Tick-borne zoonotic bacteria in wild and domestic small mammals in northern Spain.
ABSTRACT The prevalence and diversity of tick-borne zoonotic bacteria (Borrelia spp., Anaplasma phagocytophilum, Coxiella burnetii, and spotted fever group rickettsiae) infecting 253 small mammals captured in the Basque Country (Spain) were assessed using PCR and reverse line blot hybridization. Trapping sites were selected around sheep farms (study 1, 2000 to 2002) and recreational parks (study 2, 2003 to 2005). The majority of the studied mammals (162) were wood mice (Apodemus sylvaticus), but six other different species were also analyzed: yellow-necked mice (Apodemus flavicollis), shrews (Crocidura russula and Sorex coronatus), bank voles (Clethrionomys glareolus), domestic mice (Mus domesticus), and moles (Talpa europaea). The results showed an infection rate ranging from 10.7% to 68.8%, depending on the small mammal species. One C. russula shrew and one A. sylvaticus mouse gave positive reactions for A. phagocytophilum, and C. burnetii was detected in two domestic mice and one A. sylvaticus mouse in a farm. The DNA of Borrelia spp. was detected in 67 animals (26.5%), most of them presenting positive hybridization with the probe for Borrelia sp. strain R57, the new Borrelia species previously detected in small mammals in our region. Furthermore, a second PCR and reverse line blot hybridization specific for B. burgdorferi sensu lato revealed the presence of Borrelia afzelii in 6.3% of C. glareolus voles and 14.3% of S. coronatus shrews. All small mammals were negative for spotted fever group rickettsiae. These results highlight the relevance of small mammals as reservoirs of some zoonotic bacteria.
- SourceAvailable from: nhm.ac.uk[show abstract] [hide abstract]
ABSTRACT: Ixodes ricinus ticks infected with Borrelia burgdorferi sensu lato were numerous on the edges of paths and roads in a recreational park in south-western Ireland. The abundance of ticks at different sites was related to the presence of deer, but a negative relationship was shown between tick abundance and tick infection rates. This is thought to be due to the deposition of large numbers of uninfected ticks by deer, which are apparently not good reservoir hosts of B. burgdorferi s.l. Blood meal analysis only detected deer DNA in uninfected nymphs. Reservoir competent rodents, Apodemus sylvaticus and Clethrionomys glareolus, were abundant at all sites and a high proportion of captured specimens were infested with larval ticks. However, very few rodents were infected with B. burgdorferi s.l. and none of the unfed infected nymphs analysed for the identity of their larval blood meal had fed on rodents. The spirochaetes detected in I. ricinus in the study area may be poorly adapted to rodents or are not transmitted readily because of the absence of nymphal infestation. The majority of spirochaetes in these ticks were apparently acquired from non-rodent hosts, such as birds.Enperimental and Applied Acarology 10/1999; 23(9):717-29. · 1.85 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The role of small mammals as reservoir hosts for Borrelia burgdorferi was investigated in several areas where Lyme disease is endemic in northern Spain. A low rate of infestation by Ixodes ricinus nymphs was found in the small mammal populations studied that correlated with the near-absence of B. burgdorferi sensu lato in 184 animals tested and with the lack of transmission of B. burgdorferi sensu lato to I. ricinus larvae that fed on them. In contrast, questing ticks collected at the same time and in the same areas were found to carry a highly variable B. burgdorferi sensu lato repertoire (B. burgdorferi sensu stricto, Borrelia garinii, Borrelia valaisiana, and Borrelia afzelii). Interestingly, the only isolate obtained from small mammals (R57, isolated from a bank vole) grouped by phylogenetic analyses with other Borrelia species but in a separate clade from the Lyme disease and relapsing fever organisms, suggesting that it is a new species. This new agent was widely distributed among small mammals, with infection rates of 8.5 to 12% by PCR. Moreover, a high seroprevalence to B. burgdorferi sensu lato was found in the animal sera, suggesting cross-reactivity between B. burgdorferi sensu lato and R57. Although small mammals do not seem to play an important role as reservoirs for B. burgdorferi sensu lato in the study area, they seem to be implicated in the maintenance of spirochetes similar to R57.Applied and Environmental Microbiology 04/2005; 71(3):1336-45. · 3.68 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: