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Members of the family Parvoviridae are well recognized infectious agents of companion, livestock and wild animals as well, whose relevance on production, health, welfare and conservation is often high. Nevertheless, the knowledge of their epidemiology in wild populations is scarce or fragmentary. In this study, the presence and features of two parvoviruses, Carnivore protoparvovirus 1 and Amdoparvovirus, were evaluated in the red fox population resident in the Dolomites area, Northern Italy, and compared with the scenario of other countries and Italian regions. Six out of 117 spleen samples (5.13%: 95CI: 1.91-10.83%) tested positive to Carnivore protoparvovirus 1 and were molecularly characterized as Canine parvovirus (CPV). Infection frequency was comparable with that observed in wild carnivore populations present in Southern Italian regions, although in that case, Feline parvovirus (FPV) was predominant. No evidence of infection-related clinical signs was reported and viral loads were invariably low, suggesting the subclinical nature of the infection, the persistent carrier status or the detection of traces of viral DNA. No samples tested positive to Amdoparvovirus genus-specific PCR. The present study provides the first evidence of CPV circulation in the Northern Italy fox population. Unfortunately, the inevitable convenience nature of the sampling prevents definitive conclusions. Therefore, a more coordinated and standardized approach should be applied, at least in neighbouring geographic areas, to study these viral infections and their relevance in wildlife.

BackgroundSurveillance of E. multilocularis at the edge of its range is hindered by fragmented distributional patterns and low prevalence and burden in definitive hosts. Thus, tests with adequate levels of sensitivity are especially important for discriminating between infected and non-infected areas.AimWe reassessed the prevalence of E. multilocularis at the southern border of its distribution in Alto Adige (Italy), to improve surveillance in wildlife and provide more accurate estimates of exposure risk.MethodsWe compared results from the diagnostic test currently implemented for surveillance (based on Coproscopy+Multiplex PCR - CMPCR), against a real-time quantitative PCR (qPCR) for 235 fox faeces collected in 2019-2020. The performances of the two tests were estimated using a scraping technique (SFCT) as the gold standard applied to the small intestines of a subsample (n=123) of the same hosts. True prevalence was calculated and sample size required by each faecal test for the detection of the parasite was then estimated.ResultsTrue prevalence of E. multilocularis in foxes (14.3%) was definitely higher than reported in the last decade (never >5% from 2012 to 2018). The qPCR also had a higher sensitivity (83%) compared to CMPCR (21%). Agreement with the gold standard was far higher for qPCR (0.816) than CMPCR (0.298) as well, determining a smaller sample size required to detect the disease.ConclusionsAlto Adige should be considered a highly endemic area. Surveillance at the edges of E. multilocularis distribution should adopt qPCR diagnostics on definitive hosts on a small geographic scale.AUTHOR SUMMARYEchinococcus multilocularis is an intestinal flatworm, whose adult stage in Europe is harboured mainly by the red fox (Vulpes vulpes), which spreads parasite’s eggs by faeces. This parasite is the agent of a severe and potentially lethal zoonosis, the alveolar echinococcosis, affecting humans after accidental ingestion of parasite’s eggs. In the Italian Alpine area, which represents the southernmost border of E. multilocularis European range, surveillance is hindered by a fragmented distributional pattern, where presence in foxes has been consistently reported only in few isolated foci in Alto Adige (Bolzano province – Italy) of low prevalence. In order to improve the efficiency of monitoring efforts, we tested the performances of two diagnostic protocols on fox faeces (sedimentation, filtration, counting technique followed by standard PCR, and whole stool real-time PCR) against a benchmark technique on fox intestines (scraping, filtration, counting technique - considered as the gold standard). This allowed not only to determine qPCR as a far more sensitive and sample-efficient diagnostic tool for E. multilocularis detection in marginally affected areas, but also to re-assess its prevalence in Alto Adige, which should be considered a highly endemic area. Consequent actions in the field and modifications in the surveillance strategy should be therefore considered.

In Europe, Ixodes ricinus is the main vector for tick-borne pathogens (TBPs), the most common tick species in Italy, particularly represented in pre-alpine and hilly northern areas. From 2011 to 2017, ticks were collected by dragging in Belluno province (northeast Italy) and analyzed by molecular techniques for TBP detection. Several species of Rickettsia spp. and Borrelia spp. Anaplaspa phagocitophilum, Neoerlichia mikurensis and Babesia venatorum, were found to be circulating in the study area carried by I. ricinus (n = 2668, all stages). Overall, 39.1% of screened pools were positive for at least one TBP, with a prevalence of 12.25% and 29.2% in immature stages and adults, respectively. Pathogens were detected in 85% of the monitored municipalities, moreover the presence of TBPs varied from one to seven different pathogens in the same year. The annual TBPs prevalence fluctuations observed in each municipality highlights the necessity of performing continuous tick surveillance. In conclusion, the observation of TBPs in ticks remains an efficient strategy for monitoring the circulation of tick-borne diseases (TBDs) in a specific area.

