MAJOR ARTICLE

Molecular Epidemiology of Francisella tularensis

in the United States
Kiersten J. Kugeler, Paul S. Mead, Aimee M. Janusz, J. Erin Staples, Kristy A. Kubota, Linda G. Chalcraft,
and Jeannine M. Petersen
Centers for Disease Control and Prevention, Division of Vector-Borne Infectious Diseases, Fort Collins, Colorado

Background. In the United States, tularemia is caused by Francisella tularensis subsps. tularensis (type A) and
holarctica (type B). Molecular subtyping has further divided type A into 2 subpopulations, A1 and A2. Significant
mortality differences were previously identified between human infections caused by A1 (14%), A2 (0%) and type
B (7%). To verify these findings and to further define differences among genotypes, we performed a large-scale

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molecular epidemiologic analysis of F. tularensis isolates from humans and animals.
Methods. Pulsed-field gel electrophoresis with PmeI was performed on 302 type A and 61 type B isolates.
Pulsed-field gel electrophoresis pattern and epidemiologic analyses were performed. Logistic regression was used
to assess factors associated with human mortality.
Results. Pulsed-field gel electrophoresis typing identified 4 distinct type A genotypes, A1a, A1b, A2a, and A2b,
as well as type B. Genotypic and geographic divisions observed among isolates from humans were mirrored among
isolates from animals, specifically among animal species that are linked to human infection and to enzootic
maintenance of tularemia. Significant differences between human infections caused by different genotypes were
identified with respect to patient age, site of organism recovery, and mortality. Human infections due to A1b
resulted in significantly higher mortality (24%) than those caused by A1a (4%), A2 (0%), and type B (7%).
Conclusions. Three type A genotypes, A1a, A1b, and A2, were shown to be epidemiologically important. Our
analysis suggests that A1b strains may be significantly more virulent in humans than A1a, A2, or type B strains.
These findings have important implications for disease progression, disease prevention, and basic research programs.

Tularemia is a zoonotic disease of the northern hemi-
sphere. The etiologic agent Francisella tularensis has
been recovered from numerous animal species [1–3]
and can be transmitted to humans through arthropod
bites, inhalation or ingestion of the organism, and di-
rect skin contact with infected tissues [1, 4, 5]. Illness
severity varies and depends on route of infection, dose,
and infecting subspecies.
In the United States, tularemia is caused by F. tu-
larensis subsps. tularensis (type A) and holarctica (type
B). Type A strains have been divided into 2 distinct
subpopulations, A1 and A2 (A.I. or A-east and A.II. or
Received 11 September 2008; accepted 8 December 2008; electronically
published 26 February 2009.
Reprints or correspondence: Dr. Jeannine M. Petersen, Centers for Disease
Control and Prevention, Div. of Vector-Borne Infectious Diseases, 3150 Rampart
Rd., Fort Collins, CO 80521 (nzp0@cdc.gov).
Clinical Infectious Diseases 2009; 48:863–70
This article is in the public domain, and no copyright is claimed.
1058-4838/2009/4807-0003$15.00
DOI: 10.1086/597261
A-west, respectively) [6, 7]. This division has been dem-
onstrated using several typing methods, including mul-
tilocus variable number tandem repeat analysis, PFGE,
and analysis of single nucleotide polymorphisms [6–
12], which confirms that the difference observed be-
tween the A1 and A2 subpopulations is not an artifact
of a specific typing method.
Type A strains have historically been considered to
be more pathogenic than type B strains [1, 4, 13–15];
however, a preliminary analysis of human tularemia in
the United States identified differences in mortality as-
sociated with infections caused by A1 (14%), A2 (0%),
and type B (7%) strains [7]. This mortality difference
among infections due to F. tularensis type A was un-
expected, but interpretation of these results was limited,
because only 20% of the type A isolates in this study
were genotyped, whereas the remaining 80% were clas-
sified as A1 or A2 solely on the basis of geographic
origin [7].
We used PFGE to genotype all type A and a portion
of type B isolates (from humans and animals) sub-

