Research in Veterinary Science 97 (2014) 631–636

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Research in Veterinary Science

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Investigation of the presence of canine adenovirus (CAdV) in owned

dogs in Northern Italy
A. Balboni, C. Mollace, M. Giunti, F. Dondi, S. Prosperi, M. Battilani *
Department of Veterinary Medical Sciences, Alma Mater Studiorum, University of Bologna, Via Tolara di Sopra 50, Ozzano dell’Emilia, Bologna 40064, Italy

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

Article history: The use of a modified live canine adenovirus (CAdV) vaccine has greatly reduced the incidence of infec-
Received 9 January 2014 tious canine hepatitis (ICH) in dogs. Nevertheless, cases of CAdV type 1 and 2 (CAdV-1 and CAdV-2) infection
Accepted 27 October 2014 have been recently reported posing questions about the epidemiological situation of CAdV in dogs. In
order to assess the presence of CAdV, samples from 51 dogs presented at a Veterinary Teaching Hospi-
Keywords: tal in Bologna, Italy, for reasons unrelated with CAdV infection, were tested with a polymerase chain reaction
Canine adenovirus
(PCR) assay for CAdV. Thirty dogs (58.8%) were PCR positive for CAdV-2 infection and four of them (7.8%)
Coinfection
were positive for CAdV-1. Sequence analysis performed on the obtained PCR products suggests that a
Dog
Molecular epidemiology genetically stable CAdV-1 strain and different CAdV-2 strains circulate in the canine population exam-
Italy ined and that coinfections are relatively frequent.
© 2014 Elsevier Ltd. All rights reserved.

1. Introduction ICH in domestic canine population (Abdelmagid et al., 2004; Bass

Two types of canine adenovirus (CAdV) are distinguishable by
genetic, antigenic and pathogenetic characteristics: CAdV type 1
(CAdV-1) and CAdV type 2 (CAdV-2) (King et al., 2011).
In dogs, CAdV-1 is the aetiologic agent of the infectious canine
hepatitis (ICH), a severe disease mainly characterised by acute
necrohaemorragic hepatitis, but also by corneal edema (“blue eye”),
uveitis and interstitial nephritis, that may occur after the acute stage
of the disease as a consequence of circulating immune complex de-
position (Greene, 2012). Central nervous system (CNS), lung and
intestine are rarely target of injury in dogs, while encephalitis is the
main pathological finding of CAdV-1 infection in wild canids (epi-
zootic fox encephalitis) (Decaro et al., 2008; Woods, 2001). The viral
shedding occurs through saliva, urine and faeces for the first 10–
15 days post-infection (PI) but, in absence of chronic hepatic fibrosis,
it was reported that viral shedding continues only with urine for
at least 6–9 months PI because kidney represents the main site of
persistence (Decaro et al., 2008; Greene, 2012). The mortality rate
from ICH normally ranges from 10% to 30% (Cabasso, 1962), but it
was reported to increase in the presence of coinfections with other
viruses (Decaro et al., 2007; Kobayashi et al., 1993; Pratelli et al.,
2001).
In the last decades, the widespread use of a modified live CAdV-2
vaccine in countries like Italy has greatly reduced the incidence of
* Corresponding author. Department of Veterinary Medical Sciences, Alma Mater
Studiorum, University of Bologna, Via Tolara di Sopra 50, Ozzano dell’Emilia, Bologna
40064, Italy. Tel.: +39 051 2097081; fax: +39 051 2097039.
E-mail address: mara.battilani@unibo.it (M. Battilani).
et al., 1980; Decaro et al., 2008). Thus, CAdV-1 is nowadays con-
sidered a neglected canine virus and veterinary practitioners rarely
take it into account as causative agent of disease. Nevertheless, case
of hepatitis secondary to CAdV-1 infection have been documented
in recent years in dogs (Caudell et al., 2005; Decaro et al., 2007;
Headley et al., 2013; Müller et al., 2010; Pratelli et al., 2001). These
findings support the hypothesis that the CAdV-1 continues to cir-
culate and to be pathogenic in dogs. Furthermore, widespread
infection of CAdV-1 in wild carnivores may be a source of infec-
tion to the domestic canine population (Balboni et al., 2013;
Thompson et al., 2010; Truyen et al., 1998; Woods, 2001).
Canine adenovirus type 2 is implicated in the aetiopathogenesis
of the canine infectious respiratory disease (CIRD), or infectious tra-
cheobronchitis (ITB), a mild self-limiting acute upper respiratory
disease, which has a high prevalence in canine communities (Ford,
2012). Canine adenovirus type 2 is also associated with lower re-
spiratory tract diseases and enteritis, and it was detected in the brain
of dogs with neurological signs (Almes et al., 2010; Benetka et al.,
2006; Decaro et al., 2004; Hamelin et al., 1985; Macartney et al.,
1988). Dogs vaccinated with the modified live CAdV-2 vaccine are
considered protected from the clinical manifestation of CAdV-2 in-
fection (Appel et al., 1973, 1975; Bass et al., 1980; Cornwell et al.,
1982; Decaro et al., 2008). Nevertheless, canine adenovirus type 2
is still actively circulating in canids, whether or not immune to CAdV,
and, as for the CAdV-1, clinical reports of infection in both domes-
tic and wildlife animals are documented (Almes et al., 2010; Balboni
et al., 2013; Benetka et al., 2006; Buonavoglia and Martella, 2007;
Decaro et al., 2004; Headley et al., 2013; Kalinowski et al., 2012).
The recent update on CAdV-1 and CAdV-2 circulation in the wild
carnivores, and the sporadic outbreaks reported in the domestic

