Virulence difference between the prototypic Schu S4 strain (A1a) and Francisella tularensis A1a, A1b, A2 and type B strains in a murine model of infection

ArticleinBMC Infectious Diseases 14(1):67 · February 2014with26 Reads
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Abstract

The use of prototypic strains is common among laboratories studying infectious agents as it promotes consistency for data comparability among and between laboratories. Schu S4 is the prototypic virulent strain of Francisella tularensis and has been used extensively as such over the past six decades. Studies have demonstrated virulence differences among the two clinically relevant subspecies of F. tularensis, tularensis (type A) and holarctica (type B) and more recently between type A subpopulations (A1a, A1b and A2). Schu S4 belongs to the most virulent subspecies of F. tularensis, subspecies tularensis. In this study, we investigated the relative virulence of Schu S4 in comparison to A1a, A1b, A2 and type B strains using a temperature-based murine model of infection. Mice were inoculated intradermally and a hypothermic drop point was used as a surrogate for death. Survival curves and the length of temperature phases were compared for all infections. Bacterial burdens were also compared between the most virulent type A subpopulation, A1b, and Schu S4 at drop point. Survival curve comparisons demonstrate that the Schu S4 strain used in this study resembles the virulence of type B strains, and is significantly less virulent than all other type A (A1a, A1b and A2) strains tested. Additionally, when bacterial burdens were compared between mice infected with Schu S4 or MA00-2987 (A1b) significantly higher burdens were present in the blood and spleen of mice infected with MA00-2987. The knowledge gained from using Schu S4 as a prototypic virulent strain has unquestionably advanced the field of tularemia research. The findings of this study, however, indicate that careful consideration of F. tularensis strain selection must occur when the overall virulence of the strain used could impact the outcome and interpretation of results.

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Available from: Brook M Yockey, Apr 10, 2014
RES E AR C H A R T I C L E Open Access
Virulence difference between the prototypic Schu
S
4
strain (A1a) and Francisella tularensis A1a, A1b,
A2 and type B strains in a murine model of
infection
Claudia R Molins
1*
, Mark J Delorey
1
, Brook M Yockey
1
, John W Young
1
, John T Belisle
2
, Martin E Schriefer
1
and Jeannine M Petersen
1
Abstract
Background: The use of prototypic strains is common among laboratories studying infectious agents as it promotes
consistency for data comparability among and between laboratories. Schu S
4
is the prototypic virulent strain of
Francisella tularensis and has been used extensively as such over the past six decades. Studies have demonstrated
virulence differences among the two clinically relevant subspecies of F. tularensis, tularensis (type A) and holarctica
(type B) and more recently between type A subpopulations (A1a, A1b and A2). Schu S
4
belongs to the most virulent
subspecies of F. tularensis,subspeciestularensis.
Methods: In this study, we investigated the relative virulence of Schu S
4
in comparison to A1a, A1b, A2 and type B
strains using a temperature-based murine model of infection. Mice were inoculated intradermally and a hypothermic
drop point was used as a surrogate for death. Survival curves and the length of temperature phases were compared for
all infections. Bacterial burdens were also compared between the most virulent type A subpopulation, A1b, and Schu
S
4
at drop point.
Results: Survival curve comparisons demonstrate that the Schu S
4
strain used in this study resembles the virulence of
type B strains, and is significantly less virulent than all other type A (A1a, A1b and A2) strains tested. Additionally, when
bacterial burdens were compared between mice infected with Schu S
4
or MA00-2987 (A1b) significantly higher burdens
were present in the blood and spleen of mice infected with MA00-2987.
Conclusions: The knowledge gained from using Schu S
4
as a prototypic virulent strain has unquestionably advanced
the field of tularemia research. The findings of this study, however, indicate that careful consideration of F. tularensis
strain selection must occur when the overall virulence of the strain used could impact the outcome and interpretation
of results.
