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Veterinary Microbiology 118 (2006) 223–229
Torque teno virus (TTV) is highly prevalent in the European
wild boar (Sus scrofa)
Laura Martınez a,1, Tuija Kekarainen a,1,*, Marina Sibila a, Francisco Ruiz-Fons c,Dolors Vidal c, Christian Gortazar c, Joaquim Segales a,b
a Centre de Recerca en Sanitat Animal (CReSA), Universitat Autonoma de Barcelona, 08193 Bellaterra, Barcelona, Spainb Departament de Sanitat i d’Anatomia Animals, Facultat de Veterinaria, Universitat Autonoma de Barcelona, Bellaterra, Barcelona, Spain
c Instituto de Investigacion en Recursos Cinegeticos, IREC (CSIC-UCLM-JCCM), Ciudad Real, Spain
Received 16 May 2006; received in revised form 17 July 2006; accepted 27 July 2006
Abstract
The present study represents the first survey of Torque teno virus (TTV) prevalence in European wild boar (Sus scrofa). The
prevalence of two distinct TTV genogroups in 178 Spanish wild boar sera from different geographic regions, management
conditions, gender and age was determined by a nested PCR method. The overall prevalence of TTV genogroups was 84% (58% for
genogroup 1 and 66% for genogroup 2), and differences between genogroup prevalence were observed depending on the
geographical region analysed. Significantly higher prevalence for TTV genogroup 2 was found in fenced managed wild boar,
juvenile animals and females. No other significant differences in TTV genogroup prevalence were observed. The phylogenetic
analysis of nucleotide sequences obtained from the untranslated region of selected samples revealed that the same TTV genogroups
are infecting wild boar and domestic pig. The results indicate that TTVis apparently ubiquitous in European wild boar populations.
# 2006 Elsevier B.V. All rights reserved.
Keywords: Torque teno virus (TTV); Anellovirus; European wild boar (Sus scrofa); Nested polymerase chain reaction; Spain
1. Introduction
Torque teno virus (TTV) belongs to the floating
genus Anellovirus. TTV is a small non-enveloped virus
which contains a single-stranded, negative sense,
* Corresponding author. Tel.: +34 935814620;
fax: +34 935814490.
E-mail address: [email protected]
(T. Kekarainen).1 These authors contributed equally to this work.
0378-1135/$ – see front matter # 2006 Elsevier B.V. All rights reserved
doi:10.1016/j.vetmic.2006.07.022
circular DNA genome. Up to now, two distinct
genogroups have been identified in the domestic pig
(Niel et al., 2005). Virus detection by genogroup 1
specific PCR in serum samples from different geo-
graphic regions (Canada, China, Korea, Spain, France,
Thailand and USA) has revealed a prevalence ranging
from 33% to 100% (McKeown et al., 2004; Bigarre
et al., 2005). The prevalence of genogroups 1 and 2 in
Spain is 60% and 77%, respectively (Kekarainen et al.,
2006). No other data are available regarding TTV
genogroup 2 prevalence.
.
L. Martınez et al. / Veterinary Microbiology 118 (2006) 223–229224
In Spain, as well as in other European countries,
number and density of wild boar have largely increased
in the last three decades (Gortazar et al., 2006). Besides
the obvious existence of free-living animals, wild boar
populations are increasingly managed by high-wire
fencing, artificial feeding and restocking with farm bred
individuals. As a result, some wild boar hunting estates
resemble extensive pig breeding facilities, with high
densities but minimal or no sanitary care (Vicente et al.,
2004).
The domestic pig and wild boar share common
pathogens and thus wild boar could constitute a disease
or infection reservoir for the domestic pig (Gortazar
et al., 2002; Fernandez-de-Mera et al., 2003; Vicente
et al., 2004, 2005). Due to the ubiquitous nature of TTV
in domestic pigs and the abundance of European wild
boar (Sus scrofa) in Spain, we decided to determine the
presence of TTV genogroups 1 and 2 in the wild boar
population in Spain, to compare the prevalence of the
virus in animals of different ages, gender and under
different management conditions, and to determine the
genetic divergence of wild boar TTV to previously
published domestic pig TTV genogroups.