http://dx.doi.org/10.1016/j.rvsc.2014.10.010
0034-5288/© 2014 Elsevier Ltd. All rights reserved.
632 A. Balboni et al./Research in Veterinary Science 97 (2014) 631–636

canine population raise some concern about the real epidemiologi- Tissue Mini Kit (Macherey-Nagel, Düren, Germany) according to the
cal situation of the canine adenovirus in dogs. In order to assess the manufacturer’s instructions. The extracted DNA was eluted in 100 μl
presence of canine adenovirus in domestic dogs, patients were of elution buffer and stored at −20 °C. A fragment of the E3 gene
sampled at a veterinary hospital in Northern Italy. and flanking regions was amplified using two conserved primers
that produce fragments of different lengths which allowed differ-
2. Materials and methods entiation between the two adenovirus types, 508 bp for CAdV-1 and
1030 bp for CAdV-2, respectively (Chaturvedi et al., 2008; Hu et al.,
2.1. Study design and sampling 2001). The PCR assay was carried out using the HotStar HiFidelity
Polymerase Kit (QIAGEN, Hilden, Germany), in a total volume of 50 μl.
The study was conducted at the Veterinary Teaching Hospital of The HotStar HiFidelity Polymerase Kit contains a hot-start proof-
the University of Bologna (VTH-UB), Italy, on client-owned dogs. In reading enzyme that allows to reduce the error rate in DNA
the absence of epidemiological data about the prevalence of ad- duplication increasing the reaction fidelity. The mixtures were am-
enovirus in the canine population that would allow to predefine an plified as follows: an initial denaturation step at 95 °C for 5 min, 45
adequate number of subjects to be sampled, it was decided to include cycles of amplification with 1 cycle at 94 °C for 30 s, at 58 °C for
all dogs referred to the veterinary hospital during a 5-week period 1 min, and at 72 °C for 1 min, followed by a final elongation at 72 °C
(May 2012–June 2012). Selection of the subjects was done by one for 10 min. Standard precautions were taken to avoid PCR contam-
of the investigators (CM) on voluntary basis of the owners by in- ination, and the ultrapure water negative control included in all PCR
terviewing them in the waiting room of the VTH-UB during regular assays did not show false positive results. The DNA extracted from
hours of first opinion referrals (Monday–Friday 9 am–4 pm). The a CAdV-2 attenuated strain vaccine (Canigen CEPPi/L, Virbac, Carros,
owners were informed about the aim of the project and asked to France) and the DNA extracted from the paraffin embedded liver
sign a consent for the inclusion of their dog in the study. Rectal swabs of a dog that died from CAdV-1 infection (strain 313–2010-Lparaffin,
(RS) and urine samples (UR) were then collected by the same KF676977) were used as positive controls. Five microlitres of the
investigator after being obtained data related to breed, sex, age, vac- amplicons was electrophoresed in 2% (w/v) agarose gel stained with
cination status and reason for medical referral. ethidium bromide in 1X standard tris-acetate-EDTA (TAE) buffer and
Fifty-one dogs were sampled and data on signalment, vaccina- visualised by UV light, using a GeneRuler 100 bp Plus DNA Ladder
tion status, clinical presentation and positivity to CAdV are reported (Fermentas, Burlington, Ontario, Canada) to check the presence of
in Table 1. Rectal swabs were taken from all the dogs, while urine amplicons of the expected size.
were available from 20 (39.2%) dogs due to inability to collect spon-
taneous voiding urine samples at the time of the inclusion. Samples 2.3. Statistical analysis
were stored at −80 °C upon analysis. The study was approved by the
Scientific and Ethical Committee of the University of Bologna. Results were analysed using descriptive statistics. Data compar-
ison between subgroups (breed, sex, age and vaccination status) was
2.2. PCR for canine adenovirus detection performed by chi-square test. Statistical significance was set at
p < 0.05. An inter-rater agreement statistic (Cohen’s kappa coeffi-
Canine adenovirus screening was carried out on DNA extracts cient) was calculated to compare the results obtained by the two
using polymerase chain reaction (PCR) assay. Viral DNA extraction different biological matrices. Statistical analysis was performed using
from rectal swabs and urine was performed using the NucleoSpin commercially available statistical software (MedCalc Statistical Soft-
ware version 12.7.5 – MedCalc Software bvba, Ostend, Belgium;
http://www.medcalc.org; 2013).
Table 1
Tested dogs presented in subgroups and results of canine adenovirus testing. 2.4. Sequencing and sequence analysis
Subgroup Tested CAdV-1 CAdV-2
