Molecular Detection of Viral and Bacterial Pathogens in Red Foxes (Vulpes vulpes) from Italy

Simple Summary Wild animals play an important role in the transmission and maintenance of infectious diseases. In this study, molecular assays were used to detect and genetically characterise Protoparvovirus carnivoran 1 (PPVC-1), Canine adenovirus type 1 and 2 (CAdV-1 and CAdV-2), Circovirus canine (CanineCV), Canine distemper virus (CDV), and Leptospira spp. in different matrices collected from 126 red foxes (Vulpes vulpes) in Italy from 2022 to 2023. A total of 39 of 126 (30.9%; 95%CI: 23.5–39.5) animals were infected with at least one pathogen. A red fox was coinfected with feline panleukopenia virus (FPV) and canine parvovirus type 2b (CPV-2b) showing quasispecies dynamics. Unique genetic characteristics were identified in CanineCV. The first detection of L. interrogans ST198 serogroup Australis in red foxes was also reported. The genetic analysis of the identified PPVC-1, CAdV-1, and Leptospira spp. support a wild-to-domestic animals (or vice versa) transmission, while the genetic characteristics of the identified CanineCV confirm a host predilection or limited interspecies transmission of this virus. Further studies are necessary to understand the role of red foxes in the maintenance of these pathogens not only in the wild but also in urban and peri-urban environments. Abstract Animals, including wildlife, are part of One-Health concept since many infectious diseases can affect both humans and animals. In this study, 126 red foxes (Vulpes vulpes) from Northern Italy in 2022–2023 were tested by molecular assays for Protoparvovirus carnivoran 1 (PPVC-1), Canine adenovirus type 1 and 2 (CAdV-1 and CAdV-2), Circovirus canine (CanineCV), Canine distemper virus (CDV), and Leptospira spp. A total of 39 of 126 (30.9%) red foxes were infected with at least one pathogen and five of these were coinfected: 20/126 (15.9%) red foxes tested positive for PPVC-1, 3/126 (2.4%) for CAdV, 20/126 (15.9%) for CanineCV, and 2/126 (1.6%) for Leptospira spp. DNA. No foxes tested positive for CDV RNA. The pathogens identified were genetically analysed. New findings were reported such as a fox with multiple feline panleukopenia virus (FPV) and canine parvovirus type 2b (CPV-2b) infection associated with quasispecies dynamics, typical genetic characteristics of the identified CanineCV, and the first detection in red foxes of Leptospira ST198 related to L. interrogans serogroup Australis. Further studies are necessary to investigate the transmission between domestic animals and wildlife and to understand the role of red foxes in the maintenance of these pathogens not only in the wild but also in urban and peri-urban environments.


Introduction
Animals, including wildlife, are part of the One-Health concept since many infectious diseases can affect both humans and animals [1].On the other hand, domestic animals may also be responsible for introducing infectious agents into wildlife, threatening the potential extinction of some animal species [2,3].The factors that mostly influence the diffusion of infectious diseases in European wild animals are globalisation and global warming.Because natural habitats in decline no longer constitute a suitable environment, many wild animal species tend to move closer to anthropised areas, colonising urban and peri-urban ones [1,[4][5][6][7].The red fox (Vulpes vulpes) is a wild carnivore capable of adapting to different environments that can be found in urban, suburban, and rural areas.These behavioural characteristics determine a crucial role in the epidemiology of infectious diseases [8].However, even red foxes living in exclusively wild environments could be infected by infectious diseases of domestic animals and could represent an important sentinel species [9].If viral and bacterial pathogens such as Protoparvovirus carnivoran 1 (PPVC-1), Canine mastadenovirus (CAdV), Circovirus canine (CanineCV), Morbillivirus canis or Canine distemper virus (CDV), and Leptospira spp.were to become established in red foxes, it could cause severe diseases affecting the conservation of the species and potentially serve as a source of infection for domestic animals or humans.
CanineCV, of the Circoviridae family [10], was discovered for the first time in 2011 in serum samples from several dogs [35] and successively reported in wild animals, including foxes [36], wolves, and badgers [37].To date, its pathogenic role in domestic and wild carnivores is not clarified, but its detection was associated with gastrointestinal and neurological alterations [38,39].
Leptospirosis is a worldwide zoonosis affecting numerous wild and domestic mammalian species [51], caused by pathogenic and highly motile spirochete of the genus Leptospira, of the Leptospiraceae family [52].Leptospires are maintained in nature by several subclinical wild and domestic reservoir hosts that serve as exposure sources to wildlife, livestock, domestic animals, and humans [53][54][55][56], and they are transmitted by direct or indirect contact with material, especially soil or water, that has been contaminated with urine from an infected animal [57].Interestingly, leptospirosis detection has been documented in accidental hosts such as domestic dogs [55,58] and wild animals [59,60], where the clinical signs could be severe and sometimes fatal.
The aim of this study was to investigate the frequency of infection with viral and bacterial infectious agents causing severe diseases in red foxes in Italy that can be transmitted to domestic animals or humans.For this purpose, a molecular detection of PPVC-1, CAdV, CanineCV, CDV, and Leptospira spp. was carried out from internal organs of red foxes in Northern Italy (Emilia Romagna region) in 2022-2023.Genetic characterisation of the identified pathogens was carried out.

