Wild ungulates as sentinels of flaviviruses and tick-borne zoonotic pathogen circulation: an Italian perspective.

BACKGROUND: Vector-borne zoonotic diseases are a concerning issue in Europe. Lyme disease and tick-borne encephalitis virus (TBEV) have been reported in several countries with a large impact on public health; other emerging pathogens, such as Rickettsiales, and mosquito-borne flaviviruses have been increasingly reported. All these pathogens are linked to wild ungulates playing roles as tick feeders, spreaders, and sentinels for pathogen circulation. This study evaluated the prevalence of TBEV, Borrelia burgdorferi sensu lato, Rickettsia spp., Ehrlichia spp., and Coxiella spp. by biomolecular screening of blood samples and ticks collected from wild ungulates. Ungulates were also screened by ELISA and virus neutralization tests for flaviviral antibody detection. RESULTS: A total of 274 blood samples were collected from several wild ungulate species, as well as 406 Ixodes ricinus, which were feeding on them. Blood samples tested positive for B. burgdorferi s.l. (1.1%; 0-2.3%) and Rickettsia spp. (1.1%; 0-2.3%) and showed an overall flaviviral seroprevalence of 30.6% (22.1-39.2%): 26.1% (17.9-34.3%) for TBEV, 3.6% (0.1-7.1%) for Usutu virus and 0.9% (0-2.7%) for West Nile virus. Ticks were pooled when possible and yielded 331 tick samples that tested positive for B. burgdorferi s.l. (8.8%; 5.8-11.8%), Rickettsia spp. (26.6%; 21.8-31.2%) and Neoehrlichia mikurensis (1.2%; 0-2.4%). TBEV and Coxiella spp. were not detected in either blood or tick samples. CONCLUSIONS: This research highlighted a high prevalence of several tick-borne zoonotic pathogens and high seroprevalence for flaviviruses in both hilly and alpine areas. For the first time, an alpine chamois tested positive for anti-TBEV antibodies. Ungulate species are of particular interest due to their sentinel role in flavivirus circulation and their indirect role in tick-borne diseases and maintenance as Ixodes feeders and spreaders.

Use of cigarettes and heated tobacco products during pregnancy and maternal-fetal outcomes: a retrospective, monocentric study.

PURPOSE: To compare the effects of using heated tobacco products (HTP) or traditional cigarettes (C) on maternal and neonatal outcomes. METHODS: This is a retrospective, monocentric study conducted at San Marco Hospital from July 2021 to July 2022. We compared a cohort of pregnant patients who smoked HTP (HS), with pregnant women smoking cigarettes (CS), ex-smoker (ES) and non-smoker (NS) pregnant women. Biochemistry, ultrasound, and neonatal evaluations were performed. RESULTS: In total, 642 women were enrolled, of which 270 were NS, 114 were ES, 120 were CS, and 138 were HS. CS had the greatest weight gain and had more difficulty getting pregnant. Smokers and ES experienced more frequently threats of preterm labor, miscarriages, temporary hypertensive spikes, and higher rates of cesarean sections. Preterm delivery was more associated with CS and HS groups. CS and HS had lower awareness of the risks to which the mother and the fetus are exposed. CS were more likely to be depressed and anxious. Biochemical parameters did not show significant differences between the groups. CS had the greatest difference in days between the gestational age calculated based on the last menstrual period and the one based on the actual ultrasound age. The average percentile newborn weight range of CS was lower, as well as the mean 1st minute and the 5th minute Apgar scores. CONCLUSION: The comparison of the data obtained between CS and HS underlines the greater danger of C. Nevertheless, we do not recommend HTP because the maternal-fetal outcomes are not superimposable to the NS outcomes.

SARS-CoV-2 and Swabs: Disease Severity and the Numbers of Cycles of Gene Amplification, Single Center Experience.

