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The bacterium Treponema paraluisleporidarum causes syphilis in Lagomorphs. In a set of 1,095 samples from four species – European brown hare (Lepus europaeus), mountain hare (Lepus timidus), Corsican hare (Lepus corsicanus) and European rabbit (Oryctolagus cuniculus) – we genotyped the strains that infect wild lagomorphs. Samples originate from Sweden, the Netherlands, the United Kingdom, Germany, the Czech Republic and Italy. The phylogenetic analyses of two informative gene targets (tp0488 and tp0548) showed high genetic diversity among the lagomorph-infecting treponemes. More specifically, we found a high number of nucleotide variants and various short repeat units in the tp0548 locus which have not been described for human syphilis and primate yaws causing Treponema pallidum. While the functional aspect of these short repeat units remains subject to ongoing investigations, it likely enables the pathogen to better survive in its lagomorph host. Our data did not support any geographic clustering which is equally reflected in the host population genetics as shown by mitochondrial genome data corresponding to the sampled lagomorph populations. This is unexpected and in contrast with what has been shown for nonhuman primate infection with T. pallidum. In the future, the combination of multi-locus sequence typing and WGS from modern and ancient samples from a wide geographic range and multiple lagomorph species will contribute to a better understanding of the epidemiology and evolutionary path of lagomorph-infecting treponemes. In conclusion, our current study demonstrates a high genetic variation of the syphilis-causing pathogen in a higher number of positively PCR-tested European lagomorphs (n=496/1095).

Deliverable D.4.3.3 reports information on coronavirus (CoV) contamination in wildlife by collecting samples from different species, in order to improve our knowledge on the spread of CoVs in wildlife and to predict the potential risk of cross-species interactions and early and reverse zoonotic adaptations. Different groups of wild animals, such as bats, foxes, marmots, deer, rodents, etc. were sampled and tested for CoVs by pan-cov PCR and subsequent sequencing.

Serosurveillance among animals, including pets, plays an important role in the current coronavirus disease 2019 (COVID-19) pandemic, because severe acute respiratory coronavirus 2 (SARS-CoV-2) infections in animal populations could result in the establishment of new virus reservoirs. Serological assays that offer the required sensitivity and specificity are essential. In this study, we evaluated the diagnostic performance of three different commercially available immunoassays for the detection of SARS-CoV-2 antibodies in pets, namely two ELISA tests for the detection of antibodies against SARS-CoV-2 nucleocapsid [ID Screen SARS CoV-2 double antigen multispecies (Double antigen) and ID Screen® SARS-CoV-2-N IgG indirect ELISA (Indirect)] and one test for the detection of neutralizing antibodies against SARS-CoV-2 receptor-binding-domain [surrogate virus neutralization test (sVNT)]. The obtained results were compared with those of conventional virus neutralization test (VNT), which was regarded as reference method. A total of 191 serum samples were analysed. Thirteen (6.8%) samples showed VNT-positive results. The overall sensitivity was higher for sVNT (100%) compared to nucleocapsid-based ELISA assays (23% for Double antigen and 60% for Indirect). The specificity was 100% for Indirect ELISA and sVNT, when a higher cut-off (30%) was used compared to the one previously defined by the manufacturer (20%), whereas the other test showed lower value (99%). The sVNT test showed the highest accuracy and agreement with VNT, with a perfect agreement when the higher cut-off was applied. The agreement between each nucleocapsid-based ELISA test and VNT was 96% for Indirect and 94% for Double antigen. Our findings showed that some commercially available serological tests may lead to a high rate of false-negative results, highlighting the importance of assays validation for the detection of SARS-CoV-2 antibodies in domestic animals.

In Italy, the West Nile Virus surveillance plan considers a multidisciplinary approach to identify the presence of the virus in the environment (entomological, ornithological, and equine surveillance) and to determine the risk of infections through potentially infected donors (blood and organ donors). The costs associated with the surveillance program for the Lombardy Region between 2014 and 2018 were estimated. The costs of the program were compared with a scenario in which the program was not implemented, requiring individual blood donation nucleic acid amplification tests (NAT) to detect the presence of WNV in human samples throughout the seasonal period of vector presence. Considering the five-year period, the application of the environmental/veterinary surveillance program allowed a reduction in costs incurred in the Lombardy Region of 7.7 million EUR. An integrated surveillance system, including birds, mosquito vectors, and dead-end hosts such as horses and humans, can prevent viral transmission to the human population, as well as anticipate the detection of WNV using NAT in blood and organ donors. The surveillance program within a One Health context has given the possibility to both document the expansion of the endemic area of WNV in northern Italy and avoid most of the NAT-related costs.