Study Design and Sampling
To evaluate the frequency of PPVC-1, CAdV, CanineCV, CDV, and Leptospira spp.infection in red foxes, animals that died in Northern Italy for reasons unrelated to the current study were sampled and tested.All the red foxes culled during the regular hunting season or found dead in the environment or dead in wildlife rescue and rehabilitation centres in the Emilia Romagna region (Bologna, Modena, and Ferrara provinces) from January 2022 to March 2023 were included in the study.Animals were collected as part of the wildlife health surveillance program set up in the Emilia Romagna region [61].No criteria of exclusion were adopted.Each fox was subjected to post-mortem examination at the Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna (IZSLER, Italy) where the intestine, spleen, kidney, and liver were sampled based on availability (the liver was sampled when the spleen was not available) using sterile instruments.Biological samples were stored at −20 • C until further processing.Signalment data (geographical origin, data of sampling, sex, and age) and gross findings were recorded for each animal.Animals were classified as young (<1 year old) or adult (≥1 year old) based on body size and weight.The presence of PPVC-1, CAdV, CanineCV, CDV, and Leptospira spp.nucleic acids was detected using quantitative molecular assays.The complete genome or informative genes of the identified pathogens were amplified, sequenced, and analysed.

Detection of Protoparvovirus carnivoran 1, Canine mastadenovirus, Circovirus canine, Canine Distemper Virus, and Leptospira spp. Nucleic Acids
The detection of the nucleic acid of the pathogens investigated was carried out from different tissue samples: PPVC-1 and CanineCV DNA from spleen, liver, and intestine samples; CAdV DNA from spleen, liver, intestine, and kidney samples; CDV RNA from spleen or liver samples; and Leptospira spp.DNA from kidney samples.
DNA extraction from all the sampled organs was performed using the NucleoSpin Tissue 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 until analysis.RNA extraction from spleen and liver samples was performed using the RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.The extracted RNA was eluted in 40 µL of RNase-free water and stored at −80 • C until analysis.
The detection of PPVC-1 (FPV and CPV-2), CAdV (CAdV-1 and CAdV-2), CanineCV, and Leptospira spp.DNA was carried out with four specific SYBR Green real-time PCR (qPCR) assays, each performed using the PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, Life Technologies, Carlsbad, CA, USA), as described in previous studies and in the Supplementary Materials [18,62].The detection of CDV RNA was carried out with a specific SYBR Green reverse transcriptase qPCR (RT-qPCR) assay, performed using the Power SYBR Green RNA-to-C T 1-Step kit (Life Technologies, Carlsbad, CA, USA), as reported by previous studies [63] and in the Supplementary Materials.Each reaction was carried out using the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, Life Technologies, Carlsbad, CA, USA).The melting experiment was performed after the last extension step by a continuous increment from 55  C for Leptospira spp.Nucleic acid copy number determination was carried out by using the standard curve method, as reported by Balboni and colleagues [18], with a limit of detection (LOD) of the reactions of 1 copy/µL for FPV, CPV-2, CAdV-1, CAdV-2, CDV, and Leptospira spp., and of 5 copies/µL for CanineCV.In each run, samples and standards were repeated in duplicate and a no template control (ultrapure water) and a negative extraction control were analysed simultaneously.

