Veterinary Microbiology 162 (2013) 551–557

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Veterinary Microbiology
journal homepage: www.elsevier.com/locate/vetmic

Molecular epidemiology of canine adenovirus type 1 and type 2 in

free-ranging red foxes (Vulpes vulpes) in Italy
Andrea Balboni a,*, Ranieri Verin b, Federico Morandi c, Alessandro Poli b,
Santino Prosperi a, Mara Battilani a
a
Department of Veterinary Medical Sciences, Alma Mater Studiorum-University of Bologna, Via Tolara di Sopra 50, 40064, Ozzano dell’Emilia (BO), Italy
b
Department of Animal Pathology, Prophylaxis and Food Hygiene, University of Pisa, Viale Delle Piagge 2, 56124, Pisa, Italy
c
Servizio gestione del territorio e sviluppo sostenibile, Monti Sibillini National Park, Piazza del Forno 1, 62039, Visso (MC), Italy

A R T I C L E I N F O A B S T R A C T

Article history: To date, no studies exist regarding the presence of canine adenovirus (CAdV) infection in
Received 27 July 2012 foxes in Italy. Furthermore, the majority of worldwide investigations regarding the
Received in revised form 26 October 2012 presence of CAdV in foxes have been carried out using common serological assays which
Accepted 7 November 2012 are unable to differentiate between CAdV type 1 and CAdV type 2. To assess the presence of
viral infection in Italian red foxes (Vulpes vulpes), thirty-two subjects shot during the
Keywords: regular hunting season in the province of Pisa (Tuscany, Italy) were sampled and tested
Adenovirus using a polymerase chain reaction (PCR) assay capable of distinguishing between CAdV
Fox
type 1 and type 2. Two subjects were positive for CAdV-1 infection and one other for CAdV-
Canine
2 infection. Sequence analysis of the two CAdV-1 viruses showed complete identity
Molecular epidemiology
Italy
between them and a high genetic similarity with all reference strains sequenced in dogs in
the last twenty years, indicating the presence of genetically stable CAdV-1 in red foxes in
Italy which could easily be transmitted from the wild animal population to domestic dogs.
Therefore, this is the first reliable identification of CAdV-2 in foxes, and cloning of the virus
detected has revealed a possible coinfection involving two different CAdV-2 strains,
raising new questions about the pathogenic role of CAdV-2 in wildlife. The presence of
CAdV-1 and CAdV-2 infection in foxes could represent a problem for both wild animals and
domestic dogs, and emphasises the central role of red foxes in maintaining these viruses in
the territory.
ß 2012 Elsevier B.V. All rights reserved.

1. Introduction Canine adenovirus type 1 is the aetiologic agent of a

Canine Adenovirus (CAdV), family Adenoviridae, genus
Mastadenovirus, is a non-enveloped dsDNA virus (Fauquet
et al., 2005) which infects numerous mammalian carni-
vores, including dogs. There are two types of canine
adenovirus, CAdV type 1 (CAdV-1) and CAdV type 2 (CAdV-
2), distinguishable by genetic, antigenic and pathogenetic
characteristics which, however, show cross-reaction.
* Corresponding author. Tel.: +39 051 2097084; fax: +39 051 2097039.
E-mail address: a.balboni@unibo.it (A. Balboni).
severe disease in dogs, infectious canine hepatitis (ICH),
characterised by acute necrohaemorragic hepatitis (Green,
2006a). Corneal oedema (blue eye), uveitis and interstitial
nephritis may occur after the acute stage of the disease as a
consequence of circulating immune complex deposition.
The histologically striking finding is the presence of large
eosinophilic intranuclear inclusion bodies, mainly within
hepatocytes surrounding the necrotic foci but also in
epithelial and endothelial cells in other organs. During the
acute stages of the disease, it can be isolated from any
animal tissue, secretions and excretions (saliva, urine,
faeces) while, 10–15 days postinfection (PI), the virus can