Pediatric COVID-19 determines a mild clinical picture, but few data have been published about the correlation between disease severity and PCR amplification cycles of SARS-CoV-2 from respiratory samples. This correlation is clinically important because it permits the stratification of patients in relation to their risk of developing a serious disease. Therefore, the primary endpoint of this study was to establish whether disease severity at the onset, when evaluated with a LqSOFA score, correlated with the gene amplification of SARS-CoV-2. LqSOFA score, also named the Liverpool quick Sequential Organ Failure Assessment, is a pediatric score that indicates the severity of illness with a range from 0 to 4 that incorporates age-adjusted heart rate, respiratory rate, capillary refill and consciousness level (AVPU). The secondary endpoint was to determine if this score could predict the days of duration for symptoms and positive swabs. Our study included 124 patients aged between 0 and 18 years. The LqSOFA score was negatively correlated with the number of PCR amplification cycles, but this was not significant (Pearson's index -0.14, p-value 0.13). Instead, the correlation between the LqSOFA score and the duration of symptoms was positively related and statistically significant (Pearson's index 0.20, p-value 0.02), such as the correlation between the LqSOFA score and the duration of a positive swab (Pearson's index 0.40, p-value < 0.01). So, the LqSOFA score upon admission may predict the duration of symptoms and positive swabs; the PCR amplification of SARS-CoV-2 appears not to play a key role at onset in the prediction of disease severity.

Into the backyard: Multiple detections of PCV-2e in rural pig farms of Northern Italy. An unexpected ecological niche?

Porcine circovirus type 2 (PCV-2) is among the most burdensome viruses of the swine industry globally. Several genotypes have been periodically emerging, but just three of them (PCV-2a, PCV-2b, and PCV-2d) seem to circulate worldwide and be associated with the disease. Conversely, the spatial-temporal distribution of minor genotypes appears limited and their clinical relevance is still unclear. Recently PCV-2e was incidentally detected for the first time in Europe in a breeding farm in Northeastern Italy, while no connection could be established with countries where this genotype had been previously detected. To investigate circulating genotypes in the neglected rural context and provide a comparison with the most explored industrial context, a molecular survey was performed on samples collected in rural (n = 72) and industrial farms (n = 110) located in the same geographic area. Phylogenetic analysis surprisingly evidenced PCV-2e circulation only in pigs reared in backyard farms (n = 5), while major genotypes (PCV-2a, -2b, -2d) circulate in both rearing contexts. However, the close genetic similarity between the herein detected PCV-2e strains and the previously reported one testify that, although unusual, such rural-to-industrial strains exchange affected also PCV-2e. The greater genetic and phenotypic diversity of PCV-2e genotype compared to other ones might threaten the protection granted by current vaccines. The present study suggests the rural context as an ecological niche for the circulation of PCV-2e, and even of other minor genotypes. PCV-2e detection in pigs with outdoor access further stresses the epidemiological role of backyard farms as interfaces for pathogen introduction, potentially ascribable to the different rearing approaches, lower managerial and biosecurity capabilities, and easier contacts with wildlife.

Effective Methods of Estimation of Pathogen Prevalence in Pooled Ticks.

Since tick-borne diseases (TBDs) incidence, both in human and animal populations, is increasing worldwide, there is the need to assess the presence, distribution and prevalence of tick-borne pathogens. Reliable estimates on tick-borne pathogens (TBPs) prevalence represent the public health foundation to create risk maps and take effective prevention and control actions against TBDs. Tick surveillance consists of collecting and testing (usually in pools) thousands of specimens. Construction and analysis of tick pools represent a challenge due to the complexity of tick-borne pathogens and tick-borne diseases ecology. The aim of this study is to provide a practical guideline on appropriate pooling strategies and statistical analysis of infection prevalence through: (i) reporting the different pooling strategies and statistical methodologies commonly used to calculate pathogen prevalence in tick populations and (ii) practical comparison between statistical methods utilising a real dataset of infection prevalence in ticks collected in Northern Italy. Reporting detailed information on tick pool composition and size is as important as the correct TBPs prevalence estimation. Among the prevalence indexes, we suggest using maximum-likelihood estimates of pooled prevalence instead of minimum infection rate or pool positivity rate given the merits of the method and availability of software.