Treponema paraluisleporidarum infects both rabbits (ecovar Cuniculus) and hares (ecovar Lepus). While the occurrence of the bacterium has previously been reported for European brown hares (Lepus europaeus) and domestic rabbits (Oryctolagus cuniculus f. domestica), there are no data available that report infection in the European context. We tested a total of 1,995 serum samples and 287 genital swabs from opportunistically sampled European brown hares (Lepus europaeus; n = 2135), Mountain hares (Lepus timidus; n = 4), European rabbits (Oryctolagus cuniculus; n = 138), and pet rabbits (O. cuniculus f. domestica; n = 5). The samples originated from eight European countries. In case only serum was available, we tested the samples for the presence of anti-treponemal antibodies. For this, we utilized the Treponema pallidum-particle agglutination test (TP-PA), which is suited for the use in lagomorphs due to the antigenic cross-reactivity of anti-T. pallidum and anti-T. paraluisleporidarum antibodies. In addition, the results of 380 sera were confirmed using the fluorescent-Treponema antibody absorption test (FTA-ABS). In all cases where swab samples were available, DNA was extracted and tested using quantitative PCR to test for the presence of the lagomorph syphilis-bacterium. We were able to detect antibodies in 825 of 1,995 lagomorph sera (41.4%; brown hare: 825/1,868; rabbit: 0/127) and obtained positive qPCR results from 182 of 287 swab samples (63.4%; European brown hare: 167/267; mountain hare: 4/4; rabbit: 11/16). While all rabbit sera (n = 127) tested negative for anti-treponemal antibodies, the presence of the bacterium was confirmed in eight wild (n = 8/11) and three domestic rabbits (n = 3/5) from Germany using qPCR.

Wild birds play an important role in the circulation and spread of pathogens that are potentially zoonotic or of high economic impact on zootechnical production. They include, for example, West Nile virus (WNV), Usutu virus (USUV), avian influenza virus (AIV), and Newcastle disease virus (NDV), which, despite having mostly an asymptomatic course in wild birds, have a strong impact on public health and zootechnical production. This study investigated the presence of these viruses in several wild bird species from North Italy during the biennium 2019–2020. Wild birds derived from 76 different species belonging to 20 orders. Out of 679 birds, 27 were positive for WNV (lineage 2) with a prevalence of 4%, all birds were negative for USUV, one gull was positive for H13N6 influenza virus, and 12 samples were positive for NDV with a prevalence of 2%. Despite the low prevalence observed, the analyses performed on these species provide further data, allowing a better understanding of the diffusion and evolution of diseases of both economic and zoonotic importance.

To date, no evidence supports the fact that animals play a role in the epidemiology of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus infectious disease 2019 (COVID-19). However, several animal species are naturally susceptible to SARS-CoV-2 infection. Besides pets (cats, dogs, Syrian hamsters, and ferrets) and farm animals (minks), different zoo animal species have tested positive for SARS-CoV-2 (large felids and non-human primates). After the summer of 2020, a second wave of SARS-CoV-2 infection occurred in Barcelona (Spain), reaching a peak of positive cases in November. During that period, four lions (Panthera leo) at the Barcelona Zoo and three caretakers developed respiratory signs and tested positive for the SARS-CoV-2 antigen. Lion infection was monitored for several weeks and nasal, fecal, saliva, and blood samples were taken at different time-points. SARS-CoV-2 RNA was detected in nasal samples from all studied lions and the viral RNA was detected up to two weeks after the initial viral positive test in three out of four animals. The SARS-CoV-2 genome was also detected in the feces of animals at different times. Virus isolation was successful only from respiratory samples of two lions at an early time-point. The four animals developed neutralizing antibodies after the infection that were detectable four months after the initial diagnosis. The partial SARS-CoV-2 genome sequence from one animal caretaker was identical to the sequences obtained from lions. Chronology of the events, the viral dynamics, and the genomic data support human-to-lion transmission as the origin of infection. info:eu-repo/semantics/publishedVersion