Molecular Epidemiology of F. tularensis • CID 2009:48 (1 April) • 863

Table 1. Number of Francisella tularensis isolates from source genotyped. In addition, we wished to further define genotypic
patients and animals, by state and genotype. and epidemiologic differences among type A isolates and to
determine if genotypic divisions observed among isolates from
Genotype, no. of isolates
humans were mirrored among isolates from animals, specifi-
A1
Type cally those animal species that are linked to human infection
US state A1a A1b Non-A1a, non-A1b A2 B
and enzootic maintenance of tularemia [2, 16–20].
Alaska … 1 … 1 …
Arizona … … … 4 7 METHODS
Arkansas 10 6 1 … …
California 1 2 1 1 19 Isolates. F. tularensis isolates submitted to or recovered at the
Colorado 1 1 38 31 CDC during 1964–2004 (from humans) and 1963–2005 (from
Delaware 1 2 … … …
animals) were analyzed. Isolates were confirmed to be F. tu-
Georgia 3 3 … … 1
Idaho … 2 … … 2
larensis, and identified as either type A or type B as described
Illinois 3 … … … 6 elsewhere [3, 7]. Clinical information was extracted from ac-
Indiana 1 … … … 7 companying submission forms and included age, sex, onset
Iowa 2 … … … 1 date, site of isolate recovery, underlying illness, outcome, state
Kansas 15 12 … … 3 and county of residence, state and county of exposure (if dif-
Kentucky 1 1 … … 20
ferent than residence), and mode of transmission. Species and

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Louisiana 2 3 1 … …
Maryland 2 4 … … …
county of infection for isolates from animals was extracted from
Massachusetts 1 4 … … … submission forms.
Michigan … 1 … … 9 PFGE analysis. F. tularensis cells were embedded in agarose
Minnesota … … … … 1 plugs and lysed. Genomic DNA was restricted with PmeI, and
Mississippi 2 … … … 1
electrophoresis was performed [7]. Salmonella enterica serotype
Missouri 9 4 … … 8
Montana … … … … 1
Braenderup strain H9812 restricted with XbaI was used for gel
Nebraska 5 2 … … 1 normalization [7]. PFGE patterns were analyzed using Bio-
Nevada … … … 11 … Numerics, version 3.5 (Applied Maths). Dendrograms were
New Jersey … 1 … … … constructed using Dice similarity coefficients and unweighted
New Mexico … … … 19 … pair group method with averages.
New York … 8 … … 3
Statistical methods and maps. Epidemiologic analyses
North Carolina … 11 1 … …
North Dakota 3 … … … 5
were performed with SAS, version 9.1 (SAS Institute). x2 tests
Ohio 1 … … … 1 were used for categorical data, and Wilcoxon rank-sum tests
Oklahoma 19 9 … 1 3 were used for age distribution comparisons. P ! .05 was con-
Oregon 1 1 … 6 34 sidered to be significant in all analyses. Logistic regression anal-
Pennsylvania 2 … … … …
ysis was used to identify factors associated with human mor-
South Dakota 6 3 … … 9
Tennessee 4 … … … …
tality and to calculate adjusted ORs. Age, sex, immune status,
Texas 3 1 … 1 1 and infecting genotype were assessed to determine their rela-
Utah 1 … … 7 … tionships with mortality. Age was converted into a categorical
Vermont … … … … 1 variable by quartiles. Because the isolation of an organism from
Virginia 1 7 1 … 1 an invasive site is indicative of disseminated infection and thus
Washington … … … … 8
could be part of the biologic pathway to death, it was excluded
Wyoming … … … 16 4
Unknown 1 … … 2 7
from multivariable analysis. Characteristics of type B infections
Total 100 89 6 107 195 in humans, as described elsewhere, were included in multivar-
iable analysis [7]. The multivariable model included variables
NOTE. The US state is listed with respect to source of F. tularensis infec-
tion. Type A genotypes were determined by PFGE. Three nonviable type A with P ! .1 in the crude analysis. Maps were generated by plot-
isolates were excluded from this table. All type B isolates from humans were ting isolates randomly within county of exposure with use of
described elsewhere [7]. Non-A1a, non-A1b isolates were from 5 humans and
1 domestic cat. ArcGIS, version 9 (ESRI).