dogs (n = 51) positive (n = 4) positive (n = 30) The amplicons of the expected size and with a sufficient amount
Breed of DNA product to allow direct sequencing were purified using the
Purebred 36 3 (8.3%) 22 (61.1%) High Pure PCR Product Purification Kit (ROCHE, Mannheim,
Crossbred 15 1 (6.7%) 8 (53.3%) Germany) according to the manufacturer’s protocol, and the nucleo-
Sex tide sequences were determined with an ABI 3730 DNA Analyzer
Male 25 2 (8%) 16 (64%)
(Applied Biosystems, Foster City, CA, USA) using both forward and
Female 26 2 (7.7%) 14 (53.9%)
Age (months) reverse primers. Samples yielding PCR amplification of two differ-
≤12 [puppies] 12 1 (8.3%) 6 (50%) ent DNA fragments, attributable to CAdV-1 and CAdV-2 respectively,
13–48 [young] 9 1 (11.1%) 5 (55.6%) were purified from agarose gel to separate the two products and
49–120 [adults] 21 2 (9.5%) 14 (66.7%)
directly sequenced.
>120 [elderly] 9 0 (0%) 5 (55.6%)
Vaccinationa The nucleotide sequences obtained were assembled and trans-
Yes 44 4 (9.1%) 26 (59.1%) lated into amino acid sequences using BIOEDIT sequence alignment
Yes from less than 1 year 33 4 (12.1%) 21 (63.6%) editor version 7.0.9. The assembled nucleotide sequences were
Never 7 0 (0%) 4 (57.1%) aligned with 14 reference sequences of canine adenovirus and bat
Symptoms
adenovirus, available from the GenBank nucleotide database (http://
Dermatologic signs 4 0 (0%) 2 (50%)
Gastrointestinal signs 6 2 (33.3%) 5 (83.3%) www.ncbi.nlm.nih.gov/genbank), using the ClustalW method
Musculoskeletal signs 3 0 (0%) 1 (33.3%) implemented in BIOEDIT sequence alignment editor version 7.0.9.
Neurologic signs 1 0 (0%) 1 (100%) Three sequences of field CAdV identified in Italian dogs during
Respiratory signs 4 0 (0%) 4 (100%)
years 2010–2011 in our labs were also included in the alignment
Urinary signs 3 1 (33.3%) 2 (66.7%)
Other signs 2 0 (0%) 1 (50%) (CAdV-1: 313-2010-Lparaffin and CAdV-2: 60-2011-OFS and
No symptoms (routine 28 1 (3.6%) 14 (50%) S1-2011-OFS).
check/vaccination) The phylogenetic relationships of the viruses detected with canine
a
Yes: dogs that have been vaccinated at least once with CAdV-2 vaccine. adenovirus and bat adenovirus reference sequences were evalu-
Never: dogs that have never been vaccinated. ated using MEGA version 5.05 (Tamura et al., 2011). The best-fit
 A. Balboni et al./Research in Veterinary Science 97 (2014) 631–636
model of nucleotide substitution was determined using the Find Best
DNA/Protein Model function implemented in MEGA and Kimura two-
parameter model with gamma distribution resulted optimal for all
the sequence data (including reference strains). Phylogenetic tree
was constructed using the neighbor-joining method and boot-
strap values were determined by 1000 replicates to assess the
confidence level of each branch pattern.
3. Results
3.1. Detection of CAdV in dog samples
A PCR fragment positive for CAdV was detected in 30 out of 51
sampled dogs (58.8%). A DNA fragment of 1030 bp, corresponding
to CAdV-2, was present in all the PCR positive dogs and four of these
also showed a fragment of 508 bp, corresponding to CAdV-1 (Hu
et al., 2001). Thus, a mixed infection with CAdV-1 and CAdV-2 was
detected in 7.8% of the sampled dogs.
A PCR product specific for CAdV was detected in 17 out of 31
dogs with only rectal swabs available, including two dogs coinfected
by CAdV-1 and CAdV-2, and in 13 out of 20 dogs with both rectal
swabs and urine samples: faeces only (n = 8, including two dogs
coinfected by CAdV-1 and CAdV-2), urine only (n = 3) and both faeces
and urine (n = 2) (Table 2).
3.2. Statistical analysis
Dogs included in the study were sub-grouped according to: breed
(purebred, mongrel), sex, age (puppy: ≤12 months, young: 12–48,
adult: 49–120, elderly: >120), vaccination status and clinical pre-
sentation at the time of sample collection (Table 1). All vaccinated
dogs received a modified live vaccine containing a CAdV-2 strain
(TorontoA26/71). Forty of the 51 sampled dogs were from the geo-
graphical area of Bologna, while the remaining 11 dogs were from
other areas.
There was no significant difference of CAdV positive subjects
between breed, sex, age and vaccination status subgroups of animals
(Table 1). In particular, the prevalence of CAdV infection was 59.1%
(26/44) in dogs that had undergone at least one vaccination and 57.1%
(4/7) in dogs that had never been vaccinated. In addition, 63.6% (21/
33) of dogs vaccinated less than 1 year before sampling, were positive
to CAdV. This included the four subjects infected by CAdV-1. The
majority of sampled dogs were completely asymptomatic (54.9%)
and only 23 dogs showed mild clinical signs (Table 1). A poor agree-