Sequencing and Analysis of the Viruses Identified
For viruses identified in qPCRs, either the complete genome or the informative genes were amplified, sequenced, and analysed.Each end-point PCR assay was performed using the Phusion Hot Start II DNA Polymerase (Thermo Fisher Scientific, Life Technologies, Carlsbad, CA, USA), containing a high-fidelity DNA polymerase, according to the manufacturer's instruction.A positive control, consisting of a laboratory positive sample, and a no template control underwent analysis simultaneously (Supplementary Materials).
For the identified PPVC-1, CAdV, and CanineCV, a fragment of the VP2 gene, the complete hexon and fibre genes, and the complete viral genome were amplified [18] and sequenced by the Sanger method (BioFab Research, Rome, Italy), respectively.
Nucleotide sequences obtained from the PCR product of one identified PPVC-1 (lab ID: 829/2022-intestine) showed an unusually high number of ambiguities, suggesting a mixed viral population.Therefore, the amplification product was cloned into the pCR 4/TOPO vector using the pCR 4-TOPO TA kit (Life Technologies, Carlsbad, CA, USA) and transformed into Escherichia coli DH5α-competent cells according to the manufacturer's protocol.The recombinant clones obtained were sequenced after plasmid purification with the PureLink Quick Plasmid Miniprep (Life Technologies, Carlsbad, CA, USA).
Phylogeny was carried out on nucleotide sequences obtained in this study and reference sequences obtained in the GenBank database (https://www.ncbi.nlm.nih.gov/nucleotide/, accessed on 25 January 2024) using MEGA 11 software, version 11.0.10 [64].Subsequently, a Neighbour-Joining phylogenetic tree of the partial VP2 gene of PPVC-1 was constructed using the Tamura 3-parameter model with gamma distribution.Additionally, a Maximum Likelihood phylogenetic tree of the multiple gene sequences (concatenated hexon and fibre gene sequences) of CAdV was constructed using the Hasegawa-Kishino-Yano model with gamma distribution and invariable sites.A Maximum Likelihood phylogenetic tree of the complete genome of CanineCV was constructed using the General Time Reversible model with gamma distribution and invariable sites.One thousand replicates of bootstrap analysis were performed to evaluate the robustness of the phylogenetic trees.
A phylogenetic analysis was conducted on the concatemers of the seven MLST genes in MEGA X software version 10.1.8[67], using the Maximum Likelihood method and Kimura 2-parameter model with a bootstrap analysis based on 1000 replicates.The phylogeny was inferred with some reference sequences downloaded from BIGSdb [68] related to the most common STs found in Italy.

Statistical Analysis
Data were evaluated using standard descriptive statistics and were analysed using the Fisher's exact test or Pearson's Chi-squared test.Not available data were excluded from statistical analysis.Statistical significance was set at p < 0.05.Statistical analysis was carried out using the MedCalc Statistical Software version 16.8.4(MedCalc Software bvba).

Study Population and Sampling
During the study period, 126 red foxes were included (Table 1): 62/126 (49.3%) were male and 56/126 (44.4%) were female, while for the 8/126 (6.3%) remaining animals, this information was not available.Regarding age, 28/126 (22.2%) red foxes were classified as juveniles, 57/126 (45.2%) as adults, and for 41/126 (32.6%), the data were not available.A total of 55 out of 126 (43.6%) red foxes were from the province of Bologna, 66/126 (52.4%) from the province of Modena, and 5/126 (4%) from the province of Ferrara.Carcass examination revealed macroscopic lesions compatible with culling in 79/126 (62.7%) red foxes and with trauma in 30/126 (23.8%), most likely due to motor vehicle collision.In the remaining 17/126 (13.5%) animals, macroscopic findings were mostly lymphadenomegaly, hepatomegaly, splenomegaly, mange, and neurological, gastrointestinal, or renal lesions.Due to the state of conservation of the carcasses, frequently subjected to autolysis of tissues, histological examinations were not carried out.An intestine sample was collected from all the 126 red foxes included in the study; spleen and kidney samples were available for 123/126 and 117/126 red foxes, respectively; and the liver was collected from the 3/126 animals from which the spleen was not collected.