An overview of Fintech applications to solve the puzzle of health care funding: state-of-the-art in medical crowdfunding.

Crowdfunding is emerging as an alternative form of funding for medical purposes, with capital being raised directly from a broader and more diverse audience of investors. In this paper, we have systematically researched and reviewed the literature on medical crowdfunding to determine how crowdfunding connects with the health care industry. The health care industry has been struggling to develop sustainable research and business models for economic systems and investors alike, especially in pharmaceuticals. The research results have revealed a wealth of evidence concerning the way crowdfunding is applied in real life. Patients and caregivers utilize web platform-based campaigns all over the world to fund their medical expenses, generally on a spot basis, using donation-based or even reward-based schemes, regardless of the health care system archetype (public, private insurance-based or hybrid). Academics have also focused on funding campaigns and the predictors of success (which range from social behaviour and environment to the basic demographics of the campaigners and their diseases) and on social and regulatory concerns, including heightened social inequality and stigma. While equity crowdfunding is disrupting the way many ventures/businesses seek capital in the market, our research indicates that there are no relevant or consistent data on the practice of medical equity crowdfunding in health care, apart from a few anecdotal cases.

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.

High Prevalence of Tick-Borne Zoonotic Rickettsia slovaca in Ticks from Wild Boars, Northeastern Italy.

Tick-borne rickettsiae are emerging pathogens that are becoming widespread in Europe. Rickettsiae are endemic in Italy, but epidemiological data are currently scarce. This study aimed to improve our knowledge about rickettsial infections in tick and wild boar populations. Blood and ticks were collected from 102 wild boars in 2010 and 2018. Ticks were also collected from the vegetation in the area. All of the samples were examined using real-time PCR targeting the gltA gene to detect Rickettsia DNA. Positivity was confirmed by PCR amplifying the gltA and/or ompB genes. A total of 254 ticks and 89 blood samples were analyzed. Zoonotic rickettsiae were detected in the ticks but not in the blood samples. Rickettsia slovaca (R. slovaca) was the most prevalent in ticks and was found in 23.7% of Dermacentor marginatus (D. marginatus) and in 3.4% of Ixodes ricinus (I. ricinus). Other zoonotic species were identified, such as Rickettsia monacensis, which was detected in 12% of I. ricinus ticks, and Rickettsia helvetica which was found in 3.4% of questing I. ricinus ticks and in 1.1% of D. marginatus collected from wild boars. This study highlights a high prevalence of zoonotic rickettsiae, particularly that of R. slovaca, in northeastern Italy. As rickettsioses are underreported and underdiagnosed in human medicine, both clinicians and researchers should pay more attention to this topic.

Detection and Molecular Characterization of a Novel Species of Circovirus in a Tawny Owl (Strix aluco) in Southern Italy.

Thanks to recent developments in molecular methods, many new species have been discovered within the genus Circovirus, which comprises viruses of veterinary relevance found in a broad range of hosts. In particular, several circoviruses are known to infect birds, often causing immunosuppression and feathering disorders. Nonetheless, nothing is known about their circulation in birds of prey. In this study, samples from 61 birds of prey representing ten different species, recovered by a wildlife rescue centre in Southern Italy, were taken at necropsy and analysed by PCR with pan-Circovirus primers. Only one sample, collected from a tawny owl (Strix aluco), tested positive. Its genome, sequenced by primer walking, displays the typical features of circoviruses. Based on demarcation criteria, the detected strain qualifies as a novel species, which was named "tawny owl-associated circovirus" (ToCV). Phylogenetically, ToCV clustered with mammalian rather than avian circoviruses, and its closeness to a rodent circovirus suggests that its host may have been a micromammal eaten by the tawny owl. On the other hand, its detection in the spleen fits with the tropism of other avian circoviruses. Little can be therefore said on its biology and pathogenicity, and further efforts are needed to better characterize its epidemiology.

