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Veterinary Microbiology 132 (2008) 129–134

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Natural and experimental Salmonella Typhimurium

infections in foxes (Vulpes vulpes)
Kjell Handeland a,*, Live L. Nesse b, Atle Lillehaug a,
Turid Vikøren a, Berit Djønne b, Bjarne Bergsjø b
a
National Veterinary Institute, Section for Wildlife Diseases, P.O. Box 8156 Dep., N-0033 Oslo, Norway
b
Section for Bacteriology, P.O. Box 8156 Dep., N-0033 Oslo, Norway
Received 15 January 2008; received in revised form 25 April 2008; accepted 5 May 2008

Abstract

The red fox (Vulpes vulpes) can be considered as a relevant indicator species for Salmonella in the local environment and
Salmonella faecal carriage was investigated in 215 red foxes in Norway shot during the winters 2002/2003 and 2003/2004.
Fourteen (6.5%) of the foxes carried Salmonella. Four isolates were determined as serovars Kottbus (n = 2) and Hessarek (n = 2)
of Salmonella enterica subspecies enterica, and one as S. enterica subspecies IIIb:61:k:1,5,(7). The remaining nine isolates were
S. enterica subspecies enterica serovar Typhimurium 4,12:i:1,2 and all displayed the same pulsed-field gel electrophoresis
(PFGE) profile designated A2. This serovar regularly causes disease outbreaks amongst small passerine birds during winter and
most of these outbreaks are associated with the PFGE profile A2. The results strongly indicated that the Salmonella
Typhimurium infections in red foxes were primarily acquired through ingestion of infected small passerines. To investigate
the capability of the A2 strain to establish a true intestinal infection in the fox an inoculation experiment with an A2 isolate from
small passerines was carried out in farmed silver foxes (V. vulpes). The experiment also included one strain with an uncommonly
occurring profile (X201) from small passerines. To highlight possible differences in capability of the two inoculation strains to
pass the acid gastric juice in the fox, in vitro studies of their acid tolerance was carried out. Also their catalase activity and biofilm
production were studied. All three foxes inoculated with the A2 strain developed sub-clinical intestinal infection of 2 weeks
duration, whereas none of the three foxes inoculated with the X201 strain shed this bacterium. The X201 strain displayed a much
lower capability, than the A2 strain, to survive at pH 3 in vitro. The low acid tolerance probably made it difficult for the X201
strain to pass the stomach and establish an intestinal infection in the experimental foxes. Reduced catalase activity and biofilm
production were found for the X201 strain, indicating that the low acid tolerance was caused by a defect in the stationary-phase
stress response system.
# 2008 Elsevier B.V. All rights reserved.

Keywords: Salmonella Typhimurium; Red fox; Vulpes vulpes; Wildlife; Acid tolerance; Indicator species

* Corresponding author. Tel.: +47 23216350; fax: +47 23216095.

E-mail address: kjell.handeland@vetinst.no (K. Handeland).

0378-1135/$ – see front matter # 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetmic.2008.05.002
130 K. Handeland et al. / Veterinary Microbiology 132 (2008) 129–134
1. Introduction
Salmonellosis is a zoonosis of world-wide concern
and more than 2500 serovars of the Salmonella
bacteria have been identified (Popoff and Le Minor,
1992). In Norway, the human incidence of salmo-
nellosis is less than 1/3 of the overall incidence in
Europe (Anonymous, 1997–2006; Thorns, 2000). This
favourable situation is linked to the fact that
Norwegian farm animals are virtually free from
Salmonella (Anonymous, 1998–2006), except for
Salmonella enterica subspecies IIIb (S. IIIb) in sheep
(Alvseike and Skjerve, 2002). The most common
serovars of Salmonella found in Norwegian patients
are Salmonella enterica subspecies enterica serovars
Enteritidis and Typhimurium (Anonymous, 1997–
2006). Two endemic wildlife reservoirs for human S.
