One health surveillance strategy for coronaviruses in Italian wildlife.

The recent reinforcement of CoV surveillance in animals fuelled by the COVID-19 pandemic provided increasing evidence that mammals other than bats might hide further diversity and play critical roles in human infectious diseases. This work describes the results of a two-year survey carried out in Italy with the double objective of uncovering CoV diversity associated with wildlife and of excluding the establishment of a reservoir for SARS-CoV-2 in particularly susceptible or exposed species. The survey targeted hosts from five different orders and was harmonised across the country in terms of sample size, target tissues, and molecular test. Results showed the circulation of 8 CoV species in 13 hosts out of the 42 screened. Coronaviruses were either typical of the host species/genus or normally associated with their domestic counterpart. Two novel viruses likely belonging to a novel CoV genus were found in mustelids. All samples were negative for SARS-CoV-2, with minimum detectable prevalence ranging between 0.49% and 4.78% in the 13 species reaching our threshold sample size of 59 individuals. Considering that within-species transmission in white-tailed deer resulted in raising the prevalence from 5% to 81% within a few months, this result would exclude a sustained cycle after spillback in the tested species.

Main causes of death of free-ranging bats in Turin province (North-Western Italy): gross and histological findings and emergent virus surveillance.

BACKGROUND: Bats are recognized as reservoir species for multiple viruses. However, little is known on bats' health and mortality. Thus, this study aimed to investigate the main causes of death of bats from Turin province (North-western Italy) and to describe gross and histopathological lesions potentially associated with the presence of selected bat viruses. RESULTS: A total of 71 bats belonging to 9 different species of the families Vespertilionidae and Molossidae were necropsied and samples of the main organs were submitted to histopathological examination. Also, aliquots of the small intestine, liver, spleen, lung, and brain were collected and submitted to biomolecular investigation for the identification of Coronaviridae, Poxviridae, Reoviridae (Mammalian orthoreovirus species), Rhabdoviridae (Vaprio ledantevirus and Lyssavirus species) and Kobuvirus. The majority of bats died from traumatic lesions due to unknown trauma or predation (n = 40/71, 56.3%), followed by emaciation (n = 13/71,18.3%). The main observed gross lesions were patagium and skin lesions (n = 23/71, 32.4%), forelimbs fractures (n = 15/71, 21.1%) and gastric distension (n = 10/71,14.1%). Histologically, the main lesions consisted of lymphoplasmacytic pneumonia (n = 24/71, 33.8%), skin/patagium dermatitis (n = 23/71, 32.4%), liver steatosis and hepatitis (n = 12, 16.9%), and white pulp depletion in the spleen (n = 7/71, 9.8%). Regarding emergent bat viruses, only poxvirus (n = 2, 2.8%) and orthoreovirus (n = 12/71, 16.9%) were detected in a low percentage of bats. CONCLUSIONS: Trauma is the main lesion observed in bats collected in Turin province (North-western Italy) associated with forelimb fractures and the detected viral positivity rate seems to suggest that they did not represent a threat for human health.

Molecular characterization of Dictyocaulus nematodes in wild red deer Cervus elaphus in two areas of the Italian Alps.

Nematodes of the genus Dictyocaulus are the causative agents of parasitic bronchitis and pneumonia in several domestic and wild ungulates. Various species have been described in wild cervids, as the case of Dictyocaulus cervi in red deer, recently described as a separate species from Dictyocaulus eckerti. In Italy, information on dictyocaulosis in wildlife is limited and often outdated. In this work, 250 red deer were examined for the presence of Dictyocaulus spp. in two areas of the Italian Alps (n = 104 from Valle d'Aosta, n = 146 from Stelvio National Park), and the retrieved lungworms were molecularly characterized. Lungworms were identified in 23 and 32 animals from Valle d'Aosta and Stelvio National Park, respectively. The nematodes, morphologically identified as D. cervi, were characterized molecularly (18S rDNA, ITS2, and coxI). Consistently, almost all specimens were found to be phylogenetically related to D. cervi. Three individuals, detected from both study sites and assigned to an undescribed Dictyocaulus sp., clustered with Dictyocaulus specimens isolated from red deer and fallow deer in previous studies. Within each of D. cervi and the undescribed Dictyocaulus sp., the newly isolated nematodes phylogenetically clustered based on their geographical origin. This study revealed the presence of D. cervi in Italian red deer, and an undetermined Dictyocaulus sp. that should be more deeply investigated. The results suggest that further analyses should be focused on population genetics of cervids and their lungworms to assess how they evolved, or co-evolved, throughout time and space and to assess the potential of transmission towards farmed animals.

