137Cesium (137Cs) assessment in wild boars from northwestern Italy.

Radionuclide contamination is a serious health issue caused by nuclear experiments and plant accidents, as seen for the Chernobyl and Fukushima nuclear plants. Italy has been especially interested in northwestern alpine regions, as have several other nations. The aim of this work was to indagate 134Cs and 137Cs contamination in wild boars, which were considered bioindicators sampled in the Chisone/Germanasca Valley and the Pellice Valley districts (Piedmont, Italy) in two hunting seasons (2014 and 2016). In the 2014 season, only the livers of the animals (n = 48) were sampled, whereas in 2016, five different anatomical sampling sites were sampled for each animal (n = 16). The analyses were conducted in an accredited laboratory (Agenzia Regionale per la Protezione dell'Ambiente-ARPA) by the aid of an HPGe detector (Ortec) with a relative efficiency of 50%. In general, the contamination levels registered in 2014 were under the detection limit for 134Cs and low for 137Cs (Chisone/Germanasca valley: min: 0.0, max: 23.9 median 11.0 Bq/kg vs Pellice valley: min 0, max: 31.7, median: 9.6 Bq/kg) and no health concern can be supposed. In the first-year samples, the liver showed a negative correlation between age and contamination level. In the second year of sampling, low levels were confirmed (min: 3.1 Bq/kg, max: 113.3; median 17.7 Bq/kg). Multiple sampling from the same animal showed that the diaphragm (median = 27.7 Bq/kg) kidney (27.4) and tongue (27.6) were more contaminated than the liver (17.7) and spleen (15.3). Moreover, a linear mixed model revealed a negative organ-by-age interaction, meaning that interorgan differences in contamination level were greater in younger (5-11 months) than in older (18-36 months) animals. Different feeding habits can be the explanation. Our paper shows that muscle sites (diaphragm and tongue) can be useful for radionuclide pollution surveillance in wild boar populations and that younger animals show more interorgan variability in contamination levels than older animals. More investigations are needed to confirm this correlation and to fulfill the request for more data to achieve better risk assessment.

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.

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.

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.

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.

Brain-Derived Neurotrophic Factor, Kynurenine Pathway, and Lipid-Profiling Alterations as Potential Animal Welfare Indicators in Dairy Cattle.

Complete animal welfare evaluation in intensive farming is challenging. With this study, we investigate new biomarkers for animal physical and mental health by comparing plasma expression of biochemical indicators in dairy cows reared in three different systems: (A) semi-intensive free-stall, (B) non-intensive tie-stall, and (C) intensive free-stall. Additionally, protein levels of mature brain-derived neurotrophic factor (mBDNF) and its precursor form (proBDNF) and indoleamine 2,3-dioxygenase (IDO1) specific activity were evaluated in brain samples collected from 12 cattle culled between 73 and 138 months of age. Alterations in plasma lipid composition and in the kynurenine pathway of tryptophan metabolism were observed in the tie-stall-reared animals. The total plasma BDNF concentration was higher in tie-stall group compared to the two free-housing groups. Brain analysis of the tie-stall animals revealed a different mBDNF/proBDNF ratio, with a higher level of proBDNF (p < 0.001). Our data are similar to previous studies on animal models of depression, which reported that inhibition of the conversion of proBDNF in its mature form and/or elevated peripheral kynurenine pathway activation may underlie cerebral biochemical changes and induce depressive-like state behavior in animals.

Snow Metrics as Proxy to Assess Sarcoptic Mange in Wild Boar: Preliminary Results in Aosta Valley (Italy).

The widespread diffusion of the wild boar on the Italian territory and its consistent use for hunting have created the possibility to conduct multiple studies on the pathologies afflicting this ungulate. Nevertheless, in the last two decades, only some pathologies such as classical and African Swine Fever, Tuberculosis, Brucellosis from Brucella suis have benefited from substantial public funding and the consequent great interest from the scientific world, while less attention was addressed to parasitic diseases including sarcoptic mange. Therefore, to fill this gap, the purpose of this study was to contribute to the knowledge of sarcoptic mange in the wild boar population in Aosta Valley in the Northwest of Italy, including sympatric species as foxes. Due to past field surveys, it has been possible to find a possible role of snow metrics in the spread of this pathogen. Even if there are only empirical evidence and the mechanism remain unknown remote sensing analysis considering snow metrics were performed to provide to veterinarians, foresters, biologists, and ecologists new tools to better understand wield board dynamics and join to ordinary tool an instrument to enhance management and planning strategies. The snow metrics (SM) were derived from USGS NASA Landsat 8 L2A retrieved from Theia CNES platform and processed in Orfeo Toolbox LIS extension package. The relationship between SM and the disease spread was tested per each Aosta Valley municipality obtaining LISA maps for each hunting season. The results have showed that this parasite is present in an endemic form even if with rather low prevalence values, equal to 1.2% in the season hunting season 2013/2014, and equal to 7.5% in the hunting season 2014/2015. Moreover, within simultaneous given values of SM, sarcoptic mange seem to find good conditions for spreading.