mitted to the Centers for Disease Control and Prevention
(CDC) during a 40-year period. Our aim was to determine
whether previously identified geographic and clinical differ-
ences among infections due to F. tularensis A1, A2, and type
B were observed when all type A isolates in the dataset were
RESULTS
PFGE genotyping. A total of 500 F. tularensis isolates were
identified for analysis: 316 isolates from humans (208 type A
and 108 type B) from 38 US states and 184 isolates from animals
(97 type A and 87 type B) from 24 US states (table 1). Three

864 • CID 2009:48 (1 April) • Kugeler et al.

type A isolates from humans were not viable; the remaining
type A isolates (n p 302; 205 from humans and 97 from an-
imals) were genotyped using PFGE. Only a portion of the type
B isolates (n p 61; 22 from humans and 39 from animals) were
genotyped using PFGE, because PFGE typing had previously
indicated little to no diversity among type B isolates [7].
PFGE patterns grouped into primary clusters that corre-
sponded with type A and type B (figure 1). Among type A
isolates, patterns divided into the A1 and A2 genotypes, with
195 isolates (65%) classified as A1 (139 from humans and 56
from animals) and 107 isolates (35%) classified as A2 (66 from
humans and 41 from animals) (figure 1). Among the A1 iso-
lates, 2 distinct clusters were evident and were designated A1a
and A1b. The A1a genotype consisted of 100 isolates (74 from
humans and 26 from animals), and the A1b genotype consisted
of 89 isolates (60 from humans and 29 from animals). Limited
pattern diversity was observed among the A1a and A1b clusters,

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with 11 patterns comprising each genotype. Patterns for 6 A1
isolates (5 from humans and 1 from an animal) did not cluster
with the A1a or A1b genotypes or with each other and were
designated non-A1a and non-A1b (figure 1). PFGE patterns
for A2 isolates also divided into 2 clusters, which were desig-
nated A2a and A2b (figure 1). The A2a genotype consisted of
65 isolates (45 human, 20 animal), and the A2b genotype con-
sisted of 42 isolates (21 human, 21 animal). Pattern diversity
was observed within the A2a and A2b clusters, with 28 and 17
patterns comprising these genotypes, respectively.
Human infections. Epidemiologic analyses were performed
for all type A clusters identified by PFGE genotyping (A1 and
A2, A1a and A1b, A2a and A2b). Characteristics of type B
infections have been described elsewhere [7]. Human infections
due to either A1 or A2 generally segregated east or west, re-
spectively, of the 100th meridian (figure 2, top; table 1) [6, 7].
However, a small number (n p 9 ; 6%) of infections due to A1
were identified west of the 100th meridian along the coast of
California and Oregon and in areas of Idaho, Utah, and Col-
orado. A single infection due to A2 was identified east of the Figure 1. Dendrogram depicting 363 isolates of Francisella tularensis
100th meridian. Geographic distributions overlapped for in- from humans and animals (302 type A and 61 type B isolates) genotyped
fections due to A1a and A1b strains (figure 2, top) and for by PFGE. A 1.5% optimization and position tolerance setting was used
infections due to A2a and A2b strains (data not shown). to calculate Dice similarity coefficients. Clusters corresponding to type
A, type B, and A1, A2, A1a, A1b, A2a, and A2b genotypes are indicated.
Age and sex were known for 191 (93%) and 205 (100%)
patients, respectively. Age distributions differed among patients
with infection due to type A strains; patients with infection due The majority (71%–80%) of type A infections across all ge-
to A2 strains (mean age, 33 years; median age, 31 years) were notypes occurred in men. An immunocompromising condition
younger than patients with infection due to A1 strains (mean was reported for 5 (3.6%) of 139 patients with infection due
age, 41 years; median age, 44 years) (P ! .02). Patients with to an A1 strain, 0 (0%) of 66 patients with infection due to
infection due to A1a strains (mean age, 37 years; median age, an A2 strain, 1 (1%) of 74 patients with infection due to an
40 years) were younger than patients with infection due to A1b A1a strain, and 3 (5%) of 60 patients with infection due to an
strains (mean age, 44 years; median age, 44 years) (P ! .01) A1b strain.
(table 2). No difference was noted with regard to age distri- Most infections (72%) occurred between May and Septem-
butions between patients with infection due to A2a and A2b. ber. Month of onset did not differ among infections caused by

