tularensis. Nat Immunol 2010;11:
385 93.
[147] Jones CL, Weiss DS. TLR2 signaling contributes to rapid inflam-
masome activation during F. novicida infection. PLoS One
2011;6:e20609.
[148] Wickstrum JR, Bokhari SM, Fischer JL, Pinson DM, Yeh HW,
Horvat RT, et al. Francisella tularensis induces extensive
caspase-3 activation and apoptotic cell death in the tissues of
infected mice. Infect Immun 2009;77:4827 36.
[149] Levine B, Mizushima N, Virgin HW. Autophagy in immunity
and inflammation. Nature 2011;469:323 35.
[150] Checroun C, Wehrly TD, Fischer ER, Hayes SF, Celli J.
Autophagy-mediated reentry of Francisella tularensis into the
endocytic compartment after cytoplasmic replication. Proc Natl
Acad Sci U S A 2006;103:14578 83.
2008 PART | 17 Animal and Ectoparasitic Source Infections
[151] Cremer TJ, Amer A, Tridandapani S, Butchar JP. Francisella
tularensis regulates autophagy-related host cell signaling path-
ways. Autophagy 2009;5:125 8.
[152] Edwards JA, Rockx-Brouwer D, Nair V, Celli J. Restricted
cytosolic growth of Francisella tularensis subsp. tularensis
by IFN-gamma activation of macrophages. Microbiology
2010;156:327 39.
[153] Akimana C, Al-Khodor S, Abu Kwaik Y. Host factors required for
modulation of phagosome biogenesis and proliferation of
Francisella tularensis within the cytosol. PLoS One 2010;5:e11025.
[154] Meibom KL, Charbit A. Francisella tularensis metabolism and
its relation to virulence. Front Microbiol 2010;1:140.
[155] Bridges K, Seligman P. Disorders of iron metabolism.
In: Handlin RI, Lux SE, Stossel TP, London JB, editors. Blood
principles and practice of hematology. Lippincott Company;
1995. p. 1433 72.
[156] Braun V. Iron uptake mechanisms and their regulation in patho-
genic bacteria. Int J Med Microbiol 2001;291:67 79.
[157] Wandersman C, Stojiljkovic I. Bacterial heme sources: the role
of heme, hemoprotein receptors and hemophores. Curr Opin
Microbiol 2000;3:215 20.
[158] Mulero V, Brock JH. Regulation of iron metabolism in murine
J774 macrophages: role of nitric oxide-dependent and -indepen-
dent pathways following activation with gamma interferon and
lipopolysaccharide. Blood 1999;94:2383 9.
[159] Sullivan JT, Jeffery EF, Shannon JD, Ramakrishnan G.
Characterization of the siderophore of Francisella tularensis and
role of fslA in siderophore production. J Bacteriol 2006;188:
3785 95.
[160] Miethke M, Marahiel MA. Siderophore-based iron acquisition
and pathogen control. Microbiol Mol Biol Rev 2007;71:413 51.
[161] Kiss K, Liu W, Huntley JF, Norgard MV, Hansen EJ.
Characterization of operon mutants of Francisella novicida
U112. FEMS Microbiol Lett 2008;285:270 7.
[162] Ramakrishnan G, Meeker A, Dragulev B. fslE is necessary for
siderophore-mediated iron acquisition in Francisella tularensis
Schu S4. J Bacteriol 2008;190:5353 61.
[163] Larsson P, Oyston PC, Chain P, Chu MC, Duffield M, Fuxelius
HH, et al. The complete genome sequence of Francisella tular-
ensis, the causative agent of tularemia. Nat Genet
2005;37:153 9. Epub 2005 Jan 09.
[164] Lindgren H, Honn M, Golovlev I, Kadzhaev K, Conlan W,
Sjostedt A. The 58-kilodalton major virulence factor of
Francisella tularensis is required for efficient utilization of iron.
Infect Immun 2009;77:4429 36.
[165] Sen B, Meeker A, Ramakrishnan G. The fslE homolog, FTL_0439
(fupA/B), mediates siderophore-dependent iron uptake in
Francisella tularensis LVS. Infect Immun 2010;78:4276 85.
[166] Cartron ML, Maddocks S, Gillingham P, Craven CJ, Andrews SC.
Feo transport of ferrous iron into bacteria. Biometals
2006;19:143 57.
[167] Su J, Yang J, Zhao D, Kawula TH, Banas JA, Zhang JR.