SARS‐CoV‐2 positive or seropositive owned cats have been reported worldwide. The detection of seropositive stray cats in the proximity of farms of infected minks, coupled with the demonstration of cat‐to‐cat transmission in experimental settings, raise the question whether stray cats may have an epidemiological role in the COVID‐19 pandemic and may act as sentinel for the circulation of SARS‐CoV‐2. The aim of this study was to evaluate the presence of SARS‐CoV‐2 RNA and anti‐SARS‐CoV‐2 antibodies in free roaming cats belonging to colonies located in an area highly affected by the COVID‐19 pandemic and to correlate the results with the positivity rate in people sharing the same area. Interdigital, cutaneous, oropharyngeal, nasal and rectal swabs, as well as blood samples, were collected from 99 cats living in colonies and admitted to our hospital for neutering. This caseload corresponds to the 24.2% of the feline population living in the 25 sampled colonies and to the 5.6% of all the free‐roaming registered cats. The presence of SARS‐CoV‐2 RNA in swabs was assessed using real time RT‐PCR. Anti‐SARS‐CoV‐2 serum antibodies were assessed using commercially available ELISA kits and confirmed by serum virus neutralization. In people, the SARS‐CoV‐2 positivity rate ranged from 3.0% to 5.1% (mean rate: 4.1%) and the seropositive rate from 12.1% to 16.3% (mean rate: 14.2%). Most of the colonies were in urban areas and resident cats had frequent contacts with external cats or people. A COVID‐19 positive caretaker was found, whereas all the cats were negative for SARS‐CoV‐2 RNA and seronegative. Although the negative results cannot exclude previous infections followed by decrease of antibodies, this study suggests that colony cats do not have an important epidemiological role in SARS‐CoV‐2 transmission dynamics. Further studies on larger caseloads are warranted, also in the light of the emerging new viral variants, on a One Health perspective.

Cryptosporidium is a widespread apicomplexan protozoan of major zoonotic importance, characterized by a wide host range, and with relevant economic implications and potential negative effects on livestock and wildlife population dynamics. Considering the recent strong demographic increase of alpine ungulates, in this study, carried out in the Italian Northwestern Alps, we investigated the occurrence of Cryptosporidium spp. in these species and their potential involvement in environmental contamination with Cryptosporidium spp. oocysts. The immune-enzymatic approach revealed a Cryptosporidium prevalence of 1.7% (5/293), 0.5% (1/196) and 3.4% (4/119) in alpine chamois (Rupicapra rupicapra), red deer (Cervus elaphus) and roe deer (Capreolus capreolus), respectively. Positive samples were subjected to polymerase chain reaction (PCR) amplification for the COWP and gp60 genes. The presence of Cryptosporidium was confirmed in one chamois and four roe deer. Sequences obtained clustered within Cryptosporidium ubiquitum, currently recognized as an emerging zoonotic species. This finding represents the first detection of zoonotic C. ubiquitum in chamois and in the Alpine environment. Despite the low observed prevalences, environmental contamination by oocysts could play a role as a potential source of infections for humans and livestock.

Wildlife is frequently infected by intestinal protozoa, which may threaten their fitness and health. A diverse community of Eimeria species is known to occur in the digestive tract of mountain-dwelling ungulates, including chamois (genus Rupicapra). However, available data on Eimeria diversity in these taxa is at times inconsistent and mostly dated. In the present study, we aimed to revisit the occurrence of Eimeria spp. in the Alpine subspecies of the Northern chamois (Rupicapra rupicapra rupicapra) and the Apennine subspecies of the Southern chamois (Rupicapra pyrenaica ornata) in Italy, using an integrated approach based on a hierarchical cluster analysis (HCPC) applied to oocyst morphology and morphometry. A total of 352 fecal samples were collected from R. r. rupicapra (n = 262) and R. p. ornata (n = 90). Overall, 85.3% (300/352) of the animals tested microscopically positive to Eimeria spp. Based on morphological analysis, we identified all the eimerian species described in chamois. Through the HCPC method, five clusters were generated, corresponding to E. suppereri, E. yakimoffmatschoulskyi, E. riedmuelleri (two different clusters), and E. rupicaprae morphotypes. The well-defined clusters within E. riedmuelleri support the existence of two distinct morphological groups, possibly referable to different taxonomic units. This study suggests that combining a morphometrical approach with a powerful statistical method may be helpful to disentangle uncertainties in the morphology of Eimeria oocysts and to address taxonomic studies of eimeriid protozoa at a specific host taxon level.