Typhimurium infection have been identified in Nor-
way; hedgehogs, a source of S. Typhimurium
4,5,12:i:1,2 infection (Søbstad et al., 2000; Handeland
et al., 2002), and non-migratory small passerine birds,
a source of S. Typhimurium 4,12:i:1,2 (Kapperud
et al., 1998; Refsum et al., 2002a). Hedgehogs and
small passerines also constitute a potential source of
infection for domestic animals in Norway (Refsum
et al., 2002b). The human cases of S. Typhimurium
4,12:i:1,2 infection are linked to bird feeding places
and disease outbreaks in small passerines during
winter (Kapperud et al., 1998). Disease in small
passerines has been associated with six pulsed-field
gel electrophoresis (PFGE) profiles for this serovar
(Refsum et al., 2002b), and the infection seems to be
maintained through the presence of healthy carrier
birds (Refsum et al., 2003). Sporadic cases of
Salmonella infection, most often due to S. Typhimur-
ium, have also been diagnosed in other wildlife
species in Norway (Anonymous, 1965–2007). Oppor-
tunistic birds, like gulls, are well-known carriers and
indicator species for a variety of Salmonella serovars
that may be present, both in a local and geographically
wider environment (Refsum et al., 2002a; Nesse et al.,
2005). Mammalian opportunists, like the widely
distributed red fox (Vulpes vulpes), can also be
considered a relevant local indicator species.
The present study reports the prevalence of
Salmonella infections in hunted red foxes in Norway.
The study also included an infection experiment in
silver foxes (Vulpes vulpes) with two different PFGE
profiles of S. Typhimurium from small passerines, and
an in vitro acid tolerance testing of these strains. The
latter studies were carried out to highlight the
capability of these small passerine strains to pass
the acid gastric juice and establish an intestinal
infection in the fox.
2. Materials and methods
2.1. Natural infections in red foxes
Two hundred and fifteen free-ranging red foxes
primarily collected for parasitological studies (David-
son et al., 2006), were included in the study. The foxes
were shot during ordinary hunting from November
2002 to April 2003, and from November 2003 to April
2004, and came from different regions of Norway. The
carcasses were sent to the laboratory accompanied by
a standard form completed by the hunters. In the
laboratory, the carcasses were autopsied, and faecal
samples of about 10 g were taken. The samples
collected in 2004 were analyzed fresh, whereas those
collected in 2002 and 2003 were stored at 18 8C until
examination at the end of the sampling period (2004).
2.2. Experimental infections in silver foxes
The experiment was carried out in accordance with
Norwegian ethical guidelines and authorized by the
Norwegian Committee for Experimental Animals
(Licence number 2004-36646-1). Six male and six
female silver foxes, aged 4–5 months, were purchased
from a local farm. During the experiment they were
housed indoors in separate wire cages, and fed a
commercially produced wet-feed for foxes. Plastic
sheets were placed under each cage allowing
individual, daily faecal collection. The faecal samples
were subsequently frozen at 18 8C, and examined
bacteriologically at the end of the experiment.
Fourteen days after arrival (Day 0), two groups;
each containing three randomly selected foxes; were
inoculated with each their isolate of S. Typhimurium
4,12:i:1,2 obtained from small passerines that had died
due to salmonellosis during the winter in Norway. The
remaining six foxes served as controls. The two
isolates used were of the PFGE profile A2 (Salmo-
nella-isolate 88005562) and X201 (Salmonella-isolate
 K. Handeland et al. / Veterinary Microbiology 132 (2008) 129–134
A201-72), respectively, and both were low passage
number isolates. A2 is the most common PFGE profile
associated with fatal disease outbreaks in small
passerines in Norway, representing more than 50%
of all cases examined at the National Veterinary
Institute 1969–2000 (Refsum et al., 2002b). The X201
profile has only been identified once. During the
experiment, the foxes were observed daily, and any
sign of clinical disease was recorded. All foxes were
euthanized by electrocution 41 days post inoculation.