2. Materials and methods
2.1. Case selection
A total of 178 wild boar serum samples collected
by the ‘‘Instituto de Investigacion en Recursos
Table 1
Prevalence of TTV genogroups in European wild boar (Sus scrofa)
Area Genogroups 1 or 2
TTV
Genogroup 1 TT
Pos Prevalence Pos Prev
Albacete (n = 11) 10 90.1 a,b,c 7 63.6
Cuenca (n = 11) 9 81.8 a,b,c 9 81.8
Guadalajara (n = 7) 5 71.4 a,b,c 4 57.1
Guadiana (n = 45) 40 88.9 a,b 34 75.6
M. Toledo (n = 37) 28 75.7 b,c 18 48.6
Ruidera (n = 12) 6 50.0 c 1 8.3
S. Morena (n = 41) 39 95.1 a 20 48.8
Toledo (n = 14) 13 92.9 a,b 10 71.4
Total 150 84.3 103 57.8
Number of analysed sera in each geographic region (n), total amount of pos
b, c) within the same column mean statistical significant differences in r
Cinegeticos’’ (IREC, Ciudad Real, Spain) were used.
Samples were obtained between years 2000 and 2005,
and corresponded to eight south-central Spanish
geographic regions (Table 1). Available sera per
studied region ranged from 7 to 45 (Table 1). Used
samples were balanced with regard to different ages
(juveniles [7–12 months of age], n = 60; sub-adults
[12–24 months], n = 58; and adults [over 2 years];
n = 60), gender (female, n = 89; male, n = 86, from
three animals the gender was unknown) were
recorded. At each sampling site, management
conditions to which the wild boars were subjected
were recorded: open (n = 59), without any manage-
ment; fenced (n = 59), areas with fencing, transloca-
tions and artificial feeding; intensively managed
(n = 60), which were fenced with livestock-like
management system.
2.2. Nested polymerase chain reaction (nPCR)
DNA from 200 ml of serum samples was extracted
using the NucleoSpin Blood DNA extraction Kit
(Nucleospin1 Blood, Macherey-Nagel GmbH & Co
KG Duren) and eluted in 100 ml elution buffer. Presence
or absence of TTV genogroups 1 and 2 in wild boar
samples was determined using a published nested PCR
(nPCR) method (Kekarainen et al., 2006). Briefly, for
TTV genogroup 1, first round 20 ml PCR reactions
contained 4 ml of serum DNA, 20 pmol primer forward-
1 (50-TACACTTCCGGGTTCAGGAGGCT-30) and
reverse-1 (50-ACTCAGCCATTCGGAACCTCAC-30),
V Genogroup 2 TTV Genogroups 1 and 2
TTV
alence Pos Prevalence Pos Prevalence
a,b 8 72.7 a,b 5 45.5 a,b
a,b 0 0 c 0 0 c
a,b 2 28.6 b,c 1 14.3 b,c
a 30 66.7 b 24 53.3 a
b 23 62.2 b 13 35.1 b
c 5 41.7 b 0 0 c
b 37 90.2 a 18 43.9 a,b
a,b 13 92.9 a 10 71.4 a
118 66.3 71 39.9
itive animals (Pos) and prevalence in percentage. Different letters (a,
egional TTV prevalence when comparing one region to another.