ment between the two biological matrices was shown by the value
of Cohen’s kappa coefficient (<0.20).
3.3. Sequence data
Nucleotide sequencing was performed on 10 of the CAdV-2
viruses detected. Nucleotide sequencing was also performed for the
CAdV-1 and CAdV-2 fragments obtained from the four dogs with
coinfection. The nucleotide sequences of the four CAdV-1 viruses
were 462 bp in length and comprised the last 285 bp of E3 gene
Table 2
Positivity to CAdV in rectal swabs (RS) and urine (UR) samples.
Only rectal swab Urine samples
samples
Positive Negative
No urine samples
Positive in rectal swab 17 2 8
Negative in rectal swab 14 3 7
633
(corresponding to the last 94 amino acidic codons of E3 proteins).
Instead, nucleotide sequences of the 14 CAdV-2 viruses were 870 bp
in length and comprised the last 743 bp of E3 gene (correspond-
ing to the last 246 amino acidic codons of E3 proteins).
The nucleotide alignment showed complete identity between the
four obtained CAdV-1 sequences. Comparison of the 14 obtained
CAdV-2 sequences allowed to distinguish three genogroups on the
basis of only one nucleotide mutation at position 230 (correspond-
ing to nucleotide 582 of the entire E3 gene) that does not involve
substitutions in the amino acid sequences. In particular, eight se-
quences (internal lab codes: 249-2012-RS, 243-2012-UR, 244-2012-
RS, 252-2012-RS, 260-2012-RS, 270-2012-RS, 275-2012-RS and
303-2012-RS) showed nucleotide 230C; three sequences (internal
lab codes: 272-2012-RS, 286-2012-RS and 300-2012-RS) showed
nucleotide 230T and the last three sequences (internal lab codes:
253-2012-RS, 257-2012-RS and 296-2012-RS) showed an ambi-
guities in the electroferogram at position 230, characterised by a
double peak 230C/T, compatible with the presence of two differ-
ent CAdV-2 strains in the same subject.
The comparison of the obtained nucleotide sequences with ref-
erence sequences has allowed to calculate: (A) an identity of 100%
between the four obtained CAdV-1 sequences with five CAdV-1 ref-
erence strains: one virus identified in an Italian dog in 2010 (313-
2010-Lparaffin, KF676977), two viruses identified in Italian red foxes
in 2011 (09-13F, JX416838 and 113-5L, JX416839, Balboni et al.,
2013), the vaccine strain GLAXO (M60937) and one strain identi-
fied in the UK in 1996 (RI261, Y07760). (B) An identity of 100%
between the obtained CAdV-2 sequences with nucleotide 230C with
one CAdV-2 virus identified in an Italian red fox in 2011 (113-3F-
c04, JX416842, Balboni et al., 2013). (C) An identity of 100% between
the obtained CAdV-2 sequences with nucleotide 230T with four
CAdV-2 reference strains: two viruses indentified in Italian dogs in
2011 (60-2011-OFS, KF676978 and S1-2011-OFS, KF676979), the
currently used vaccine strain (TorontoA26/71, CAU77082) and the
strain Manhattan (S38212).
In the phylogenetic tree constructed from the detected viruses
with reference strains, the nucleotide sequences of 462 bp identi-
fied in dogs 275-2012, 286-2012, 296-2012 and 300-2012 were
grouped in the CAdV-1 cluster whereas the CAdV-2 cluster in-
cluded all other obtained sequences (Fig. 1).
4. Discussion and conclusions
In this study, a survey on the presence of CAdV-1 and CAdV-2
in 51 dogs presented at the VTH-UB, Italy, during a 5-week period
in May and June 2012, is reported. The reduced number of dogs in-
cluded in the study and the lack of a predefined stratification did
not allow to make epidemiological evaluations but to obtain pre-
liminary data concerning the presence of CAdV in the tested dogs.
Using a PCR assay, prevalence of CAdV-2 and CAdV-1 infection were
58.8% and 7.8%, respectively. The four dogs infected with CAdV-1
were also coinfected with CAdV-2.
The high prevalence of CAdV infection detected is in agree-
ment with the prevalence of CAdV infection recently observed in
serologic surveys carried out on domestic dogs and free-ranging
canids (Akerstedt et al., 2010; Belsare and Gompper, 2013; Gür and
Acar, 2009; Wright et al., 2013), which confirm the ubiquitous dif-
fusion of CAdV in receptive hosts in different parts of the world. The
significant prevalence of CAdV-1 and CAdV-2 infection in the dogs
analysed in this study support the hypothesis of a widespread cir-
culation of these viruses in the canine population.
However, the clinical relevance of these findings need to be clari-
fied, since, at the time of sampling, 12 positive dogs showed only
mild clinical signs (gastrointestinal, neurologic, respiratory and
urinary signs) potentially related to CAdV infection, including 3 out
of 4 dogs positives to CAdV-1 manifesting only mild intestinal or
634 A. Balboni et al./Research in Veterinary Science 97 (2014) 631–636