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.

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.

Detection and Characterization of a Novel Picornavirus in European Badger (Meles meles).

The recent development of unbiased metagenomic next-generation sequencing has provided a richer view of the wild animal virome making it necessary to expand the knowledge about virus diversity in wildlife, as well as to monitor their potential transmission to domestic animals or humans. In the present study, by screening collections of enteric specimens from wild animals, a novel picornavirus was identified in the intestinal content of a badger (Meles meles). By enrichment with a sequence-independent single-primer amplification (SISPA) approach and deep sequencing with Oxford Nanopore Technologies (ONT) platform, the genome sequence of a novel picornavirus strain, Badger/3A-2019/ITA, was reconstructed. On comparison based on the polyprotein sequences, the virus was distantly related (58.7% and 59.7% sequence identity at the nucleotide and amino acid level, respectively) to the feline picornavirus strain FFUP1, identified in 2012 in Portugal and classified into genus Sakobovirus within the species Sakobuvirus A. Upon phylogenetic, pairwise homology, and distance analyses performed on the P1, 2Chel, 3Cpro, and 3Dpol proteins and the complete genomic sequence, the badger picornavirus may be considered a member of a new sakobuvirus species, which we propose as Sakobuvirus B.

Characterisation of Yersinia Enterocolitica Strains Isolated from Wildlife in the Northwestern Italian Alps.

Introduction: Yersiniosis is a zoonosis causing gastroenteritis, diarrhoea, and occasionally reactive arthritis and septicaemia. Cases are often linked to meat consumption and the most common aetiological agent is the Gram-negative bacilliform Yersinia enterocolitica bacterium. The occurrence of Yersinia spp. among wild animals has mostly been studied in wild boar, but it has seldom been in other species. Material and Methods: A total of 1,868 faecal samples from animals found dead or hunted were collected between 2015 and 2018 in the Valle d'Aosta region of the northwestern Italian Alps. Alpine ibex faecal samples were collected during a health monitoring program in 2018. Bacteria were isolated via PCR and confirmed as Y. enterocolitica biochemically. Strain antimicrobial susceptibility was tested by Kirby-Bauer disc diffusion, and the presence of virulence factors and antimicrobial resistance genes was investigated using whole-genome sequencing. Results: Yersinia enterocolitica strains of biotype 1A were detected in six faecal samples from red deer (0.93%), roe deer (0.49%) and red foxes (0.7%). Strains found in beech martens (3.57%) and Alpine ibex (2.77%) belonged to biotypes 1B and 5, respectively and harboured the pYPTS01 plasmid that had only been detected in Y. pseudotuberculosis PB1/+. All the isolates were resistant to ampicillin and erythromycin. Conclusion: The biovar 1A strains exhibited different virulence factors and behaved like non-pathogenic commensals. The strain from an Alpine ibex also harboured the self-transmissible pYE854 plasmid that can mobilise itself and the pYPTS01 plasmid to other strains. The beech marten could be considered a sentinel animal for Y. enterocolitica. Phenotypic resistance may account for the ability of all the strains to resist ß-lactams.

An Integrated, Tentative Remote-Sensing Approach Based on NDVI Entropy to Model Canine Distemper Virus in Wildlife and to Prompt Science-Based Management Policies.