During 1998-1999, Ixodes ricinus (L.) populations were investigated in three different biotopes (deciduous, mixed, coniferous forest) situated in popular recreational areas in Poznań, Poland. In total, 1,123 questing ticks (1,002 nymphs, 69 males, 52 females) were collected by flagging vegetation. Additionally, in 1998 between May and September small rodents were trapped and inspected for feeding ticks. Altogether, 213 rodents of three species: Apodemus agrarius Pall., A. flavicollis Melchior, Clethrionomys glareolus Schreber were captured. Of 323 engorged ticks, 304 were larvae and 19 nymphs. All ticks collected from vegetation, as well as from rodents were examined for the presence of Borrelia burgdorferi Johnson, Schmid, Hyde, Steigenwalt & Brenner s.l. spirochetes by indirect immunofluorescence assay (IFA) using PAB 1B29. The seasonal pattern of activity of questing I. ricinus was always bimodal (May/June and August/September). The most abundant tick population occurred in the deciduous forest. The total infection rate in questing ticks was 16.2%. Differences in mean infection prevalence of host-seeking ticks between three biotopes each year were not significant. On average more larvae parasitized on the genus of Apodemus than on C. glareolus. 17.8% of larvae and 31.6% of nymphs fed on rodents harbored spirochetes. The three rodent species contributed to a different degree in to transmission of the pathogen to subadult stages. Approximately 27% of larvae infested on A. agrarius, 22% on C. glareolus, and only 4.2% on A. flavicollis contained spirochetes. The results suggest that the prevalence of A. agrarius and C. glareolus in disturbed urban forests used for leisure activities seems to be crucial for the maintenance of B. burgdorferi s.l. in I. ricinus populations.Journal of Medical Entomology 09/2003; 40(5):690-7. · 1.86 Impact Factor
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 2007, p. 6166–6171
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 73, No. 19
Tick-Borne Zoonotic Bacteria in Wild and Domestic Small Mammals
in Northern Spain?
J. F. Barandika,1A. Hurtado,1C. Garcı ´a-Esteban,2† H. Gil,2R. Escudero,2M. Barral,1I. Jado,2
R. A. Juste,1P. Anda,2and A. L. Garcı ´a-Pe ´rez1*
NEIKER, Instituto Vasco de Investigacio ´n y Desarrollo Agrario, Department of Animal Health and Production, 48160 Derio,
Bizkaia, Spain,1and Centro Nacional de Microbiologı ´a, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid, Spain2
Received 14 March 2007/Accepted 26 July 2007
The prevalence and diversity of tick-borne zoonotic bacteria (Borrelia spp., Anaplasma phagocytophilum,
Coxiella burnetii, and spotted fever group rickettsiae) infecting 253 small mammals captured in the Basque
Country (Spain) were assessed using PCR and reverse line blot hybridization. Trapping sites were selected
around sheep farms (study 1, 2000 to 2002) and recreational parks (study 2, 2003 to 2005). The majority of the
studied mammals (162) were wood mice (Apodemus sylvaticus), but six other different species were also
analyzed: yellow-necked mice (Apodemus flavicollis), shrews (Crocidura russula and Sorex coronatus), bank voles
(Clethrionomys glareolus), domestic mice (Mus domesticus), and moles (Talpa europaea). The results showed an
infection rate ranging from 10.7% to 68.8%, depending on the small mammal species. One C. russula shrew and
one A. sylvaticus mouse gave positive reactions for A. phagocytophilum, and C. burnetii was detected in two
domestic mice and one A. sylvaticus mouse in a farm. The DNA of Borrelia spp. was detected in 67 animals
(26.5%), most of them presenting positive hybridization with the probe for Borrelia sp. strain R57, the new
Borrelia species previously detected in small mammals in our region. Furthermore, a second PCR and reverse
line blot hybridization specific for B. burgdorferi sensu lato revealed the presence of Borrelia afzelii in 6.3% of
C. glareolus voles and 14.3% of S. coronatus shrews. All small mammals were negative for spotted fever group
rickettsiae. These results highlight the relevance of small mammals as reservoirs of some zoonotic bacteria.
Ticks are important vectors of various pathogenic bacteria,
protozoa, and viruses that cause disease in humans and ani-
mals worldwide. Some of these agents, such as Coxiella burnetii,
are now recognized as important emerging vector-borne
pathogens as well as agents of bioterrorism (3). Ehrlichia spe-
cies, rickettsiae, and some Borrelia species have been reported
across the world and have also been associated with disease in
animals and humans (33, 35).
