We report the detection of canine adenovirus type 1 DNA by real-time PCR technique in an oral sample of an Italian wolf (Canis lupus italicus). Genetic characterization of the virus revealed a strict relationship with viruses detected in dogs (Canis lupus familiaris), wolves, and red foxes (Vulpes vulpes), suggesting that transmission between wild animals and dogs had occurred.

Canine adenovirus (CAdV) type 1 (CAdV-1), the etiologic agent of infectious canine hepatitis in the domestic dog (Canis lupus familiaris), is also widespread in wildlife. Evidence of exposure to CAdV is reported in free-ranging and captive wolves (Canis lupus) in several geographic areas (Stephenson et al. 1982; Millán et al. 2016; Pizzurro et al. 2017; Dowgier et al. 2018). We describe the genetic characterization of CAdV-1 detected in an oral sample of an Italian wolf (Canis lupus italicus; subject no. 874/2014) found dead in Tuscany, Italy (43°38′40″N, 11°12′34″E) in December 2014.

A complete postmortem examination showed the cause of death to be a road traffic accident with an evident skull fracture. The wolf was in good body condition as determined by adequate fat deposits and body weight. The wolf's age was estimated to be second class (ranging between 12 mo and 24 mo) according to dental development, body size, and weight (Mörner et al. 2005). Tissue samples were collected for animal genotyping and to assess the presence of infectious agents infecting wolves and dogs. Genetic analyses were performed on DNA extracted from the lingual muscular tissue stored in 95% ethanol, as previously described (Caniglia et al. 2014; Randi et al. 2014), which confirmed the carcass belonged to a male, pure Italian wolf (Montana et al. 2017). To investigate the presence of infectious pathogens, total DNA and RNA extractions from the sampled tissues were performed with the NucleoSpin Tissue Mini Kit (Macherey-Nagel, Düren, Germany) and the RNeasy Mini Kit (Qiagen, Hilden, Germany), respectively.

Samples were screened for the presence of canine distemper virus RNA (lung; Frisk et al. 1999), canine adenovirus types 1 and 2 DNA (liver; Hu et al. 2001), Leishmania spp. DNA (spleen; Galletti et al. 2011), and Leptospira spp. DNA (kidney; Stoddard et al. 2009). The wolf was negative for the presence of nucleic acids for all the investigated pathogens, with the exception of a positive PCR product corresponding to CAdV-1 (Hu et al. 2001) detected in the liver DNA extract.

To genetically characterize the identified CAdV-1, a DNA extract of the superficial portion of the tongue sample stored in 95% ethanol was used because the tissues previously tested were not stored in the laboratory and were not available for further investigation. We detected and quantified CAdV-1 DNA using a real-time PCR assay (Balboni et al. 2015). Subsequently, amplification, nucleotide sequencing, and assembly of hexon and fiber CAdV genes were performed according to Balboni et al. (2017). The sequences obtained were aligned with reference sequences of canine adenoviruses from Gen-Bank and translated into amino acids with BioEdit 7.2.5 software (Hall 1999). Phylogenetic relationships among the multiple gene sequences (concatenated hexon and fiber genes sequences) were evaluated with MEGA version 6.0.6 software (Tamura et al. 2013).

We identified CAdV-1 DNA in the tongue sample with a quantity of 1.6×106 DNA copies per gram. The CAdV-1 hexon (2,718 base pairs; GenBank no. MH105809) and fiber (1,632 base pairs; GenBank no. MH105810) genes sequences differed from all the CAdV-1 reference sequences both at the nucleotide and the amino acid levels (Table 1). Position 388 of the predicted CAdV-1 hexon protein differentiated the Italian sequences from the other reference sequences by having serine instead of asparagine. One other CAdV-1 strain identified outside Italy, in a captive wolf in France (Wolf/835/2015/FRA, MH048659; Dowgier et al. 2018), showed the same amino acid substitution. Several nucleotide and amino acid mutations in the hexon and fiber genes allowed clustering with the CAdV-1 showing 388-Ser in the predicted hexon protein in two groups, each of which consisted of viruses identified both in wild carnivores and in dogs. Furthermore, the CAdV-1 identified in this study showed a unique amino acid change in position 138 (alanine-proline) in the predicted hexon protein, which has never, to our knowledge been previously reported. Phylogenetic tree confirmed the clustering in two groups of the CAdV-1 sequences with 388-Ser in the predicted hexon protein (Fig. 1).

