Research in Veterinary Science 97 (2014) 631–636 Contents lists available at ScienceDirect Research in Veterinary Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / r v s c 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 Article history: Received 9 January 2014 Accepted 27 October 2014 Keywords: Canine adenovirus Coinfection Dog Molecular epidemiology Italy A B S T R A C T The use of a modified live canine adenovirus (CAdV) vaccine has greatly reduced the incidence of infectious canine hepatitis (ICH) in dogs. Nevertheless, cases of CAdV type 1 and 2 (CAdV-1 and CAdV-2) infection 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 Hospital in Bologna, Italy, for reasons unrelated with CAdV infection, were tested with a polymerase chain reaction (PCR) assay for CAdV. Thirty dogs (58.8%) were PCR positive for CAdV-2 infection and four of them (7.8%) were positive for CAdV-1. Sequence analysis performed on the obtained PCR products suggests that a genetically stable CAdV-1 strain and different CAdV-2 strains circulate in the canine population examined and that coinfections are relatively frequent. © 2014 Elsevier Ltd. All rights reserved. 1. Introduction 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 deposition (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 (epizootic 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: [email protected] (M. Battilani). http://dx.doi.org/10.1016/j.rvsc.2014.10.010 0034-5288/© 2014 Elsevier Ltd. All rights reserved. ICH in domestic canine population (Abdelmagid et al., 2004; Bass et al., 1980; Decaro et al., 2008). Thus, CAdV-1 is nowadays considered 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 circulate and to be pathogenic in dogs. Furthermore, widespread infection of CAdV-1 in wild carnivores may be a source of infection 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 tracheobronchitis (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 respiratory 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 infection (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 domestic 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 632 A. Balboni et al./Research in Veterinary Science 97 (2014) 631–636 canine population raise some concern about the real epidemiological situation of the canine adenovirus in dogs. In order to assess the presence of canine adenovirus in domestic dogs, patients were sampled at a veterinary hospital in Northern Italy. 2. Materials and methods 2.1. Study design and sampling The study was conducted at the Veterinary Teaching Hospital of the University of Bologna (VTH-UB), Italy, on client-owned dogs. In the absence of epidemiological data about the prevalence of adenovirus in the canine population that would allow to predefine an adequate number of subjects to be sampled, it was decided to include all dogs referred to the veterinary hospital during a 5-week period (May 2012–June 2012). Selection of the subjects was done by one of the investigators (CM) on voluntary basis of the owners by interviewing them in the waiting room of the VTH-UB during regular hours of first opinion referrals (Monday–Friday 9 am–4 pm). The owners were informed about the aim of the project and asked to sign a consent for the inclusion of their dog in the study. Rectal swabs (RS) and urine samples (UR) were then collected by the same investigator after being obtained data related to breed, sex, age, vaccination status and reason for medical referral. Fifty-one dogs were sampled and data on signalment, vaccination status, clinical presentation and positivity to CAdV are reported in Table 1. Rectal swabs were taken from all the dogs, while urine were available from 20 (39.2%) dogs due to inability to collect spontaneous voiding urine samples at the time of the inclusion. Samples were stored at −80 °C upon analysis. The study was approved by the Scientific and Ethical Committee of the University of Bologna. 2.2. PCR for canine adenovirus detection Canine adenovirus screening was carried out on DNA extracts using polymerase chain reaction (PCR) assay. Viral DNA extraction from rectal swabs and urine was performed using the NucleoSpin Table 1 Tested dogs presented in subgroups and results of canine adenovirus testing. Subgroup Breed Purebred Crossbred Sex Male Female Age (months) ≤12 [puppies] 13–48 [young] 49–120 [adults] >120 [elderly] Vaccinationa Yes Yes from less than 1 year Never Symptoms Dermatologic signs Gastrointestinal signs Musculoskeletal signs Neurologic signs Respiratory signs Urinary signs Other signs No symptoms (routine check/vaccination) Tested dogs (n = 51) CAdV-1 positive (n = 4) CAdV-2 positive (n = 30) 36 15 3 (8.3%) 1 (6.7%) 22 (61.1%) 8 (53.3%) 25 26 2 (8%) 2 (7.7%) 16 (64%) 14 (53.9%) 12 9 21 9 1 (8.3%) 1 (11.1%) 2 (9.5%) 0 (0%) 6 (50%) 5 (55.6%) 14 (66.7%) 5 (55.6%) 44 33 7 4 (9.1%) 4 (12.1%) 0 (0%) 26 (59.1%) 21 (63.6%) 4 (57.1%) 4 6 3 1 4 3 2 28 0 (0%) 2 (33.3%) 0 (0%) 0 (0%) 0 (0%) 1 (33.3%) 0 (0%) 1 (3.6%) 2 (50%) 5 (83.3%) 1 (33.3%) 1 (100%) 4 (100%) 2 (66.7%) 1 (50%) 14 (50%) a Yes: dogs that have been vaccinated at least once with CAdV-2 vaccine. Never: dogs that have never been vaccinated. Tissue Mini Kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions. The extracted DNA was eluted in 100 μl of elution buffer and stored at −20 °C. A fragment of the E3 gene and flanking regions was amplified using two conserved primers that produce fragments of different lengths which allowed differentiation between the two adenovirus types, 508 bp for CAdV-1 and 1030 bp for CAdV-2, respectively (Chaturvedi et al., 2008; Hu et al., 2001). The PCR assay was carried out using the HotStar HiFidelity Polymerase Kit (QIAGEN, Hilden, Germany), in a total volume of 50 μl. The HotStar HiFidelity Polymerase Kit contains a hot-start proofreading enzyme that allows to reduce the error rate in DNA duplication increasing the reaction fidelity. The mixtures were amplified as follows: an initial denaturation step at 95 °C for 5 min, 45 cycles of amplification with 1 cycle at 94 °C for 30 s, at 58 °C for 1 min, and at 72 °C for 1 min, followed by a final elongation at 72 °C for 10 min. Standard precautions were taken to avoid PCR contamination, and the ultrapure water negative control included in all PCR assays did not show false positive results. The DNA extracted from a CAdV-2 attenuated strain vaccine (Canigen CEPPi/L, Virbac, Carros, France) and the DNA extracted from the paraffin embedded liver of a dog that died from CAdV-1 infection (strain 313–2010-Lparaffin, KF676977) were used as positive controls. Five microlitres of the amplicons was electrophoresed in 2% (w/v) agarose gel stained with ethidium bromide in 1X standard tris-acetate-EDTA (TAE) buffer and visualised by UV light, using a GeneRuler 100 bp Plus DNA Ladder (Fermentas, Burlington, Ontario, Canada) to check the presence of amplicons of the expected size. 2.3. Statistical analysis Results were analysed using descriptive statistics. Data comparison between subgroups (breed, sex, age and vaccination status) was performed by chi-square test. Statistical significance was set at p < 0.05. An inter-rater agreement statistic (Cohen’s kappa coefficient) was calculated to compare the results obtained by the two different biological matrices. Statistical analysis was performed using commercially available statistical software (MedCalc Statistical Software version 12.7.5 – MedCalc Software bvba, Ostend, Belgium; http://www.medcalc.org; 2013). 