Detection and molecular analysis of Pseudorabies virus from free-ranging Italian wolves (Canis lupus italicus) in Italy - a case report.

BACKGROUND: The only natural hosts of Pseudorabies virus (PRV) are members of the family Suidae (Sus scrofa scrofa). In mammals, the infection is usually fatal and typically causes serious neurologic disease. This study describes four Aujeszky's disease cases in free-ranging Italian wolves (Canis lupus italicus). In Italy, the wolf is a strictly protected species and is in demographic expansion. CASE PRESENTATION: Three wolves (Wolf A, B, and C) were found in a regional park in Northern Italy, and one (Wolf D) was found in Central Italy. Wolf A and D were alive at the time of the finding and exhibited a fatal infection with epileptic seizures and dyspnoea, dying after a few hours. Wolf B presented scratching lesions under the chin and a detachment of the right earlobe, whilst Wolf C was partially eaten. The wolves showed hepatic congestion, diffuse enteritis, moderate pericardial effusion, severe bilateral pneumonia, and diffuse hyperaemia in the brain. The diagnostic examinations included virological analyses and detection of toxic molecules able to cause serious neurological signs. All four wolves tested positive for pseudorabies virus (PrV). The analysed sequences were placed in Italian clade 1, which is divided into two subclades, "a" and "b". The sequences of Wolf A, B, and C were closely related to other Italian sequences in the subclade b, originally obtained from wild boars and hunting dogs. The sequence from Wolf D was located within the same clade and was closely related to the French hunting dog sequences belonging to group 4. CONCLUSION: Results showed the presence of PrV strains currently circulating in wild boars and free-ranging Italian wolves. The genetic characterisation of the PrV UL44 sequences from the four wolves confirmed the close relationship with the sequences from wild boars and hunting dogs. This fact supports a possible epidemiological link with the high PrV presence in wild boars and the possibility of infection in wolves through consumption of infected wild boar carcasses or indirect transmission. To the best of our knowledge, this study is the first detection of Pseudorabies virus in free-ranging Italian wolves in northern and central Italy.

Isolation of Batborne Neglected Zoonotic Agent Issyk-Kul Virus, Italy.

We isolated Issyk-Kul virus (ISKV) from a bat sampled from Italy in 2021 and conducted ISKV-specific surveillance in bats collected in Italy during 2017-2023. ISKV circulation among synanthropic and sedentary species of bat, such as Savi's pipistrelle bat (Hypsugo savii) in northern Italy, may have public health implications in this region.

Parasite-mediated manipulation? Toxoplasma gondii infection increases risk behaviour towards culling in red deer.

Parasites can modify host behaviour to increase their chances of survival and transmission. Toxoplasma gondii is a globally distributed protozoan whose ability to modify host behaviour is well known in taxa such as rats and humans. Less well known are the effects on the behaviour of wild species, with the exception of a few studies on primates and carnivores. Taking advantage of a culling activity conducted in Stelvio National Park (Italy), the serological status of T. gondii was studied in 260 individuals of red deer Cervus elaphus with respect to the risk of being culled. A temporal culling rank index was fitted as a response variable, and T. gondii serological status as the main explanatory variable in linear models, accounting for covariates such as sex, age, jaw length, bone marrow fat and culling location. The overall seroprevalence of T. gondii was 31.5%, and the selected models suggested that seropositive deer were culled earlier than seronegative ones, but this effect was only evident in females, in individuals with medium-good body condition, and in areas with greater human presence. Our results suggest that T. gondii may be involved in risk behaviour in large herbivores, supporting its role as a facilitator of predation risk.

Analysis of seroprevalence data on Hepatitis E virus and Toxoplasma gondii in wild ungulates for the assessment of human exposure to zoonotic meat-borne pathogens.

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.

Spread and genotype of Toxoplasma gondii in naturally infected alpine chamois (Rupicapra r. rupicapra).

The complex life cycle of Toxoplasma gondii involves many animal species, raising zoonotic, economic, and conservation issues. This complexity is reflected in the molecular structure of T. gondii, whose different genotypes differ in pathogenicity. Among the intermediate hosts of T. gondii, wild ungulates may be a source of human infection. Despite intense hunting activity and the consumption of raw or undercooked meat, little information is available on the spread of T. gondii and the distribution of its genotypes in these species, including the alpine chamois (Rupicapra r. rupicapra). Ninety-three sera and 50 brain tissues from chamois were sampled (1) to investigate the spread of T. gondii with serological and molecular analyses, and (2) to genotype the strains with a restriction fragment length polymorphism analysis of the SAG2 locus. The prevalence of T. gondii was low on both serological (3.2 %) and molecular (2 %) analyses, and infections were concentrated in individuals >1 year old. These findings demonstrate the sporadic presence of the protozoan in this species on consistent diagnostic tests. Horizontal transmission seems to be the main route of infection, and cats are the only definitive host in the study area. This prevalence suggests that the environment of the chamois is less contaminated with oocysts than environments close to human settlements. The SAG2 type II genotype was detected in this species for the first time. Although this genotype is predominant in human toxoplasmosis, these results suggest that the chamois is a minor source of human infection.