Table 1

Amino acid mutations allowing differentiation of the hexon and fibre sequences of a Canine adenovirus type 1 (CAdV-1) identified in an oral sample of an Italian wolf (Canis lupus italicus), with amino acid mutations obtained from the worldwide CAdV-1 reference sequences identified in wolves (Canis lupus), red foxes (Vulpes vulpes), and dogs (Canis lupus familiaris).

Amino acid mutations allowing differentiation of the hexon and fibre sequences of a Canine adenovirus type 1 (CAdV-1) identified in an oral sample of an Italian wolf (Canis lupus italicus), with amino acid mutations obtained from the worldwide CAdV-1 reference sequences identified in wolves (Canis lupus), red foxes (Vulpes vulpes), and dogs (Canis lupus familiaris).
Amino acid mutations allowing differentiation of the hexon and fibre sequences of a Canine adenovirus type 1 (CAdV-1) identified in an oral sample of an Italian wolf (Canis lupus italicus), with amino acid mutations obtained from the worldwide CAdV-1 reference sequences identified in wolves (Canis lupus), red foxes (Vulpes vulpes), and dogs (Canis lupus familiaris).
Figure 1

Rooted phylogenetic tree constructed with the multiple gene approach: concatenated nucleotide sequences of the hexon and fiber genes of Canine adenovirus type 1 (CAdV-1) identified in this study in an oral sample of an Italian wolf (Canis lupus italicus) killed by a motor vehicle in 2014 and the canine adenovirus reference sequences retrieved from GenBank. The best-fit model of nucleotide substitution was determined for the sequence alignment using the Find Best DNA/Protein Model function implemented in MEGA 6.0.6. The phylogenetic tree was constructed using the maximum-likelihood method, whereas the Hasegawa-Kishino-Yano model was used for nucleotide substitution. Bootstrap values were determined by 1,000 replicates to assess the confidence level of each branch pattern. (a) The obtained tree. (b) An enlarged portion of the obtained tree to better visualize the phylogenetic relationships existing between the CAdV-1 nucleotide sequences and the bootstrap values (values >70% are indicated on the respective branches). For some viruses, two GenBank accession numbers are reported (the hexon and fiber genes sequences, respectively). CAdV-1 sequences showing amino acid serine in position 388 of the predicted hexon protein are depicted in bold. The nucleotide sequences generated in this study are underlined. The two clusters of CAdV-1 sequences showing 388-Ser in the predicted hexon protein are highlighted with two different shades of gray.

Figure 1

Rooted phylogenetic tree constructed with the multiple gene approach: concatenated nucleotide sequences of the hexon and fiber genes of Canine adenovirus type 1 (CAdV-1) identified in this study in an oral sample of an Italian wolf (Canis lupus italicus) killed by a motor vehicle in 2014 and the canine adenovirus reference sequences retrieved from GenBank. The best-fit model of nucleotide substitution was determined for the sequence alignment using the Find Best DNA/Protein Model function implemented in MEGA 6.0.6. The phylogenetic tree was constructed using the maximum-likelihood method, whereas the Hasegawa-Kishino-Yano model was used for nucleotide substitution. Bootstrap values were determined by 1,000 replicates to assess the confidence level of each branch pattern. (a) The obtained tree. (b) An enlarged portion of the obtained tree to better visualize the phylogenetic relationships existing between the CAdV-1 nucleotide sequences and the bootstrap values (values >70% are indicated on the respective branches). For some viruses, two GenBank accession numbers are reported (the hexon and fiber genes sequences, respectively). CAdV-1 sequences showing amino acid serine in position 388 of the predicted hexon protein are depicted in bold. The nucleotide sequences generated in this study are underlined. The two clusters of CAdV-1 sequences showing 388-Ser in the predicted hexon protein are highlighted with two different shades of gray.