0378-1135/$ – see front matter ß 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.vetmic.2012.11.015
552 A. Balboni et al. / Veterinary Microbiology 162 (2013) 551–557
only be isolated from the kidney (Green, 2006a). Indeed, it
has been said that hepatocytes and vascular endothelial
cells of many tissues are the main targets of viral
localisation and injury but, in the absence of chronic
hepatic fibrosis, the kidney represents the main site of
persistence, and CAdV-1 is excreted in the urine for at least
6–9 months PI (Green, 2006a).
Canine adenovirus type 2 is one of the viral agents
implicated in the aetiopathogenesis of infectious tracheo-
bronchitis (ITB), also known as kennel cough, a mild self-
limiting acute upper respiratory disease in dogs (Buona-
voglia and Martella, 2007; Green, 2006b) which has a high
prevalence in canine communities. Canine adenovirus type
2 infection has not only been associated with mild
respiratory disease but it has also been isolated in dogs
which died after clinical manifestation of pneumonia and
enteritis (Macartney et al., 1988; Almes et al., 2010).
Canine adenovirus type 1 has been considered to be
effectively controlled, although not eradicated, in the
domestic dog population as a result of vaccination (Green,
2006a), primarily with modified live CAdV-2 vaccine,
which also protects against CAdV-2 infection. On the other
hand, CAdV-1 is widespread in wildlife, primarily as a
subclinical infection, and can cause epizootic episodes in
wild carnivores belonging to the Canidae, Mustelidae and
Ursidae families (Woods, 2001; Fauquet et al., 2005).
In foxes, CAdV-1 infection was reported as a cause of
illness (epizootic fox encephalitis) for the first time in 1925
(Green, 1925; Green et al., 1930). Successively, evidence of
exposure to CAdV has been reported for different fox
species in several geographic areas (Amundson and Yuill,
1981; McCue and O’Farrell, 1988; Davidson et al., 1992;
Garcelon et al., 1992; Truyen et al., 1998, Riley et al., 2004;
Robinson et al., 2005; Clifford et al., 2006; Gerhold et al.,
2007; Akerstedt et al., 2010; Thompson et al., 2010) but, to
date, there are no reports on CAdV infection or clinical
manifestation of epizootic encephalitis in foxes in Italy.
To the best of author’s knowledge, the majority of studies
regarding the presence of CAdV in foxes have been carried
out using serology and only a few studies by means of viral
isolation. Due to cross reactions which characterise the two
types of canine adenovirus, common serological methods are
unable to differentiate between CAdV type 1 and CAdV type
2. Only the inhibition of haemagglutination assay is capable
of distinguishing between the two types of CAdV (Marusyk
and Yamamoto, 1971). As a result, serological surveys carried
out on wild animal populations in recent years have been
able to evaluate the presence of adenovirus but have not
been able to evaluate the real prevalence of the two viral
types. Furthermore, only one short genome sequence of
CAdV-1 identified in a grey fox (Urocyon cinereoargenteus) is
present in the GenBank nucleotide database (GenBank ID:
EF611185, Gerhold et al., 2007). Therefore, no data exist on
the molecular epidemiology of the virus in foxes.
The aim of this paper was to describe the first survey
regarding the presence of CAdV-1 and CAdV-2 in red foxes
(Vulpes vulpes) in Italy. Thirty-two subjects shot during the
regular hunting season in the province of Pisa (Tuscany,
Italy) were sampled and tested for the presence of CAdV
using polymerase chain reaction (PCR). The study per-
mitted the identification of three foxes positive for CAdV
infection; two were positive for CAdV-1 while the other
was positive for CAdV-2. Furthermore, the sequence
analysis carried out on the identified CAdV-2 revealed a
possible coinfection with two different viruses.
2. Materials and methods
2.1. Sampling and histopathology
We examined thirty-two red foxes (V. vulpes) of both
genders (19 females and 13 males) shot during the regular
hunting season in the province of Pisa (438N 10–118E,
Tuscany, Italy) in two different time periods: 15 in
February 2011 and 17 in October 2011 (Table 1). Seventeen
animals were over 1 year of age and 15 were juveniles. Age
was determined by crystalline lens weight as previously
reported (Cavallini and Santini, 1995); body weight,
ranging from 3.5 kg to 8.1 kg, was also recorded. The
sampling areas were characterised by wooded areas and
hilly sites with a high population density of red foxes (1.2–
1.6 foxes/km2; Verin et al., 2010).
A complete post mortem examination was carried out
on all subjects, and different samples were collected and
stored at 80 8C for further investigation. During the first
sampling, faecal samples were taken from all 15 foxes. In
addition to the faeces, parts of the liver and kidneys were
collected from the 17 foxes shot in October, because they
represent the main site of persistence of the virus.
Representative portions of the same organs were also
fixed in 10% buffered formalin, processed and embedded in
paraffin. Four-micrometre serial sections from paraffin-
embedded organs were stained with haematoxylin and
eosin for general examination.
2.2. DNA extraction
Viral DNA extraction from the faeces and organs was
performed using the NucleoSpin Tissue Mini Kit
(Macherey-Nagel, Düren, Germany) according to the
manufacturer’s instructions. The extracted DNA was eluted
in 100 ml of elution buffer and stored at 20 8C.
2.3. PCR for canine adenovirus detection
Canine adenovirus screening was carried out on DNA
extracts using polymerase chain reaction (PCR) assay. A
fragment of the E3 gene and flanking regions (Hu et al.,
2001; Fauquet et al., 2005) were amplified using two
conserved primers: forward: 50 -CGC GCT GAA CAT TAC TAC
CTT GTC-30 and reverse: 50 -CCT AGA GCA CTT CGT GTC CGC
TT-30 (Hu et al.,2001; Chaturvedi et al., 2008). This PCR
assay produces fragments of different lengths which
allowed differentiation between the two adenovirus types,
508 bp for CAdV-1 and 1030 bp for CAdV-2, respectively.
The PCR assay was carried out using the Taq DNA
Polymerase Kit (QIAGEN, Hilden, Germany) in a total
volume of 50 ml. The DNA extract of the Canigen CEPPi/L
vaccine (Virbac, Carros, France) containing a CAdV-2
attenuated strain and the DNA extracted from the paraffin
embedded liver of a dog died for CAdV-1 infection were
used as positive controls. The mixtures were amplified as
 A. Balboni et al. / Veterinary Microbiology 162 (2013) 551–557 553