L. Martınez et al. / Veterinary Microbiology 118 (2006) 223–229 225
2.5 mM dNTP, 2 mM MgCl and 0.75 U Taq DNA
polymerase (Ecogen). The amplification was initiated
by heating for 5 min at 94 8C, followed by 35 cycles of
20 s at 94 8C, 15 s at 52 8C, 20 s at 72 8C and a final
extension for 5 min at 72 8C. From this reaction, 4 ml of
amplification product was used as a template for nPCR
using primers pair forward nested-1 (50-CAATTTGGC-
TCGCTTCGCTCGC-30) and reverse nested-1 (50-TACTTATATTCGCTTTCGTGGGAAC-30) 50 pmol
each primer, 2.5 mM dNTP, 2 mM MgCl and 0.75 U
Taq DNA polymerase initiated for 5 min at 94 8C,
followed by 35 cycles of 20 s at 94 8C, 15 s at 55 8C,
20 s at 72 8C and a final extension for 5 min at 72 8C.
Finally, 15 ml of nPCR (260 bp) product was run on 2%
TAE-agarose gel. For TTV genogroup 2, the amplifica-
tion was carried out as described above using primers
pairs forward-2 (50-AGTTACACATAACCACCAAA-
CC-30) and reverse-2 (50-ATTACCGCCTGCCCGA-
TAGGC-30) for the first round and forward nested-2 (50-CCAAACCACAGGAAACTGTGC-30) and reverse
nested-2 (50-CTTGACTCCGCTCTCAGGAG-30) for
the nPCR (260 bp).
2.3. Sequencing and phylogenetic studies
Sixteen amplified products (6 from TTV gen-
ogroup 1 and 10 from TTV genogroup 2) were
excised from the agarose gel and purified using
QIAquick gel extraction kit (Qiagen). Sequencing
reactions were done using Big Dye Terminator v3.1
cycle sequencing Kit (Applied Biosystems) and run
using ABI Prism 3100 sequence analyser (Perkin-
Elmer). Sequences were edited using ChromasPro
and aligned with the Clustal W programs. Align-
ments containing about 130 bp overlapping gen-
ogroup sequences were used to calculate
phylogenetic distances. Phylogenetic and molecular
evolutionary analyses were conducted using the
MEGA version 3.1 (Kumar et al., 2004). Sequences
from TTV genogroups 1 and 2 from Spanish
domestic pigs (Kekarainen et al., 2006) as well as
those considered reference strains for the mentioned
TTV genogroups (AB076001 for genogroup 1 and
AY823991 for genogroup 2) were included in the
phylogenetic analyses. The nucleotide sequences
obtained in this study were deposited in the GenBank
under the accession numbers from DQ523801 to
DQ523816.
2.4. Statistical analyses
Statistical analyses were performed with the SAS
system for windows version 9.1 (SAS Institute Inc.,
Cary, North Carolina, USA). Contingency tables
(PROC FREQ procedure) to compare genogroups 1
and 2 detection among different geographic areas, age
groups, gender and management conditions were
performed. Post hoc comparisons were addressed using
the Bonferroni correction and the level of statistical
significance was set at p < 0.05. Confidence intervals
for standard errors of prevalence were estimated with
the expression S.E.95% CI = 1.96[p(1 � p)]/n1/2.
3. Results
A total of 84.3% (150/178) of the wild boars were
positive for one or the other TTV genogroups. There
was no significant difference on the prevalence of
genogroups 1 and 2, being 57.8% (103/178) and
66.3% (118/178), respectively. From all the studied
animals, 39.9% (71/178) were co-infected with both
TTV genogroups. Analysing the samples for the
presence of only one genogroup, it was observed a
significantly lower amount of animals infected solely
with TTV genogroup 1 (17.9%, 32/178) than with
genogroup 2 (26.4%, 47/178).
The prevalence results of wild boar TTV gen-
ogroups in eight different geographical regions in
Spain are shown in Table 1. The distribution of TTV
positive animals varied depending on the genogroup
and geographical region. For genogroup 1, prevalence
values ranged from 8.3% (1/12 in Ruidera) to 81.8%
(9/11 in Cuenca). For genogroup 2, prevalence ranged
from 0% (0/11 in Cuenca) to 92.9% (13/14 in Toledo).
Significant differences in prevalence among regions
are shown in Table 1.