Examining insurance companies' use of technology for innovation.

The insurance industry is innovating. Business models, services and processes are rapidly evolving, largely backed by technological developments. The particular historical context of COVID-19 provides a suitable case to understand the relevance of exploiting technology to react quickly to traditional and emerging risks. Focusing on the initiatives put in place by the most influential insurance companies at the global level, we have framed the innovation mechanisms in the industry, highlighting four rationales underpinning these initiatives (Adaption, Expansion, Reaction and Aggression), which differ according to the relevance of the technology in use and innovation to the portfolio of risks covered. Overall, it emerges that insurance companies have the room and capability to innovate, in many cases using technological applications to cover new and existing risks. While the initiatives studied concern the entire value chain, basic primary activities, such as product development, sales and claims management, show that innovation based on new or existing technology determines the success and competitiveness of the business.

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.

Impact of viral features, host jumps and phylogeography on the rapid evolution of Aleutian mink disease virus (AMDV).

Aleutian mink disease virus (AMDV) is one the most relevant pathogens of domestic mink, where it can cause significant economic losses, and wild species, which are considered a threat to mink farms. Despite their relevance, many aspects of the origin, evolution, and geographic and host spreading patterns of AMDV have never been investigated on a global scale using a comprehensive biostatistical approach. The present study, benefitting from a large dataset of sequences collected worldwide and several phylodynamic-based approaches, demonstrates the ancient origin of AMDV and its broad, unconstrained circulation from the initial intercontinental spread to the massive among-country circulation, especially within Europe, combined with local persistence and evolution. Clear expansion of the viral population size occurred over time until more effective control measures started to be applied. The role of frequent changes in epidemiological niches, including different hosts, in driving the high nucleotide and amino acid evolutionary rates was also explored by comparing the strengths of selective pressures acting on different populations. The obtained results suggest that the viral passage among locations and between wild and domesticated animals poses a double threat to farm profitability and animal welfare and health, which is particularly relevant for endangered species. Therefore, further efforts must be made to limit viral circulation and to refine our knowledge of factors enhancing AMDV spread, particularly at the wild-domestic interface.

Exposure to Major Vector-Borne Diseases in Dogs Subjected to Different Preventative Regimens in Endemic Areas of Italy.

Vector-borne diseases (VBDs) are globally widespread arthropod-transmitted diseases with a significant impact on animal and human health. Many drivers have recently spurred the geographic spread of VBDs in dogs. This study has evaluated the exposure to most important VBDs in dogs under different preventative treatments in different regions of Italy, i.e., Veneto, Friuli Venezia-Giulia, Umbria, Giglio Island (Tuscany), Abruzzo and Latium. Serological analyses were performed to detect antibodies against Leishmania infantum, Babesia canis, Anaplasma phagocytophilum/Anaplasma platys, Ehrlichia canis/Ehrlichia ewingii, Borrelia burgdorferi, Rickettsia conorii and the circulating antigen of Dirofilaria immitis. Dogs were categorized according to the treatment schedule usually received, and the association between seropositivity and possible risk factors was statistically evaluated. Overall, 124/242 (51.2%) dogs tested positive for at least one pathogen, while 34 (14.0%) were exposed to two or more pathogens. The most detected seropositivity was against R. conorii, followed by Anaplasma spp., L. infantum, B. canis, and the other pathogens under study. Significant statistical associations were found according to geographical provenance, history of tick infestation, lifestyle and inadequate prophylactic treatments. Random/irregular treatments have been identified as a clear risk factor. These results show that adequate prophylactic treatment protocols are overlooked by dog owners, despite the availability of several effective products, with possible implications in veterinary medicine and on public health.

Imaging a unicuspid aortic valve with transillumination.