With several human cases reported annually since 2008 and the unapparent risk of infection of blood donors, the West Nile virus (WNV) is emerging as an important health issue in Europe. Italy, as well as other European countries, experienced a recrudescence of the virus circulation in 2018, which led to an increased number of human cases. An integrated surveillance plan was activated in the Emilia-Romagna and Lombardy regions (Northern Italy) since 2008 in order to monitor the intensity and timing of WNV circulation. A fundamental part of this plan consists in entomological surveillance. In 2018, the surveillance plan made it possible to collect 385,293 mosquitoes in 163 stations in the two Regions. In total 269,147 Culex mosquitoes were grouped into 2,337 pools and tested for WNV, which was detected in 232 pools. Circulation started in the central part of the Emilia-Romagna region in the middle of June, about one month before the previous seasons. Circulation suddenly expanded to the rest of the region and reached the Lombardy region in the middle of July. WNV circulated more intensively in the eastern part of the surveyed area, as confirmed by the highest number of human cases. A relationship between the number of mosquitoes collected and the virus incidence emerged, but the data obtained highlighted that the probability of detecting the virus in a given site was less than expected with a higher number of collected mosquitoes. A significant relationship was observed between the temperature recorded one week before the sampling and the number of collected mosquitoes, as well as between the estimated number of WNV-positive mosquitoes and the temperature recorded two weeks before the sampling. The two weeks delay in the influence of temperature on the positive mosquitoes is in line with the time of the virus extrinsic incubation in the mosquito. This finding confirms that temperature is one of the principal drivers in WNV mosquito infection. The surveillance system demonstrated the ability to detect the virus circulation early, particularly in areas where circulation was more intense. This allowed evaluating the effect of mosquito abundance and weather factors on virus circulation.

Simple Summary Most of the newly emerging infections arise from animal reservoirs, frequently represented by wildlife species. Western European hedgehogs (Erinaceus europaeus) are mammalian hibernators, mainly nocturnal and insectivorous, living in natural open and green spaces as well as artificial, rural and urban, areas. They are generalist predators of macro-invertebrates, but they may also eat meat, bird eggs and on occasion pet food. These ecological and feeding habits, along with their high population densities, notable synanthropic attitudes, frequent contacts with sympatric wild and domestic species, including humans, implicate the possibility of intra- and interspecies interactions accounting for the possible involvement of E. europaeus in the ecology of several potentially emerging pathogens, including coronaviruses. Using PCR-based and virus isolation methods, we found that 58.3% of 24 hedgehogs’ fecal samples were PCR-positive for Erinaceus coronaviruses (EriCoVs). We did not observe any clinical disease related to the EriCoV infection in hedgehogs. However, the high mutation rates characterizing members of the Coronaviridae family and their potential successful interspecies host jumps—as that likely occurred in the Novel coronavirus (2019-nCoV) emergence—should be considered in the management of hedgehogs admitted to multi-species wildlife rehabilitation centers, recommending their return back to the original recovery areas. Abstract The Western European Hedgehog (Erinaceus europaeus) is one of the four hedgehog species belonging to the genus Erinaceus. Among them, E. amurensis is extant in East Asia’s areas only, whereas E. europaeus, E. roumanicus and E. concolor are mainly found in Europe. E. europaeus is endemically distributed from western to central and southern Europe, including Italy. Western European hedgehogs’ ecological and feeding habits, along with their high population densities, notable synanthropic attitudes, frequent contacts with sympatric wild and domestic species, including humans, implicate the possible involvement of E. europaeus in the ecology of potentially emerging viruses, such as coronaviruses, influenza A and influenza D viruses, canine distemper virus, pestiviruses and Aujeszky’s disease virus. We examined 24 E. europaeus individuals found injured in urban and rural areas of Northern Italy. Of the 24 fecal samples collected and tested for the above-mentioned pathogens by both PCR-based and virus isolation methods, 14 were found PCR-positive for betacoronaviruses belonging to lineage C and related to the known Erinaceus coronaviruses (EriCoVs), as determined by partial sequencing of the virus genome. Our findings suggest that hedgehogs could be considered natural reservoirs of CoVs, and also act as chronic shedding carriers of these potentially emerging RNA viruses.