A wide range of mammalian reservoir hosts, including ro-
dents, are involved in the natural cycle of various bacterial
diseases. Different species of small mammals, mainly mice
(Apodemus spp.) and voles (Microtus spp. and Clethrionomys
glareolus), are recognized vertebrate reservoirs of tick-borne
bacterial zoonoses such as Lyme disease (borreliosis) and hu-
man granulocytic ehrlichiosis, among others. In Europe, sev-
eral rodent species seem to be the natural reservoirs of Borrelia
burgdorferi sensu lato (35), Anaplasma phagocytophilum (7, 25),
and C. burnetii (44), but there are few reports on the role of
such species as possible reservoirs of tick-borne zoonotic bac-
teria in Spain. The role of small mammals in the biological
cycle of B. burgdorferi sensu lato was recently investigated in
areas of northern Spain where Lyme disease is endemic (14),
and a low prevalence of infection was found (0.5%), but a new
Borrelia sp. (Borrelia sp. strain R57), whose clinical and patho-
genic importance remains unknown, was widely distributed
among small mammals (12.5%). A limited number of small
mammals from the same area was also analyzed for the pres-
ence of DNA of A. phagocytophilum, but all the specimens
were negative (31). All these results were explained by the low
number of Ixodes nymphs parasitizing small mammals in this
area, with a 1:450 ratio of infestation of nymphs to that of
larvae (14). The study area is particularly relevant due to the
endemicity of Q fever pneumonia (29) and its proximity to an
area where a new species of Rickettsia causing human disease,
Rickettsia monacensis, has recently been identified in a patient
(20a). However, no data are available regarding the presence
of C. burnetii or Rickettsia spp. in small mammals in Spain. All
these data create an interest in the search for potential reser-
voirs of such organisms.
PCR-based methods have become widely used as rapid and
effective tools for the detection and identification of tick-borne
pathogens in ticks and animal reservoirs. Increased sensitivity
and specificity can be achieved by combining PCR with a spe-
cific hybridization by means of reverse line blot (RLB) hybrid-
ization, a macroarray that is able to identify mixed infections
(39, 40). This study was undertaken to investigate the preva-
lence of tick-borne pathogens in domestic and wild small mam-
mals in northern Spain. The main aim of the study was to
determine the risk of disease in areas of potential contact
between humans and small mammals carrying pathogens. In
recreational areas, large numbers of human beings might come
in contact with ticks and tick-borne pathogens from wildlife.
Sheep farms, on the other hand, were selected since sheep are
the most abundant livestock species in the area. Peridomestic
small mammals were selected to investigate the role of these
* Corresponding author. Mailing address: NEIKER, Instituto Vasco
de Investigacio ´n y Desarrollo Agrario, 48160 Derio (Bizkaia), Spain.
Phone: 34 94 403 4312. Fax: 34 94 403 4310. E-mail: agarcia@neiker
† Present address: Hospital Universitario de Getafe, 28905 Getafe
?Published ahead of print on 10 August 2007.
species as reservoirs of some tick-borne bacteria affecting
sheep (C. burnetii and A. phagocytophilum) that might put at
risk professionals living and working around farms. Rapid
screening of a selection of bacteria infecting small-mammal
tissues was carried out using multiplex PCR, followed by RLB
hybridization using genus-specific or species-specific probes.
Using this approach, we report herein the infection rates in
small mammals for A. phagocytophilum, C. burnetii, spotted
fever group (SFG) rickettsiae, B. burgdorferi sensu lato, and
Borrelia sp. strain R57, with the purpose of providing an as-
sessment of the role of small mammals as reservoir hosts for
MATERIALS AND METHODS
Study area and small-mammal sampling. The Basque Country is a 7,200-km2
region located in Atlantic northern Spain where ticks are abundant (4), there is
a maritime climate with mild winters, and annual rainfall ranges from 600 to
1,500 mm. Livestock, raised at pasture several months a year, is abundant as well
as wildlife, which consists mainly of rodents, foxes, badgers, wild boar, roe deer,
and red deer.
Small mammals were captured between years 2000 and 2005 in two consecu-
tive studies carried out at 15 different sites in the Basque Country. In the first
study (study 1), carried out between April 2000 and November 2002, domestic
and wild rodents were sampled in areas surrounding five sheep farms and in
forested areas nearby. Study 2 started in September 2003 and finished in May
2005, and small mammals were captured in 10 recreational areas where, as
previously described (4), the tick population was abundant.
After the authority’s permission was obtained, Sherman traps (7.6 cm by 8.9
cm by 22.9 cm; Tallahassee, FL) and INRA traps (5 cm by 5 cm by 15 cm; BTS
Mechanique, Besanc ¸on, France) were used for the live capture of rodents.
Captures were carried out throughout the year, but mainly in the spring and
autumn months. In study 1, 120 traps (20 Sherman traps placed inside sheep
farms and 100 INRA traps placed outdoors) were set for two consecutive nights,
and 100 INRA traps were set overnight in study 2. In study 1, moles were also
captured using 25 pincer traps (Michel Touchard et Fils, Grainville Langannerie,
France) per sampling. To compare the abundances of these animals between
studies, the small-mammal abundance index (SAI) was calculated as follows:
SAI ? (SC ? 100)/(T ? N), where SC is the number of small mammals captured,
T is the number of traps, and N is the number of nights.