Detection of Viral and Bacterial Infectious Agents
A total of 39 out of 126 (30.9%; 95% confidence interval CI: 23.5-39.5)red foxes tested positive for at least one of the pathogens screened: 34 were positive to one pathogen only and 5 were coinfected.Regarding the coinfected animals, four were positive for two viruses (PPVC-1 and CanineCV) and one was positive for three viruses (PPVC-1, CAdV-2, and CanineCV) (Table S1).Nevertheless, at post-mortem examination, none of these aforementioned animals showed lesions typically attributable to infection with the identified pathogens.No statistical association was found regarding positivity to all pathogens investigated and sex, age, and geographical origin (Table 1).
Specifically, 20 out of 126 (15.9%; 95%CI: 10.5-23.3)red foxes tested positive for PPVC-1 DNA (Table 1): 8 were positive only in the intestine, 6 only in the spleen, and 1 only in liver samples, whereas 5 red foxes were positive in both spleen and intestine samples (Table S1).The overall median quantity of PPVC-1 DNA was 1.2 × 10 1 copies of target DNA per microliter of extracted DNA (copies/µL) (range 1.9 × 10 0 -4.1 × 10 2 ).A total of 3 out of 126 (2.4%; 95%CI: 0.8-6.7)red foxes tested positive for CAdV DNA (Table 1): 2 were positive for CAdV-1 only in the spleen sample (lab IDs: 1784/2022 and 1798/2022) and 1 was positive for CAdV-2 only in the intestine sample (lab ID: 1422/2022) (Table S1).All foxes tested negative for CAdV DNA in the kidney sample.The overall median quantity of CAdV DNA was 1.2 × 10 2 copies/µL (range 4.4 × 10 1 -5.2 × 10 2 ).A total of 20 out of 126 (15.9%; 95%CI: 10.5-23.3)red foxes tested positive for CanineCV DNA (Table 1): 1 was positive only in the intestine and 4 only in spleen samples, whereas 15 were positive in both the spleen and intestine samples (Table S1).The overall median quantity of CanineCV DNA was 3.1 × 10 2 copies/ µL (range 6.4 × 10 0 -1.3 × 10 7 ).CDV RNA was never detected in spleen or liver samples (Table 1).A total of 2 out of 126 (1.6%; 95%CI: 0.4-5.6)red foxes were positive for Leptospira spp.DNA in kidney samples (Tables 1 and S1), with an overall median quantity of target DNA of 5.6 × 10 1 copies/µL (range 2.4 × 10 1 -8.9 × 10 1 ).The nucleotide sequences of CPV-2b 829/2022-cl03 and cl06 were identical to one another and with the CPV-2b detected in the other red foxes, and FPV 829/2022-cl07 was identical to the FPV identified in red fox 481/2023.The putative VP2 amino acid sequence of CPV-2a 830/2022 had the two distinctive amino acid residues 324-isoleucine (Ile) and 440-alanine (Ala).The putative VP2 amino acid sequence of all the CPV-2b strains identified in this study displayed the two distinctive amino acid residues 371-glicine (Gly) and 418-threonine (Thr) and were identical to each other, with the exception of the 829/2022-cl02 that had the substitution of tryptophan (Trp) to arginine (Arg) in position 414.Phylogenetic analyses showed that the CPV-2a identified in this study was closely related to some CPV-2a strains known as "Asian-like", which share a typical amino acid composition of the VP2 protein (Figure 1).All the CPV-2b strains identified in this study clustered with viruses detected in dogs and wild carnivores from Italy in the last decade, known as "New Italian CPV-2b".The FPV-like viruses detected were related to FPV reported in cats and wild carnivores from Europe.

Genetic Analysis of the Pathogens Identified
Given the low amount of CAdV DNA detected, only incomplete hexon and fibre gene nucleotide sequences of 1600 nts (from nt 676 to 2275 of CAdV-1 reference strain 113-5L, GenBank ID: KP840545) and 1549 nts (from nt 84 to 1632 of CAdV-1 reference strain 113-5L, GenBank ID: KP840544) in length, respectively, were obtained for one CAdV-1 strain (lab ID: 1798/2022).No end-point PCR products were obtained from the other two CAdV strains detected by qPCR.In the phylogenetic tree constructed from the concatenated nucleotide sequences of hexon and fibre genes, CAdV-1 1798/2022 clustered with other CAdV-1 strains identified in domestic and wild canids from Italy and France, characterised by the distinctive residue 388-serine (Ser) in the deduced hexon protein and residue 110-glutamate (Glu) in the deduced fibre protein (Figure 2).
The complete genome of In the latter four CanineCV genomes, the nucleotide deletion involved a guanine in position 959 of reference strain 214, GenBank ID: JQ821392.This deletion was in the non-coding 3' IR and did not modify the structure of the ORF1 and ORF2.All the CanineCV strains sequenced were different from one another, showing a nucleotide identity of 97-99.9%.In the phylogenetic tree, five groups of CanineCV nucleotide sequences were well distinguishable and all the viruses sequenced in this study clustered in group 5 with viruses identified in foxes and one wolf in Europe (Figure 3).In particular, they were strictly related to a CanineCV strain identified in a wolf in Italy in 2017 (MW829203).
Leptospiral DNA was detected in the kidney sample of two red foxes (lab ID: 1805/2022 and 1806/2022).The identified leptospires were genotyped by MLST analysis and a complete MLST profile was obtained for 1806/2022, while for 1805/2022, a partial profile was defined (Table 2).All two infecting Leptospira strains belonged to ST198 that clustered with strain 367/2012, reported as the L. interrogans serogroup Australis, serovar Australis (Figure 4).As depicted in Figure 4, ST198 resulted as being very similar to ST24, referred to as the L. interrogans serogroup Australis serovars Jalna and Bratislava, having all identical alleles except for the pntA gene.