Passive surveillance of wild fauna in northwestern Italy: a possible cognitive approach through geographical district study and fur elementary analysis.

The analysis of animal hair is a useful and non-invasive investigation method for monitoring metal content, whose beginning dates to a few decades ago. This study addresses the issue of wildlife mortality in Piedmont and Aosta Valley by linking the evidence to the characteristics of the territories and to hair elemental profile. The considered animal species were badger, fox, marten, and wolf. The quantitative data for 11 trace metals and 16 lanthanides were evaluated considering animal location and death causes regarding impacts, whose relevant number was confined to the Turin province and to the badger and fox species. The evaluation of the analytical results was performed after the out of bounds sample identification by mean of statistics. For trace metals, some areas have thus been identified, mainly in Turin province, in which the large excess of As, Cd, and Pb was related to district pollution. Moreover, the lower contents of Fe, Mn, and Al in Aosta Valley badgers' hair seem influenced by the different characteristics of the subsoil, in comparison to Piedmont, and a relationship with the living environment and the habits was suggested. Regarding lanthanides, Biella province represents an area in which environmental contamination and abundance of rare-earth elements was recorded.

Molecular detection of canine bufaviruses in wild canids.

Novel protoparvoviruses genetically related to human and non-human primate bufaviruses (BuVs) have been detected recently in respiratory and enteric specimens collected from dogs and cats. In this study, by molecular screening of archival collections of faecal samples from wolves and foxes, we detected BuVs with a rate of 17.1% (7/41) and 10.5% (9/86), respectively. Sequence analysis of a portion of the ORF2 gene region of nine positive samples showed that the viruses in these samples were closely related to BuVs (97.5-99.0% nucleotide sequence identity) found in domestic carnivores.

First molecular identification of kobuviruses in wolves (Canis lupus) in Italy.

Canine kobuviruses (CaKoVs) were first identified in diarrhoeic and asymptomatic dogs in 2011 in the USA. Subsequent studies have demonstrated a worldwide distribution of these viruses, but it is not clear if CaKoVs play a role as enteric pathogens of dogs. More recently, CaKoV RNA has been detected in wild carnivores, including red fox, golden jackal, side-striped jackal and spotted hyena. In this study, we addressed the hypothesis that wolves are susceptible to CaKoV infections. A total of 185 wolf stool samples were collected from necropsied animals and from transects in the Liguria, Piemonte and Valle D'Aosta regions of Italy, and CaKoV RNA was identified in two of these specimens. Both samples were obtained from necropsied wolves, with a prevalence rate of 4.9% (2/41). Sequence analysis of the full-length VP1 region showed that these strains displayed the highest nucleotide (nt) sequence identity (86.3-98.5%) to canine strains identified in the UK and Africa, and to kobuviruses that were previously detected in other African wild carnivores. This suggests that genetically related CaKoV strains circulate in domestic and wild carnivores, with interspecies transmission being not uncommon among carnivores of different ecosystems.

Serological survey in wild boar (Sus scrofa) in Switzerland and other European countries: Sarcoptes scabiei may be more widely distributed than previously thought.

BACKGROUND: Sarcoptic mange has recently emerged in wild boar in Switzerland, raising the question of the origin of the infection. The main aim of this study was to assess the extent of exposure of the wild boar populations to Sarcoptes scabiei in Switzerland, prior to and after the detection of mange cases, to determine whether the mite has been recently introduced into the populations concerned. We performed a serological survey using a commercially available ELISA and 1056 archived blood samples of free-ranging wild boar from Switzerland. To facilitate the interpretation of the obtained data, we additionally estimated seroprevalence in wild boar populations of four other European countries (1060 samples), both from areas with confirmed clinical cases of mange and from areas without reported cases in wild boar. Lastly, we revised the evaluation of the commercial ELISA when used with wild boar sera. RESULTS: Seropositive reactions were observed for samples from all five countries and from 15 of the 16 study areas. The obtained apparent seroprevalences ranged from 0.0% (0/82; 95% confidence interval [CI]: 0.0-4.4) to 17.4% (8/46; 95% CI: 7.8-31.4). Wild boar from study areas with known clinical cases and those ≤60 kg were four times more likely to be seropositive than wild boar from areas without reported cases and > 60 kg, respectively. Optical density values did not differ between the two types of study areas among seropositive samples but were significantly lower among seronegative samples from areas without than from areas with clinical cases. No difference was observed between the two sampling periods in Switzerland. The revised ELISA specificity was 96.8% (984/1017; 95% CI: 95.5-97.7) when wild boar from areas without history of mange were considered truly negative. CONCLUSIONS: Seropositivity to S. scabiei is more frequent and occurs over a larger geographic range than expected. Data suggest that the parasite is endemic within the wild boar populations of Switzerland and other European countries but that its presence is not necessarily associated with disease occurrence. Extrinsic factors which trigger disease emergence in infected populations remain to be investigated. The applied ELISA represents a promising tool for future studies.