Table 1
Table of foxes sampled in the province of Pisa (Tuscany, Italy) in 2011.

No. of foxes Date shot Sampling areas Sex Weight (kg) Age (years) Type of sample

09-1 02 Feb 2011 Coltano (Pisa) M 4.1 >1 F

09-2 03 Feb 2011 Coltano (Pisa) F 3.5 <1 F
09-3 04 Feb 2011 Coltano (Pisa) F 4.2 >1 F
09-4 04 Feb 2011 Coltano (Pisa) F 3.7 <1 F
09-5 04 Feb 2011 Coltano (Pisa) F 4.5 >1 F
09-6 04 Feb 2011 Coltano (Pisa) M 4.7 >1 F
09-7 04 Feb 2011 Coltano (Pisa) F 3.8 <1 F
09-8 08 Feb 2011 San Miniato (Pisa) M 6.4 >1 F
09-9 08 Feb 2011 San Miniato (Pisa) F 5.2 >1 F
09-10 08 Feb 2011 San Miniato (Pisa) F 5.3 >1 F
09-11 08 Feb 2011 San Miniato (Pisa) M 4.3 <1 F
09-12 08 Feb 2011 San Miniato (Pisa) M 4.7 <1 F
09-13 08 Feb 2011 San Miniato (Pisa) F 6.1 >1 F
09-15 15 Feb 2011 Coltano (Pisa) M 4.7 <1 F
09-16 15 Feb 2011 Coltano (Pisa) M 4.9 <1 F
113-1 01 Oct 2011 Coltano (Pisa) F 4.6 <1 F, L, K
113-2 01 Oct 2011 Coltano (Pisa) M 7.6 >1 F, L, K
113-3 01 Oct 2011 Coltano (Pisa) F 4.7 <1 F, L, K
113-4 01 Oct 2011 San Frediano (Pisa) F 5.3 >1 F, L, K
113-5 01 Oct 2011 San Frediano (Pisa) F 4.4 >1 F, L, K
113-6 01 Oct 2011 San Frediano (Pisa) F 5 >1 F, L, K
113-7 01 Oct 2011 San Frediano (Pisa) F 4.1 <1 F, L, K
113-8 01 Oct 2011 San Frediano (Pisa) M 6.2 >1 F, L, K
113-9 01 Oct 2011 San Frediano (Pisa) F 4.8 <1 F, L, K
113-10 01 Oct 2011 Coltano (Pisa) M 8.1 >1 F, L, K
113-11 01 Oct 2011 Coltano (Pisa) F 4.1 <1 F, L, K
113-12 01 Oct 2011 Coltano (Pisa) M 7.2 >1 F, L, K
113-13 01 Oct 2011 Coltano (Pisa) F 3.8 <1 F, L, K
113-14 01 Oct 2011 Coltano (Pisa) M 7 >1 F, L, K
113-15 01 Oct 2011 Coltano (Pisa) F 4.7 <1 F, L, K
113-17 01 Oct 2011 Coltano (Pisa) F 5.1 >1 F, L, K
113-19 01 Oct 2011 Coltano (Pisa) M 5.2 <1 F, L, K