CAdV-1_field strain_IN_2006_dog_EF057101
CAdV-1_strain Utrecht_NL_1992*_dog_S38238
CAdV-1_vaccine strain GLAXO_1991*_M60937
CAdV-1_field strain RI261_UK_1996_dog_Y07760
CAdV-1_field strain 09-13F_IT_2011_fox_JX416838
CAdV-1_field strain 113-5L_IT_2011_fox_JX416839
CAdV-1
91 CAdV-1_field strain 313-2010-Lparaffin_IT_2010_dog_KF676977

100 CAdV-1_300-2012-RS_KF676980
CAdV-1_vaccine strain CLL_1996*_U55001
CAdV-1_field strain B579_IN_2006_dog_GQ340423
CAdV-2_field strain 113-3F-c01_IT_2011_fox_JX416841
CAdV-2_strain Toronto A26/61_CA_1961_dog_CAU77082
CAdV-2_strain Manhattan_US_1992*_dog_S38212
100
CAdV-2_field strain 60-2011-OFS_IT_2011_dog_KF676978
CAdV-2_field strain 113-3F-c04_IT_2011_fox_JX416842 CAdV-2
87
CAdV-2_field strain S1-2011-OFS_IT_2011_dog_KF676979
CAdV-2_249-2012-RS_KF676981
CAdV-2_272-2012-RS_KF676982
BtAdV_field strain TJM_CN_2009_bat_GU226970
99 BtAdV-2_field strain PPV1_DE_2011_bat_JN252129