Evidence of circulation of the novel border disease virus genotype 8 in chamois.

Evidence of association between the novel putative border disease virus genotype 8 (BDV-8) and fatal disease in an Alpine chamois (Rupicapra rupicapra) is reported. Diagnostically, we also demonstrated, as already previously reported, the failure of BDV-specific primers (PDB1 and PDB2) to detect BDV-8.

Coronavirus and paramyxovirus in bats from Northwest Italy.

BACKGROUND: Bat-borne virus surveillance is necessary for determining inter-species transmission risks and is important due to the wide-range of bat species which may harbour potential pathogens. This study aimed to monitor coronaviruses (CoVs) and paramyxoviruses (PMVs) in bats roosting in northwest Italian regions. Our investigation was focused on CoVs and PMVs due to their proven ability to switch host and their zoonotic potential. Here we provide the phylogenetic characterization of the highly conserved polymerase gene fragments. RESULTS: Family-wide PCR screenings were used to test 302 bats belonging to 19 different bat species. Thirty-eight animals from 12 locations were confirmed as PCR positive, with an overall detection rate of 12.6% [95% CI: 9.3-16.8]. CoV RNA was found in 36 bats belonging to eight species, while PMV RNA in three Pipistrellus spp. Phylogenetic characterization have been obtained for 15 alpha- CoVs, 5 beta-CoVs and three PMVs; moreover one P. pipistrellus resulted co-infected with both CoV and PMV. A divergent alpha-CoV clade from Myotis nattereri SpA is also described. The compact cluster of beta-CoVs from R. ferrumequinum roosts expands the current viral sequence database, specifically for this species in Europe. To our knowledge this is the first report of CoVs in Plecotus auritus and M. oxygnathus, and of PMVs in P. kuhlii. CONCLUSIONS: This study identified alpha and beta-CoVs in new bat species and in previously unsurveyed Italian regions. To our knowledge this represents the first and unique report of PMVs in Italy. The 23 new bat genetic sequences presented will expand the current molecular bat-borne virus databases. Considering the amount of novel bat-borne PMVs associated with the emergence of zoonotic infections in animals and humans in the last years, the definition of viral diversity within European bat species is needed. Performing surveillance studies within a specific geographic area can provide awareness of viral burden where bats roost in close proximity to spillover hosts, and form the basis for the appropriate control measures against potential threats for public health and optimal management of bats and their habitats.

Detection and genetic characterization of hepatitis E virus (HEV) genotype 3 subtype c in wild boars in Italy.

Hepatitis E virus (HEV) was detected in stools collected from wild boars in Italy, with an overall prevalence of 1.5 % (3/196). The sequence of a ~3.0-kb portion at the 3' end of the genome of one such strain, HEV/WB/P6-15/ITA, was determined. In the full-length ORF2, which encodes the capsid protein, the virus was genetically closest to wild boar and human HEV strains currently classified as genotype 3 subtype c. Interestingly, the 3' end of ORF2 of the WB/P6-15/ITA matched the 340-nucleotide (nt) sequence (94.0 % nt identity) of the human strain PeGe, identified in 2015 from a patient with acute hepatitis E in Genoa, Italy, suggesting that similar HEV strains are circulating in the same geographical setting in humans and animals.

Molecular detection of kobuviruses in European roe deer (Capreolus capreolus) in Italy.

Kobuvirus RNA was found in 6.6 % (13/198) of stool specimens from roe deer (Capreolus capreolus) captured during the regular hunting season. Upon sequence analysis of a fragment of the 3D gene, nine strains displayed the highest nucleotide sequence identity (91.2-97.4 %) to bovine kobuviruses previously detected in either diarrhoeic or asymptomatic calves. Interestingly, four strains were genetically related to the newly discovered caprine kobuviruses (84.2-87.6 % nucleotide identity) identified in black goats in Korea.

Molecular evidence of kobuviruses in free-ranging red foxes (Vulpes vulpes).

Red foxes (Vulpes vulpes) are susceptible to viral diseases of domestic carnivores. In this study, by screening rectal swabs collected from 34 red foxes in Italy, we identified kobuvirus RNA in five samples. Based on analysis of partial RdRp and full-length VP1 genes, all of the strains shared the highest identity with canine kobuviruses (CaKVs) recently detected in the US, the UK and Italy. These findings provide the first evidence of the circulation of these novel viruses in foxes.