Processing of small mammals. Trapped animals were immediately transported
to the laboratory and examined for attached ticks and other ectoparasites. Live
captured rodents were anesthetized with ether and with ketamine hydrochloride
(Imalgene 500; Merial, France) at a dose of 10 mg/kg of body weight intramus-
cularly and euthanized in a CO2chamber. At necropsy, samples from different
tissues were collected (ear, urinary bladder, spleen, liver, kidney, lung, and brain)
and stored at ?80°C. Small mammals were classified by external morphological
data and skull features (1, 6, 32). Collected ticks were identified using taxonomic
keys (15, 26).
DNA extraction and multiplex PCR. DNA was extracted from pools of tissues
and ear samples by using a QIAamp DNA mini kit (QIAGEN, Hilden, Ger-
many), with a previous treatment with proteinase K for 3 h. A negative control
was included for every 10 samples. The DNA concentration was determined for
each sample with a NanoDrop ND-1000 spectrophotometer (NanoDrop Tech-
DNA was subjected to two multiplex PCR amplifications, one for the detection
of C. burnetii and A. phagocytophilum and a second one for the detection of SFG
rickettsiae and Borrelia spp. The oligonucleotide sequences of the primers, the
gene targets, the concentration of each primer, and the annealing temperature of
the PCR are shown in Table 1. Extracted DNA (100 to 200 ng) was used in each
One amplicon from each of the genes targeted in this study was routinely
cloned, sequenced, and used as a positive control. Briefly, the amplified products
were purified with a GFX PCR kit (Amersham Biosciences, Uppsala, Sweden),
cloned into a pCR4-TOPO vector, and introduced into Escherichia coli according
to the manufacturer’s instructions (TOPO TA cloning kit for sequencing; In-
vitrogen, CA). Recombinant plasmid DNA was purified using a FlexiPrep kit
(Amersham Biosciences) and subjected to automatic dye terminator cycle se-
quencing. The different plasmids were used to calculate the absolute detection
limit of the technique. The concentration of each plasmid was calculated spec-
trophotometrically, and the plasmids were serially diluted in Tris-EDTA buffer
to reach concentrations ranging from 108to 1 copies per microliter.
The prevention of cross-contamination and false-positive results was managed
by using plugged tips, setting PCRs in a room separate from that used for DNA
extraction, and including a negative (water) control in each run.
RLB hybridization. To increase the detection limit of the PCR assay, PCR
amplicons were hybridized to DNA probes specific for C. burnetii, A. phagocy-
tophilum, Borrelia sp. strain R57, SFG rickettsiae, and Borrelia spp. by RLB
hybridization. The probes, synthesized by MWG Biotech AG (Germany) with a
C6amino linker, were as listed in Table 1. The preparation of RLB membranes
and hybridization were carried out as previously described by Gubbels et al. (17)
with the following adaptations: the complete amplification reaction mixture (25
TABLE 1. Oligonucleotide sequences of primers and probes used in PCR and RLB hybridization for detection
and identification of different pathogens
Target speciesTarget gene
Primer/probe sequence (5? to 3?)
C. burnetiihtpAB Trans 1
TAT GTA TCC ACC GTA GCC AGT C
biotin-CCC AAC AAC ACC TCC TTA TTC
CCA GCG TTT AGC AAG ATA AGA G
biotin-GCC CAG TAA CAA CAT CAT AAG C
ATG GCG AAT ATT TCT CCA AAA
biotin-AGT GCA GCA TTC GCT CCC CCT
CGC TGG CAG TGC GTC TTA A
biotin-GCG GCT GCT GGC ACG TAA TTA GC
ACC ATA GAC TCT TAT TAC TTT GAC CA
biotin-GAG AGT AGG TTA TTG CCA GGG
A. phagocytophilummsp20.4 46
SFG rickettsiae ompA 0.4 46 36
Borrelia spp.16S rRNA0.4 14
B. burgdorferi sensu lato5S-23S rRNA 0.257 39
amino-GCA AGA ATA CGG ACT CAC GA
amino-GGT TAC GAG CGC TTC AAG ACC
amino-GGC AAA AGC TTA ACT TTA AA
amino-GAG GAA TAA GCT TTG TAG GAA ATG
amino-AGT CAT TAA AGA TGT TTA ATG
amino-CTT TGA CCA TAT TTT TAT CTT CCA
amino-AAC ACC AAT ATT TAA AAA ACA TAA
amino-AAC ATT TAA AAA ATA AAT TCA AGG
amino-AAC ATG AAC ATC TAA AAA CAT AAA
amino-CAT TAA AAA AAT ATA AAA AAT AAA
TTT AAG G
amino-TCT ATT TTA TTT TTT ATA TTT TTT T
Borrelia sp. strain R57
B. burgdorferi sensu lato
B. burgdorferi sensu stricto
Borrelia R 57
B. lusitaniae16S rRNA Bb LU0.913
VOL. 73, 2007TICK-BORNE BACTERIA IN SMALL MAMMALS IN SPAIN6167
?l) was loaded onto the blotter after dilution with 2? SSPE-0.1% sodium
dodecyl sulfate (1? SSPE is 0.18 NaCl, 10 mM NaH2PO4, and 1 mM EDTA [pH
7.7]) to a total volume of 160 ?l, incubation was carried out at 48°C for 60 min,
and washing steps were performed at 40°C. After hybridization, PCR products
were stripped from the membrane as previously described by Gubbels et al. (17)
and the membrane was rinsed and stored in 20 mM EDTA (pH 8.0) at 4°C until
the next hybridization and reused a maximum of eight times.