Wildlife is frequently infected by intestinal protozoa, which may threaten their fitness and health. A diverse community of Eimeria species is known to occur in the digestive tract of mountain-dwelling ungulates, including chamois (genus Rupicapra). However, available data on Eimeria diversity in these taxa is at times inconsistent and mostly dated. In the present study, we aimed to revisit the occurrence of Eimeria spp. in the Alpine subspecies of the Northern chamois (Rupicapra rupicapra rupicapra) and the Apennine subspecies of the Southern chamois (Rupicapra pyrenaica ornata) in Italy, using an integrated approach based on a hierarchical cluster analysis (HCPC) applied to oocyst morphology and morphometry. A total of 352 fecal samples were collected from R. r. rupicapra (n = 262) and R. p. ornata (n = 90). Overall, 85.3% (300/352) of the animals tested microscopically positive to Eimeria spp. Based on morphological analysis, we identified all the eimerian species described in chamois. Through the HCPC method, five clusters were generated, corresponding to E. suppereri, E. yakimoffmatschoulskyi, E. riedmuelleri (two different clusters), and E. rupicaprae morphotypes. The well-defined clusters within E. riedmuelleri support the existence of two distinct morphological groups, possibly referable to different taxonomic units. This study suggests that combining a morphometrical approach with a powerful statistical method may be helpful to disentangle uncertainties in the morphology of Eimeria oocysts and to address taxonomic studies of eimeriid protozoa at a specific host taxon level.

Seroprevalence data for Toxoplasma gondii and Hepatitis E virus (HEV) in wild boar (Sus scrofa), roe deer (Capreolus capreolus), red deer (Cervus elaphus), mouflon (Ovis aries/musimon) and chamois (Rupicapra rupicapra) hunted/culled in northern Italy were used to fit seroprevalence distributions describing the exposure and co-exposure of the species to the two pathogens. The higher proportion of T. gondii and HEV seropositive animals was observed in wild boars with point estimate seroprevalence of 49% (N = 331) and 15% (N = 326) respectively. Data allowed comparisons by area (pre-Alpine Vs Alpine environment) for roe deer, red deer and mouflons. Contrasts between the distributions describing the uncertainty in seroprevalence suggest roe deer, red deer and mouflons have higher probability of being seropositive to T. gondii in pre-Alps. When considering HEV, few seropositive animals were detected and contrasts were symmetrically centred to zero for roe deer and red deer; mouflons shown higher probability of being seropositive in Alpine environment. HEV seropositive animals also included chamois (P = 5.1%, N = 97) in the Alpine districts, confirming circulation of HEV in remote areas. Evidence of HEV and T. gondii co-exposure was limited except for wild boars where it was observed in 30 samples representing 60% of the overall HEV-positive samples. Seroprevalence data of single infection and co-infection are extremely useful to investigate circulation of zoonotic pathogens in wild animals and estimate the foodborne risk of human exposure, however, these type of data do not directly translate into the presence/absence of the pathogen in seropositive and seronegative animals. At benefit of future development of quantitative risk assessments aiming at estimating the risk of human infection/co-infection via consumption of game meat, we developed and made available an online application that allows estimating the probability of the pathogen(s) being present as a function of seroprevalence data.