Keywords: Prototypic strains, Murine model, Fever, Francisella tularensis,Tularemia,SchuS
4
,A1a
Background
Tularemia, a disease caused by the Gram-negative bacter-
ium Francisella tularensis, was first confirmed as a human
infection in 1913 in a 21 year-old meat cutter from Ohio
who developed ulcerative conjunctivitis and lymphadenitis
after handling infected meat [1]. Approximately 25 years
later, Foshay isolated F. tularensis (previously named
Bacterium tularense) strain S chu f rom a finger ulcer of
a patient in Ohio [2]. Virulence studies conducted in
variousanimalmodelsshowedthatthisstrain(alsore-
ferred to as strain Sm) was of high virulence with an
LD
100
in rabbits of 1100 bacterial cells [3-5]. Add-
itionally, time to d eath experiments in guinea pigs and
mice re vea led that animals infected with this strain died
earlier than those infected with a type B strain by one
day when infe cting mice and 3 to 4 days when infecting
* Correspondence: CMolins@cdc.gov
1
Division of Vector-Borne Diseases, Centers for Disease Control and Prevention,
Bacterial Diseases Branch, 3156 Rampart Road, Fort Collins, CO 80521 USA
Full list of author information is available at the end of the article
© 2014 Molins et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
Molins et al. BMC Infectious Diseases 2014, 14:67
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Page 1
guinea pigs [4]. In 1951, Eigelsbach et al. conducted a
study using the Schu strain to determine if F. tularensis
colony morphology correlated with pathogenicity and
immunogenic properties [6]. This study revealed colony
morphologies that varied in co lor (buff, blue or grey),
texture (smooth o r nonsmooth) and LD
100
(range from
110 to avi rulent). One colony type, designated Schu
S
4,
for smooth (S) variant clone #4, was described as a
smooth blue colony with a watery consistency, and
highly virulent in mice with an LD
100
of 110 organisms.
Schu S
4
has subsequently served as the representative
strain for pathogenic studies of F. tularensis subspecies
tularensis (type A), the most virulent of the three F. tular-
ensis subspecies, tularensis (type A), holarctica (type B)
and mediasiatica. Given its extensive use as a prototypic
type A strain, the complete genome for Schu 4 was
sequenced in 2005 [7].
Within the last decade, it has become increasingly
clear that type A strains do not represent a uniform
population. Multi-locus varia ble number tandem repeat
(MLVA), pulsed field gel electrophoresis (PFGE), whole
genome single nucleotide polymorphism (SNP) and whole
genome sequencing have all been used to demonstrate a
major split into two subpopulations, A.I (A1) and A.II
(A2) [8-13]. Additional PFGE analyses have further identi-
fied two A1 subpopulations, A1a and A1b [14]. Although
type A strains are well documented to be more virulent
than type B strains (LD
100
in rabbits of 1100 bacterial
cells for type A strains as compared to an LD
100
of 10
9
for
type B strains) [4,15], more recent studies have revealed
differing levels of virulence between the type A subpopula-
tions A1a, A1b and A2 [14,16,17]. An epidemiological
analysis of culture-confirmed human tulare mia in t he
United States showed that infec tions caused by A1b
strains resulted in significantly higher mortality (24%)
than infections caused by A1a (4%) or A2 (0%). Logistic
regression analysis of A1b infections further indicated
that this higher mortality rate was not linked to host
characteristics , suggesting tha t A1b strains have an
intrinsic characteristic which makes them more virulent
than A1a or A2 strains [14]. Additionally, a virulence
study using a m urine model of infection found that
mice infected with A1b died significantly earlier than mice
infected with A1a or A2 [16]. These result s c orrelate
with epidemiological findings for type A infections in
humans and demonstrate the utility of murine models
for identifying virulence differences among F. tularensis
type A subpopulations.
Schu S
4
has been classified by PFGE and SNP analysis
as a type A strain belonging to the A1a and A.I.Br.SCHU
S4 subpopulations, respectively [13,14] indicating the
prototypic type A strain does not fall within the PFGE
group of type A strains with highest virulence. Previous
analyses indicate mice are a suitable system to monitor
differences in virulence if survival curves opposed to LD
50
are analyzed [16-18]. Additionally, most studies requiring
an animal model for the study of F. tularensis are con-
ducted in mice due to their ease of use. We therefore
investigated the relative virulence of S chu S
4
in com-
parison to A1a, A1b, A2 and type B strains isolated from
more recent human cases using a murine model of infec-
tion and generated survival curves based on subcutaneous
temperature measurements [18]. Our results re vealed
that the Schu S
4
strain used in this study most closely
resembles the virulence of type B strains, and is less
virulent than all other type A strains tested here; A1a,
A1b, and A2 strains.