To exclude false-positive results, negative controls included during DNA ex-
traction and PCR amplification were also subjected to RLB hybridization. The
specificities of the probes were tested against those of the previously constructed
plasmids, which were also used as positive controls in each assay.
Identification of species of Borrelia. Samples positive for Borrelia spp. by
multiplex PCR (16S rRNA gene) and RLB hybridization were analyzed using a
PCR specific for B. burgdorferi sensu lato, targeting the 5S-23S rRNA intergenic
spacer, followed by RLB hybridization with specific probes described previously
(13, 14, 39, 40) for B. burgdorferi sensu lato, B. burgdorferi sensu stricto, B. afzelii,
B. garinii, B. valaisiana, and B. lusitaniae (Table 1). RLB hybridization was
performed as previously described (14).
Statistical analysis. The prevalence of each bacterial species was analyzed
according to independent variables, such as host species, study (study 1 and study
2), and date of sampling (season and year), by chi-square or Fisher’s exact test by
using the SAS statistical package (version 8.0; SAS Institute Inc., Cary, NC).
Significance was set at a P value of ?0.05. Different logistic regression models
including the same variables were performed on the data for the only agent
(Borrelia sp. strain R57) that had a large enough set of positive results, and the
model that better fitted the rates of infections was used.
Small mammals captured and tick infestation. A total of
6,580 trap nights were used over the two studies (3,680 in study
1 and 2,900 in study 2), and 334 small mammals belonging to
seven species were trapped (186 in study 1 and 148 in study 2),
with a global SAI of 5.1 (5.05 for study 1 and 5.10 for study 2).
During study 1, 450 pincer traps were also placed and 30 moles
(Talpa spp.) were captured. A. sylvaticus was the most fre-
quently found species (69.8%) and the most ubiquitous in both
One hundred twenty-four of the 334 animals captured har-
bored at least one tick, with a maximum of 202 ticks observed
in one A. sylvaticus mouse. The total amount of collected ticks
was 1,961, accounting for 1,934 larvae (98.6%), 26 nymphs
(1.3%), and one adult (an Ixodes acuminatus female). The
percentage of small mammals parasitized and the number of
ticks collected varied between studies, and the individual tick
infestation followed a Poisson distribution. Whereas in study 1
the percentage of animals infested with ticks was low (9.1%), in
study 2, 1,883 larvae were collected from 72.3% of the cap-
tured animals. Very few nymphs were found over the study
periods, and all of them were collected in study 2 and consisted
of 26 nymphs that were detached from 10 A. sylvaticus mice
and one C. glareolus vole, giving a 1:72 average ratio of infes-
tation of nymphs to that of larvae.
Ixodes ricinus was the most abundant tick species found,
corresponding to 89.3% of larvae (100% in study 1 and 89.1%
in study 2) and 96.2% of nymphs (all from study 2) collected.
Three other species (Ixodes trianguliceps, Rhipicephalus turani-
cus, and Haemaphysalis concinna) were sporadically collected.
I. ricinus larval infestation varied strongly among captured
small mammal species. A. sylvaticus was the most parasitized
species with 7.3 larvae/animal, followed by C. glareolus with 1.1
larvae/animal. Other small mammal species harbored very few
larvae, i.e., Apodemus flavicollis with 0.7, Sorex coronatus with
0.5, and Crocidura russula with 0.2, whereas Mus domesticus
and Talpa europaea had none.
Tick-borne bacterial infection in small mammals. All the
nucleotide probes designed for hybridization assays gave pos-
itive results with their corresponding positive controls and did
not show any cross-reaction. The sensitivity of the hybridiza-
tion assay was assessed by RLB hybridization processing seri-
ally diluted PCR products and was found to be 1 or 2 orders of
magnitude higher than that calculated for the multiplex PCR
only. The absolute limit of detection for the combined PCR
and subsequent RLB hybridization procedures using gene
clones as templates was between 6 and 60 gene molecules for
the different multiplex PCR amplifications.
