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.

Corrigendum: Comparing copromicroscopy to intestinal scraping to monitor red fox intestinal helminths with zoonotic and veterinary importance.

[This corrects the article DOI: 10.3389/fvets.2022.1085996.].

Investigation of Carnivore protoparvovirus 1 and Amdoparvovirus infections in red fox populations of the Italian Dolomites.

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.

Description of a Sarcoptic Mange Outbreak in Alpine Chamois Using an Enhanced Surveillance Approach.

Since 1995, the Alpine chamois (Rupicapra r. rupicapra) population of the Dolomites has been affected by sarcoptic mange with considerable management concerns. In this study, 15 years (2006-2020) of passive surveillance and demographic data were analyzed in order to describe a mange outbreak. Furthermore, an enhanced passive surveillance protocol was implemented in order to evaluate the efficiency of ordinary vs. enhanced surveillance protocol in identifying dead chamois in the field and in reaching a correct diagnosis. Our results confirm the role of mange as a determining factor for chamois mortality, while stressing the importance of a wider view on the factors affecting population dynamics. The enhanced passive surveillance protocol increased the probability of carcass retrieval and identification of the cause of death; however, its adoption may be too costly if applied for long periods on a wide scale. Passive surveillance, in both ordinary and enhanced surveillance protocol, should encompass the use of other strategies in the future to study the eco-epidemiology of the disease in wild Caprinae.

A highly endemic area of Echinococcus multilocularis identified through a comparative re-assessment of prevalence in the red fox (Vulpes vulpes), Alto Adige (Italy: 2019-2020).

Surveillance of Echinococcus multilocularis at the edge of its range is hindered by fragmented distributional patterns and low prevalence in definitive hosts. Thus, tests with adequate levels of sensitivity are especially important for discriminating between infected and non-infected areas. In this study we reassessed the prevalence of E. multilocularis at the southern border of its distribution in Province of Bolzano (Alto Adige, northeastern Alps, Italy), to improve surveillance in wildlife and provide more accurate estimates of exposure risk. We compared the diagnostic test currently implemented for surveillance based on coproscopy and multiplex PCR (CMPCR) to a real-time quantitative PCR (qPCR) in 235 fox faeces collected in 2019 and 2020. The performances of the two tests were estimated using a scraping technique (SFCT) applied to the small intestines of a subsample (n = 123) of the same foxes as the reference standard. True prevalence was calculated and the sample size required by each faecal test for the detection of the parasite was then estimated. True prevalence of E. multilocularis in foxes (14.3%) was markedly higher than reported in the last decade, which was never more than 5% from 2012 to 2018 in the same area. In addition, qPCR showed a much higher sensitivity (83%) compared to CMPCR (21%) and agreement with the reference standard was far higher for qPCR (0.816) than CMPCR (0.298) meaning that for the latter protocol, a smaller sample size would be required to detect the disease. Alto Adige should be considered a highly endemic area. Routine surveillance on definitive hosts at the edges of the E. multilocularis distribution should be applied to smaller geographic areas, and rapid, sensitive diagnostic tools using directly host faeces, such as qPCR, should be adopted.

Clinical Tick-Borne Encephalitis in a Roe Deer (Capreolus capreolus L.).

Tick-borne encephalitis virus (TBEV) is the causative agent of tick-borne encephalitis (TBE), a severe zoonosis occurring in the Palearctic region mainly transmitted through Ixodes ticks. In Italy, TBEV is restricted to the north-eastern part of the country. This report describes for the first time a case of clinical TBE in a roe deer (Capreolus capreolus L.). The case occurred in the Belluno province, Veneto region, an area endemic for TBEV. The affected roe deer showed ataxia, staggering movements, muscle tremors, wide-base stance of the front limbs, repetitive movements of the head, persistent teeth grinding, hypersalivation and prolonged recumbency. An autopsy revealed no significant lesions to explain the neurological signs. TBEV RNA was detected in the brain by real-time RT-PCR, and the nearly complete viral genome (10,897 nucleotides) was sequenced. Phylogenetic analysis of the gene encoding the envelope protein revealed a close relationship to TBEV of the European subtype, and 100% similarity with a partial sequence (520 nucleotides) of a TBEV found in ticks in the bordering Trento province. The histological examination of the midbrain revealed lymphohistiocytic encephalitis, satellitosis and microgliosis, consistent with a viral etiology. Other viral etiologies were ruled out by metagenomic analysis of the brain. This report underlines, for the first time, the occurrence of clinical encephalitic manifestations due to TBEV in a roe deer, suggesting that this pathogen should be included in the frame of differential diagnoses in roe deer with neurologic disease.