Methods
Strains and culture conditions
The Schu S
4
strain tested here is available through BEI
(catalog number NR-643) and was de posited by the
Centers for Disease Control. Schu S
4
was grown from
frozen stocks (70°C) on cysteine heart agar supplemented
with 9% sheep blood (CHAB) at 35°C for 48 h, followed by
subculture onto CHAB for 24 h at 35°C. A bacterial sus-
pension for inoculations was prepar ed in sterile saline. Col-
ony forming units (CFU) in the inoculum was verified by
spotting 50 μl of the inoculum onto each quadrant of
two CHAB quad plates (8 replicates total) and letting
the plates d ry without sprea ding. CFU were cou nted
after 4872 hours of growth at 35°C. The Schu S
4
strain
used in this study was typed using PFGE as previously
described [11,14].
Additional F. tularensis strains (n = 8) (see Additional
file 1), two A1a (MO02 -4195 and OK01-2528), two A1b
(MA00-2987 and MD00-2970), two A2 (WY96-3418
and NM99-1823), and two type B (KY99-3387 and
MI00-1730) strains, used in this study were described
previously [16,18]. Several of these strains are available
through BEI (WY96-3418, MA00-2987 and KY99-3387,
and their respective catalog numbers are NR-644, NR-
645 and NR-647).
Mice and experimental protocol
Specific pathogen-free female C57BL/6 J mice, (The
Jackson La boratory, Bar Harbor, ME) 89 weeks of age,
were used. For Schu S
4
infections , mice (n = 7) were
anesthetized by inhalation of isoflurane to effe ct and
infected intradermally with 1020 CFU (50 μl) via the
tail dermis. Control mice (n = 7) were inoculated with
50 μl of saline. Seven mice were infe cted with Schu S
4
to ensure sufficient power t o detect a sc ale shift in the
fitted survival cur ve corresponding to a mean survival
time of at least one day. The temperature of infec ted
mice wa s monitored every 1 2 hours (minimum of 12
observations per day) until they reached their drop point.
The drop point is the time at which a febrile mouses
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temperature first drops below the mean temperature of
the normal (early afebrile) phase [18]. Therefore, the mean
temperature of the normal phase was determined for each
mouse and mice were euthanized once the drop point was
observed. Mice were given food at libitum and an exercise
wheel was provided in every cage. All animal procedures
were approved by the Division of Vector-Borne Infectious
Diseases Institutional Animal Care and Use Committee
(protocol number 08012) and performed in accordance
with the guidelines on the care and use of laboratory ani-
mals [19]. All animal experiments with F. tularensis were
conducted in ABL3 facilities. Data for mice infected with
strains representing each of the four F. tularensis subpop-
ulations (A1a, A1b, A2 and type B) was previously pub-
lished [16,18]. Mice were infected with Schu S
4
during
Round 2 of the previously published experiments [16].
In Round 2 experiments , seven mice were infected with
strains MA00-2987 (A1b), MO02-419 5 (A1a), NM99 -
1823 (A2 ) and MI00-1730 (type B ), in addition to the
seven control mice and se ven Schu S
4
infected mice de-
scribed here. An additional infection of se ven mice with
MA00-2987 was also performed and these mice were
euthanized at drop point.
Comparative virulence
Survival times for Schu S
4
infected mice were modeled
according to a Weibull distribution (allowing both the
scale and shape parameters to differ by strain) using the
drop point as a surrogate of death. Previous experiments
demonstrated that statistical inference from a compari-
son of survival curves generated for F. tularensis strains
based on drop point were the same as those obtained
using observed time to death [18]. Additionally, drop
point is an ethical experimental endpoint, as mice are
still responsive to stimuli and taking food and water.