2.4. Sequencing and sequence analysis The amplicons of the expected size and with a sufficient amount of DNA product to allow direct sequencing 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. Samples yielding PCR amplification of two different DNA fragments, attributable to CAdV-1 and CAdV-2 respectively, were purified from agarose gel to separate the two products and directly sequenced. The nucleotide sequences obtained were assembled and translated into amino acid sequences using BIOEDIT sequence alignment editor version 7.0.9. The assembled nucleotide sequences were aligned with 14 reference sequences of canine adenovirus and bat adenovirus, available from the GenBank nucleotide database (http:// www.ncbi.nlm.nih.gov/genbank), using the ClustalW method implemented in BIOEDIT sequence alignment editor version 7.0.9. Three sequences of field CAdV identified in Italian dogs during years 2010–2011 in our labs were also included in the alignment (CAdV-1: 313-2010-Lparaffin and CAdV-2: 60-2011-OFS and S1-2011-OFS). The phylogenetic relationships of the viruses detected with canine adenovirus and bat adenovirus reference sequences were evaluated 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 twoparameter model with gamma distribution resulted optimal for all the sequence data (including reference strains). Phylogenetic tree was constructed using the neighbor-joining method and bootstrap 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 presentation 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 geographical 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 agreement 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. Positive in rectal swab Negative in rectal swab Only rectal swab samples No urine samples Urine samples Positive Negative 17 14 2 3 8 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 (corresponding 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 (corresponding to nucleotide 582 of the entire E3 gene) that does not involve substitutions in the amino acid sequences. In particular, eight sequences (internal lab codes: 249-2012-RS, 243-2012-UR, 244-2012RS, 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 ambiguities in the electroferogram at position 230, characterised by a double peak 230C/T, compatible with the presence of two different CAdV-2 strains in the same subject. The comparison of the obtained nucleotide sequences with reference sequences has allowed to calculate: (A) an identity of 100% between the four obtained CAdV-1 sequences with five CAdV-1 reference strains: one virus identified in an Italian dog in 2010 (3132010-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 identified 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 (113-3Fc04, 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 identified in dogs 275-2012, 286-2012, 296-2012 and 300-2012 were grouped in the CAdV-1 cluster whereas the CAdV-2 cluster included 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 included in the study and the lack of a predefined stratification did not allow to make epidemiological evaluations but to obtain preliminary 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 agreement 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 diffusion 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 circulation of these viruses in the canine population. However, the clinical relevance of these findings need to be clarified, 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 91 CAdV-1_field strain 313-2010-Lparaffin_IT_2010_dog_KF676977 CAdV-1_300-2012-RS_KF676980 100 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 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 BtAdV-2_field strain PPV1_DE_2011_bat_JN252129 CAdV-1 CAdV-2 99 0.1 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 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: 2752012-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 immunity 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 adenovirus 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 biological 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 reported 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 different 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 investigated 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 previous 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. Furthermore, 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 variants 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, distinguishable 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 recently 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 complete identity of the CAdV-2 sequences including 230T with the vaccine strain Toronto A26/61shown in six dogs could be explained 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 gastrointestinal 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 shedding. Moreover, concern about the pathogenic role of CAdV-2 in canids is dictated by a recent case report of hepatitis in a maned wolf associated 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 representing 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 frequency (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 different 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 concomitant 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 response 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 encountered 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 currently 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 vaccination 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 analysis 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 manifestation 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 infections 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 geographical 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 sequenced to assess whether the high degree of genetic conservation observed in this study is also present in other genes coding important 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 References Abdelmagid, O.Y., Larson, L., Payne, L., Tubbs, A., Wasmoen, T., Schultz, R., 2004. Evaluation of the efficacy and duration of immunity of a canine combination vaccine against virulent parvovirus, infectious canine hepatitis virus, and distemper virus experimental challenges. 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