No statistical differences were detected on the
overall (TTV genogroup 1 or 2 open [24/59, 40.7%],
fenced [27/60, 45.0%] and intensive [20/59, 33.9%])
or TTV genogroup 1 prevalence among different
management conditions (open [37/59, 62.7%], fenced
[32/60, 53.3%] and intensive [34/59, 57.6%] Fig. 1A).
However, TTV genogroup 2 prevalence was signifi-
cantly lower in open (37/59, 62.7%) and intensive
conditions (31/59, 52.5%) than in fenced areas (50/60,
83.3%).
L. Martınez et al. / Veterinary Microbiology 118 (2006) 223–229226
Fig. 1. Mean prevalence (and 95% CI for standard errors) of TTV
genogroup 1 (grey bar) and genogroup 2 (black bar) in wild boars from
(A) different management conditions, (B) age classes and (C) gender.
TTV genogroup prevalence were studied by age
class and gender. The prevalence varied among age
classes depending on the genogroup (Fig. 1B).
Significantly more juveniles (80.0%, 48/60) were
nPCR positive for genogroup 2 than sub-adults
(60.3%, 35/58) and adults (58.3%, 35/60). No
differences by age were observed for TTV genogroup
1. Significantly higher amount of females (74.2%, 66/
89) than males (57.0%, 49/86) tested positive for TTV
genogroup 2 (Fig. 1C), but no differences in
genogroup 1 prevalence were observed between sexes.
Analysis of the obtained sequences revealed a low
genetic diversity within TTV genogroup variants but
high genetic diversity between genogroups, thus
forming different clusters in the phylogenetic analysis
(Fig. 2). The pair wise comparison of nucleotide
sequences within a genogroup showed high percentage
of homology (89–96% within TTV genogroup 1, and
95–100% within TTV genogroup 2). The percentage of
sequence identity among different variants was not
apparently associated with the geographical region,
gender, age or management conditions of the wild boar.
Diversity between genogroups 1 and 2 sequences was
high, showing overall sequence identities of only 49–
58%. Comparison of TTV sequences from wild boars
and domestic pigs revealed high identities in average,
93% and 96% for genogroups 1 and 2, respectively.
Genetic distances were similar to percentage identity
values between wild boar and domestic pig TTV
variants and, therefore, thosevariants did not segregated
into different clusters (Fig. 2).
4. Discussion
The present study represents the first description of
TTV infection in the European wild boar. Results
indicated a high prevalence of TTV in wild boar in
Spain, similar to that observed in domestic pigs
(Kekarainen et al., 2006). Moreover, in both species,
genogroup 2 is more prevalent than genogroup 1. In
domestic pig, TTV infections with genogroup 2 have
been shown to be more common in pigs affected by
postweaning multisystemic wasting syndrome
(PMWS), a porcine circovirus type 2 (PCV2) disease,
than non-PMWS affected pigs (Segales et al., 2005;
Kekarainen et al., 2006). The significance of the higher
incidence of genogroup 2 in wild boar remains unclear
since the health status of the wild boars analysed was
unknown and TTV seems to be a non-pathogenic virus,
at least for the domestic pig. The present study did not
allow assessing if wild boar TTV genogroups were
associated or not to any disease status.
While for some viruses like classical swine fever
virus, Aujeszky’s disease virus and bovine viral
diarrhoea virus the wild boar is considered a potential
reservoir, in the case of PCV2 it is suggested that the
domestic pig is the main reservoir (Albina et al., 2000;
Laddomada, 2000; Vicente et al., 2004, 2005).
According to data presented here, due to its ubiquitous
nature in both domestic pig and wild boar, it is likely
that TTV has adapted to both species and is circulating
within these species with similar prevalence.
L. Martınez et al. / Veterinary Microbiology 118 (2006) 223–229 227
Fig. 2. Phylogenetic tree constructed based on the nucleotide sequences of untranslated region of TTV from wild boar (Ss) and domestic pig
(GenBank accession numbers AB076001 and AY823991 are considered the reference strains for pig TTV genogroups 1 and 2, respectively). The
reference number of each sequenced strain refers to the year followed by identification code of animals; small ‘‘a’’ (genogroup 1) or ‘‘b’’
(genogroup 2) has been used to differentiate TTV sequences derived from the same animal. The bootstrap values adjacent to the main nodes
represent the percentage of 1000 trees that support the clustering; only bootstrap values >50% are shown.