0.1

Fig. 1. Phylogenetic tree constructed with nucleotide sequences of canine and bat adenoviruses. The phylogenetic tree was constructed with the nucleotide sequences gen-
erated 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 adenovirus reference strains included in the phylogenetic analysis are named with: acronym of viral species/
type, strain name, acronym of nation, year of identification and host species, plus the GenBank accession number. (* year of identification not available and substituted
with the year of submission in the GenBank database.) The obtained sequences included in the phylogenetic analysis are: CAdV-1_300-2012-RS (identical to CAdV-1: 275-
2012-RS, 286-2012-RS and 296-2012-RS), CAdV-2_249-2012-RS (identical to CAdV-2: 243-2012-UR, 244-2012-RS, 252-2012-RS, 260-2012-RS, 270-2012-RS, 275-2012-RS
and 303-2012-RS) and CAdV-2_272-2012-RS (identical to CAdV-2: 286-2012-RS and 300-2012-RS). In bold: Italian nucleotide reference sequences. Underlined: Nucleotide
sequences generated in this study.

urinary signs. This finding could be explained by the protective im-
munity in vaccinated dogs (44/51, 86.3%) or by a subclinical infection,
frequently reported for these two viruses, which normally cause more
severe clinical manifestation once bacterial or viral co-infection
occurs. Since the association between clinical symptoms and ad-
enovirus was not the aim of this study, further studies addressing
the clinical relevance of CAdV infection in canine population are
warranted.
The poor agreement in the detection of CAdV in urine and faecal
samples could be justified by a different viral load in the two bio-
logical matrices. Faecal material seems to be preferable to urine and
it is even more practical to collect. However it is still advisable to
test both biological matrices to potentially reduce the number of
false negative dogs.
Although a higher incidence of CAdV infection have been re-
ported in young dogs, aged less than 1 year, in unvaccinated dogs
and, potentially, in elderly dogs aged over 10 years (Greene, 2012),
our result did not show any preferential distribution within the dif-
ferent population categories, including breed, sex, age and vaccination
status. Moreover, all dogs coinfected by CAdV-1 and CAdV-2 had
been vaccinated since less than 1 year. However, the small number
of dogs sampled in this study and the nature of the population in-
vestigated do not allow to properly assess differences in prevalence
in relation to breed, sex, age and vaccination status. Further studies
on a more heterogeneous population of domestic dogs will be needed
to investigate the current situation in Italy.
Vaccination protects from the development of the disease, but
from our findings, it does not seem to protect from infection and
shedding of CAdV-1 as well as CAdV-2. This is in contrast with pre-
vious studies showing that CAdV-2 vaccination prevents subclinical
CAdV-2 infection in respiratory tissue (Bass et al., 1980) or allow
very low viral shedding via respiratory route (Cornwell et al., 1982).
Furthermore, clinical cases of upper respiratory tract infections have
already been reported in regularly vaccinated dogs (Kalinowski et al.,
2012).
The sequence analysis carried out on the four identified CAdV-1
viruses shows a complete identity between them and the strains
detected in Italian foxes and dogs in the last years. It is reasonable
to suppose that a genetically stable strain, or very similar strains,
of CAdV-1 circulates in the Italian territory, and that this virus is
transmitted between domestic dogs and wild canids. Further-
more, the high identity found with all the reference strains suggests
that CAdV-1 has maintained over time a remarkable stability.
Therefore, the recent outbreaks of CAdV infection reported in
dogs are probably not related to the introduction of new viral vari-
ants able to elude the immunity induced by vaccination, but
confirm that the virus is still circulating in the territory and
being able to cause infection and clinical manifestation of the disease
in more susceptible subjects not following a regular immune
prophylaxis.
Two different strains of CAdV-2 were identified, distinguish-
able on the basis of nucleotide 230. Three dogs were simultaneously
infected with both strains. The obtained CAdV-2 sequences showing
nucleotides 230C and 230T were identical to CAdV-2 strains re-
cently identified in an Italian fox and in Italian dogs, respectively.
Therefore, it is likely that different CAdV-2 strains circulate in the
Italian territory among domestic dogs and wild canids. The com-
plete identity of the CAdV-2 sequences including 230T with the
vaccine strain Toronto A26/61shown in six dogs could be ex-
plained by the detection of the vaccine virus in these subjects.