Standard diagnostics including residual plots and good-
ness of fit tests were used to validate the model fits. The
survival curve gener ated for Schu S
4
infected mice was
compared to survival curves previously generated for
eight F. tularensis strains; two A1a, two A1b, two A2
and two type B [ 16,18]. Survival curves for F. tularensis
A1a, A1b, A2 and type B infected mice were also fitted
to drop points, although mice were allowed to expire in
order to establis h and validate the drop point temp-
erature model [18]. Differences in the parameter estimates
(shape and scale) of the survival curves were considered
statistically significant if p < 0.008, with this level of signifi-
cance determined using the Bonferroni adjustment for
multiple comparisons.
Survival function parameters were simulated from the
estimated parameter distributions. From these, 10,000
sur vival times were simulated a nd used to compute the
probability that a mouse infected with one strain fails
before a mouse infected with a second strain for each
F. tularensis group (A1a , A1b, A2 and type B a s well as
Schu S
4
). This was done 1,000 times to create distributions
for the estimated probabilities from which 95% confidence
inter vals were obtained.
Comparisons between the lengths of time spent in the
normal and febrile phases by Schu S
4
infected mice and
mice infected with A1a, A1b, A2 and type B strains were
performed using a weighted ANOVA followed by mul-
tiple comparisons using Tukeys method with an overall
Type I error of 0.05.
Quantitative bacteriology and mouse and organ weights
Mice infected with Schu S
4
(A1a) were euthanized at
drop point and whole organs (spleen, liver and lungs)
and blood were removed aseptically. Whole organs and
blood were also taken from control, non-infected mice
and from mice infected with strain MA00-2987 (A1b) and
euthanized at drop point [16,18]. Each organ was weighed
and sterile saline was added (5 ml to spleen and lungs
and 10 ml to liver) prior to homogenization using a
Stomacher 80 micro Biomaster (Seward, Bohemia, NY).
Homogenized samples were serially diluted in sterile saline
and aliquots (50 μl) were spotted in duplicate onto CHAB
agar quadrant plates without spreading. Colonies were
counted after 4872 h of growth at 35°C. Whole blood
was collected from the abdominal aorta and diluted into
sterile saline and plated as described [16]. Bacterial load
comparisons were performed using MANOVA with an
overall Type I error rate of α = 0.05 and confidence inter-
vals for Pearsons correlation were computed using Fishers
z-transformation. Mice were weighed prior to infection
and at drop point.
Results
Virulence comparison between Schu S
4
and other
A1a strains
The estimated shape (i.e. symmetry of pattern of time to
drop point) and scale (i.e. mean and variability of time
to drop point) parameters for the survival curve for mice
infected with S chu S
4
were compared to those from
sur vival curves estimated previously for mice infected
with two other A1a strains, OK01-2528 and MO02-4195
(Figure 1). Survival curves were generated using drop
point a s t he surrogate for death. Drop point has been
previously described and is the f irst temperature below
the mean temperature of the normal phase [18]. Surpris-
ingly, a significant difference (p < 0.008) was observed in
both the scale and shape parameters for survival curves of
mice infected with OK01-2528 or MO02-4195 compared
to Schu S
4
. In comparison, as previously demonstrated,
there was no difference (p > 0.008) between the scale and
shape parameter for survival curves of mice infected with
OK01-2528 and MO02-4195 [16,18 ]. The shift of the
survival cur ve reflected that mice infected with Schu S
4
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reached drop point significantly later than mice infected
with other A1a strains, while the shape of the curve indi-
cated that mice infected with Schu S
4
reached drop point
within a significantly narrower timeframe as compared to
mice infected with the two other A1a strains. As shown in
Figure 1, mice infected with A1a strains OK01-2528 and
MO02-4195 had all reached their drop point as mice
infected with Schu S
4
were beginning to reach drop point.
Additionally, temperature measurements showed that the
length of time in the normal phase (non-febrile, asymp-
tomatic phase) was statistically longer for Schu S
4
infected
mice as compared to A1a infected mice by 27 hours (95%
CI 12 to 42 hours) on average. Although the length of the
febrile phase was not statistically different between Schu
S
4
and A1a infected mice, Schu S
4
infected mice were in
the febrile phase slightly longer (average of 47 hours) than
A1a infected mice (average of 40 hours) [18]. At the time
of infections, the average inoculation doses were deter-
mined to be similar among all three A1a strains, at 16 ±
3 CFU for OK01-2528, 11 ± 4 CFU for MO02-4195 and
14 ± 2 CFU for Schu S
4
, indicating that the significant
differences observed for the Schu S
4
survival curve were
not directly attributable to dosage disparities [16].