Recombination between non-structural and structural genes as a mechanism of selection in lagoviruses: The evolutionary dead-end of an RHDV2 isolated from European hare.

The genus Lagovirus, belonging to the family Caliciviridae, emerged around the 1980s. It includes highly pathogenic species, rabbit hemorrhagic disease virus (RHDV/GI.1) and European brown hare syndrome virus (EBHSV/GII.1), which cause fatal hepatitis, and nonpathogenic viruses with enteric tropism, rabbit calicivirus (RCV/GI.3,4) and hare calicivirus (HaCV/GII.2). Lagoviruses have evolved along two independent genetic lineages: GI (RHDV and RCV) in rabbits and GII (EBHSV and HaCV) in hares. To be emphasized is that genomes of lagoviruses, like other caliciviruses, are highly conserved at RdRp-VP60 junctions, favoring intergenotypic recombination events at this point. The recombination between an RCV (genotype GI.3), donor of non-structural (NS) genes, and an unknown virus, donor of structural (S) genes, likely led to the emergence of a new lagovirus in the European rabbit, called RHDV type 2 (GI.2), identified in Europe in 2010. New RHDV2 intergenotypic recombinants isolated in rabbits in Europe and Australia originated from similar events between RHDV2 (GI.2) and RHDV (GI.1) or RCV (GI.3,4). RHDV2 (GI.2) rapidly spread worldwide, replacing RHDV and showing several lagomorph species as secondary hosts. The recombination events in RHDV2 viruses have led to a number of viruses with very different combinations of NS and S genes. Recombinant RHDV2 with NS genes from hare lineage (GII) was recently identified in the European hare. This study investigated the first RHDV2 (GI.2) identified in Italy in European hare (RHDV2_Bg12), demonstrating that it was a new virus that originated from the recombination between RHDV2, as an S-gene donor and a hare lagovirus, not yet identified but presumably nonpathogenic, as an NS gene donor. When rabbits were inoculated with RHDV2_Bg12, neither deaths nor seroconversions were recorded, demonstrating that RHDV2_Bg12 cannot infect the rabbit. Furthermore, despite intensive and continuous field surveillance, RHDV2_Bg12 has never again been identified in either hares or rabbits in Italy or elsewhere. This result showed that the host specificity of lagoviruses can depend not only on S genes, as expected until today, but potentially also on some species-specific NS gene sequences. Therefore, because RHDV2 (GI.2) infects several lagomorphs, which in turn probably harbor several specific nonpathogenic lagoviruses, the possibility of new speciation, especially in those other than rabbits, is real. RHDV2 Bg_12 demonstrated this, although the attempt apparently failed.

Molecular and Serological Detection of Bovine Coronaviruses in Marmots (Marmota marmota) in the Alpine Region.

In this study, virological surveillance focused on coronaviruses in marmots in the Alpine region in 2022, captured as part of a population control reduction program in the Livigno area. Seventy-six faecal samples were randomly collected from marmots at the time of capture and release and tested for genome detection of pan-coronavirus, pan-pestivirus, canine distemper virus, and influenza A and D virus. Nine faecal samples were positive in the Pan-CoV RT-PCR, while all were negative for the other viruses. Pan-coronavirus positives were further identified using Illumina's complete genome sequencing, which showed the highest homology with Bovine Coronavirus previously detected in roe deer in the Alps. Blood samples (n.35) were collected randomly from animals at release and tested for bovine coronavirus (BCoV) antibodies using competitive ELISA and VNT. Serological analyses revealed that 8/35 sera were positive for BCoV antibodies in both serological tests. This study provides molecular and serological evidence of the presence of BCoV in an alpine marmot population due to a likely spillover event. Marmots share areas and pastures with roe deer and other wild ruminants, and environmental transmission is a concrete possibility.

Presence and Characterisation of Porcine Respirovirus 1 (PRV1) in Northern Italy.

