www.elsevier.com/locate/vetmic

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: Tuija.Kekarainen@cresa.uab.es

(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.

References

Albina, E., Mesplede, A., Chenut, G., Le Potier, M.F., Bourbao, G.,

Le Gal, S., Leforban, Y., 2000. A serological survey on classical

swine fever (CSF), Aujeszky’s disease (AD) and porcine repro-

ductive and respiratory syndrome (PRRS) virus infections in

French wild boars from 1991 to 1998. Vet. Microbiol. 77, 43–57.

Bigarre, L., Beven, V., de Boisseson, C., Grasland, B., Rose, N.,

Biagini, P., Jestin, A., 2005. Pig anelloviruses are highly pre-

valent in swine herds in France. J. Gen. Virol. 86, 631–635.

Fernandez-de-Mera, I.G., Gortazar, C., Vicente, J., Hofle, U., Fierro,

Y., 2003. Wild boar helminths: risks in animal translocations.

Vet. Parasitol. 115, 335–341.

Gortazar, C., Acevedo, P., Ruiz-Fons, F., Vicente, J., 2006. Disease

risks and overabundance of game species. Eur. J. Wildl. Res. 52,

81–87.

Gortazar, C., Vicente, J., Fierro, Y., Leon, L., Cubero, M.J., Gon-

zalez, M., 2002. Natural Aujeszky’s disease in a Spanish wild

boar population. Ann. N.Y. Acad. Sci. 969, 210–212.

Kekarainen, T., Sibila, M., Segales, J., 2006. Prevalence of swine

Torque teno virus in post-weaning multisystemic wasting syn-

drome (PMWS)-affected and non-PMWS-affected pigs in

Spain. J. Gen. Virol. 87, 833–837.

Komatsu, H., Inui, A., Sogo, T., Kuroda, K., Tanaka, T., Fujisawa, T.,

2004. TTV infection in children born to mothers infected with

TTV but not with HBV, HCV, or HIV. J. Med. Virol. 74, 499–506.

Kumar, S., Tamura, K., Nei, M., 2004. MEGA3: integrated software

for molecular evolutionary genetics analysis and sequence

alignment. Brief Bioinform. 5, 150–163.

Laddomada, A., 2000. Incidence and control of CSF in wild boar in

Europe. Vet. Microbiol. 73, 121–130.

Lin, C.-L., Kyono, W., Tongson, J., Chua, P.K., Easa, D., Yanagi-

hara, R., Nerurkar, V.R., 2000. Fecal excretion of a novel human

circovirus, TT virus, in healthy children. Clin. Diagn. Lab.

Immunol. 7, 960–963.

Maggi, F., Fornai, C., Morrica, A., casula, F., Vatteroni, M.L.,

Marchi, S., Ciccorossi, P., Riente, L., Pistello, M., Bendinelli,

M., 1999. High prevalence of TT virus viremia in Italian

L. Martınez et al. / Veterinary Microbiology 118 (2006) 223–229 229

patients, regardless of age, clinical diagnosis and previous

interferon treatment. J. Infect. Dis. 180, 838–842.

Maggi, F., Pistello, M., Vatteroni, M., Presciuttini, S., Marchi, S.,

Isola, P., Fornai, C., Fagnani, S., Andreoli, E., Antonelli, G.,

Bendinelli, M., 2001. Dynamics of persistent TT virus infection,

as determined in patients treated with alpha interferon for

concomitant hepatitis C virus infection. J. Virol. 75, 11999–

12004.

McKeown, N.E., Fenaux, M., Halbur, P.G., Meng, X.J., 2004.

Molecular characterization of porcine TT virus, an orphan virus,

in pigs from six different countries. Vet. Microbiol. 104, 113–

117.

Moen, E.M., Sagedal, S., Bjøro, K., Degre, M., Opstad, P.K., Grinde,

B., 2003. Effect of immune modulation on TT virus (TTV) and

TTV-like-mini-virus (TLMV) viremia. J. Med. Virol. 70, 177–

182.

Niel, C., Diniz-Mendes, L., Devalle, S., 2005. Rolling-circle ampli-

fication of Torque teno virus (TTV) complete genomes from

human and swine sera and identification of a novel swine TTV

genogroup. J. Gen. Virol. 86, 1343–1347.

Ohto, H., Ujiie, N., Takeuchi, C., Sato, A., Hayashi, A., Ishiko, H.,

Nishizawa, T., Vertical Transmission of Hepatitis Viruses Col-

laborative Study Group, H.O., 2002. TT virus infection during

childhood. Transfusion 42, 892–898.

Ross, R.S., Viazov, S., Runde, V., Schaefer, U.W., Roggendorf, M.,

1999. Detection of TT virus DNA in specimens other than blood.

J. Clin. Virol. 13, 181–184.

Saback, F., Gomes, S.A., de Paula, V., da Silva, R.R., Lewis-

Ximenez, L.L., Niel, C., 1999. Age-specific prevalence and

transmission of TT virus. J. Med. Virol. 59, 318–322.

Segales, J., Allan, G.M., Domingo, M., 2005. Porcine circovirus

diseases. Anim. Health Res. Rev. 6, 119–142.

Vicente, J., Ruiz-Fons, F., Vidal, D., Hofle, U., Acevedo, P., Villa-

nua, D., Fernandez-de-Mera, I.G., Martin, M.P., Gortazar, C.,

2005. Serosurvey of Aujeszky’s disease virus infection in Eur-

opean wild boar in Spain. Vet. Rec. 156, 408–412.

Vicente, J., Segales, J., Hofle, U., Balasch, M., Plana-Duran, J.,

Domingo, M., Gortazar, C., 2004. Epidemiological study on

porcine circovirus type 2 (PCV2) infection in the European wild

boar (Sus scrofa). Vet. Res. 35, 243–253.