An autopsy was carried out on all fox carcasses
following standard procedures. Samples of ileum,
mesenteric lymph nodes, gall bladder and faeces were
collected for bacteriology. Tissue specimens of the
heart, lung, liver, ileum, colon, and mesenteric lymph
nodes were fixed in 10% neutral buffered formalin and
embedded in paraffin, sectioned at 5 mm, and stained
with hematoxylin and eosin, van Gieson, and Gram for
histological examination (Culling et al., 1985).
2.3. Bacteriological analyses and culture
conditions
Bacteriological analyses of faeces from the hunted
red foxes, and of faeces and tissue samples from the
experimental silver foxes were performed by pre-
enrichment in phosphate-buffered peptone water at
37  1 8C for 18 h, subsequently selective enrichment
in Rappaport-Vassiliadis soya peptone broth (Oxoid,
Denmark) at 41  0.5 8C for 24 h, and plating out on
red violet bile agar plates (Oxoid, Denmark) and
bromthymol blue lactose sucrose agar. The agar plates
were incubated at 37  1 8C for 18 h. Typical colonies
were isolated by sub-cultivation and tested biochemi-
cally API20E (bioMérieux, Marcy l’Etoile, France)
and serologically for confirmation. All strains were
stored at 80 8C in Brain Heart Infusion broth (Difco,
BD, Franklin Lakes, NJ, USA) supplemented with
15% glycerine (Merck KGaA, Darmstadt, Germany)
and recovered on bloodagar (sheepblood) at 37  1 8C
overnight.
The two inoculation strains were transferred into
PBS and incubated at 37  1 8C for 16 h. The cultures
were then kept chilled until inoculation (same day),
which was performed by pouring 1 mL bacterial
suspension over the feed of each fox just prior to
feeding. At the same time, the number of cfu/mL of
each culture was determined by parallel serial dilutions
131
and spreading on BGA plates incubated at 37  1 8C
for 18 h. The inoculation doses were 4.1  108 cfu/
animal given the PFGE-profile A2 and 3.4  108 cfu/
animal given the PFGE-profile X201.
Pulsed-field gel electrophoresis was performed
according to Kaufmann (1998). For DNA restriction,
plugs were digested with XbaI (Roche, Mannheim,
Germany) according to the manufacturer’s instruc-
tions. PFGE banding patterns were identified by
computer analysis (BioNumerics, Applied Maths,
Kortrijk, Belgium).
2.4. In vitro studies of inoculation strains: acid
tolerance, biofilm production, catalase activity
Prior to the acid tolerance study, the two inoculation
strains were inoculated in 20 mL heart infusion broth
(HIB; Difco, BD, Franklin Lakes, NJ, USA) and
incubated at 37  1 8C for 19 h. Then 1.0 mL was
mixed with 99.0 mL HIB which was adjusted to pH 3.0
with HCl and preheated to 37  1 8C. The mixtures
were incubated at 37  1 8C. Samples were taken from
the broth after 0, 1, and 2 h and the number of cfu/mL of
each culture was determined by parallel serial dilutions
and spreading on BGA plates incubated at 37  1 8C
for 18 h.
The ability to produce biofilm on agar was
examined, mainly as described by Römling and
Rohde (1999). Colonies were inoculated onto LB agar
without salt, containing 40 mg/mL Congo Red (Merck
KGaA, Darmstadt, Germany) and 20 mg/mL Coo-
massie blue (Sigma–Aldrich, St. Luis, MO) (CR
plates). The CR plates were incubated at 20  1.0 8C
for 8 days, and visually examined after 2, 4, 6 and 8
days.
The catalase response was studied by semiquanti-
tative visual examination after adding H2O2 to the
bacterial colony mass derived from a nutrient agar
plate. The extent of bubbling was compared to that of
the positive control S. Typhimurium ATCC 14028.