Porcine Respirovirus 1 (PRV1) is an enveloped, single-stranded, negative-sense RNA virus belonging to the genus Respirovirus within the Paramyxoviridae family. Since its first detection in China in 2013, PRV1 has been identified in several American and European countries. Although its pathogenicity is uncertain, recent studies have suggested that it may play a role in the Porcine Respiratory Disease Complex (PRDC) because of its capacity to replicate in the upper and lower respiratory tracts. This study aimed to determine the spread of PRV1 in Northern Italy and the phylogeny of the isolates. Therefore, PRV1 was investigated using real-time RT-PCR in 902 samples collected from September 2022 to September 2023 from pigs with respiratory symptoms in North Italy. Fourteen (1.55%) samples tested as PRV1-positive. The full-length fusion (F) gene, which codifies for a major surface protein, was amplified and used for phylogenetic analysis to help carry out molecular epidemiological studies on this virus. In addition, swine influenza virus (SIV) and porcine reproductive and respiratory syndrome virus (PRRSV) infections were detected in most of the PRV1-positive samples. In conclusion, we report the detection of PRV1 in Italy and discuss its potential role as a co-factor in causing the Porcine Respiratory Disease Complex.

High prevalence and genetic diversity of Treponema paraluisleporidarum isolates in European lagomorphs.

IMPORTANCE: Syphilis is an ancient disease of humans and lagomorphs caused by two distinct but genetically closely related bacteria (>98% sequence identity based on the whole genome) of the genus Treponema. While human syphilis is well studied, little is known about the disease in the lagomorph host. Yet, comparative studies are needed to understand mechanisms in host-pathogen coevolution in treponematoses. Importantly, Treponema paraluisleporidarum-infected hare populations provide ample opportunity to study the syphilis-causing pathogen in a naturally infected model population without antibiotic treatment, data that cannot be obtained from syphilis infection in humans. We provide data on genetic diversity and are able to highlight various types of repetitions in one of the two hypervariable regions at the tp0548 locus that have not been described in the human syphilis-causing sister bacterium Treponema pallidum subsp. pallidum.

Isolation and Molecular Characterisation of Respirovirus 3 in Wild Boar.

Paramyxoviruses are important pathogens affecting various animals, including humans. In this study, we identified a paramyxovirus in 2004 (180608_2004), isolated from a sample of the femoral marrow bone of a wild boar carcass imported from Australia. Antigenic and morphological characteristics indicated that this virus was similar to members of the family Paramyxoviridae. The complete genome phylogenetic analysis grouped this virus into genotype A of bovine parainfluenza virus type 3 (BPIV-3), recently renamed bovine respirovirus type 3 (BRV3), which also includes two swine paramyxoviruses (SPMV)-Texas-81 and ISU-92-isolated from encephalitic pigs in the United States in 1982 and 1992, respectively. The wild boar 180608_2004 strain was more closely related to both the BRV3 shipping fever (SF) strain and the SPMV Texas-81 strain at the nucleotide and amino acid levels than the SPMV ISU-92 strain. The high sequence identity to BRV3 suggested that this virus can be transferred from cattle to wild boars. The potential for cross-species transmission in the Respirovirus genus makes it essential for intensified genomic surveillance.

Spatiotemporal Distribution of Salmonella enterica in European Hedgehogs in Northern Italy.

Growing attention is being given to the European hedgehog (Erinaceus europaeus) because of its synanthropic behaviour and its potential role in harbouring parasites, viruses, fungi and bacteria and disseminating them to several animals and humans. Salmonella are the most frequently detected zoonotic bacteria that hedgehogs could transmit through contaminating water and food sources with faeces. This study aimed to determine the prevalence and distribution of Salmonella spp. in wild hedgehogs in the Emilia-Romagna region (northern Italy). From 2019 to 2022, 212 European hedgehogs that died naturally were tested for Salmonella spp. through culture isolation. Positive samples were subjected to serological typing. A total of 82 samples tested positive for Salmonella spp., with the overall Bayesian posterior estimated prevalence ranging from 35% (95% CI: 23-47%) to a maximum of 45% (95% CI: 31-59%) during the years considered and with an overall prevalence calculated at 39% (95% CI: 33-45%). Salmonella enterica Enteritidis and Veneziana were the most prevalent detected serovars in 65% and 17% of the positive samples, respectively. Since 2021, S. Typhimurium, S. Typhimurium Monofasica, S. Zaiman, S. Hessarek, S. Muenster, S. Isangi serovars, S. enterica subsp. Diarizonae and S. enterica subsp. Houtenae have been detected. These findings show a high prevalence of Salmonella spp. in tested hedgehogs, suggesting an important role of this animal species in the epidemiology of potentially zoonotic serovars circulating in the Emilia-Romagna region.