Discussion
Wild animals play an important epidemiological role in the maintenance and transmission of infectious diseases, whose causative infectious agents represent a direct threat not only for wild animals but also for domestic animals and humans' health [1,2,4].In this study, 39/126 (30.9%) red foxes, that were culled, found dead, or dead in wildlife rescue and rehabilitation centres in Northern Italy (Emilia Romagna region), tested positive to at least one viral or bacterial infectious agent.Post-mortem examination showed no lesions clearly attributable to the pathogens detected, but the advanced state of autolysis, which frequently affected the carcasses, did not allow us to carry out histological examinations and to draw conclusions.
PPVC-1 and CanineCV were the most frequently detected pathogens, both identified in 20/126 (15.9%) red foxes.In five red foxes, the two viruses were detected, confirming the association already reported for domestic dogs, wolves, and badgers [18,37,70,71].In dogs, some authors did not observe an aggravation of clinical conditions in animals coinfected by parvovirus and CanineCV [72], while Anderson and colleagues [73] reported an increase in mortality.
Regarding PPVC-1, a lower frequency of positivity in red fox Italian populations was recently reported (2.8% and 5.1%) [13,74].Conversely, Balboni and colleagues [18] detected a higher frequency of infection in wolves from the same geographical area in Italy (21/23), suggesting that wolves may be more susceptible to infection than foxes.For 11 red foxes that tested positive, a fragment of the PPVC-1 VP2 gene was sequenced, allowing us to identify one CPV-2a virus with a putative amino acid sequence attributable to so-called "Asian-like" viruses never reported before in foxes [75], five CPV-2b viruses with putative amino acid sequences typical of viruses recently circulating in Italy [18], and four FPVlike viruses phylogenetically related to FPV circulating in cats and wild carnivores from Europe.Previous surveys carried out in red foxes in Italy detected only CPV-2 [74] or FPV [13].These results might suggest a different geographical or temporal distribution of parvoviruses in wildlife.In red fox 829/2022, a multiple infection of FPV and CPV-2b was detected and associated with nucleotide sequence variability of the CPV-2b viral popula-