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.

Background Echinococcus multilocularis is a small tapeworm affecting wild and domestic carnivores and voles in a typical prey-predator life cycle. In Italy, there has been a focus of E. multilocularis since 1997 in the northern Italian Alps, later confirmed in red foxes collected from 2001 to 2005. In this study, we report the results of seven years of monitoring on E. multilocularis and other cestodes in foxes and describe the changes that occurred over time and among areas (eco-regions) showing different environmental and ecological features on a large scale. Methods Eggs of cestodes were isolated from feces of 2872 foxes with a sedimentation/filtration technique. The cestode species was determined through multiplex PCR, targeting and sequencing ND1 and 12S genes. Analyses were aimed to highlight variations among different eco-regions and trends in prevalence across the study years. Results Out of 2872 foxes, 217 (7.55%) samples resulted positive for cestode eggs at coproscopy, with differences of prevalence according to year, sampling area and age class. Eight species of cestodes were identified, with Taenia crassiceps (2.65%), Taenia polyacantha (1.98%) and E. multilocularis (1.04%) as the most represented. The other species, Mesocestoides litteratus, Taenia krabbei, T. serialis, T. taeniaeformis and Dipylidium caninum, accounted for < 1% altogether. Echinococcus multilocularis was identified in foxes from two out of six eco-regions, in 30 fecal samples, accounting for 1.04% within the cestode positives at coproscopy. All E. multilocularis isolates came from Bolzano province. Prevalence of cestodes, both collectively and for each of the three most represented species (T. crassiceps, T. polyacantha and E. multilocularis), varied based on the sampling year, and for E. multilocularis an apparent increasing trend across the last few years was evidenced. Conclusions Our study confirms the presence of a focus of E. multilocularis in red foxes of northeast Italy. Although this focus seems still spatially limited, given its persistence and apparent increasing prevalence through the years, we recommend research to be conducted in the future on the ecological factors that, on a smaller scale, allow this zoonotic species to persist. On the same scale, we recommend a health education campaign to inform on the measures to prevent this zoonosis, targeted at people living in the area, especially hunters, dog owners, forestry workers and other potentially exposed categories.

Canine distemper virus (CDV) represents an important threat for both wild and domestic carnivores. Since 2006, the North-Eastern regions in Italy have been experiencing severe and widespread recurring outbreaks of CDV affecting the wild carnivore population. In this study we performed an extensive phylogeographic analysis of CDV strains belonging to the Wildlife-Europe genetic group identified between 2006 and 2018 in Veneto, Trentino Alto Adige and Friuli Venezia Giulia regions. Our analysis revealed that viruses from the first (2006-2009) and the second (2011-2018) epidemic wave cluster separately, suggesting the introduction of two distinct genetic variants. These two events were characterized by different diffusion rates and spatial distribution, thus suggesting the existence of a connection between infection spread and host population dynamics. We also report the first spillover event of this strain to a non-vaccinated dog in a rural area of Friuli Venezia Giulia. The increasing prevalence of the infection in wildlife population, the broad host range of CDV circulating in the Alpine wildlife and the first reported transmission of a wild-adapted strain to a domestic dog in this region raise concerns over the vulnerability of wildlife species and the exposure of our pets to new threatening strains. Understanding the dynamic of CDV epidemics will also improve preparedness for re-emerging diseases affecting carnivore species.

Sarcoptic mange represents the most severe disease for wild Caprinae individuals and populations in Europe, raising concerns for both conservation and management of these ungulates. To date, this disease has been investigated in different wild caprine species and under many different perspectives including diagnostics, epidemiology, impact on the host populations, and genetics of both hosts and parasite, with the aim to disentangle the host-Sarcoptes scabiei relationship. Notwithstanding, uncertainty remains and basic questions still need an answer. Among these are the effect of immune responses on mange severity at an individual level, the main drivers in host-parasite interactions for different clinical outcomes, and the role of the immune response in determining the shift from epidemic to endemic cycle. A deeper approach to the pathology of this disease seems therefore advisable, all the more reason considering that immune response to S. scabiei in wild Caprinae, generally classified as a hypersensitivity, remains poorly understood. In this paper, we reviewed the pathological features associated to sarcoptic mange in wildlife, exploring different kinds of hypersensitivity and outcomes, with the objective of highlighting the major drivers in the different responses to this disease at an individual level and proposing some key topics for future research, with a particular attention to Alps-dwelling wild caprines.