Genome-wide identification of Francisella tularensis virulence
determinants. Infect Immun 2007;75:3089 101.
[168] Weiss DS, Brotcke A, Henry T, Margolis JJ, Chan K, Monack
DM. In vivo negative selection screen identifies genes required
for Francisella virulence. Proc Natl Acad Sci USA
2007;104:6037 42.
[169] Taylor MW, Feng GS. Relationship between interferon-gamma,
indoleamine 2,3-dioxygenase, and tryptophan catabolism.
FASEB J 1991;5:2516 22.
[170] Zegarra-Moran O, Folli C, Manzari B, Ravazzolo R, Varesio L,
Galietta LJ. Double mechanism for apical tryptophan depletion
in polarized human bronchial epithelium. J Immunol
2004;173:542 9.
[171] Chu P, Rodriguez AR, Arulanandam BP, Klose KE. Tryptophan
prototrophy contributes to Francisella tularensis evasion of gamma
interferon-mediated host defense. Infect Immun 2011;79:
2356 61.
[172] Peng K, Monack DM. Indoleamine 2,3-dioxygenase 1 is a lung-
specific innate immune defense mechanism that inhibits growth
of Francisella tularensis tryptophan auxotrophs. Infect Immun
2010;78:2723 33.
[173] Rohmer L, Fong C, Abmayr S, Wasnick M, Larson Freeman TJ,
Radey M, et al. Comparison of Francisella tularensis genomes
reveals evolutionary events associated with the emergence of
human pathogenic strains. Genome Biol 2007;8:R102.
[174] De Pascalis R, Taylor BC, Elkins KL. Diverse myeloid and lym-
phoid cell subpopulations produce gamma interferon during early
innate immune responses to Francisella tularensis live vaccine
strain. Infect Immun 2008;76:4311 21.
[175] Leiby DA, Fortier AH, Crawford RM, Schreiber RD, Nacy CA.
In vivo modulation of the murine immune response to
Francisella tularensis LVS by administration of anticytokine
antibodies. Infect Immun 1992;60:84 9.
[176] Boehm U, Klamp T, Groot M, Howard JC. Cellular responses to
interferon-gamma. Annu Rev Immunol 1997;15:749 95.
[177] Virgin HW, Levine B. Autophagy genes in immunity. Nat
Immunol 2009;10:461 70.
[178] Elkins KL, Rhinehart-Jones TR, Culkin SJ, Yee D, Winegar RK.
Minimal requirements for murine resistance to infection with
Francisella tularensis LVS. Infect Immun 1996;64:3288 93.
[179] Woolard MD, Wilson JE, Hensley LL, Jania LA, Kawula TH,
Drake JR, et al. Francisella tularensis-infected macrophages
release prostaglandin E2 that blocks T cell proliferation and pro-
motes a Th2-like response. J Immunol 2007;178:2065 74.
[180] Wilson JE, Katkere B, Drake JR. Francisella tularensis induces
ubiquitin-dependent major histocompatibility complex class II deg-
radation in activated macrophages. Infect Immun 2009;77:4953 65.
[181] Bosio CM, Dow SW. Francisella tularensis induces aberrant
activation of pulmonary dendritic cells. J Immunol 2005;175:
6792 801.
[182] Elkins KL, Cooper A, Colombini SM, Cowley SC, Kieffer TL.
In vivo clearance of an intracellular bacterium, Francisella tular-
ensis LVS, is dependent on the p40 subunit of interleukin-12
(IL-12) but not on IL-12 p70. Infect Immun 2002;70:1936 48.
[183] O’Farrell AM, Liu Y, Moore KW, Mui AL. IL-10 inhibits mac-
rophage activation and proliferation by distinct signaling
mechanisms: evidence for Stat3-dependent and -independent
pathways. EMBO J 1998;17:1006 18.
[184] Mahnke K, Schmitt E, Bonifaz L, Enk AH, Jonuleit H.
Immature, but not inactive: the tolerogenic function of immature
dendritic cells. Immunol Cell Biol 2002;80:477 83.
[185] Fontenot JD, Rudensky AY. A well adapted regulatory contriv-
ance: regulatory T cell development and the forkhead family
transcription factor Foxp3. Nat Immunol 2005;6:331 7.

[186] Cyktor JC, Turner J. Interleukin-10 and immunity against pro-
karyotic and eukaryotic intracellular pathogens. Infect Immun
2011;79:2964 73.