Pools of tissues from 253 animals (127 from study 1 and 126
from study 2), belonging to A. sylvaticus (162; 64%) and six
other species, were subjected to the two multiplex PCRs de-
scribed above and RLB hybridization. The prevalences of in-
fection were different between animal species (Table 2). The
most frequently infected species was C. glareolus, with 68.8% of
the studied specimens (11/16) harboring DNA from some of
the bacteria investigated, followed by A. sylvaticus (33.3%), C.
russula (16.7%), S. coronatus (14.3%), and M. domesticus
(10.7%). All the moles and A. flavicollis were negative, though
only three specimens of the latter were examined. The preva-
lences were similar between studies (Table 2), with 26.0% of
the analyzed animals being positive in study 1 and 30.1% in
Borrelia sp. DNA was detected in 67 (26.5%) animals, and
most of these positive animals also hybridized with the specific
probe for Borrelia sp. strain R57 (62/67). A total of 37.5% of
small mammals trapped in spring were positive, whereas the
prevalences among summer, autumn, and winter captures were
20.0, 15.0, and 21.1%, respectively. Statistical differences were
observed only between spring and autumn prevalences (P ?
0.05). Infection rates varied significantly among species (P ?
0.0001), from 3.6% in M. domesticus to 68.7% in C. glareolus,
while none of the T. europaea, A. flavicollis, and C. russula
strains analyzed harbored DNA of Borrelia spp. The best lo-
gistic regression model included study, host species, season,
and Ixodes larval number and showed that C. glareolus had an
odds ratio to be infected that was 5.62 (95% confidence inter-
val [CI], 1.63 to 19.33) for A. sylvaticus, 29.22 (95% CI, 5.43 to
157.10) for M. domesticus, and 16.95 (95% CI, 2.54 to 113.13)
for S. coronatus. Also, the odds ratio was 3.37 (95% CI, 1.62 to
6.99) higher in the spring than in the fall. Five animals hybrid-
ized with the generic probe for Borrelia spp. but not with that
for Borrelia sp. strain R57 after RLB analysis targeting the 16S
rRNA gene. To check whether B. burgdorferi sensu lato could
be found in these five animals and in mixed infections with
Borrelia sp. strain R57, all Borrelia-positive samples were sub-
jected to 5S-23S rRNA RLB hybridization. B. burgdorferi sensu
lato was detected in two S. coronatus shrews (14.3%) and one
C. glareolus vole (6.3%), and B. afzelii was the genospecies
identified. In one animal (C. glareolus), B. afzelii was detected
in a mixed infection with Borrelia sp. strain R57. Other geno-
species such as B. garinii, B. valaisiana, B. lusitaniae, and B.
burgdorferi sensu stricto were not detected. Three animals pos-
itive for Borrelia spp. did not hybridize with any of the specific
Two domestic mice (M. domesticus) captured in the spring
6168 BARANDIKA ET AL.APPL. ENVIRON. MICROBIOL.
and winter in 2000 in the premises of a sheep farm (study 1)
had positive hybridization with the C. burnetii probe. The re-
maining captures of that period were negative for C. burnetii
(seven A. sylvaticus mice, two C. glareolus voles, one A. flavi-
collis mouse, and one S. coronatus shrew). In the spring of
2001, one of the seven A. sylvaticus mice captured in a forest
area near the same farm was also positive, whereas other small
mammals trapped in this season (five M. domesticus mice and
two C. glareolus voles) were negative. These results indicate
that 28.6% of domestic mice (M. domesticus) and 7.1% of
wood mice (A. sylvaticus) were infected in this farm. A. phago-
cytophilum was detected in one C. russula shrew captured in
study 1 and one A. sylvaticus mouse from study 2, which rep-
resent 16.7% and 0.6% infection rates for these species, re-
spectively, and an overall infection rate of 0.8%. Rickettsia was
the only pathogen included in this study that was never de-
tected in any of the small mammals tested. Coinfection with
more than one pathogen was found in two small mammal
species and accounted for 0.8% (2/253) of the animals ana-
lyzed (Table 2). The combinations were those formed by B.