Virulence differences between F. tularensis strain Schu S
4
and F. tularensis A1b, A2 and type B strains
To determine if statistical differences in survival were
also observed between Schu S
4
and other type A as well
as type B strains, the survival curve for Schu S
4
infected
mice was compared to survival curves for mice infected
with F. tularensis A1b, A2 or type B strains (Figure 2)
[16,18]. Two strains were used for each of the three
F. tularensis groups; A1b, A2 and type B, with resu lts
combined to generate one sur vival curve per group.
For reference, the combined survival cur ve for the two
A1a strains from Fi gure 1 is shown (Figure 2).
Significant differences (p < 0.008) were observed in both
the scale and shape parameters of the survival curve for
Schu S
4
infected mice when compared to the survival
curves of mice infected with either A1b or A2 strains, and
in the shape parameter when compared to the survival
curve of mice infected with type B strains (Figure 2).
There was no significant difference in the scale parameters
between the survival curves of mice infected with Schu S
4
as compared to type B strains (0.05 < p < 0.10). These data
indicate that the mice infected with Schu S
4
reached drop
point significantly later than mice infected with A1b or A2
strains and that there was no statistical difference in the
time to drop point between mice infected with Schu S
4
as
compared to type B strains. Additionally, as observed in
the comparison with A1a strains, mice infected with Schu
S
4
reached drop point within a significantly narrower
timeframe as compared to mice infected with A1b, A2 or
type B strains.
Estimated survival distributions were used to determine
whether mice infected with Schu S
4
are likely to reach
drop point after mice infected with A1a, A1b, A2 or type
B strains (Table 1). The probability of a mouse infected
with A1a, A1b or A2 strains reaching drop point before a
mouse infected with Schu S
4
was found to be 0.99, 0.99
and 0.79, respectively. This probability dropped to 0.33 for
mice infected with type B strains.
Time to drop point (hours)
Proportion surviving
0 50 100 150 200 250
Schu S4
OK01-2528
MO02-4195
Figure 1 Survival curve comparison of mice infected with A1a strains OK01-2528, MO02-4195, and Schu S
4
. C57BL/6 J mice (n = 7/strain)
were challenged intradermally with 1020 CFU of F. tularensis A1a strains OK01-2528 ( III ; red), MO02-4195 (;blue)andSchuS
4
(▬▬; orange) and
survival curves were modeled according to a Weibull dist ribution. The drop point for ea ch mouse is shown in orange circl es for mice
infected with st rain Schu S
4
, red tria ngles for mice infect ed with OK01-2528 and blu e squa res for mi ce inf ected with MO02-4195.
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Differences in the length of time that mice spent in
the normal and febrile phase of infection [18] revealed
that the normal phase was statistically longer for mice
infected with Schu S
4
as compared to mice infected with
A1b by an average of 42 hours (95% CI 18 to 65 hours). No
statistical differences were observed between the normal
phases of Schu S
4
, A2 and type B infected mice. When the
length of time in the fever stage was compared, no statis-
tical differences between Schu S
4
and A1b, A2 or type B
were observed. Similar to Schu S
4
infected mice, the aver-
age time that type B infected mice were in the normal
phase differed significantly from that of A1a and A1b in-
fected mice. Type B infected mice were in the normal phase
on average 47 hours (95% CI 15 to 80 hours) longer com-
pared to A1a infected mice and on average 62 hours (95%
CI 25 to 99 hours) longer compared to A1b infected mice.
Bacterial load comparison between mice infected with
A1a strain Schu S
4
and A1b strain MA00-2987
Because F. tularensis A1b strains are the most virulent
among type A strains and Schu S
4
is used as a prototypic
virulent type A strain, the bacterial burden at drop point
was quantified within the blood, spleen, liver and lungs of
mice infected with Schu S
4
(A1a) and MA00-2987 (A1b)
(Figure 3). The mean bacterial burden was statistically
higher in the blood and spleen (p = 0.004 and p < 0.0001,
respectively) of mice infected with the A1b strain MA00-
2987 as compared to mice infected with the A1a strain
Schu S
4
. No statistical difference was observed between
the bacterial burden in the lungs and liver of mice infected
with these two strains (p = 0.21 and p = 0.72, respectively).