Changes in land use and land cover as well as feedback on the climate deeply affect the landscape worldwide. This phenomenon has also enlarged the human-wildlife interface and amplified the risk of potential new zoonoses. The expansion of the human settlement is supposed to affect the spread and distribution of wildlife diseases such as canine distemper virus (CDV), by shaping the distribution, density, and movements of wildlife. Nevertheless, there is very little evidence in the scientific literature on how remote sensing and GIS tools may help the veterinary sector to better monitor the spread of CDV in wildlife and to enforce ecological studies and new management policies in the near future. Thus, we perform a study in Northwestern Italy (Aosta Valley Autonomous Region), focusing on the relative epidemic waves of CDV that cause a virulent disease infecting different animal species with high host mortality. CDV has been detected in several mammalian from Canidae, Mustelidae, Procyonidae, Ursidae, and Viverridae families. In this study, the prevalence is determined at 60% in red fox (Vulpes vulpes, n = 296), 14% in wolf (Canis lupus, n = 157), 47% in badger (Meles meles, n = 103), and 51% in beech marten (Martes foina, n = 51). The detection of CDV is performed by means of real-time PCR. All the analyses are done using the TaqMan approach, targeting the chromosomal gene for phosphoprotein, gene P, that is involved in the transcription and replication of the virus. By adopting Earth Observation Data, we notice that CDV trends are strongly related to an altitude gradient and NDVI entropy changes through the years. A tentative model is developed concerning the ground data collected in the Aosta Valley region. According to our preliminary study, entropy computed from remote-sensing data can represent a valuable tool to monitor CDV spread as a proxy data predictor of the intensity of fragmentation of a given landscape and therefore also to monitor CDV. In conclusion, the evaluation from space of the landscape variations regarding the wildlife ecological corridors due to anthropic or natural disturbances may assist veterinarians and wildlife ecologists to enforce management health policies in a One Health perspective by pointing out the time and spatial conditions of interaction between wildlife. Surveillance and disease control actions are supposed to be carried out to strengthen the usage of geospatial analysis tools and techniques. These tools and techniques can deeply assist in better understanding and monitoring diseases affecting wildlife thanks to an integrated management approach.

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.

Molecular Identification and Characterization of a Genotype 3 Hepatitis E Virus (HEV) Strain Detected in a Wolf Faecal Sample, Italy.

Hepatitis E virus (HEV) infection is a major health problem worldwide. In developed countries, zoonotic transmission of HEV genotypes (Gt) 3 and 4 is caused by the ingestion of raw or undercooked meat of infected pigs and wild boars, the main reservoirs of HEV. However, additional animals may harbour HEV or HEV-related strains, including carnivores. In this study, we investigated the molecular epidemiology of orthohepeviruses in wild canids by screening a total of 136 archival faecal samples, collected from wolves (42) and red foxes (94) in Northwestern Italy. Orthohepevirus RNA was identified in a faecal specimen, collected from a wolf carcass in the province of La Spezia (Liguria Region, Italy). The nearly full-length (7212 nucleotides) genome of the strain HEV/81236/Wolf/2019/ITA (GenBank accession no. MZ463196) was determined by combining a sequence-independent single-primer amplification (SISPA) approach with the Oxford Nanopore Technologies sequencing platform. Upon phylogenetic analysis, the HEV detected in wolf was segregated into clade HEV-3.1, displaying the highest nucleotide (nt) identity (89.0-93.3%) to Gt3 strains belonging to subtype c. Interestingly, the wolf faecal sample also contained porcine astrovirus sequences, endorsing the hypothesis of a dietary origin of the HEV strain due to preying habits.

Canine Circovirus in Foxes from Northern Italy: Where Did It All Begin?

Canine circovirus (CanineCV) is a recently identified virus affecting both domestic and wild carnivores, including foxes, sometimes in presence of severe clinical signs. Its circulation in wild animals can thus represent a potential threat for endangered species conservation and an infection source for dogs. Nevertheless, no data were available on its circulation in the Alps region of Northern Italy. In the present study, samples collected from 186 foxes in the period 2009-2020 from Valle d'Aosta and Veneto regions were tested using a real-time PCR assay, demonstrating a viral circulation of approximatively 2-5%, depending on the considered regions. Two complete or almost complete genome sequences were obtained, highlighting that the detected strains were part of a so defined "fox only" clade, which suggests that, despite common contact opportunities, Alps foxes are not involved in frequent transmission events to domestic dogs. Such genetic isolation could be at least partially attributed to some sort of independent evolution occurred in the foxes, leading to species barrier. Additionally, CanineCV strains in foxes from Italy were unexpectedly related to those previously identified in foxes from the United Kingdom and Scandinavian area. Combining the history of fox distribution in Europe since the last glacial maximum (LGM) with the viral history allowed us to speculate a long-standing coexistence between European canine circovirus and this host, justifying the peculiar geographic distribution and evolutionary paths of the fox infecting clade.