Infectious bronchitis virus (IBV) control is mainly based on wide vaccine administration. Although effective, its efficacy is not absolute, the viral circulation is not prevented and some side effects cannot be denied. Despite this, the determinants of IBV epidemiology and the factors affecting its circulation are still largely unknown and poorly investigated. In the present study, 361 IBV QX (the most relevant field genotype in Italy) sequences were obtained between 2012 and 2016 from the two main Italian integrated poultry companies. Several biostatistical and bioinformatics approaches were used to reconstruct the history of the QX genotype in Italy and to assess the effect of different environmental, climatic and social factors on its spreading patterns. Moreover, two structured coalescent models were considered in order to investigate if an actual compartmentalization occurs between the two integrated poultry companies and the role of a third “ghost” deme, representative of minor industrial poultry companies and the rural sector. The obtained results suggest that the integration of the poultry companies is an effective barrier against IBV spreading, since the strains sampled from the two companies formed two essentially-independent clades. Remarkably, the only exceptions were represented by farms located in the high densely populated poultry area of Northern Italy. The inclusion of a third deme in the model revealed the likely role of other poultry companies and rural farms (particularly concentrated in Northern Italy) as sources of strain introduction into one of the major poultry companies, whose farms are mainly located in the high densely populated poultry area of Northern Italy. Accordingly, when the effect of different environmental and urban parameters on IBV geographic spreading was investigated, no factor seems to contribute to IBV dispersal velocity, being poultry population density the only exception. Finally, the different viral population pattern observed in the two companies over the same time period supports the pivotal role of management and control strategies on IBV epidemiology. Overall, the present study results stress the crucial relevance of human action rather than environmental factors, highlighting the direct benefits that could derive from improved management and organization of the poultry sector on a larger scale.

Hepatitis E virus (HEV) is a worldwide public health concern, with an increase in human autochthonous cases in Europe. Although domestic pigs and wild boar (

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.

Cryptosporidium is a widespread apicomplexan protozoan of major zoonotic importance, characterized by a wide host range, and with relevant economic implications and potential negative effects on livestock and wildlife population dynamics. Considering the recent strong demographic increase of alpine ungulates, in this study, carried out in the Italian Northwestern Alps, we investigated the occurrence of Cryptosporidium spp. in these species and their potential involvement in environmental contamination with Cryptosporidium spp. oocysts. The immune-enzymatic approach revealed a Cryptosporidium prevalence of 1.7% (5/293), 0.5% (1/196) and 3.4% (4/119) in alpine chamois (Rupicapra rupicapra), red deer (Cervus elaphus) and roe deer (Capreolus capreolus), respectively. Positive samples were subjected to polymerase chain reaction (PCR) amplification for the COWP and gp60 genes. The presence of Cryptosporidium was confirmed in one chamois and four roe deer. Sequences obtained clustered within Cryptosporidium ubiquitum, currently recognized as an emerging zoonotic species. This finding represents the first detection of zoonotic C. ubiquitum in chamois and in the Alpine environment. Despite the low observed prevalences, environmental contamination by oocysts could play a role as a potential source of infections for humans and livestock.

Mammalian orthoreoviruses (MRVs) are emerging infectious agents that may affect wild animals. MRVs are usually associated with asymptomatic or mild respiratory and enteric infections. However, severe clinical manifestations have been occasionally reported in human and animal hosts. An insight into their circulation is essential to minimize the risk of diffusion to farmed animals and possibly to humans. The aim of this study was to investigate the presence of likely zoonotic MRVs in wild ungulates. Liver samples were collected from wild boar, red deer, roe deer, and chamois. Samples originated from two areas (Sondrio and Parma provinces) in Northern Italy with different environmental characteristics. MRV detection was carried out by PCR, confirmation by sequencing and typing for MRV type 3, which has been frequently associated with disease in pigs, were carried out for positive samples. MRV prevalence was as high as 45.3% in wild boars and 40.6% in red deer in the Sondrio area, with lower prevalence in the Parma area (15.4% in wild boars). Our findings shed light on MRV occurrence and distribution in some wild species and posed the issue of their possible role as reservoir.

Canine distemper (CD) is a fatal, highly contagious disease of wild and domestic carnivores. In the Alpine territory, several outbreaks have occurred in the past few decades within wild populations. This study investigated the presence of canine distemper virus (CDV) infections in wild carnivores in Lombardy, relating to the different circulating genotypes. From 2018 to 2020, foxes, badgers, and martens collected during passive surveillance were subjected to necropsy and histological examination, showing classical signs and microscopic lesions related to CDV. Pools of viscera from each animal were analysed by molecular methods and immunoelectron microscopy. Total prevalences of 39.7%, 52.6%, and 14.3% were recorded in foxes, badgers, and stone martens, respectively. A phylogenetic analysis showed that the sequences obtained belonged to the European 1 lineage and were divided into two different clades (a and b) according to the geographical conformation of alpine valleys included in the study. Clade a was related to the European outbreaks originating from Germany in 2006&ndash, 2010, while clade b was closely related to the CDV sequences originating from northeastern Italy during the 2011&ndash, 2018 epidemic wave. Our results suggest that CDV is currently well adapted to wild carnivores, mostly circulating with subclinical manifestations and without severe impact on the dynamics of these populations.