afzelii and Borrelia sp. strain R57 in one C. glareolus vole and by
C. burnetii and Borrelia sp. strain R57 in one A. sylvaticus
To investigate the role of small mammals as potential res-
ervoir hosts for tick-borne bacterial zoonoses in northern
Spain, small mammals were captured between years 2000 and
2005 in two consecutive studies. Almost half of the captured
mammals harbored at least one attached tick, but the number
of parasitized animals and ticks collected was higher in study 2
than in study 1, probably because sampling sites in study 2 were
selected on the basis of tick abundance, whereas in study 1,
sampling sites were nearby lowland sheep farms where the
presence of ticks was normally scarce. I. ricinus was the pre-
dominant tick species (91.6% of larvae and 79.4% of nymphs)
feeding on small mammals. This observation is consistent with
the tick collection rates obtained by blanket dragging the veg-
etation in this region (4). A. sylvaticus was clearly the most
abundant species trapped over the two studies, indicating that
this species is predominant in woodland areas of northern
Spain, which is in agreement with reports from La Rioja, a
neighboring region in north-central Spain (11). A. sylvaticus
was also the rodent species most heavily infested with I. ricinus
larvae and nymphs, in agreement with other reports from Eu-
rope (14, 16, 23). The different tick infection levels observed
for different animal species (C. glareolus versus A. sylvaticus)
were explained by acquired immunity to I. ricinus larvae after
repeated infestations in C. glareolus (9).
The mean number of I. ricinus larvae in A. sylvaticus in both
studies (7.3) is clearly lower than in studies from Sweden (49.1)
(42) but higher than in several European studies, e.g., 1.7 in
Switzerland (25), 2.0 in La Rioja, Spain (11), and 2.1 in Ireland
(16). In addition, few nymphs (26 nymphs) were collected,
providing a 72:1 overall ratio of larvae to nymphs. This low
nymphal infestation may limit the capacity of transmission of
tick-borne bacteria to small mammals. This could be especially
true when transovarial transmission of pathogens in ticks does
not exist or occurs at a very low level and infection must be
transmitted in the course of feeding by infected nymphs, as is
the case for B. burgdorferi. However, the observed 72:1 ratio of
larvae to nymphs was markedly lower than the one reported by
Gil et al. (14) in the same region (450:1), but the prevalence of
B. burgdorferi sensu lato in small mammals was higher (0.8% in
study 1 and 1.6% in study 2) than in the former study (0.5%),
suggesting an increase in nymphal infestation in recent years.
Interestingly, this is the first report of B. afzelii infection of
small mammals in Spain and the first time that S. coronatus has
been involved as a reservoir of Lyme borreliosis. The preva-
lence values of B. afzelii infection found in S. coronatus
(14.3%) and C. glareolus (6.3%) were markedly lower than
those found in shrews and rodents in Central Europe (20, 22,
47). In any case, our results confirm reports by other groups
that propose small mammals as reservoirs of B. afzelii (18, 19).
Despite being the most abundant species captured and ana-
TABLE 2. Results of RLB analysis for the two studies and the different species of small mammals
No. (%) of infected animals
RLB hybridization results
Borrelia sp. strain R57
B. afzelii/Borrelia sp.
C. burnetii/Borrelia sp.
1 (0.8)0 1 (0.4)1 (0.6)000000
Total single and
33 (26.0)38 (30.2) 71 (28.1)54 (33.3)0 11 (68.8)2 (14.3)1 (16.7)3 (10.7)0
Results by genus
aThe first number in parentheses represents the number of animals captured, and the second number represents the number of animals analyzed.
VOL. 73, 2007 TICK-BORNE BACTERIA IN SMALL MAMMALS IN SPAIN6169
lyzed, none of the A. sylvaticus specimens was positive for B.
burgdorferi sensu lato, suggesting its low contribution in the
transmission of these spirochetes to subadult tick stages. These
findings are in accordance with previous studies that found
lower numbers of ticks and higher infection prevalences in
bank voles than in Apodemus mice (23, 28). The absence of B.
burgdorferi sensu stricto, B. garinii, and B. valaisiana in the
analyzed small mammals contrasts with the results reported for
questing ticks from the vegetation in our region, where B.
burgdorferi sensu stricto, B. garinii, and B. valaisiana and not B.
afzelii are the most prevalent genospecies (5, 10, 14). Hence,
the main reservoir hosts for B. burgdorferi sensu lato in north-
ern Spain remain unknown, and other mammal species, liz-
ards, or birds may play a major role in the maintenance of this
spirochete in the natural environment.
The novel Borrelia sp. strain R57, a spirochete closely related
to the genus Borrelia but forming a clade separate from that of
the Lyme disease agent and relapsing fever organisms (14), was
widely distributed in rodents and shrews, both in sheep farms
boundaries (22.1%) and in recreational parks (27.8%). Espe-
cially noteworthy were the detection of Borrelia sp. strain R57
in a domestic mouse and its high prevalence in bank voles
(68.8%). These two species of small mammals and most of the
animals captured in study 1 had low tick infestation levels,
suggesting that Borrelia sp. strain R57 might be transmitted by
ectoparasites other than ticks. This was already proposed by
Gil et al. (14), who found that all the ticks collected from
Borrelia sp. strain R57-positive small mammals were negative.