Discussion
Wild animals play an important epidemiological role in the maintenance and transmission of infectious diseases, whose causative infectious agents represent a direct threat not only for wild animals but also for domestic animals and humans' health [1,2,4].In this study, 39/126 (30.9%) red foxes, that were culled, found dead, or dead in wildlife rescue and rehabilitation centres in Northern Italy (Emilia Romagna region), tested positive to at least one viral or bacterial infectious agent.Post-mortem examination showed no lesions clearly attributable to the pathogens detected, but the advanced state of autolysis, which frequently affected the carcasses, did not allow us to carry out histological examinations and to draw conclusions.
PPVC-1 and CanineCV were the most frequently detected pathogens, both identified in 20/126 (15.9%) red foxes.In five red foxes, the two viruses were detected, confirming the association already reported for domestic dogs, wolves, and badgers [18,37,70,71].In dogs, some authors did not observe an aggravation of clinical conditions in animals coinfected by parvovirus and CanineCV [72], while Anderson and colleagues [73] reported an increase in mortality.
Regarding PPVC-1, a lower frequency of positivity in red fox Italian populations was recently reported (2.8% and 5.1%) [13,74].Conversely, Balboni and colleagues [18] detected a higher frequency of infection in wolves from the same geographical area in Italy (21/23), suggesting that wolves may be more susceptible to infection than foxes.For 11 red foxes that tested positive, a fragment of the PPVC-1 VP2 gene was sequenced, allowing us to identify one CPV-2a virus with a putative amino acid sequence attributable to so-called "Asian-like" viruses never reported before in foxes [75], five CPV-2b viruses with putative amino acid sequences typical of viruses recently circulating in Italy [18], and four FPVlike viruses phylogenetically related to FPV circulating in cats and wild carnivores from Europe.Previous surveys carried out in red foxes in Italy detected only CPV-2 [74] or FPV [13].These results might suggest a different geographical or temporal distribution of parvoviruses in wildlife.In red fox 829/2022, a multiple infection of FPV and CPV-2b was detected and associated with nucleotide sequence variability of the CPV-2b viral population, resembling the quasispecies distribution already reported for cats [76].The quasispecies distribution represents a selective advantage favouring viral evolution and the generation of new variants [77], and it could be consistent with the role suggested for wild carnivores in the evolution of carnivore parvoviruses [78,79].
The frequency of CanineCV DNA detection in red foxes obtained in this study was higher than the values reported in Southern Italy by Zaccaria and colleagues (0%) [37] and in Northern Italy by Franzo and colleagues (2-5%) [80].Contrary to this, an equal or higher prevalence was reported in red foxes from Norway by Urbani and colleagues (16.9%) [36] and from the United Kingdom by Bexton and colleagues (65%) [39], or in wolves from Italy [18,37,81].The numerous surveys carried out suggest that CanineCV circulates with variable intensity in the different populations of wild canids, with variations probably linked to the investigated host species and to the considered geographical area.Interestingly, the CanineCV genetic group 1 appears mainly widespread in dogs and wolves, whereas CanineCV genetic group 5 appears mainly widespread in foxes, confirming a prevalent species specificity of the different viral genotypes, or a limited transmissibility between different animal species [18,36,37].Four sequenced CanineCV strains had a nucleotide deletion in the non-coding 3' intergenic region never reported before and apparently not affecting the functionality of the two main ORFs.
Differently, CAdV and Leptospira spp.infections appeared less frequently in red foxes in the investigated area, as they were detected in 3/126 (2.4%) and 2/126 (1.6%) animals, respectively.The frequency of CAdV DNA detection reported in this study is lower than the results of previous surveys conducted in red foxes from neighbouring geographical areas and in wolves from the same geographical area in Italy (9.4% and 17.4%) [18,31].This finding may reflect a different geographic or host distribution of the virus, as well as variations related to the specific population sampled and the year of investigation.Of the three red foxes that tested positive for CAdV DNA, two were positive for CAdV-1 and one was positive for CAdV-2.Furthermore, in one of the red foxes that tested positive for CAdV-2, PPVC-1 and CanineCV DNA were also detected, highlighting a possible coinfection with three viruses sporadically reported in wolves [18].The CAdV-1 identified in this study was phylogenetically grouped with all CAdV-1 strains detected in domestic dogs and wild carnivores from Italy and France and showed the residues 388-Ser in the deduced hexon protein and 110-Glu in the deduced fibre protein.These results support the hypothesis that the same viruses circulate in domestic and wild environments [18] and confirm that some amino acid positions may allow us to distinguish viruses circulating in different geographical regions [18].
The red fox, like the domestic dog, is considered an accidental host for Leptospira infection and its role was investigated considering that the main food of foxes is represented by rodents, the most important reservoir host for several Leptospira serovars [82].Leptospira spp.infection in foxes not only represents a possible source of environmental contamination [83] but may also cause illness and death [84,85].The majority of the surveys carried out to evaluate the prevalence of Leptospira spp.infection in foxes used the microscopic agglutination test (MAT), and in Europe, the predominant serogroups detected were Australis, Bataviae, Grippotyphosa, Hebdomadis, Icteroheamorrhagiae, Javanica, Pomona, and Sejroe [56,59,[86][87][88][89][90][91][92][93].Only few surveys carried out on red foxes adopted molecular-based assays such as qPCR or MLST [93][94][95][96][97]. On one hand, the MAT assay detecting specific antibodies allows us to obtain more complete indications than molecular methods regarding the circulation of leptospires in an animal population, but on the other hand, using a limited antigens panel can lead to an underestimation of the serovars.Molecular methods do not have this limitation, therefore, qPCR and MLST assays allow for the detection of serovars not included in the MAT antigen panel.The frequency of Leptospira spp.DNA detection obtained in this study is similar to that reported for red foxes from neighbouring geographical areas by Ebani and colleagues [96].Furthermore, MLST analysis allowed us to identify Leptospira ST198 related to L. interrogans serogroup Australis serovar Australis in both red foxes testing positive in this study.The Australis serogroup has been widely reported in dogs in Italy [55,62], and Leptospira ST198 was previously reported in a horse [55], in hedgehogs, and in association with severe disease in dogs in Italy [58,98].To the best of the authors' knowledge, this represents the first report of Leptospira ST198 in red foxes, suggesting that this variant is widespread in the Italian territory, involving different animal species acting as maintenance or accidental hosts, and confirming that the red fox may be a reliable sentinel for epidemiological monitoring [92].
In the present study, none of the red foxes analysed were found positive for CDV RNA.Although it cannot be ruled out that viral RNA lability in the environment may have affected the result obtained, the lack of positive findings is in line with the Italian epidemiological situation where CDV circulates in wild animals in the Alps [45,99] but is detected sporadically on the rest of the territory [49,100,101].
For PPVC-1, CAdV-1, and Leptospira spp., the amount of genomic nucleic acid detected was low, not exceeding the order of magnitude of 10 2 .These values are potentially correlated to states of subclinical or even persistent infection, typical of the carrier state or maintenance host, often hypothesised for the red fox [17,74], and compatible with the absence of characteristic lesions detected at post-mortem examination.Differently, in some foxes, the amount of CanineCV DNA detected reached up to 10 7 copies/uL.Similar quantities were also reported in intestine and spleen samples of wolves [18], suggesting an active infection dynamic for this virus, although not necessarily associated with clinical signs.
The present study has some limitations.Firstly, samples were only collected from dead animals that were often found hours or days after death, and this could have affected the integrity of the nucleic acids, resulting in an underestimation of the pathogens detected (especially CDV which has an RNA genome).Secondly, the study was based exclusively on the use of molecular methods that detect the presence of the genome of the infectious agents but not their vitality.So, positive results may not correspond to the real state of infection, but they are nevertheless indicative of the circulation of pathogens in wildlife.Furthermore, molecular tests give an indication of the presence of the agent at the time of sampling only and the combination of a serological test could have allowed us to obtain more complete information on the circulation of these infectious agents in the red fox population.