Sex: M: male; F: female. Type of sample: F: faeces; L: liver; K: kidneys.

follows: 40 cycles of amplification with 1 cycle at 96 8C for
30 s, at 58 8C for 1 min, and at 72 8C for 1 min, followed by a
final elongation at 72 8C for 10 min. Standard precautions
were taken to avoid PCR contamination, and the ultrapure
water controls included in all PCR assays did not show false
positive results.
Five microlitres of the amplicons were electrophoresed
in 2% (w/v) agarose gel stained with ethidium bromide in
1X standard tris-acetate-EDTA (TAE) buffer and visualised
by UV light and using GeneRuler 100 bp Plus DNA Ladder
(Fermentas, Burlington, Ontario, Canada) to check the
presence of amplicons of the expected size.
2.4. Sequencing and cloning
The DNA products of the expected size were purified
using the High Pure PCR Product Purification Kit (ROCHE,
Mannheim, Germany) according to the manufacturer’s
protocol, and the nucleotide sequences were determined
with an ABI 3730 DNA Analyzer (Applied Biosystems,
Foster City, CA, USA) using both forward and reverse
primers.
The PCR product obtained from faecal sample 113-3F
was quantitatively too low to allow direct sequencing.
Therefore, the amplification product was cloned into the
pCR 4/TOPO vector using the TOPO cloning kit (Invitrogen,
Carlsbad, CA, USA), and was transformed into Escherichia
coli DH5a-competent cells according to the manufac-
turer’s protocol. The recombinant clones obtained were
sequenced using both forward and reverse primers after
plasmid purification with the Turbo Kit (QBIOgene-MP
Biomedicals, Montreal, Canada).
2.5. Sequence data
The nucleotide sequences obtained were assembled
and translated into aminoacid sequences using BIOEDIT
sequence alignment editor version 7.0.9. The assembled
nucleotide sequences were analysed by BLAST web
interface (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and
multiple alignment with reference sequences available
from the GenBank nucleotide database (http://
www.ncbi.nlm.nih.gov/genbank) was carried out using
the CLUSTALW2 web interface (http://www.ebi.ac.uk/
Tools/msa/clustalw2).
The phylogenetic relationships of the viruses detected
with canine adenovirus and bat adenovirus reference
sequences available from GenBank were evaluated using
MEGA version 5.05; pairwise genetic distances were
calculated using the Tamura 3-parameter model, and a
phylogenetic tree was constructed using the neighbour-
joining method. Bootstrap values were determined by
1000 replicates to assess the confidence level of each
branch pattern.
554 A. Balboni et al. / Veterinary Microbiology 162 (2013) 551–557