Comparing copromicroscopy to intestinal scraping to monitor red fox intestinal helminths with zoonotic and veterinary importance.

The red fox acts as reservoir for several helminthic infections which are of interest for both public and animal health. Huge efforts have been made for the assessment of the sensitivity of coprological tests for the detection of Echinococcus multilocularis, while less attention has been paid to other helminthic species. This study aimed at assessing the performance of two copromicroscopic techniques in the detection and prevalence estimation of gastrointestinal helminths in the red fox. Helminths were isolated from the small intestines of 150 red foxes from Bolzano province, Italy, with a scraping, filtration and counting technique (SFCT) and morphologically identified. Rectal contents were collected and submitted to simple flotation (FT) and, only for Taenids, a method based on the concentration of eggs and identification with multiplex PCR (CMPCR). Using SFCT as a reference standard, we estimated the sensitivity of the copromicroscopic tests. Three species of nematodes (namely, Toxocara canis, Uncinaria stenocephala and Pterygodermatites sp.) and five species of cestodes (E. multilocularis, Taenia crassiceps, T. polycantha, Hydatigera taeniaeformis, Mesocestoides sp.) were identified with SFCT, whereas eggs referable to the same taxa were detected with fecal diagnostics, except for Pterygodermatites sp. and Mesocestoides sp. The sensitivity of FT was low for all taxa, ranging from 9.8 to 36.3%, with lower values for Taeniidae. CMPCR was confirmed to perform better for the detection of Taeniidae eggs (23.5%) and the multiplex PCR on retrieved eggs was effective in the identification of the species. A meta-analysis of literature also suggested that our results are consistent with existing data, indicating that copromicroscopy tends to underestimate the prevalence of helminthic infections. The extent of such underestimation varies with taxon, being higher at high prevalence levels, in particular for cestodes. Irregular dynamics of egg shedding, and routine deep freezing of red fox feces may explain the frequency of false negatives with copromicroscopy. Low sensitivity of copromicroscopic tests should be accounted for when estimating prevalence and when defining the correct sample size for the detection of the parasites.

Assessing Red Fox (Vulpes vulpes) Demographics to Monitor Wildlife Diseases: A Spotlight on Echinococcus multilocularis.

The assessment of red fox population density is considered relevant to the surveillance of zoonotic agents vectored by this species. However, density is difficult to estimate reliably, since the ecological plasticity and elusive behavior of this carnivore hinder classic methods of inference. In this study, red fox population density was estimated using a non-invasive molecular spatial capture-recapture (SCR) approach in two study areas: one in a known hotspot of the zoonotic cestode Echinococcus multilocularis, and another naïve to the parasite. Parasitological investigations on collected samples confirmed the presence of the parasite exclusively in the former area; the SCR results indicated a higher fox population density in the control area than in the hotspot, suggesting either that the relationship between fox density and parasite prevalence is not linear and/or the existence of other latent factors supporting the parasitic cycle in the known focus. In addition, fox spotlight count data for the two study areas were used to estimate the index of kilometric abundance (IKA). Although this method is cheaper and less time-consuming than SCR, IKA values were the highest in the areas with the lower molecular SCR density estimates, confirming that IKA should be regarded as a relative index only.

Occurrence and Identification of Ixodes ricinus Borne Pathogens in Northeastern Italy.

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 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.

Echinococcus multilocularis and other cestodes in red foxes (Vulpes vulpes) of northeast Italy, 2012-2018.

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.

Two waves of canine distemper virus showing different spatio-temporal dynamics in Alpine wildlife (2006-2018).

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 in Wild Caprinae of the Alps: Could Pathology Help in Filling the Gaps in Knowledge?

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.

Genetic and spatial characterization of the red fox (Vulpes vulpes) population in the area stretching between the Eastern and Dinaric Alps and its relationship with rabies and canine distemper dynamics.

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.

Pathology and Distribution of Trombiculosis in Northern Chamois ( Rupicapra rupicapra rupicapra) in the Italian Alps.

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.

Ticks are more suitable than red foxes for monitoring zoonotic tick-borne pathogens in northeastern Italy.

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.

Rabies and canine distemper virus epidemics in the red fox population of northern Italy (2006-2010).

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.

A distinct CDV genotype causing a major epidemic in Alpine wildlife.

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.