3. Results
3.1. Natural infections in red foxes
The results of the prevalence study in red foxes, by
month of sampling, are summarized in Table 1. None
132 K. Handeland et al. / Veterinary Microbiology 132 (2008) 129–134
Table 1
Prevalence of Salmonella in faecal samples from 215 Norwegian red foxes by sampling month during 2002–004
Month No. Salmonella negative
November 52 52
December 11 11
January 34 32
February 67 60
March 35 30
April 16 16
November–April 215 201
of the 63 foxes shot during November and December
were positive for Salmonella bacteria in faeces,
whereas 14 of 152 (9.2%) of those shot during the
period January to April shed Salmonella, with the
highest prevalence in February (10.5%) and March
(14.3%). The foxes sampled in January–April 2004
showed a significantly higher carrier rate (12/74;
16.2%), compared to those shot during the same
period in 2003 (2/78; 2.6%). The Salmonella positive
foxes originated from eastern, western and central
regions of Norway. The most frequently detected
serovar was S. Typhimurium 4,12:i:1,2, representing
64.3% (9/14) of all isolates. All of the S. Typhimurium
4,12:i:1,2 isolates displayed the PFGE profile A2. The
remaining five isolates were determined as S. enterica
subspecies enterica serovars Kottbus (n = 2), Hes-
sarek (n = 2), and S. IIIb:61:k:1,5,(7) (n = 1).
3.2. Experimental infections in silver foxes
Clinical signs of disease were not observed in any
of the silver foxes during the experiment, and their
appetite remained normal. All three of the foxes
inoculated with S. Typhimurium, PFGE profile A2,
shed this strain in their faeces from day 1 to days 13,
14 and 15, respectively. No Salmonella bacteria could
be detected in their faeces during the remaining 4
weeks of the experiment. None of the three foxes
inoculated with S. Typhimurium, PFGE profile X201,
shed this strain in their faeces. All faecal samples from
the six control foxes remained free from Salmonella.
Salmonella positive Salmonella serovar (No.*)
0
0
2 (5.9%) S. Hessarek (1*)
S. IIIb:61:k:1,5,(7) (1*)
7 (10.5%) S. Typhimurium 4,12:i:1,2 (5*)
S. Kottbus (1*)
S. Hessarek (1*)
5 (14.3%) S. Typhimurium 4,12:i:1,2 (4*)
S. Kottbus (1*)
0
14 (6.5%)
At autopsy, all foxes were in excellent body
condition. No specific gross or histological lesions
were found, and all bacteriological analyses of faeces
and tissue samples were negative for Salmonella.
3.3. In vitro studies of the inoculation strains:
acid tolerance, biofilm production, catalase
activity
The two inoculation strains showed considerable
differences in the ability to survive at pH 3.0. The
bacterial concentrations at the start of the experiment
were 5.3  107 cfu/mL for the A2 strain and
2.8  108 cfu/mL for the X201 strain. After 2 h
incubation, 60% of the bacteria of the A2 strain were
still viable, whereas the X201 strain had less than
0.04% viable cells left. When testing biofilm
production on CR plates, the A2 strain displayed
the morphotype rdar (red dry and rough), indicating
production of biofilm. The X201 strain displayed the
morphotype saw (smooth and wet) indicating no
biofilm. The catalase reaction of the strain A2 was of
the same magnitude as the positive control, whereas
the X201 strain showed no catalase activity in this test.
4. Discussion
The present study found that free-ranging red foxes
are commonly infected by S. Typhimurium 4,12:i:1,2
during the winter. The same serovar regularly causes
 K. Handeland et al. / Veterinary Microbiology 132 (2008) 129–134
fatal disease outbreaks among small passerines at
winter feeding places in Norway, and the months with
the highest prevalence in red foxes (February and
March) coincided with the peak incidence of disease
in the birds (Refsum et al., 2003). This strongly
indicates that the red fox infections were primarily
acquired through ingestion of salmonella infected,
sick or dead small passerines. This assumption was
further supported by demonstrating that the PFGE
profile of all S. Typhimurium isolates from red foxes,
A2, was the same as that found in more than 50% of
small passerines dying from salmonellosis during
winter (Refsum et al., 2002b). The study also showed
that an A2 strain from small passerines established a
sub-clinical infection of 2 weeks duration, in
experimental silver foxes, the domesticated counter-
part of the red fox. This finding indicated that the S.