2.6. Nucleotide sequence accession numbers 3.3. Sequence data

The nucleotide sequences obtained have been lodged
within the GenBank sequence database under accession
numbers: JX416838–JX416842.
3. Results
3.1. Gross findings and histopathology
All the foxes appeared to have been in good general
health, as determined by fat deposits and body weight,
except for 2 subjects (n. 113-7 and 113-13) which were
affected by sarcoptic mange. None of the foxes, including
those which were PCR-positive, exhibited gross and
microscopic hepatic and renal lesions. Lesions were also
absent in the upper respiratory tract.
3.2. Detection of CAdV in fox samples
Positive PCR products of the expected size of 508 bp,
corresponding to CAdV-1 (Hu et al., 2001), were detected
in 1 out of 15 faecal samples of the first sampling (n. 09-
13F), and in both the liver and kidneys of 1 out of the 17
foxes sampled in October (n. 113-5L and 113-5K,
respectively). Another positive PCR product was obtained
from a faecal sample of the second sampling (n. 113-3F),
but the obtained fragment size of 1030 bp was indicative of
a CAdV type 2 infection (Hu et al., 2001).
The cloning of amplicon 113-3F was characterised by a
low yield and only two recombinant clones, 113-3F-c01
and 113-3F-c04 respectively, were obtained.
Nucleotide sequencing was obtained for viruses
detected in the faeces of fox 09-13 (09-13F), the liver
and kidneys of fox 113-5 (113-5L and 113-5K), and both
clones obtained from the faeces of fox 113-3 (113-3F-c01
and 113-3F-c04). The sequences were assembled and
subsequently analysed by BLAST web interface. Sequences
09-13F, 113-5L and 113-5K produced significant align-
ments with CAdV-1 reference strains showing identity
values ranging from 99 to 100% whereas clones 113-3F-c01
and 113-3F-c04 produced significant alignments with
CAdV-2 reference strains showing identity values ranging
from 97 to 99%.
The nucleotide alignment showed complete identity
between the three CAdV-1 sequences, 09-13F, 113-5L and
113-5K, whereas the comparison of the two clones of the
CAdV-2 strain, 113-3F-c01 and 113-3F-c04, allowed the
calculation of an identity of 99% for both nucleotide and
translated aminoacid sequences. A total of 6 nucleotide
differences were found between the sequences obtained
from the two clones of sample 113-3F, indicating the
presence of two different viral populations in the same
fox.
A phylogenetic tree constructed from the detected
viruses with reference strains available from GenBank
showed a clear subdivision of canine adenovirus sequences
into two main clusters supported by elevated bootstrap
values: CAdV-1 clade one and CAdV-2 clade one. The
nucleotide sequences of the viruses identified in foxes 09-
13 and 113-5 were grouped in the CAdV-1 cluster whereas
the CAdV-2 cluster included the sequences of the two
clones of sample 113-3F (Fig. 1).