Calculations to determine whether the bacterial burden
within the blood and spleen of Schu S
4
and MA00-2987
infected mice correlated to drop point revealed only a
moderate correlation of 0.62 (95% CI 0.13 to 0.87) be-
tween the bacterial burden in the spleen and drop point
in S chu S
4
infected mice. Whe n comparing mouse and
organ weight between animals infected with MA00-
2987 and Schu S
4
, no significant differences were found
inboththeweightatdroppoint(p=0.58)andthe
weight of all organs (p = 0.10). The average inoculation
doses were determined to be similar for mice infec ted
with Schu S
4
and MA00-298 7 with inoculation doses of
14 ± 2 CFU and 14 ± 3 CFU, respectively.
Discussion
Schu S
4
serves as the prototypic type A strain for much
of the current F. tularensis research being performed
and is often classified as fully virulent. It has been dis-
tributed internationally among laboratories and the main-
tenance and propagation history among laboratories is
unknown. Moreover, any laboratory adaptation this strain
Time to drop point (hours)
Proportion surviving
0 50 100 150 200 250
A1a
A1b
A2
Type B
Schu S4
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Figure 2 Comparison of fitted survival curves for mice infected with F. tularensis A1a, A1b, A2, type B and Schu S
4
. Survival curves were
modeled previously [16,18] according to a Weibull distribution for mice (n = 7/strain) infected intradermally with 1020 CFU of two strains each of A1a
(; black), A1b ( I ;blue),A2(▬▬; red), and type B ( III ; green), and are compared to the survival curve of Schu S
4
infected mice (III ; orange).
The time at which each mouse reached drop point is shown below the graph as black squares for A1a infected mice, blue circles for A1b infected
mice, red triangles for A2 infected mice, green diamonds for type B infected mice and orange upside-down triangles for Schu S
4
infected mice.
Table 1 Probability of A1a, A1b, A2 and type B infected
mice reaching drop point before Schu S
4
infected mice
Event Probability Lower 95% CI
a
Upper 95% CI
A1a versus Schu S
4
0.99 0.95 1.00
A1b versus Schu S
4
0.99 0.86 0.99
A2 versus Schu S
4
0.79 0.59 0.93
Type B versus Schu S
4
0.33 0.14 0.53
a
CI: Confidenc e interval.
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has undergone over the 60+ years since it was isolated, or
genomic variation between Schu S
4
strains of different
laboratories is undetermined as only a single strain,
Schu 4, has been sequenced at the whole genome level [7].
Our present data show that this particular Schu S
4
strain
is significantly less virulent than more recent clinical A1a
strains as well as A1b and A2 strains. Beyond PFGE typing
[14], limited studies have been performed to compare
the virulence and genetic make-up between A1a strains;
however, virulence differences between A1a and A1b
strains have been reported [17]. Twine et al. previously
demonstrated a significant difference in time to death for
BALB/c mice infected intradermally or via aerosol with
Schu S
4
as compared to the type A strain, FSC033 [17].
FSC033 falls into the same canSNP group A.1.Br.001/002
as the two A1b strains tested here, MA00-2987 and
MD00-2970. Spe cifically, mice infected with FSC033,
expired significantly earlier than mice infe cted with
Schu S
4
(FSC237; Francisella Strain Collection, Swedish
Defence Research Agency, Umea , Sweden). Thus, the
difference in virulence between Schu S
4
and other type
A strains appears to be conserved among Schu S
4
strains
from different laboratories and within two mouse strains.
The Schu S
4
strain (FSC237) used in Twine et al. was the
source of DNA for the published Schu S
4
genome se-
quence. Twine et al. also reported unsuccessful attempts
to enhance the virulence of Schu S
4
by passage in mice;
after five animal passages there was no change in the
mean time to death for the Schu S
4
strain. Together these
results suggest that there is a fixed genetic basis respon-
sible for the decreased virulence observed for Schu S
4
.