Background Northeastern Italy is a hotspot for several tick-borne pathogens, transmitted to animals and humans mainly by Ixodes ricinus. Here we compare the results of molecular monitoring of ticks and zoonotic TBPs over a six-year period, with the monitoring of red foxes (Vulpes vulpes) in an endemic area. Results In the period 2011–2016, 2,578 ticks were collected in 38 sites of 20 municipalities of Belluno Province. Individual adults (264), pooled larvae (n = 330) and nymphs (n = 1984) were screened for tick-borne encephalitis virus, Borrelia burgdorferi (s.l.), Rickettsia spp., Babesia spp., Anaplasma phagocytophilum and “Candidatus Neoehrlichia mikurensis” by specific SYBR green real-time PCR assays and sequencing. The spleens of 97 foxes, culled in the period 2015–2017 during sport hunting or population control programs, were also screened. Overall, nine different pathogens were found in I. ricinus nymph and adult ticks: Rickettsia helvetica (3.69%); R. monacensis (0.49%); four species of the B. burgdorferi (s.l.) complex [B. afzelii (1.51%); B. burgdorferi (s.s.) (1.25%); B. garinii (0.18%); and B. valaisiana (0.18%)]; A. phagocytophilum (3.29%); “Candidatus N. mikurensis” (1.73%); and Babesia venatorum (0.04%). Larvae were collected and screened in the first year only and two pools (0.6%) were positive for R. helvetica. Tick-borne encephalitis virus was not found in ticks although human cases do occur in the area. The rate of infection in ticks varied widely according to tick developmental stage, site and year of collection. As expected, adults were the most infected, with 27.6% harboring at least one pathogen compared to 7.3% of nymphs. Pathogens with a minimum infection rate above 1% were recorded every year. None of the pathogens found in ticks were detectable in the foxes, 52 (54%) of which were instead positive for Babesia cf. microti (also referred to as Babesia microti-like, “Theileria annae”, “Babesia annae” and “Babesia vulpes”). Conclusions The results show that foxes cannot be used as sentinel animals to monitor tick-borne pathogens in the specific epidemiological context of northeastern Italy. The high prevalence of Babesia cf. microti in foxes and its absence in ticks strongly suggests that I. ricinus is not the vector of this pathogen. Electronic supplementary material The online version of this article (10.1186/s13071-018-2726-7) contains supplementary material, which is available to authorized users.

Hepatitis E virus (HEV) is an emerging zoonosis caused by a positive RNA single stranded virus of the Hepeviridae family. In developed countries, pigs and wild boars are the main reservoir for zoonotic genotypes 3 and 4. In spring 2017, for the first time HEV was detected in wild boars captured in the Regional Park of the Euganean Hills, in north-eastern Italy. Phylogenetic analysis of two complete viral genomes and seven partial ORF1 and ORF2 sequences of HEV viruses, selected from twelve positive animals, showed that the viruses grouped together within genotype 3 but clustered separately from previously identified subtypes, thus suggesting the identification of a novel genotype 3 subtype. The phylogenetic analysis of nine partial ORF2 sequences showed the closest similarity with wild boar/human viruses identified in central-northern Italy in 2012. The circulation of HEVs in this area, characterized by a vast man-made environment, an overpopulation of wild boars and >150 swine farms, should be considered in a public health perspective. Further investigations at the wild/domestic species and human interface are therefore necessary to gain a deeper understanding of HEV dynamics.

Trombiculosis has been reported in some wild ruminant species. We investigated the occurrence of trombiculosis in the northern chamois ( Rupicapra rupicapra rupicapra) in the Western Italian Alps, and we describe the related histopathologic changes. Superficial scrapes and skin samples were taken from 191 chamois from the Lombardy and Piedmont regions during the hunting season in September-December 2015. Numbers of eosinophils, mast cells, cluster of differentiation (CD)3-, CD79α-, CD68-positive cells were determined on immunohistochemically stained skin sections using a semiautomatic analysis system. Forty (20.9, 40/191) chamois were positive for trombiculid larvae on skin scrapings. Of the positive animals, 15 were from Lombardy and 25 from Piedmont, with similar prevalences. Macroscopic lesions were light with involvement of body regions that had contact with the ground, especially head (pinnae and areas around eyes and mouth) and limbs, where stylostome was easily formed due to thin skin. Histologically, trombiculosis caused a focal moderate dermatitis with epidermal necrosis, thin crusts, and hyperkeratosis. Inflammatory infiltrates were suggestive of a granulomatous reaction centered on a stylostome, formed by mite saliva and necrotic host tissue debris. However, we detected some difference in cutaneous immune response with some chamois showing a prevalent T-cell response and others having an increased B-cell count associated with a higher number of eosinophils, mast cells and a lower number of T cells.