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Figure 2. Geographic distribution of isolates from humans (circles) and animals (squares), by genotype. Isolates are placed randomly within the
county of exposure. For isolates from humans, the county of residence was used as the county of exposure if no travel history was recorded. For
animals, county of “residence” or “collection” was used for county of exposure. Top, Distribution of A1a (n p 48 ; light green), A1b (n p 42; dark
green), and A2 (n p 80 ; dark blue) isolates. County-level information was available for 48%, 47%, and 75% of A1a, A1b, and A2 isolates, respectively.
The location of the 100th meridian is approximated by a dotted line. Bottom, Distribution of type A (n p 178 ; yellow) and type B (n p 142; light
blue) isolates. County-level information was available for 58% and 73% of type A and type B isolates, respectively.

different type A genotypes. Distributions of type A genotypes
did not differ by decade of infection. Route of exposure was
reported for 99 (48%) type A isolates. Arthropod bites and
direct animal contact each accounted for roughly one-half of
reported exposures. Contact with domestic cats or lagomorphs
(i.e., rabbits and hares) occurred across all genotypes and ac-
counted for 40 (85%) of 47 animal exposures. The type of cat
exposure was recorded for 13 (72%) of 18 cat-associated cases:
7 exposures involved a bite, 5 involved a scratch, and 1 involved
a hand wound sustained during a necropsy of an infected cat.
Information on the anatomic site of recovery was available
for 188 isolates (92%). Overall, 69 (54%) of 127 A1 isolates
were classified as invasive (i.e., were recovered from blood, lung,
pleural fluid, or CSF; noninvasive isolates were recovered from
ulcer, wound, lymph node, or eye), compared with only 6
(10%) of 61 A2 isolates (P ! .001) (table 2). Only 30 (44%) of

866 • CID 2009:48 (1 April) • Kugeler et al.

 Table 2. Significant differences among human infections caused by Francisella tularensis type
A genotypes.

A1 vs. A2 A1a vs. A1b

Variable A1 A2 A1a A1b
Age distribution, median years (range) 44 (!1 to 86) 31 (!1 to 81) 40 (!1 to 86) 44 (1–82)
Invasive isolatea 69/127 (54) 6/61 (10) 30/68 (44) 36/54 (67)
Mortality 14/108 (13) 0/53 (0) 2/55 (4) 12/49 (24)

NOTE. Data are proportion (%) of infections, unless otherwise indicated. Statistically significant differences
(P ! .05) are shown with respect to comparisons between infections due to A1 and A2 strains and between infections
due to A1a and A1b strains. Statistically significant differences were not observed between infections caused by
A2a and A2b strains.
a
The site of isolate recovery was classified as noninvasive (ulcer, skin wound, lymph node, or eye) or invasive
(blood, lung, pleural fluid, or CSF).

68 A1a isolates were recovered from invasive sites, compared animals paralleled the distribution of type A genotypes among
with 36 (67%) of 54 A1b isolates (P ! .02 ) (table 2). Of the 6 humans (figure 2).
invasive A2 isolates, 2 were genotyped as A2a and 4 as A2b. Most (97%) animal isolates originated from 4 source groups:

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Clinical outcome was reported for 108 (78%) of 139 infec-
tions due to A1 strains and 53 (80%) of 66 infections due to
A2 strains. Consistent with previous findings, mortality differed
significantly between infections caused by A1 versus A2 strains
(13% vs. 0%; P ! .01) [7]. Mortality also differed markedly
among A1 genotypes; 12 of 49 infections due to A1b strains
were fatal, compared with only 2 of 55 infections due to A1a
strains (24% vs. 4%; P ! .003) (table 2).
For logistic regression analysis of variables contributing to
human mortality, the F. tularensis genotypes were categorized
as A1a, A1b, A2, or type B. Because no deaths occurred among
patients with infection due to genotypes A2, A2a and A2b
isolates were not analyzed separately and exact logistic regres-
sion was used. In univariate analysis, genotype and age were
each associated with mortality (P ! .1 ) and were thus included
in the multivariable model. In the multivariable model adjusted
for age, F. tularensis genotype was independently associated with
mortality. Specifically, only the A1b genotype was significantly
associated with mortality when A2 was the referent group (ad-
justed OR, 13.5; 95% CI, 2.5–⬁) (table 3). When A1b was the
referent group for F. tularensis genotype, all other genotypes
differed significantly (A1a, P p .01; A2, P ! .001; B, P p .01).
Animal infections. Epidemiologic analyses were performed
for infections due to A1 and A2, A1a and A1b, and A2a and
A2b strains and type B in animals. Geographic distribution of
type A and type B isolates from animals corresponded with the
distribution of type A and type B isolates from humans de-
scribed elsewhere [7] (figure 2, bottom). Most A1 isolates from
animals (95%) came from US states east of the 100th meridian
(figure 2, top), whereas A2 isolates were from only US states
west of the 100th meridian. A1a and A1b isolates (figure 2, top)
and A2a and A2b isolates (not shown) overlapped in distri-
bution. In all cases, the distribution of type A genotypes among
rodents, lagomorphs, domestic cats, and captive primates. Ro-
dents and lagomorphs are considered to be part of the enzootic
cycle of F. tularensis, whereas domestic cats and primates are
considered to be incidental hosts. The distribution of type A
versus type B isolates among animal groups was not random
(P ! .001); the majority of isolates from cats and lagomorphs
were type A, whereas nearly all isolates from primates and
rodents were type B (table 4). Five type B isolates were recovered
from other animal sources (i.e., a dog, birds, and weasels) (table
4).
Both cats and lagomorphs are reported sources of type A
infections in humans and accounted for 85% of reported an-
imal exposures in this study [2, 16–20]. Type A isolates from
cats were genotyped as A1 and A2; 33 (80%) of 41 isolates were
genotyped as A1. Among A1 isolates from cats, roughly one-
half (48%) were A1b. Among A2 isolates from cats, 5 (63%)
of 8 were A2b. Isolates from lagomorphs were also identified
as both A1 and A2, with 21 (40%) of 52 isolates identified as
A1. The majority (47 [90%] of 52 isolates) of lagomorph iso-
lates originated from cottontail rabbits. Among isolates for
which the rabbit species was known (n p 31), all isolates from
the eastern cottontail (Sylvilagus floridanus) were A1 (n p 8),
whereas all isolates from the desert cottontail (Sylvilagus au-
dubonii) were A2 (n p 23). Both A1a and A1b were isolated
from lagomorphs; most were genotyped as A1b (13 [62%] of
21 isolates). A2a and A2b isolates were also both isolated from
lagomorphs.
DISCUSSION
Molecular subtyping of 363 F. tularensis isolates from humans
and animals identified distinct type A genotypes A1a, A1b, A2a,
and A2b, as well as type B. Significant differences between

Molecular Epidemiology of F. tularensis • CID 2009:48 (1 April) • 867

 Table 3. Crude and adjusted exact logistic regression analysis of mortality
among infections due to Francisella tularensis in humans.

a
Characteristic OR (95% CI) P Adjusted OR (95% CI) P
Age quartiles, years
⭓60 5.0 (1.1–40.2) .04 4.4 (0.7–50.4) .13
44–59 2.0 (0.3–22.7) .71 1.8 (0.2–21.2) .83
24–43 1.8 (0.2–21.7) .80 1.5 (0.2–20.7) 1.99
⭐23 Reference …
Sex
Male 0.9 (0.29–3.5) 1.99 …
Female Reference …
Genotype
A1a 1.3 (0.1–⬁) .75 1.4 (0.2–⬁) .52
A1b 13.3 (2.8–⬁) !.001 13.5 (2.5–⬁) .001
Type B 2.8 (0.6–⬁) .2 2.7 (0.4–⬁) .26
A2 Reference …
Immune status