[187] Fortier AH, Slayter MV, Ziemba R, Meltzer MS, Nacy CA. Live
vaccine strain of Francisella tularensis: infection and immunity
in mice. Infect Immun 1991;59:2922 8.
[188] Conlan JW, Vinogradov E, Monteiro MA, Perry MB. Mice
intradermally-inoculated with the intact lipopolysaccharide, but
not the lipid A or O-chain, from Francisella tularensis LVS rap-
idly acquire varying degrees of enhanced resistance against sys-
temic or aerogenic challenge with virulent strains of the
pathogen. Microb Pathog 2003;34:39 45.
[189] Wu TH, Hutt JA, Garrison KA, Berliba LS, Zhou Y, Lyons CR.
Intranasal vaccination induces protective immunity against intra-
nasal infection with virulent Francisella tularensis biovar A.
Infect Immun 2005;73:2644 54.
[190] Shen H, Chen W, Conlan JW. Susceptibility of various mouse
strains to systemically- or aerosol-initiated tularemia by virulent
type A Francisella tularensis before and after immunization with
the attenuated live vaccine strain of the pathogen. Vaccine
2004;22:2116 21.
[191] Kieffer TL, Cowley S, Nano FE, Elkins KL. Francisella novicida
LPS has greater immunobiological activity in mice than F. tular-
ensis LPS, and contributes to F. novicida murine pathogenesis.
Microbes Infect 2003;5:397 403.
[192] Eigelsbach HT, Tulis JJ, McGavran MH, White JD. Live tulare-
mia vaccine I. : Host-Parasite Relationship in Monkeys
Vaccinated Intracutaneously or Aerogenically. J Bacteriol
1962;84:1020 7.
[193] White JD, McGavran MH, Prickett PA, Tulis JJ, Eigelsbach
HT. Morphologic and immunohistochemical studies of the path-
ogenesis of infection and antibody formation subsequent to vac-
cination of Macaca irus With an Attenuated Strain of
Pasteurella tularensis: II. Aerogenic Vaccination. Am J Pathol
1962;41:405 13.
Chapter | 108 Francisella 2009
[194] Schricker RL, Eigelsbach HT, Mitten JQ, Hall WC. Pathogenesis
of tularemia in monkeys aerogenically exposed to Francisella
tularensis 425. Infect Immun 1972;5:734 44.
[195] Lyons CR, Wu TH. Animal models of Francisella tularensis
infection. Ann N Y Acad Sci 2007;1105:238 65.
[196] Vonkavaara M, Telepnev MV, Rydén P, Sjöstedt A, Stöven S.
Drosophila melanogaster as a model for elucidating the
pathogenicity of Francisella tularensis. Cell Microbiol
2008;10:1327 38.
[197] Shapiro DS, Schwartz DR. Exposure of laboratory workers to
Francisella tularensis despite a bioterrorism procedure. J Clin
Microbiol 2002;40:2278 81.
[198] Burke DS. Immunization against tularemia: analysis of the effec-
tiveness of live Francisella tularensis vaccine in prevention of
laboratory-acquired tularemia. J Infect Dis 1977;135:55 60.
[199] Eigelsbach HT, Downs CM. Prophylactic effectiveness of live
and killed tularemia vaccines. I. Production of vaccine and eval-
uation in the white mouse and guinea pig. J Immunol
1961;87:415 25.
[200] Eneslatt K, Rietz C, Ryden P, Stoven S, House RV, Wolfraim
LA, et al. Persistence of cell-mediated immunity three decades
after vaccination with the live vaccine strain of Francisella tular-
ensis. Eur J Immunol 2011;41:974 80.
[201] Cowley SC, Elkins KL. Immunity to Francisella. Front
Microbiol 2011;2:26.
[202] Ericsson M, Sandström G, Sjöstedt A, Tärnvik A. Persistence of
cell-mediated immunity and decline of humoral immunity to the
intracellular bacterium Francisella tularensis 25 years after natu-
ral infection. J Infect Dis 1994;170:110 4.
[203] Wayne Conlan J, Oyston PC. Vaccines against Francisella tular-
ensis. Ann N Y Acad Sci 2007;1105:325 50.
[204] Hornick RB, Eigelsbach HT. Aerogenic immunization of man
with live Tularemia vaccine. Bacteriol Rev 1966;30:532 8.
[205] Marohn ME, Barry EM. Live attenuated tularemia vaccines:
Recent developments and future goals. Vaccine 2013;31:3485 91.