Close modal

Representative portions of the tongue stored in 95% ethanol were processed for histopathology and stained with H&E for general examination. Immunohistochemistry (IHC) was also performed with an avidinbiotin-peroxidase technique and a commercial goat polyclonal antibody with a broad range of cross-reactivity against numerous adenoviruses (0151-9004, AbD Serotec, Kidlington, Oxford, UK) diluted 1:1,600 in phosphatebuffered saline. Liver sections from a dog infected with CAdV-1, as confirmed by PCR and serology, were used as positive controls. Negative controls were obtained by omitting the primary antibody. No histologic lesions and no positive immunohistochemical staining consistent with CAdV-1 infection were observed in the lingual tissues (Fig. 2).

Figure 2

Tongue of an Italian wolf (Canis lupus italicus) killed by a vehicle collision in 2014. (a) H&E section, including superficial stratified squamous epithelium with lingual papillae and deeper striated muscular tissues. (b) Immunohistochemistry (adenovirus), same section presented as shown in (a), without the positive immunostain. Inset: Dog, liver, positive control showing consistent nuclear positive immunostain in infected hepatocytes and less frequently in endothelial and Kupffer cells. Bar=250 µm, with the exception of insert, in which, bar=75 µm.

Figure 2

Tongue of an Italian wolf (Canis lupus italicus) killed by a vehicle collision in 2014. (a) H&E section, including superficial stratified squamous epithelium with lingual papillae and deeper striated muscular tissues. (b) Immunohistochemistry (adenovirus), same section presented as shown in (a), without the positive immunostain. Inset: Dog, liver, positive control showing consistent nuclear positive immunostain in infected hepatocytes and less frequently in endothelial and Kupffer cells. Bar=250 µm, with the exception of insert, in which, bar=75 µm.

Close modal

The amount of the target CAdV-1 DNA detected in the tongue sample suggested that the virus might have replicated in that organ. In contrast, the absence of viral antigens revealed by IHC did not support that thesis but suggested that molecular positivity was the consequence of a contamination of the tongue surface with saliva (Decaro et al. 2008) or that the amount of viral antigen was below the sensitivity of the IHC technique in the tissues tested. Comparing the amount of CAdV-1 DNA detected in the tongue sample with the amount of viral DNA in other tissues that are characteristically targets for viral replication (such as liver or kidney; Decaro et al. 2008) would have been useful to understanding the CAdV-1 replicate in the tongue, but, unfortunately, those tissue samples were no longer available for further investigation. Therefore, additional studies with a large case series are needed to determine whether the tongue is a site of viral replication, if it is a useful specimen for screening for CAdV-1 in this species, and whether there is a relationship between positive results from tongue samples and positive results from other organs. Nucleotide and predicted amino acid sequences obtained in this study confirmed that several genetic variants of CAdV-1 are circulating in carnivores (Balboni et al. 2017), and that amino acid position 388 of the hexon protein may differentiate viruses spread in a restricted geographical region (Balboni et al. 2017). Furthermore, sequence alignment and phylogeny showed high genetic relatedness between CAdV-1 identified in different animal species and suggested that transmission of the virus from wild animals to the dog, or vice versa, has occurred. Our findings point out that transmissible infectious agents between dogs and wild carnivores could threaten the conservation of the wolf population.

We acknowledge Duccio Berzi of the association “Canislupus Italia” for providing the wolf carcass for postmortem examination.

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