Fig. 1. Phylogenetic tree constructed with nucleotide sequences of canine and bat adenoviruses. The phylogenetic tree was constructed with the nucleotide
sequences generated in this study and with sequences of CAdV-1, CAdV-2 and Bat AdV reference strains obtained from the GenBank database. Bootstrap
values greater than 80%, calculated on 1000 replicates, are indicated on the respective branches. The reference strains included in the phylogenetic analysis
are: BtAdV TJM (GU226970), BtAdV-2 PPV1 (JN252129), CAdV-1 B579 (GQ340423), CAdV-1 CLL (U55001), CAdV-1 RI261 (Y07760), CAdV-1 India1
(EF057101), CAdV-1 India2 (EF090910), CAdV-1 GLAXO (M60937), CAdV-1 Utrecht (S38238), CAdV-2 Manhattan (S38212), CAdV-2 HB1 (GQ915311),
CAdV-2 TA26/61 (CAU77082). In bold: the nucleotide sequences generated in this study are: 09-13F (JX416838), 113-5K (JX416840), 113-3F-c01
(JX416841) and 113-3F-c04 (JX416842). Because sequences 113-5L and 113-5K were identical only the sequence 113-5K was used for phylogenetic
analysis of the virus identified in fox 113-5.
 A. Balboni et al. / Veterinary Microbiology 162 (2013) 551–557
4. Discussion
In this study, we reported a survey regarding the
presence of CAdV-1 and CAdV-2 on 32 red foxes shot
during the regular hunting season in the province of Pisa
(Tuscany, Italy) in 2011. Using PCR, three foxes were
positive for CAdV infection, two for CAdV-1 (one in the
faeces and one in the liver and kidneys) and one for CAdV-2
(in the faeces). The amplicons obtained were sequenced
and analysed.
Concerning the two foxes which tested positive for
CAdV-1, it is reasonable to assume that subject 09-13 was
in the acute phase of infection. This assumption is
supported by the detection of the virus in the stool which
is compatible with active faecal shedding which mostly
occurs in the first postinfection weeks (Green, 2006a).
Therefore, fox number 113-5 was probably in the chronic
stage of the infection because viral DNA was only detected
in the liver and kidneys which are the two principal sites of
viral persistence, and not in the faeces, excluding faecal
viral shedding (Green, 2006a).
The finding of CAdV-1 infection in free-ranging red
foxes shows that foxes are exposed naturally to this virus
in Italy. Sequence analysis carried out on the two CAdV-1
viruses, detected in foxes 09-13 and 113-5 respectively,
shows the genetic stability of these viruses. Indeed, the
sequences of 09-13F, 113-5L and 113-5K obtained were
identical and were very similar to all the reference strains
sequenced in dogs and deposited in GenBank in the past
twenty years. These data point out the presence of
genetically stable CAdV-1 in red foxes which could easily
be transmitted from the wild animal population to
domestic dogs.
The presence of infected foxes could represent a
problem for both wild animals and domestic dogs. Despite
the fact that systematic vaccination has considerably
reduced the incidence of the disease in the canine
population, cases of ICH in domestic dogs have been
diagnosed in Italy in recent years (Decaro et al., 2007).
Because of CAdV immunity derived from vaccination, the
presence of the infection in vaccinated dogs is improbable
due to the genetic stability of adenoviruses. The above-
mentioned cases of ICH in domestic dogs demonstrate that
dogs susceptible to CAdV-1 were exposed to the virus.
Therefore, the cases of ICH in dogs were the consequence of
non-vaccination or the interference of maternally derived
antibodies (MDA) in puppies vaccinated too early, rather
than the emergence of new strains (Decaro et al., 2008).
Fox-dog viral transmission can occur in two ways:
unvaccinated domestic dogs could contract the infection
from infected wild species or susceptible wildlife could
contract the infection from infected domestic dogs
(Woods, 2001). Therefore, the presence of infected foxes
which live on the margins of urban areas and come into
contact with domestic animals may increase the risk of
introduction of the virus and the development of the
disease in domestic dogs, especially for animals not
properly vaccinated. On the other hand, since the disease
has not been completely eradicated in the dog population
despite widespread vaccination, the detection of positive
foxes can depend on the transmission of the virus from
555
infected dogs. An important source of infection for foxes
may also be represented by hunting dogs imported from
endemic areas having an uncertain sanitary status which
could transmit the infection to wildlife. This latter
hypothesis could represent an important route of viral
diffusion in areas previously free from CAdV-1.
To date, there is scant information regarding infections
by CAdV-2 in foxes, probably due to the low impact it has
on animal health. In particular, the finding of serologic
positivities towards CAdV in wildlife has received more
attention in view of the implications that the transmission
of CAdV-1, rather than CAdV-2, may have on dogs and
wildlife health.
To the best of author’s knowledge, this is the first
reliable report of CAdV-2 in a red fox, confirmed by the