Surprisingly, the S chu S
4
strain demonstrated a viru-
lence phenotype with greater similarity to type B than to
type A clinical stra ins. LD
50-100
experiments in rabbits
and time to death experiments in mice or guinea pigs
are lacking in the published literature for Schu S
4
,despite
its use as a prototypic type A strain. F. tularensis Schu S
4
was reported as being highly virulent based solely on an
LD
100
of 110 organisms in mice [6]. However, Bell and
Olsufiev both demonstrated that virulence of F. tularensis,
derived from LD
50
determinations, in mice does not differ-
entiate between type A and type B; both subspecies show
an LD
50
of 1 [4,20]. As no direct virulence comparisons
(LD
50
in rabbits or time to death differences in mice) have
been reported in the literature, it is unknown if the viru-
lence phenotype of the original Schu strain differs from
that of Schu S
4
.
Schu S
4
infected mice reached drop point in a signifi-
cantly narrower timeframe as compa red to mice infected
with A1a, A1b, A2 and type B strains. Mice infected with
A1a, A1b, A2 and type B consistently reached their drop
point during two time frames as opposed to just one
time frame as observed with Schu S
4
infected mice. We
hypothesize this is reflective of Schu S
4
being a single
colony selected from the original clinical isolate and la-
boratory adapted over time as compared to more recent
clinical isolates with minim al laboratory manipulation. A
few of the strains (recent clinical isolates) used in this
study are also single colony picks. However, we tested
whether this resulted in a selected phenoty pe by doing
a side-by-side comparison of survival cur ves generated
from mice infected with a single colony pick of the
MA0 0-2987 s train and the original M A0 0-2987 isolate.
There were no statistical differences between the two
survival cur ves generated for these two infections (data
not shown).
Figure 3 Bacterial burden within the blood, spleen, liver and lungs of infected mice with A1a strain Schu S
4
and A1b strain
MA00-2987. C57BL/6 J mice (n = 7/strain) were challenged intradermally with 1020 CFU of F. tularensis strain MA00-2987 (A1b) and Schu S
4
(A1a). When mice reached drop point, organs were harvested and blood was taken from each mouse. Significant differences in bacterial burden
between the two infecting strains were calculated using MANOVA with an overall Type I error rate of α =0.05 and are designated with an asterisk.
The mean bacterial burden is shown as a bar.
Molins et al. BMC Infectious Diseases 2014, 14:67 Page 6 of 8
http://www.biomedcentral.com/1471-2334/14/67
Page 6
The mechanism for the difference in virulence of Schu
S
4
is unknown. However, our results indicate that bacterial
burden at drop point was significantly higher in the blood
and spleen of mice infected with the A1b strain, MA00-
2987 as compared to mice infected with Schu S
4.
A similar
observation was noted by Twine et al. who found that the
FSC033 strain was better able to disseminate than Schu S
4
from the original inoculation site to the spleen of infected
mice [17]. Additionally, we previously showed that the
bacterial burden in the blood and spleen at time to death
did not differ between mice infected with A1a and A1b
strains [16]. This suggests that the mechanism of bacterial
dissemination may be altered in the Schu S
4
strain as
compared to MA00-2987 and other A1a strains. We
also observed that Schu S
4
infected mice remained in the
normal phas e ( asymptomatic) significantly longer than
mice infected with A1a or A1b clinical strains. Interest-
ingly, type B infected mice which had a survival curve
statistically indifferent (based on scale) from Schu S
4
infected mice also displayed a longer normal phase length
that wa s statistically different f rom those of A1a and
A1b infecte d mice. Further studies are needed to better
understand the virulence me chanisms of Schu S
4
that
differ from A1a and A1b strains and to determine how
they compare to those of A2 and type B strains.
Overall, these findings indicate that infection of C57BL/
6 J mice with Schu S
4
does not completely mimic an infec-
tion with other type A strains. While the findings with
Schu S
4
in this study and in the Twine et al. study agree,
it should be noted that the Schu S
4
strain used here may
not be equivalent to other S chu S
4
strains distributed
amongst various research institutions. Although the use
of prototypic strains are important for consistency among
research, the strain used should be appropriate for the
work being performed and researchers need to acknow-
ledge the limitations associated with this prototypic strain.