Unicuspid aortic valve (UAV) is a rare congenital malformation which portends an augmented risk of early valve degeneration. Timely detection of this cardiac valvular anomaly is crucial because a strict follow-up is warranted. Currently, the best morphological information is provided by two-dimensional echocardiography; however, its diagnostic performance has been found to be suboptimal by some anatomical features, making it tough to distinguish between UAV and bicuspid aortic valve. Transillumination is a photo-realism technique that employs the use of a virtual light source that simulates the interaction of light on 3-dimensional surfaces, improving the visualization of morphological characteristics. Our report highlights the incremental value of photo-realistic rendering and lighting source technology to better define the aortic valve morphology.

Ecotyping of Anaplasma phagocytophilum from Wild Ungulates and Ticks Shows Circulation of Zoonotic Strains in Northeastern Italy.

Anaplasma phagocytophilum (A. phagocytophilum) is a tick-borne pathogen causing disease in both humans and animals. Human granulocytic anaplasmosis (HGA) is an emerging disease, but despite the remarkable prevalence in European ticks and wild animals, human infection appears underdiagnosed. Several genetic variants are circulating in Europe, including the zoonotic ecotype I. This study investigated A. phagocytophilum occurrence in wild ungulates and their ectoparasites in an area where HGA has been reported. Blood samples from wild ungulates and ectoparasites were screened by biomolecular methods targeting the mps2 gene. The groEL gene was amplified and sequenced to perform genetic characterization and phylogenetic analysis. A total of 188 blood samples were collected from different wild ungulates species showing an overall prevalence of 63.8% (88.7% in wild ruminants and 3.6% in wild boars). The prevalence of A. phagocytophilum DNA in ticks (manly Ixodes ricinus), and keds collected from wild ruminants was high, reflecting the high infection rates obtained in their hosts. Among ticks collected from wild boars (Hyalomma marginatum and Dermacentor marginatus) no DNA was detected. Phylogenetic analysis demonstrated the presence of ecotype I and II. To date, this is the first Italian report of ecotype I in alpine chamois, mouflon, and wild boar species. These findings suggest their role in HGA epidemiology, and the high prevalence detected in this study highlights that this human tick-borne disease deserves further attention.

Porcine Gammaherpesviruses in Italian Commercial Swine Population: Frequent but Harmless.

Differently from alpha- and betaherpesviruses affecting swine, interest in the recently discovered Suid gammaherpesvirus 3, Suid gammaherpesvirus 4, and Suid gammaherpesvirus 5, also known as porcine lymphotropic herpesviruses (PLHV-1, PLHV-2, and PLHV-3), has largely focused on their role as potential zoonotic agents in cases of xenotransplantation. However, their role as primary pathogens of swine or as co-factors for other lymphotropic infections has essentially been neglected. The present study aims at filling this gap, evaluating the association between PLHVs infection and different clinical conditions and/or porcine circovirus (PCV) co-infection. One hundred seventy-six samples were obtained from different animals located in a high-density pig area of Northern Italy in the period 2017-2020. The presence of PLHVs and PCVs was tested and quantified by specific real-time PCR: PLHVs were widespread among pigs (PLHV-1, PLHV-2, and PLHV-3 prevalence was 28.97%, 10.79%, and 4.54%, respectively) and detected in all considered tissues and clinical conditions. Frequent co-infections were also observed among PLHVs and with PCVs, although a significant association was not detected with the exception of a positive interaction between PLHV-1 and PLHV-3, and a negative one between PLHV-2 and PCV-2. Significantly, no association between PLHVs, alone or in co-infection, emerged with any of the considered clinical signs, their frequency being comparable between healthy and diseased animals. Based on these pieces of evidence and despite their high prevalence, PLHVs' relevance for the swine industry appears negligible, either as primary pathogens or as predisposing factors for circovirus-induced diseases.

Bovine Coronavirus: Variability, Evolution, and Dispersal Patterns of a No Longer Neglected Betacoronavirus.