sequencing of the amplicon obtained. This finding raises
new questions regarding the presence of this pathogen in
wildlife and on its pathogenic role, especially in the light of
its identification in the faeces. In fact, CAdV-2 has only
been identified in rare cases from the faeces of domestic
dogs and it has already been reported in association with
episodes of enteritis (Hamelin et al., 1985; Macartney et al.,
1988).
Moreover, the PCR product of the CAdV-2 identified was
cloned, and sequence analysis carried out on the recombi-
nant clones obtained was indicative of the presence of two
different viral populations which led us to hypothesise the
establishment of a coinfection with two different CAdV-2
strains in the same fox. Alternatively, the finding of two
genetically different CAdV-2 strains in sample 113-3F may
be the consequence of contamination during the analysis
of the samples or the establishment of a quasispecies
structure in the animal host. However, the two latter
occurrences are highly unlikely for several reasons. First,
no other foxes tested positive for CAdV-2; therefore,
contamination of the stool specimen of 113-3 during the
autoptic procedures cannot have occurred. Second, every
precaution was taken to avoid extraction or PCR contam-
ination. Third, canine adenoviruses are extremely geneti-
cally stable viruses which evolve very slowly over time and
for which the dynamics of quasispecies have never been
reported; therefore, it is unlikely that a CAdV-2 strain
accumulated so many nucleotide mutations during the
infection of a single host.
Coinfections are frequent events associated with
adenoviruses which are also subjected to recombination
(Metzgar et al., 2005; Robinson et al., 2011), but events
such as these have not yet been reported for canine
adenoviruses. The presence of two genetically different
CAdV-2 strains in the same animal may be indicative of the
circulation of different viral strains in the fox population,
increasing the chances that different viruses may come in
contact with and infect the same fox. Additional investiga-
tion is required to assess whether a similar viral variability
is also present in dogs, also considering that CAdV-2 has
been already reported in Italy (Decaro et al., 2004).
5. Conclusion
The role of red foxes in the epidemiology of canine
adenovirus types 1 and 2 is still uncertain. It is difficult to
556 A. Balboni et al. / Veterinary Microbiology 162 (2013) 551–557
determine if, and how many, foxes manifest clinical signs
or die in consequence of the infection, but there is evidence
that the two viruses circulate in the fox population. The
lack of gross and microscopic lesions referable to CAdV and
the good health status of the subject analysed suggested
that red foxes might play a role in the maintenance of
CAdV-1 and CAdV-2 in the territory and could be a source
of viral spread in the domestic dog population.
Adenoviruses were also recently discovered in bats (Bat
AdVs) (Maeda et al., 2008) and some of these adenovirus
strains showed a strictly genetic correlation with the
canine adenovirus, suggesting a possible interspecies
transmission event of an AdV from bats to mammalian
carnivores (Jánoska et al., 2011; Kohl et al., 2012). In light
of this discovery, the role of wild carnivores in the
epidemiology of the canine adenovirus is even more
plausible because it is more likely that these animals have
served as an intermediate host for virus transmission from
bats to domestic dogs. Potentially, the adenovirus of bats
may have first adapted to replicate in a wild host, such as
the fox, and only then would it be transmitted to dogs.
This first report of CAdV-1 and CAdV-2 infections in
Italian red foxes emphasises the central role of foxes in the
maintenance of the viruses in the territory. Indeed, foxes
can transmit these viruses to domestic dogs and could be a
receptive host for canine viruses. Careful monitoring of
CAdV diffusion in wildlife could help assess the epide-
miology of these viruses and control the re-emergence of
ICH which has occurred in recent years in domestic dogs
(Decaro et al., 2007).
To improve the current knowledge regarding the
epidemiology of CAdV-1 and CAdV-2, future studies should
complement traditional serological analysis with genetic
analysis to allow better characterisation of the viruses and
a greater understanding of their evolutionary dynamics. In
addition to this, further investigations such as virus
isolation and immunohistochemistry will provide useful
data about virus viability and tissue distribution.
Acknowledgements
The authors would like to thank the province of Pisa and
the region of Toscana for having made this study possible
by providing the funding. We would especially like to
thank the hunters of the province of Pisa and their
associations (ATC 14 and 15) for their work and help-
fulness. Financial support was also provided by RFO funds
(Ricerca Fondamentale Orientata) of the Alma Mater
Studiorum-University of Bologna.
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