However, this assumption is to be discarded because the latter six
dogs had undergone the last vaccination since at least 40 days. It
has been shown that dogs vaccinated with CAdV-2 vaccine do not
shed vaccine virus from day 6 after vaccination (Bass et al., 1980;
Cornwell et al., 1982). In our study, none of the 30 dogs that tested
positive for CAdV infection had undergone the last vaccination from
less than 10 days.
Canine adenovirus type 2 is considered a common respiratory
pathogen. Only occasionally, its presence was reported in the gas-
trointestinal tract (Balboni et al., 2013; Hamelin et al., 1985; Macartney
 A. Balboni et al./Research in Veterinary Science 97 (2014) 631–636
et al., 1988). The high percentage of positivity to CAdV-2 found in
faeces (27/51, 52.9%) raises new questions about the role of this virus
as opportunist or pathogen agent of the gastrointestinal tract and
indicates that faeces represent an important route of viral shed-
ding. Moreover, concern about the pathogenic role of CAdV-2 in canids
is dictated by a recent case report of hepatitis in a maned wolf as-
sociated with administration of a modified live CAdV-2 vaccine
(Swenson et al., 2012). The detection of CAdV-2 in urine confirms the
results of a previous report by Headley et al. (2013) and suggests a
potential tropism of CAdV-2 for the renal system with urine repre-
senting another potential route of viral shedding. Further studies
investigating the possible pathogenic role played by CAdV-2 in both
gastrointestinal and kidney diseases and its potential persistence at
these sites are warranted.
Another relevant finding of the present study is the high fre-
quency (6/51, 11.8%) of multiple infections. Four dogs showed a
coinfection with CAdV-1 and CAdV-2 and one of these (296-2012)
showed a threefold infection with CAdV-1 and two different CAdV-2
strains. Moreover, two other dogs were coinfected with two differ-
ent CAdV-2 strains. Coinfections with different CAdVs have been
already reported in a fox showing a dual infection with different
CAdV-2 (Balboni et al., 2013) and in a dog presenting with con-
comitant CAdV-1, CAdV-2, canine distemper virus (CDV) and canine
parvovirus (CPV) infections (Headley et al., 2013). Coinfections are
normally originated by the entry of a second virus in a host already
infected, even though it is commonly believed that the immune re-
sponse resulting from a first canine adenovirus infection should
protect against secondary CAdV infections. However, the latter
prevention, as suggested by the high number of coinfections en-
countered in this study, seems not to be guaranteed by a primary
exposure to CAdV. This finding raises some concern about the real
protective immunity generated by the modified live vaccine cur-
rently in use, which needs to be further investigated.
In conclusion, the data reported in this study show that CAdV-1
and CAdV-2 are widely distributed in a population of Italian dogs
with no significant difference in terms of age, breed, sex and vac-
cination status. Vaccines, currently available for practitioners,
although limits the clinical manifestation of the disease, do not seem
to protect from infection and faecal shedding. Furthermore, the anal-
ysis performed on the tracts of the viral genome sequenced shows
that a genetically stable strain of CAdV-1 and different CAdV-2 strains
circulate in Italy, contributing to relatively frequent cases of
coinfections. The vaccination protocol for the CAdV is today still
essential to prevent the development of a relevant clinical mani-
festation of the infection since, in spite of what is commonly believed,
CAdV-1 still circulates in dog population.
Further studies in a wider population of dogs are needed to better
assess the prevalence of canine adenovirus in the Italian territory,
to assess the length and the epidemiological role of faecal and urine
shedding, and to correlate the presence of CAdV-1 and CAdV-2 in-
fections with mild intestinal, renal and respiratory clinical signs. To
better investigate the circulation of CAdV among the different hosts,
the survey should be conducted on dogs living in different geo-
graphical areas and different environments, including areas where
domestic animals potentially live in a close contact with wild species.
In addition, other regions of the viral genome should be se-
quenced to assess whether the high degree of genetic conservation
observed in this study is also present in other genes coding impor-
tant proteins for the immune response.
Acknowledgements
Financial support was provided by RFO funds (Ricerca
Fondamentale Orientata) of the Alma Mater Studiorum-University
of Bologna.
635
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