Bovine coronavirus (BoCV) is an important pathogen of cattle, causing severe enteric disease and playing a role in the bovine respiratory disease complex. Similar to other coronaviruses, a remarkable variability characterizes both its genome and biology. Despite their potential relevance, different aspects of the evolution of BoCV remain elusive. The present study reconstructs the history and evolution of BoCV using a phylodynamic approach based on complete genome and spike protein sequences. The results demonstrate high mutation and recombination rates affecting different parts of the viral genome. In the spike gene, this variability undergoes significant selective pressures-particularly episodic pressure-located mainly on the protein surface, suggesting an immune-induced selective pressure. The occurrence of compensatory mutations was also identified. On the contrary, no strong evidence in favor of host and/or tissue tropism affecting viral evolution has been proven. The well-known plasticity is thus ascribable to the innate broad viral tropism rather than mid- or long-term adaptation. The evaluation of the geographic spreading pattern clearly evidenced two clusters: a European cluster and an American-Asian cluster. While a relatively dense and quick migration network was identified in the former, the latter was dominated by the primary role of the United States (US) as a viral exportation source. Since the viral spreading pattern strongly mirrored the cattle trade, the need for more intense monitoring and preventive measures cannot be underestimated as well as the need to enforce the vaccination of young animals before international trade, to reduce not only the clinical impact but also the transferal and mixing of BoCV strains.

Lack of Evidence on the Susceptibility of Ticks and Wild Rodent Species to PCV3 Infection.

Porcine circovirus 3 (PCV3) is an emerging virus, first detected in 2016 and widespread in the swine industry. Although not considered a primary pathogen, PCV3 is potentially linked to several clinical conditions that threaten swine farming. Wild boars are considered the main reservoir species for PCV3 infection in the wild, but recent detection in roe deer, chamois and associated ticks has complicated our understanding of its epidemiology. Much emphasis has been placed on ticks, as competent vectors, and wild rodents, which typically feed immature tick stages. The aim of this study was to clarify whether wild rodent species and associated ticks are susceptible to PCV3 infection and involved in its spread. Wild small mammals' serum samples and hosted ticks were, therefore, collected from areas where no wild boars were present and tested by PCR, targeting the PCV3 rep gene. A total of 90 yellow-necked mice (Apodemus flavicollis), two wood mice (A. sylvaticus), 26 bank voles (Myodes glareolus) and 262 Ixodes spp. ticks were investigated. PCV3 DNA was not detected in serum or in tick samples. These findings support the hypothesis that the investigated species do not have an actual role as PCV3 reservoirs. Further studies would be necessary to state whether these species, or others that we did not test, are involved in PCV3 infection spread-in particular when susceptible species share the same habitat.

Lack of Porcine circovirus 4 Genome Detection in Pig Samples from Italy and Spain.

The genus Circovirus includes several species and mostly causes asymptomatic infections. Porcine circovirus 2 (PCV-2) and, with increasing evidence, Porcine circovirus 3 (PCV-3), have been associated with different clinical conditions all over the world. In 2019, a new porcine circovirus (PCV-4) was identified from diseased animals in China. Because of the lessons learned from PCV-2 and PCV-3, it appears mandatory to investigate the actual distribution of this new virus and its potential association with clinical outcomes. To this purpose, an exploratory study to detect PCV-4 by molecular methods was performed in Italy and Spain by testing more than 300 samples of different types (serum and tissues), collected from both healthy and diseased pigs and wild boar as well. All samples, independently from the country, type, health status and host, tested PCV-4 negative. Therefore, no evidence of PCV-4 presence was found in Italy and Spain through this exploratory study. Considering the dense pig trade among European countries, its presence in the continent can similarly be considered unlikely. The reasons behind the restricted PCV-4 distribution compared to other porcine circoviruses will require further investigations. Careful surveillance might nevertheless be important since prompt recognition of PCV-4 would allow the implementation of effective countermeasures to prevent its spreading and potential economic losses.