Information on the population dynamics of a reservoir species have been increasingly adopted to understand and eventually predict the dispersal patterns of infectious diseases throughout an area. Although potentially relevant, to date there are no studies which have investigated the genetic structure of the red fox population in relation to infectious disease dynamics. Therefore, we genetically and spatially characterised the red fox population in the area stretching between the Eastern and Dinaric Alps, which has been affected by both distemper and rabies at different time intervals. Red foxes collected from north-eastern Italy, Austria, Slovenia and Croatia between 2006-2012, were studied using a set of 21 microsatellite markers. We confirmed a weak genetic differentiation within the fox population using Bayesian clustering analyses, and we were able to differentiate the fox population into geographically segregated groups. Our finding might be due to the presence of geographical barriers that have likely influenced the distribution of the fox population, limiting in turn gene flow and spread of infectious diseases. Focusing on the Italian red fox population, we observed interesting variations in the prevalence of both diseases among distinct fox clusters, with the previously identified Italy 1 and Italy 2 rabies as well as distemper viruses preferentially affecting different sub-groups identified in the study. Knowledge of the regional-scale population structure can improve understanding of the epidemiology and spread of diseases. Our study paves the way for an integrated approach for disease control coupling pathogen, host and environmental data to inform targeted control programs in the future.

International audience; Canine distemper virus (CDV) infection represents an important conservation threat to many carnivore species and has contributed to the population decline of several wild terrestrial and aquatic mammalian species. Since 2006, the Alpine region of North-Eastern (NE) Italy has been experiencing a severe and widespread outbreak of CDV affecting the wild carnivore population. In this study we performed an extensive phylogenetic and molecular evolutionary analysis of CDV identified during the recent wildlife epidemic in the Alpine region. Our analysis yielded data on the evolutionary dynamics of the Alpine wildlife CDV epidemic and revealed the emergence and spread of a single genetic cluster of CDV. The wide distribution of the novel cluster combined with the identification of a specific amino acid mutation, which is believed to increase the ability of the virus to replicate in a wider host range, raises concerns over the possible implications of the spread of this virus on the conservation of endangered wildlife species.

Since 2006 the red fox (Vulpes vulpes) population in north-eastern Italy has experienced an epidemic of canine distemper virus (CDV). Additionally, in 2008, after a thirteen-year absence from Italy, fox rabies was re-introduced in the Udine province at the national border with Slovenia. Disease intervention strategies are being developed and implemented to control rabies in this area and minimise risk to human health. Here we present empirical data and the epidemiological picture relating to these epidemics in the period 2006-2010. Of important significance for epidemiological studies of wild animals, basic mathematical models are developed to exploit information collected from the surveillance program on dead and/or living animals in order to assess the incidence of infection. These models are also used to estimate the rate of transmission of both diseases and the rate of vaccination, while correcting for a bias in early collection of CDV samples. We found that the rate of rabies transmission was roughly twice that of CDV, with an estimated effective contact between infected and susceptible fox leading to a new infection occurring once every 3 days for rabies, and once a week for CDV. We also inferred that during the early stage of the CDV epidemic, a bias in the monitoring protocol resulted in a positive sample being almost 10 times more likely to be collected than a negative sample. We estimated the rate of intake of oral vaccine at 0.006 per day, allowing us to estimate that roughly 68% of the foxes would be immunised. This was confirmed by field observations. Finally we discuss the implications for the eco-epidemiological dynamics of both epidemics in relation to control measures.

The sarcoptic mange epizootic affecting chamois in the Dolomites Alps since 1995 has risen considerable concern in a management and conservation perspective, due to its strong impact on chamois and ibex populations. A remarkable amount of data has been collected by different wildlife research and management institutions, in order to analyze mange patterns and develop possible strategies to control the disease. The present study is aimed at providing a population-related figure of the spatial and temporal dynamics of clinical sarcoptic mange in alpine chamois, proposing an approach in which relevant basic concepts and parameters, as the definition of the epidemic front and its spreading speed, can be estimated and framed. The epidemic front was referred to the different mountain massifs, corresponding to well established management units of the chamois in the study area; moreover, the mange-related mortality peak at the massif level was used (in substitution of the index case/s) for temporal analysis of the disease spreading. Two speeds of the front have been estimated: a first raw average speed of about 3.38 km/year, and a second refined speed of 4.64 ± 3.12 km/year, more consistent to the variability in the field. The time series analysis showed that the impact of mange increases over the late winter months, reaching a peak in early spring.Our results strengthen the conclusions of previous studies, proposing a new frame to include other studies in progress on the alpine chamois-Sarcoptes interactions.Download the complete issue.