Detection and Characterization of Zoonotic Pathogens in Game Meat Hunted in Northwestern Italy.

Wildlife can represent a reservoir of zoonotic pathogens and a public health problem. In the present study, we investigated the spread of zoonotic pathogens (Salmonella spp., Yersinia enterocolitica, Listeria monocytogenes, Shiga-toxin-producing Escherichia coli (STEC), and hepatitis E virus (HEV)) considering the presence of virulence and antibiotic resistance genes in game meat from animals hunted in northwest Italy. During two hunting seasons (2020 to 2022), samples of liver and/or muscle tissue were collected from chamois (n = 48), roe deer (n = 26), deer (n = 39), and wild boar (n = 35). Conventional microbiology and biomolecular methods were used for the detection, isolation, and characterization of the investigated pathogens. Two L. monocytogenes serotype IIa strains were isolated from wild boar liver; both presented fosfomycin resistance gene and a total of 22 virulence genes were detected and specified in the text. Eight Y. enterocolitica biotype 1A strains were isolated from chamois (2), wild boar (5), and deer (1) liver samples; all showed streptogramin and beta-lactam resistance genes; the virulence genes found were myfA (8/8 strains), ymoA (8/8), invA (8/8), ystB (8/8), and ail (4/8). Our data underscore the potential role of wildlife as a carrier of zoonotic and antibiotic-resistant pathogens in northwest Italy and a food safety risk for game meat consumers.

Circoviridae Survey in Captive Non-Human Primates, Italy.

Circoviruses (CVs) and cycloviruses (CyVs), members of the family Circoviridae, have been identified only occasionally in non-human primates (NHPs). In this study, we investigated the presence and genetic features of these viruses in 48 NHPs housed in the Bioparco-Rome Zoological Garden (Italy) and in the Anima Natura Wild Sanctuary Semproniano (Grosseto, Italy), testing fecal, saliva, and serum samples with a broadly reactive consensus nested PCR able of amplifying a partial region of the replicase (Rep) gene of members of the family Circoviridae. Viral DNA was detected in a total of 10 samples, including a saliva swab and 9 fecal samples collected, respectively from five Japanese macaques (Macaca fuscata) and four mandrills (Mandrillus sphinx), with an overall prevalence of 18.7% (9/48). On genome sequencing, five strains revealed the highest nucleotide identity (98.3-98.6%) to a CyV strain (RI196/ITA) detected in the intestinal content of a Maltese wall lizard (Podarcis filfolensis) in Italy. Although the origin of the Italian NHP strains, genetically distant from previously detected NHP CyVs, is uncertain, our results also highlight that the virome of captive animals is modulated by the different dietary and environmental sources of exposure.

Causes of Admission, Mortality and Pathological Findings in European Hedgehogs: Reports from Two University Centers in Italy and Switzerland.

European hedgehogs (Erinaceus europaeus) are nocturnal insectivores frequently found in urban areas. In the last decades, their population has declined in various European countries and human activities have emerged as significant contributors to this trend. While the literature has mainly focused on trauma as the major cause of mortality, few authors have considered pathological findings. The present study is based on the results of full post-mortem examinations performed on 162 European hedgehogs in Italy and 109 in Switzerland. Unlike in previous studies, the main cause of mortality was infectious diseases (60.5%), followed by traumatic insults (27.7%). The lungs were the main organ affected, showing mostly lymphoplasmacytic (45.9%), granulomatous (18.1%) or suppurative (8.2%) pneumonia. Nematodes were detected in 57.2% of all lungs and were significantly associated with pneumonia (p-value < 0.001). To our knowledge, this is the first study to report infectious diseases as the main cause of hedgehog death, emphasizing the need for wildlife rescue centers to adopt appropriate diagnostic and therapeutic measures. Further research is necessary to determine the broad range of infectious agents that affect this species and elucidate their interplay with the host. Finally, citizen sensitization should be implemented to promote responsible behaviors that could reduce human-related traumatic events.

Monitoring of pathogenic Leptospira infection in wolves (Canis lupus) from Spain and Italy.