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Compromised 3.0 (0.5–15.3) .21 …
Healthy Reference …
a
Adjusted for other variables presented in final model.

human infections caused by A1 and A2 and also between A1a and infections due to type B strains were suggested to be more
and A1b were identified with respect to patient age, site of severe than those caused by A2 strains [7], multivariable logistic
isolate recovery, and mortality. No differences were identified regression analysis did not reveal a significant difference be-
between infections caused by A2a and A2b genotypes. Thus, 3 tween infections due to type B and A2 strains when patient age
type A genotypes—A1a, A1b, and A2—demonstrated clinical and genotype were simultaneously assessed for their association
relevance in this study. These 3 genotypes were also observed with mortality.
in cats and lagomorphs, which provides evidence that A1a, A1b, Differences among type A strains have been described else-
and A2 genotypes exist among animals commonly linked to where [21]. Comparison of type A infections due to either Schu
human type A infections (cats and lagomorphs) and those im- S4 or FSC033 strains (Georgia) in laboratory mice showed that
portant for enzootic maintenance of type A (lagomorphs). FSC033 disseminated more rapidly and was associated with a
The mortality differences among type A infections were the
most notable finding of this study. Although overall mortality
Table 4. Number of Francisella tularensis isolates, by subspe-
due to type A was 9%, mortality associated with infection due cies and animal source groups.
to A2, A1a, and A1b differed significantly, at 0%, 4%, and 24%,
respectively. Differences in mortality among infections caused Type A Type B Total
by A1a, A1b, and A2 strains are not likely attributable to dif- Source group (n p 97) (n p 87) (n p 184)
ferential recognition and treatment of tularemia in varied geo- Domestic cats 41 (93) 3 (7) 44
graphic regions, because no difference in mortality was ob- Lagomorphsa 52 (90) 6 (10) 58
served between type B infections in the eastern (8%) vs. western Primatesb 2 (7) 27 (93) 29
(6%) United States. In addition, most deaths attributable to Rodentsc 2 (4) 46 (96) 48
d
Other 0 (0) 5 (100) 5
A1b infections occurred in US states where both A1a and A1b
caused infection. NOTE:Data are no. (%) of isolates. Animal isolates were submitted to or
recovered at the Centers for Disease Control and Prevention from domestic
Logistic regression analysis of human infections caused by or captive animals (from diagnostic laboratories, veterinary practices or zoo-
A1a, A1b, A2, and type B strains indicated that some charac- logical facilities) or as part of routine surveillance of wild animals (from wildlife
teristic intrinsic to A1b strains such as a virulence factor, rather or public health agencies).
a
Cottontail rabbit and jackrabbit.
than host characteristics, is responsible for the higher mortality b
Marmoset, squirrel monkey, tamarin, capuchin monkey, DeBrazza’s mon-
observed among individuals with infection due to A1b. In ad- key, macaque, bush baby, orangutan, and gorilla.
c
Type A isolates were recovered from prairie dogs; type B isolates were
dition, although crude mortality was previously shown to differ recovered from mouse, beaver, rat, squirrel, hamster, prairie dog, and vole.
d
between infections caused by type B (7%) and A2 (0%) strains Dog, bird, and weasel.

868 • CID 2009:48 (1 April) • Kugeler et al.