Hepatitis E virus (HEV) is a worldwide public health concern, with an increase in human autochthonous cases in Europe. Although domestic pigs and wild boar (Sus scrofa) are the main reservoirs of HEV, the constant expansion of wild ruminants increases the potential for HEV transmission. We investigated HEV infection in chamois (Rupicapra rupicapra) and red deer (Cervus elaphus) in the Italian Alps using an enzyme-linked immunosorbent assay (ELISA). We detected HEV antibodies from 2013 to 2015 in both host species, with seroprevalences of 1.2% and 0.8% in chamois and red deer, respectively. All serum samples that were positive to HEV antibodies by ELISA were negative when tested by real-time reverse-transcriptase PCR to detect HEV RNA. The observed low seroprevalence of HEV suggested a sporadic circulation of HEV in the alpine environment, and it was consistent with the low seroprevalence observed in wild boar in the Alps. Our observations supported the role of chamois and red deer as spillover hosts of HEV infections in the Italian Alps.

Responses of animals to environmental changes and their interactions with other species play an important role in conservation. Sharing a common habitat may lead to interspecific competition for resources, but field assessment of these biological events is not always easily accomplished. By using a non-invasive method, we evaluated the physiological stress responses of Apennine chamois (Rupicapra pyrenaica ornata) to the presence of cattle, sheep and goat, red deer (Cervus elaphus), people (hikers), and predators to identify which factors may affect this endangered species. During September 2012, November 2012, and July 2013, a total of 318 faecal samples were collected in representative sites and analysed for faecal cortisol metabolites (FCM). FCM concentration was analysed through linear mixed-effect models. A significant increase in FCM values in Apennine chamois sharing their habitat with domestic animals was recorded during all study periods. On the contrary, stress responses to red deer and people were limited in time and emerged only during summer months, when hikers are more frequent and red deer extend their altitudinal range reaching chamois’ habitat. The observed effects of domestic animals, red deer, and hikers should be considered in future Apennine chamois management plans, which should include the regulation of pastured domestic livestock, anthropogenic disturbances, and possible interferences with other wild species within parks.

In the Italian Alps the intensification of winter sports represents an increasing threat for Black Grouse (Tetrao tetrix). During the winters 2010/2011 and 2011/2012, characterised by different amounts of snowfall, 58 droppings from as many snow burrows were collected in three areas with different human disturbance to evaluate its effects on Black Grouse stress responses. Subjects in highly disturbed area showed higher concentrations of corticosterone metabolites during the winter with high snow cover highlighting a relationship between animals' stress and regular winter sports. As human-induced stress may contribute to population decline, tourism management should be planned to support Grouse conservation.

The complex life cycle of taeniids represents an ideal model of a multi-host system. The complexity of these parasites can therefore cover the epidemiological issues of the interface between wild and domestic animals, especially once spatial overlap between wild and domestic definitive and intermediate hosts occurs. Here we use the occurrence of Taenia ovis krabbei in two model areas as an example of this epidemiological complexity. In two contiguous areas in the Italian northern Apennines, two hunted roe deer (Capreolus capreolus) showed numerous cysticerci in the muscles of their whole body and an adult tapeworm was recorded in a semi-stray dog (Canis lupus familiaris). Through molecular typing of the mitochondrial cytochrome c oxidase I (cox1) gene, cysticerci and the adult tapeworm of T. krabbei were identified. Taenia krabbei cysticercosis was recorded for the first time in Italy. Although the role of dogs in the parasite's life cycle emerges, the overlap between wild and domestic definitive hosts and the increase of wild population densities raise concerns about the temporal (old or new) introduction and the spread of this parasite by one of these canid species (wolf (Canis lupus) or dog). Although T. krabbei is not a public health issue, economic concerns emerged for hunters and meat producers, related to the damage of carcasses by cysticerci. Therefore, there is a need to evaluate the spread of T. krabbei in the intermediate and definitive host populations, and to ensure the relevant sanitary education for hunters in order to avoid practices that could favour the spread and maintenance of its life cycle.