Leptospirosis is a bacterial disease of worldwide distribution with relevant implications for animal and human health. Different large wild carnivore species can act as reservoirs of this zoonotic pathogen. This study aimed to evaluate the circulation of Leptospira spp. in free-ranging wolves (Canis lupus) from southern Europe. A total of 281 kidney samples of wolves from Spain and Italy were collected between 2017 and 2023. The presence of Leptospira DNA was analysed by real-time PCR and phylogenetic analyses were carried out using a Bayesian approach. The overall prevalence was 3.2 % (9/281; 95 %CI: 1.1-5.3). Leptospira DNA was detected in nine of the 180 wolves from Spain (5.0 %; 95 %CI: 1.8-8.2), but not in the Italian wolf population (0 %; 0/101). Molecular analyses revealed high homology between the sequences obtained in the present study and isolates of Leptospira interrogans and Leptospira borgpetersenii from different rodent and domestic ungulate species. Our results provide evidence of a low and spatially heterogeneous circulation of this pathogen in wolf populations of southern Europe. The detection of zoonotic Leptospira species in this survey supports the need to consider wolf populations in monitoring programs for leptospirosis with a One Health approach.

Molecular Surveillance for Bocaparvoviruses and Bufaviruses in the European Hedgehog (Erinaceus europaeus).

The presence of bocaparvoviruses (BoVs) and bufaviruses (BuVs) in the European hedgehog (Erinaceus europaeus) was investigated by screening duodenal and liver samples collected from 183 carcasses, delivered to wildlife rescue centers located in northwestern Italy. BoV DNA was detected in 15 animals (8.2%), with prevalences of 7.1% (13/183) and 2.7% (5/183) in intestine and liver samples, respectively. Upon the sequence analyses of the NS1 gene, two highly divergent BoVs (65.5-67.8% nt identities) were identified. Fourteen strains showed the highest identity (98.3-99.4% nt) to the hedgehog BoV strains recently detected in China in Amur hedgehogs (Erinaceus amurensis), whilst four strains were genetically related (98.9-99.4% nt identities) to the porcine BoVs identified in pigs and classified in the species Bocaparvovirus ungulate 4, which included related viruses also found in rats, minks, shrews, and mice. BuV DNA was detected in the duodenal samples of two hedgehogs, with a prevalence rate of 1.1%. The nearly full-length genome of two BuV strains, Hedgehog/331DU-2022/ITA and Hedgehog/1278DU/2019/ITA, was reconstructed. Upon phylogenetic analysis based on the NS and VP aa sequences, the Italian hedgehog BuVs tightly clustered with the BuVs recently identified in the Chinese Amur hedgehogs, within a potential novel candidate species of the genus Protoparvovirus.

Wolf Is Back: A Novel Sensitive Sentinel Rejoins the Trichinella Cycle in the Western Alps.

Trichinella is a foodborne parasite whose wildlife reservoirs are represented by carnivores and omnivores with predatory and scavenger behavior. The aim of the present study was to investigate the occurrence of Trichinella infection in grey wolves (Canis lupus) that recolonized the Western Alps from the end of the past century, and discuss the epidemiological role played by this apex predator in the early phases of its return. During the period 2017-2022, diaphragm samples were obtained from 130 individuals collected in the frame of a wolf mortality survey. Trichinella larvae were found in 15 wolves (11.53%) with a parasite intensity of 11.74 larvae per gram. Trichinella britovi was the only species identified. This is the first prevalence survey of Trichinella in wolves recolonizing the Alps. Results suggest that, in this particular biotope, the wolf has rejoined the Trichinella cycle and has the potential to play an increasingly important role as maintenance host. Arguments in favor and against this perspective are discussed and knowledge gaps highlighted. The calculated Trichinella larval biomass in the estimated wolf population roaming in Northwest Italy will serve as baseline value to explore possible shifts in the relative importance of wolves as Trichinella reservoir within the regional carnivore community. Finally, wolves re-colonizing the Alps already appear as sensitive sentinels to monitor the risk of Trichinella zoonotic transmission by infected wild boar meat.

Wildlife hair as bioindicators of metal exposure.

Animal hair is a useful biomonitoring tool for assessing the occurrence of trace elements in ecosystems. Essential (chromium, copper, iron, manganese, nickel, and zinc) and nonessential (aluminum, arsenic, cadmium, mercury, lead, palladium, platinum, rubidium, tin, and vanadium) elements were investigated in hair of badger (Meles meles), wild boar (Sus scrofa), marmot (Marmota marmota), wolf (Canis lupus), fox (Vulpes vulpes) and deer (Cervus elaphus) from Northwestern Italy. Badger was found to be the highest bioaccumulator of metals, while wolf, fox and deer recorded the lowest values. Essential elements contribute in higher degree to the sum of metals for all species except for wild boar. Results have shown that animals with omnivorous diet such as badger, marmot and wild boar have metal content (especially Al, As, Cr, Cu, Fe, Ni, V) higher than carnivores (wolf and fox) and herbivores (deer) and could represent an effective sentinel of environmental exposure to metals.