Typhimurium isolates found in the red foxes were
reflecting true intestinal infections and that the red fox
may play some role in the ecology of this bacterium.
The same could be true for other free-ranging
carnivorous and omnivorous animals.
The other PFGE profile of S. Typhimurium
included in the experimental study, X201, was not
recovered from the faeces of the experimental foxes.
The low pH of the stomach can be considered one of
the host’s first lines of defence. The X201 strain
displayed a much lower in vitro acid tolerance than the
A2 strain. A separate study also found that the X201
strain had much lower tolerance to pH 3.0 than the S.
Typhimurium ATCC strain 14028 and three wild
strains of the same serovar (unpublished results). The
low acid tolerance probably made it difficult for the
X201 strain to pass through the foxes’ stomach (pH 2–
2.5) and establish an intestinal infection. This could
also be the reason why the X201 strain has only once
been found in disease outbreaks in small passerines in
Norway. In addition to reduced acid tolerance, the
X201 strain did not express catalase activity, nor did it
produce biofilm on Congo Red agar plates; in contrast
to the majority of S. Typhimurium strains (Taylor and
Achanzar, 1972; Römling et al., 2003). The absence of
catalase activity in the X201 strain indicated a reduced
activity in the stationary-phase stress response factor
sigma S (RpoS). RpoS has previously been shown to
be involved in the regulation of both acid stress
response and biofilm production (Lee et al., 1995;
Robbe-Saule et al., 2003; White and Surette, 2006).
133
The results therefore indicated that the X201 strain
had a defect in the stationary-phase stress response
system, possibly RpoS, making it less resilient in
different environments.
The other zoonotic S. Typhimurium infection
known to have an endemic reservoir among wildlife
in Norway, S. Typhimurium 4,5,12:i:1,2 in hedgehogs
(Handeland et al., 2002), was not detected in any of the
examined red foxes. Consideration has to be given to
the fact that the hedgehog is a hibernating species and
thus will not be active during the winter-period. To
study a possible spill over of Salmonella from
hedgehogs to red foxes, it would be necessary to
sample foxes during the summer-half-year.
Other Salmonella bacteria found in the red foxes in
the present study included S. IIIb:61:k:1,5,(7), S.
Hessarek and S. Kottbus. The former is commonly
found in the intestine of sheep in Norway but the
clinical significance of the infection appears to be low
(Alvseike and Skjerve, 2002; Sandberg et al., 2002). In
Norway, sheep are kept on large un-enclosed pastures
during the summer, and some flocks are even kept
outdoors during winter. It is therefore not unlikely that
the red foxes found positive for S. IIIb:61:k:1,5,(7) had
been infected through contact with infected sheep or
carcasses. During the years of our study, S. Hessarek
was isolated from one pig, and S. Kottbus was found in
feed factories (unpublished data). However, there is no
obvious epidemiological connection between these
incidences and the findings in foxes in our study, and
there is no known reservoir of these two serovars in
domestic or wild animals in Norway. Thus, the source
of these infections in the reported red foxes remains
unknown.
Acknowledgements
The collection of red foxes was financed by the
Norwegian Research Council under the Strategic
Institute Programme (SIP): Diagnosis of parasitic
diseases and zoonoses in terrestrial animals and fish
using PCR and conventional methods (2001–2005);
Project 4: Trichinella and Echinococcus multi-
locularis infections in Norwegian red foxes.
Financial support for the bacteriological examina-
tions was provided by the Norwegian Zoonosis
Center.
134 K. Handeland et al. / Veterinary Microbiology 132 (2008) 129–134
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