Both TTV genogroups were present in wild boar of
all studied geographical regions but Cuenca, where all
animals were nPCR negative for genogroup 2.
Interestingly, the frequency of genogroups varied
greatly depending on the geographical region studied.
If this situation reflects the prevalence of TTV
genogroups in domestic pig populations in the sampling
regions is currently unknown. In fact, the prevalence of
TTV in domestic pigs has been reported to vary greatly
between and within countries (McKeown et al., 2004).
Overall, similar frequencies of TTV genogroup 1
infected animals were detected in all three management
conditions tested. Genogroup 2 seemed to be less
frequent in intensively managed, farm-like populations
than in wild boars living in open and fenced areas. This
was quite surprising, since it is believed that the main
route of TTV infection is faecal-oral (Ross et al., 1999;
Ohto et al., 2002; Komatsu et al., 2004). Consequently,
it would be expected that animals in high population
densities would be at greater infection risk than those in
L. Martınez et al. / Veterinary Microbiology 118 (2006) 223–229228
low population densities. At this time is too early to
explain such data, and therefore, further studies on the
prevalence of genogroup 2 in wild boar subjected to
different epidemiological situations are needed.
Some significant differences in prevalence of TTV
genogroup 2 were observed by age; younger animals
were more often infected than adults and sub-adults.
This would be a surprising finding, since, at least in
humans, TTV produces a long-lasting and possibly
permanent infection (Maggi et al., 2001; Moen et al.,
2003). Therefore, an increased prevalence of TTV
infection with age would have been expected (Saback
et al., 1999; Ohto et al., 2002). However, based on the
obtained data, it cannot be ruled out that TTV infection
dynamics in the wild boar would be different to that in
humans. It would be of interest to compare such
dynamics in wild boar with that in domestic pig since
the same viruses apparently infect both species.
Prevalence of both TTV genogroups was higher in
females than males, although only significant for
genogroup 2. Again, there are no comparison patterns
with domestic pigs or other species that suffer from
TTV infection. Moreover, the studies performed in
humans suggest no gender differences in terms of TTV
prevalence (Maggi et al., 1999; Lin et al., 2000).
Therefore, this result should be considered as purely
descriptive or even a potential spurious effect.
The nucleotide sequence analysis indicated low
genetic variation of wild boar TTV strains within a
genogroup. Moreover, this study indicates that wild
boar and domestic pig are infected with the same TTV
genogroups. Also, the lack of geographic clustering
suggests a common source of infection for both
species. The relatedness of wild boar and domestic pig
TTV sequences is likely reflecting the evolutionary
relationship of the infected hosts and the close contact
of these two species. However, to conclude the
suggestion that wild boar and domestic pig TTV is
identical, the full-length genomic sequence should be
determined. Also, it cannot be ruled out the existence
of other TTV genogroups in the wild boar, different
from the ones described in the domestic pig.
In summary, the obtained results show that TTV is
apparently ubiquitous in the wild boar. In addition,
differences on the TTV genogroup prevalence
according to geographic regions, management condi-
tions, age and sex were detected. Wild boar TTV
nucleotide sequences obtained in this study seem to
correspond to those genogroups currently included
under the nomenclature of swine TTV, thus suggesting
widening of the classification to include TTV from
both groups of animals.
Acknowledgements
This work was partly funded by the Project no.
513928 from the Sixth Framework Programme of the
European Commission, and by OAPN-MMA and
SDGSA-MAPA, as well as Ministerio de Educacion,
Plan Nacional de I + D, grant AGL2005-07401-GAN.
Francisco Ruiz-Fons is an FPU-MEC grantee.
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