Comparison of diagnostic performances of different serological tests for SARS-CoV-2 antibody detection in cats and dogs.

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.

Assessment of the Costs Related to West Nile Virus Monitoring in Lombardy Region (Italy) between 2014 and 2018.

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.

Torque Teno Sus Virus (TTSuV) Prevalence in Wild Fauna of Northern Italy.

Torque teno sus virus (TTSuV) is a non-enveloped circular ssDNA virus which frequently infects swine and has been associated with hepatic, respiratory, and autoimmune disorders. TTSuV's pathogenic role is still uncertain, and clear data in the literature on virus reservoirs are lacking. The aims of this study were to investigate the presence of potentially zoonotic TTSuV in wild animals in Northern Italy and to evaluate their role as reservoirs. Liver samples were collected between 2016 and 2020 during four hunting seasons from wild boars (Sus scrofa), red deer (Cervus elaphus), roe deer (Capreolus capreolus), and chamois (Rupicapra rupicapra). Samples originated from areas in Northern Italy characterized by different traits, i.e., mountains and flatland with, respectively low and high farm density and anthropization. Viral identification was carried out by end-point PCR with specific primers for TTSuV1a and TTSuVk2a species. TTSuV prevalence in wild boars was higher in the mountains than in the flatland (prevalence of 6.2% and 2.3%, respectively). In wild ruminants only TTSuVk2a was detected (with a prevalence of 9.4%). Our findings shed light on the occurrence and distribution of TTSuV in some wild animal species, investigating their possible role as reservoirs.

Absence of SARS-CoV-2 RNA and anti-SARS-CoV-2 antibodies in stray cats.

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.

Detection and Molecular Characterization of Canine Distemper Virus in Wildlife from Northern Italy.

Canine distemper virus (CDV) is a fatal, highly contagious disease found in wild and domestic carnivores. Several outbreaks have occurred in wildlife in Italy in recent years. This study aims to detect CDV in wildlife following the increasing mortality of foxes (Vulpes vulpes) in the Emilia-Romagna region (northern Italy) observed in 2021. Sixty-seven foxes and one badger (Meles meles) were subjected to necropsy followed by histological examination and were analyzed with molecular techniques to detect the presence of CDV. Of the tested animals, 16% (nine foxes and one badger) were positive for CDV. Phylogenetic analysis showed two different lineages based on complete H gene sequences. The Europe/South America-1 lineage was detected in one fox from Modena, which resembled the CDV variant associated with a previous outbreak in northern Italy in 2018, while the European Wildlife lineage was detected in animals from the Rimini province. Amino acid analysis highlighted a Y549H mutation in all sequences collected, which is commonly associated with increased virulence.

SARS-CoV-2 in a Mink Farm in Italy: Case Description, Molecular and Serological Diagnosis by Comparing Different Tests.

This study described a SARS-CoV-2 infection in minks on an Italian farm. Surveillance was performed based on clinical examination and a collection of 1879 swabs and 74 sera from dead and live animals. The farm was placed under surveillance for 4.5 months, from the end of July 2020, when a man working on the farm tested positive by RT-PCR, till mid-December 2020 when all the animals were sacrificed. Clinical examination revealed no clinical signs or increased mortality rates attributable to SARS-CoV-2, while diagnostic tests detected only four weak PCR-positive samples, but 100% of sera were positive for SARS-CoV-2 anti-S antibodies. The phylogenetic analysis of two SARS-CoV-2 sequences from two minks and the sequence of the worker showed that they belonged to different clades. It could be therefore assumed that two distinct introductions of the virus occurred on the farm, and that the first introduction probably occurred before the start of the surveillance period. From the data collected, and especially from the detection of specific antibodies through the combination of different tests, it can be postulated that syndromic surveillance combined with genome detection by PCR may not be sufficient to achieve a diagnosis in asymptomatic animals. In particular, the serological approach, especially when using tests directed towards the S protein, may be useful for improving the traceability of virus circulation in similar environments.

Mammalian Orthoreovirus (MRV) Is Widespread in Wild Ungulates of Northern Italy.

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.

Detection of Zoonotic Cryptosporidium ubiquitum in Alpine Wild Ruminants.

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.

Canine Distemper Outbreaks in Wild Carnivores in Northern Italy.

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 2006-2010, while clade b was closely related to the CDV sequences originating from northeastern Italy during the 2011-2018 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.