Molecular Survey on Kobuviruses in Domestic and Wild Ungulates From Northwestern Italian Alps.

Since the first identification in 1989 in humans, kobuviruses (KoVs) have been identified from a wide range of animal species including carnivores, rodents, birds, ungulates, rabbits, and bats. Several studies have described the identification of genetically related KoVs in the fecal virome of domestic and wild animals suggesting a mutual exchange of viruses. By screening a total of 231 fecal samples from wild and domestic ungulates, KoVs RNA was detected in wild boars (3.2%; 2/63), chamois (4.6%; 2/43), and goats (2.6%; 2/77). On phylogenetic analysis of the partial RdRp sequence, the wild boar strains clustered within the species Aichivirus C whilst the strains identified in domestic and wild ruminants grouped into the species Aichivirus B. The complete VP1 gene was obtained for chamois and goat KoVs. Interestingly, upon phylogenetic analysis the strains grouped together with a KoV of ovine origin within a distinct genetic type (B3) of the species Aichivirus B.

Surveillance Study of Hepatitis E Virus (HEV) in Domestic and Wild Ruminants in Northwestern Italy.

In industrialized countries, increasing autochthonous infections of hepatitis E virus (HEV) are caused by zoonotic transmission of genotypes (Gts) 3 and 4, mainly through consumption of contaminated raw or undercooked pork meat. Although swine and wild boar are recognized as the main reservoir for Gt3 and Gt4, accumulating evidence indicates that other animal species, including domestic and wild ruminants, may harbor HEV. Herein, we screened molecularly and serologically serum and fecal samples from two domestic and four wild ruminant species collected in Valle d'Aosta and Piemonte regions (northwestern Italy. HEV antibodies were found in sheep (21.6%), goats (11.4%), red deer (2.6%), roe deer (3.1%), and in Alpine ibex (6.3%). Molecular screening was performed using different primer sets targeting highly conserved regions of hepeviruses and HEV RNA, although at low viral loads, was detected in four fecal specimens (3.0%, 4/134) collected from two HEV seropositive sheep herds. Taken together, the data obtained document the circulation of HEV in the geographical area assessed both in wild and domestic ruminants, but with the highest seroprevalence in sheep and goats. Consistently with results from other studies conducted in southern Italy, circulation of HEV among small domestic ruminants seems to occur more frequently than expected.

Dirofilaria immitis in wolves recolonizing northern Italy: are wolves competent hosts?

BACKGROUND: Wild carnivores such as the grey wolf (Canis lupus), red fox (Vulpes vulpes) and golden jackal (Canis aureus) are recognized hosts of Dirofilaria immitis. However, few studies have focused on their actual role in the epidemiology of heartworm infection. This study describes the prevalence and distribution of D. immitis in wolves in a heartworm-endemic area in northern Italy where wolves have recently returned after long-time eradication, and investigates the fertility status of the collected adult nematodes. METHODS: In the frame of a long-term wolf monitoring programme in northwestern Italy, 210 wolf carcasses from four provinces were inspected for the presence of filarioid nematodes in the right heart and pulmonary arteries. Female heartworms were measured, and their uterine content analyzed according to a previously described "embryogram" technique. RESULTS: Three wolves, all originating from a single province (Alessandria), were positive for D. immitis (1.42%, 95% CI: 0.48-4.11%, in the whole study area; 13.6%, 95% CI: 4.7-33.3%, limited to the single province from which infected wolves originated). Mean intensity was 5 worms (range: 3-7) and the female worms measured 21-28 cm in length. Six out of 9 female worms harbored uterine microfilariae: 5 were classified as gravid; 1 showed a "discontinuous gradient"; and 3 were non-gravid. CONCLUSIONS: The present data show that heartworm infection is already prevalent in wolves that have recolonized the known heartworm-endemic area. Based on "embryogram" results, wolves were shown suitable heartworm hosts. Interestingly, investigated wolves appeared similarly exposed to heartworm infection as sympatric unprotected dogs (owned dogs that have never received any heartworm prevention treatment) sampled at the beginning of the wolf return process.

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.