Conclusions
This study reports new data on the presence of PPVC-1, CAdV, CanineCV, CDV, and Leptospira spp. in red foxes and underlines the importance of this wild carnivore in the epidemiology of several infectious agents.In particular, new findings are reported here such as a fox with multiple FPV and CPV-2b infection associated with quasispecies dynamics, typical genetic characteristics of the identified CanineCV, and the first detection of Leptospira ST198 related to the L. interrogans serogroup Australis in red foxes.Furthermore, the genetic analysis of the identified PPVC-1, CAdV-1, and Leptospira spp.supports a wild-to-domestic (or vice versa) transmission, and the genetic characteristics of the identified CanineCV confirm a host predilection or limited interspecies transmission of this virus.Further studies are necessary to investigate the transmission between domestic animals and wildlife and to understand the role of red foxes in the maintenance of these pathogens not only in the wild but also in urban and peri-urban environments.
Institutional Review Board Statement: Ethical review and approval were not needed for this study, because it was performed on biological samples from animals found dead; no samples were taken from live animals for the purposes of this study.
Informed Consent Statement: Our study was not carried out on people (humans) nor domestic animals (with owners).As reported in the manuscript, it was conducted on wild foxes that died for reasons not related to the study and were collected as part of the wildlife health surveillance program set up in the Emilia Romagna region [61].For this reason, the study did not require an informed consent statement.
Data Availability Statement: All data generated or analysed during this study are included in this published article and its supplementary information files.The nucleotide sequences generated and analysed during the current study are available in the International Nucleotide Sequence Database Collaboration repository (INSDC, http://www.insdc.org/)with the IDs PP480614-PP480626 and PP551635-PP551666.

Figure 1 .
Figure 1.Phylogenetic tree constructed on the partial VP2 nucleotide sequences of Protoparvo carnivoran 1 (PPVC-1) obtained in this study and reference strains in the GenBank database, the Neighbour-Joining method and Tamura 3-parameter model with gamma distribution.One sand replicates of bootstrap analysis were performed to evaluate the robustness of the phyloge trees and bootstrap values ≥50% are indicated.Highlighted in black: sequences of canine parvo

Figure 1 .
Figure 1.Phylogenetic tree constructed on the partial VP2 nucleotide sequences of Protoparvovirus carnivoran 1 (PPVC-1) obtained in this study and reference strains in the GenBank database, using the Neighbour-Joining method and Tamura 3-parameter model with gamma distribution.One thousand replicates of bootstrap analysis were performed to evaluate the robustness of the phylogenetic trees and bootstrap values ≥ 50% are indicated.Highlighted in black: sequences of canine parvovirus type 2 (CPV-2) and feline panleukopenia virus (FPV) generated in this study.Highlighted in grey: sequences of recombinant clones obtained from red fox 829/2022 in this study.To the right of the figure, the groups evidenced in this study are indicated.

type 2 (
CPV-2) and feline panleukopenia virus (FPV) generated in this study.Highlighted in grey: sequences of recombinant clones obtained from red fox 829/2022 in this study.To the right of the figure, the groups evidenced in this study are indicated.