shorter time to death than was Schu S4. Schu S4 is an A1a
strain, and in our study, genotypes of isolates from Georgia
were found to be both A1a and A1b, which raises the possibility
that FSC033 may be an A1b strain. Future experiments will be
important to determine whether pathogenicity differences
among infections due to A1a, A1b, and A2 strains can be ob-
served in a mouse model.
The geographic distribution of infections attributed to all
genotypes was found to overlap between humans and animals.
Because their movement is limited in comparison to humans,
animals are more reliable indicators of exposure locations. In-
fections due to A1 strains predominate in the eastern United
States, with a small number of cases identified in the west.
Multiple lines of evidence support that A1 strains are endemic
in the western United States. There was no evidence that the
patients from the west who were infected with A1 strains had
traveled to the eastern United States. In addition, identification
of A1 isolates in animals (both domestic and wild) in the west
provides evidence of enzootic transmission. Moreover, infec-
tions due to A1 strains were identified in both humans and
lagomorphs in an outbreak of tularemia in Utah in 2007 [22].
In comparison, A2 strains appear to be restricted to the western
United States. A single infection in a human and no infections
in animals due to A2 strains were identified east of the 100th
meridian. Although the single infection in a human was at-
tributed to exposure while traveling in Oklahoma, the patient
resided in New Mexico; our findings suggest that this patient
may have acquired the infection in New Mexico.
Factors important to the geographic distribution of F. tu-
larensis genotypes in the United States require further study.
Geographic differences and strain variation could reflect ad-
aptation to different animal hosts or vector species. Animal
host differences for type A and type B strains have been de-
scribed anecdotally in historical literature [2, 14, 15]. In our
study, we found type B strains more frequently associated with
rodents, whereas type A strains were more commonly associated
with lagomorphs; both rodents and lagomorphs are considered
to be important in enzootic maintenance of tularemia. Host
species differences were also observed among type A isolates.
The desert cottontail S. audubonii was associated with A2 iso-
lates, whereas the eastern cottontail S. floridanus was associated
with A1 isolates.
This study is subject to potential limitations. By analyzing
only cases of human infection in which an isolate was recovered,
our dataset may be biased toward severe cases. Isolate sub-
mission should not be biased by genotype, because it was un-
known at the time of submission. Thus, relative mortality dif-
ferences observed among genotypes should not be affected by
an overall bias toward more severe cases. The site of organism
recovery may be biased by differences in clinical practice. For
example, it is unlikely that all noninvasive isolates were not
present in blood, but it is more likely that a blood sample may
not have been obtained. In addition, exposure information was
unavailable for most infections due to type A strains, and where
it was known, it was limited to the county level. Finally, these
isolates represent a convenience sample. Most isolates were
submitted as a result of proximity or established relationships
with other laboratories, with the number of isolates analyzed
in this study representing only 4% of the total reported cases
among humans for this time period.
This is the first demonstration that A1 strains can be divided
into 2 distinct genotypes. Preliminary whole genome single
nucleotide polymorphisms analysis of F. tularensis strains also
identified these 2 distinct A1 genotypes [12]. Identification of
genotypes A1a and A1b by PFGE and single nucleotide poly-
morphism analysis, but not by multilocus variable number tan-
dem repeat analysis, highlights the strengths and weaknesses of
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various subtyping methods. Multilocus variable number tan-
dem repeat analysis is based on rapidly evolving tandem repeats,
whereas both PFGE and single nucleotide polymorphism anal-
ysis are based on slowly occurring genome changes [23]. Al-
though high-resolution methods that examine hypermutable
regions of the genome are powerful for distinguishing individ-
ual strains in time and space, this level of differentiation may
at times be too fine for the identification of epidemiologic
clusters.
In conclusion, genetic diversity exists among type A strains
of F. tularensis and correlates with significant differences in
clinical outcome and geographic distribution. Although type A
strains have generally been considered to be equivalent in terms
of virulence, we demonstrate that a subset of type A strains
(genotype A1b) are associated with significantly higher mor-
tality in humans. This finding has important implications with
regard to disease progression, disease prevention, and basic
research projects, including diagnostic assay and vaccine de-
velopment. Further studies are required to elucidate the epi-
demiologic, genetic, and ecologic differences between A1a, A1b,
and A2 strains. Genotyping of isolates from animals may be a
useful surveillance tool for predicting where human infections
with specific type A genotypes may occur. Because cats are a
source of human infection with A1b strains, veterinary edu-
cation efforts to promote recognition of tularemia in domestic
cats is warranted. In areas where infection with A1b strains
occurs, physician education efforts to enhance early clinical
recognition may help prevent deaths among humans.
Acknowledgments
We thank Brad Biggerstaff, Alison Hinckley, Larissa Minicucci, state and
local health departments, and organizations that submitted animal speci-
mens for testing.
Financial support. Centers for Disease Control and Prevention.
Molecular Epidemiology of F. tularensis • CID 2009:48 (1 April) • 869
 Potential conflicts of interest. All authors: no conflicts.
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