Conclusions
In summary, we have evaluated the relative virulence of
Schu S
4
as compared to more recent A1a, A1b, A2 and
type B clinical isolates of F. tularensis using a murine
model. We demonstrated that Schu S
4
is less virulent as
compared to other type A strains and more closely re-
sembles the virulence of type B strains. Schu S
4
is still
the prototypic pathogenic strain used and although this
strain has a permanent role for such research, the obser-
vations from this study call into question the benefits of
using more recent F. tularensis type A stra ins in addition
to using the S chu S
4
strain. The use of Schu S
4
for
studying F. tularensis has a dvantages, including the ex-
pediting of research because this strain is more readily
available than non-prototypic strains, Schu S
4
is well
characterized and the data that results from the use of this
strain can be directly compared between laboratories.
However, the choice of Schu S
4
as a fully virulent strain
and as the sole type A challenge strain in murine models
that evaluate the efficacy of human vaccine and therapeu-
tics against F. tularensis infection, should be reconsidered.
Additional file
Additional file 1: This file contains a table that lists the F. tularensis
strains used in this study.
Abbreviations
CFU: Colony forming units; CHAB: Cysteine heart agar supplemented with
9% sheep blood; CI: Confidence interval; LD: Lethal dose; MLVA: Multi-locus
variable number tandem repeat; PFGE: Pulsed field gel electrophoresis;
SNP: Single nucleotide polymorphism.
Competing interests
The authors declare that they do not have any competing interests.
Authors contributions
CRM performed the animal work, data analyses and drafted the manuscript.
MJD performed the statistical analysis. BMY assisted with the mouse work
and performed the culture work. JWY assisted in the design of the study and
helped perform the mouse work. JTB assisted in the design of the study and
helped draft the manuscript. MES assisted in the design of the study. JMP
assisted in the design of the study and helped draft the manuscript. All
authors read and approved the final manuscript.
Acknowledgements
The authors would like to thank Animal Care Staff for their support with the
animals. Claudia Molins was funded by the American Society for Microbiology
and the Coordinating Center for Infectious Diseases as a postdoctoral fellow
while the work was being performed. Funding for John Belisle was provided by
the National Institutes of Health, National Institute of Allergy and Infectious
Diseases grant number U54AIO65357-01. The findings and conclusions in this
report are those of the authors and do not necessarily represent the views of
the Centers for Disease Control and Prevention or the Department of Health
and Human Services.
Author details
1
Division of Vector-Borne Diseases, Centers for Disease Control and Prevention,
Bacterial Diseases Branch, 3156 Rampart Road, Fort Collins, CO 80521 USA.
2
Department of Microbiology, Immunology, and Pathology, Colorado State
University, Fort Collins, CO 80523 USA.
Received: 26 September 2013 Accepted: 28 January 2014
Published: 6 February 2014
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doi:10.1186/1471-2334-14-67
Cite this article as: Molins et al.: Virulence difference between the
prototypic Schu S
4
strain (A1a) and Francisella tularensis A1a, A1b, A2
and type B strains in a murine model of infection. BMC Infectious Diseases
2014 14:67.
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Page 8
    • "Because of its low infectious dose, multiple routes of infection, and high morbidity and mortality rates, F. tularensis has been designated as a Tier 1 Select Agent by the U.S. Centers for Disease Control and Prevention (CDC), highlighting concerns over its potential use a bioterrorism agent (Dennis et al., 2001). Type A strains are the most virulent (ID 50 < 10 CFU via multiple infection routes in many animals, including humans; Ellis et al., 2002; Molins et al., 2010), with the human ulcer isolate, Schu S4, being the most commonlyused strain in BSL3 laboratories (Molins et al., 2014). Type B strains are highly-infectious to mice and guinea pigs (pulmonary and intradermal ID 50 < 10 CFU; Ellis et al., 2002; Molins et al., 2010) but higher doses are needed to infect rabbits (10 6 –10 9 CFU subcutaneously) and humans (<10 3 via multiple routes; Ellis et al., 2002; Petersen and Molins, 2010). "
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