The overall prevalence of Borrelia sp. strain R57 obtained in
this study was two times higher than that previously reported
by Gil et al. for regions in northern Spain where Lyme disease
is endemic (14). The only methodological difference among
both studies was probe concentration, which was much higher
in the present study (16 versus 0.4 ?M), thus increasing the
sensitivity of the assay. Interestingly, Borrelia sp. strain R57
and B. afzelii, the only genospecies of B. burgdorferi found in
northern Spain, were found as a mixed infection in a C. glareo-
lus specimen, which suggests that an exclusion phenomenon
does not exist between these two species, although the influ-
ence of such a possibility on the lack of distribution of other
Borrelia genospecies should be further investigated.
In the United States, rodents are implicated as natural res-
ervoirs for A. phagocytophilum (43). In Europe, this pathogen
has been detected in several species of rodents such as A.
flavicollis (25, 41), Apodemus agrarius, Rattus rattus (8), C.
glareolus, A. sylvaticus (25), and shrews (Sorex araneus) (25),
but their role as reservoirs is not clear. In our area, this agent
has been associated with ovine and bovine abortions in several
mountainous areas (12, 21) and in roe deer (31), but it was not
detected among a small number of small mammals tested,
probably due to the relatively low number of nymphs found to
be infesting them (31). In the present study, we detected A.
phagocytophilum in A. sylvaticus (0.6%) and C. russula (16.7%).
Here, the relatively higher proportion of nymphs observed
would explain the higher prevalence in a manner similar to that
described above for B. burgdorferi. Also interesting was the
detection of A. phagocytophilum in C. russula, since this is the
first time that this shrew species has been involved as a possible
reservoir. Nevertheless, the overall prevalence (0.8%) found in
this study is clearly lower than in studies from Bulgaria (7.7%)
(8) and Switzerland (8 to 10%) (25), where rodents seem to
represent important reservoirs for this agent.
C. burnetii has a worldwide distribution, and many wild and
domestic mammals (mainly sheep, cattle, and goats), birds, and
arthropods, such as ticks, are considered their reservoirs. How-
ever, domestic ruminants represent the most frequent source
of human infection. The disease occurs throughout Spain, and
the incidence of its respiratory manifestations is especially high
in our area (Basque Country) (29), where the largest series of
Q fever pneumonia in Europe have been reported (27). Fur-
thermore, this agent has a high importance as an abortifacient
agent in sheep flocks in northern Spain (30). There are several
serological studies that implicate small mammals in the wild
and domestic cycles of Q fever (24, 38, 44), but very few studies
demonstrate the presence of C. burnetii DNA in small mam-
mals by molecular techniques (41). Therefore, the molecular
detection of C. burnetii DNA in A. sylvaticus (0.6%) and M.
domesticus (7.1%) captured in a sheep farm with previous
reports of abortion (P. Gabiria, personal communication) is an
interesting finding. These results suggest that mice would have
acquired the infection by direct contact with infected sheep or
with sheep fetuses or placentae inside the farm or in the pas-
ture and that in the study region, C. burnetii developed in a
peridomestic cycle rather than in a wild cycle and associated
with infected flocks. Consequently, the risk of transmission to
humans is associated mainly with domestic ruminants, and
control and surveillance of C. burnetii in the animal reservoir
environment are therefore needed to avoid human infection.
Ixodidae family ticks may act as vectors, reservoirs, and
amplifiers of SFG rickettsiae (34), and small rodents have been
shown to be susceptible to infection by several Rickettsia spe-
cies (37). However, the DNA of the SFG rickettsiae was not
detected in any of the animals analyzed in this study, suggest-
ing that these animals are not involved as reservoirs of these
pathogens in this area. This is in accordance with available data
on the scarce number of Rickettsia-positive tick specimens in
the same areas (J. F. Barandika, unpublished data) and with
the low incidence of human rickettsiosis in the studied area (2).
In summary, a better knowledge of the wild and peridomes-
tic cycles of tick-borne bacteria has been achieved and ques-
tions have been raised concerning the ecology of these zoo-
notic organisms. The complex cycle of these agents and the
variations that could suffer over time suggest the need for
continuous environmental surveillance to detect variations and
prevent risks for transmission to humans.
This work was conducted under financial support from FEDER, FIS
G03/057 and PI051873, INIA RTA02-001, and the Department of
Agriculture and Fisheries and Food of the Basque Government.
We thank the EDEN Sixth Framework EU project (contract refer-
ence no. 010284-2) for providing resources for ongoing work and an
expert tick and tick-borne disease forum for discussions.
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