Figure 2 .
Figure 2. Phylogenetic tree constructed with the multiple gene approach (concatenated nucleotide sequences of the hexon and fibre genes) of Canine mastadenovirus (CAdV) obtained in this study and reference strains in the GenBank database, using the Maximum Likelihood method and Hasegawa-Kishino-Yano (HKY) model with gamma distribution and invariable sites.One thousand replicates of bootstrap analysis were performed to evaluate the robustness of the phylogenetic trees and bootstrap values ≥60% are indicated.On the top of the figure, a portion of the obtained tree is enlarged to better visualise the phylogenetic relationships existing between the CAdV-1 nucleotide sequences and the bootstrap values.For some viruses, two GenBank accession numbers are reported (the hexon and fibre genes sequences, respectively).Highlighted in black: nucleotide sequence generated in this study.The amino acid residues in position 388 for the deduced hexon protein and in position 110 for the deduced fibre protein are reported.

Figure 2 .
Figure 2. Phylogenetic tree constructed with the multiple gene approach (concatenated nucleotide sequences of the hexon and fibre genes) of Canine mastadenovirus (CAdV) obtained in this study and reference strains in the GenBank database, using the Maximum Likelihood method and Hasegawa-Kishino-Yano (HKY) model with gamma distribution and invariable sites.One thousand replicates of bootstrap analysis were performed to evaluate the robustness of the phylogenetic trees and bootstrap values ≥ 60% are indicated.On the top of the figure, a portion of the obtained tree is enlarged to better visualise the phylogenetic relationships existing between the CAdV-1 nucleotide sequences and the bootstrap values.For some viruses, two GenBank accession numbers are reported (the hexon and fibre genes sequences, respectively).Highlighted in black: nucleotide sequence generated in this study.The amino acid residues in position 388 for the deduced hexon protein and in position 110 for the deduced fibre protein are reported.

Figure 3 .
Figure 3. Phylogenetic tree constructed on the complete genome nucleotide sequences of Circovirus canine (CanineCV) obtained in this study and reference strains in the GenBank database, using the Maximum Likelihood method and General Time Reversible (GTR) model with gamma distribution and invariable sites.One thousand replicates of bootstrap analysis were performed to evaluate the robustness of the phylogenetic trees and bootstrap values ≥60% are indicated.Highlighted in black: sequences of CanineCV generated in this study.To the right of the figure, the groups evidenced in this study are indicated and correspond to the clusters proposed by Niu et al. [69] and Urbani et al. [36].

Figure 3 .
Figure 3. Phylogenetic tree constructed on the complete genome nucleotide sequences of Circovirus canine (CanineCV) obtained in this study and reference strains in the GenBank database, using the Maximum Likelihood method and General Time Reversible (GTR) model with gamma distribution and invariable sites.One thousand replicates of bootstrap analysis were performed to evaluate the robustness of the phylogenetic trees and bootstrap values ≥ 60% are indicated.Highlighted in black: sequences of CanineCV generated in this study.To the right of the figure, the groups evidenced in this study are indicated and correspond to the clusters proposed by Niu et al. [69] and Urbani et al. [36].

Figure 4 .
Figure 4. Phylogenetic tree constructed on the concatenated sequences of the seven multi-locus sequence typing (MLST) loci (3111 nts) of Leptospira spp. in accordance with the scheme proposed by Boonsilp and colleagues [65].Phylogeny was conducted using the Maximum Likelihood method and Kimura 2-parameter model.One thousand replicates of bootstrap analysis were performed to evaluate the robustness of the phylogenetic trees and bootstrap values ≥60% are indicated.The reference sequences are indicated with ST, species, serogroup, serovar, and strain.

Figure 4 .
Figure 4. Phylogenetic tree constructed on the concatenated sequences of the seven multi-locus sequence typing (MLST) loci (3111 nts) of Leptospira spp. in accordance with the scheme proposed by Boonsilp and colleagues [65].Phylogeny was conducted using the Maximum Likelihood method and Kimura 2-parameter model.One thousand replicates of bootstrap analysis were performed to evaluate the robustness of the phylogenetic trees and bootstrap values ≥ 60% are indicated.The reference sequences are indicated with ST, species, serogroup, serovar, and strain.

Table 2 .
Multi-locus sequence typing results of the Leptospira spp.identified.