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The aim of the study was to estimate the diagnostic sensitivity (DSe) and specificity (DSp) of an agar gel immunodiffusion (AGID) assay for detection of myxoma virus (MYXV) in the classical form of myxomatosis and to compare its diagnostic performance to that of molecular methods (IAC-PCR, OIE PCR, and OIE real-time PCR).

Issyk-Kul virus (ISKV), family Nairoviridae, was first isolated in 1970 from a noctule bat (Nyctalus noctule) trapped near Lake Issyk-Kul, Kyrgyzstan (1). ISKV was subsequently detected in bats of several [...]

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.

Abstract Background In Italy, malaria was endemic until the 1970s, when it was declared eradicated by WHO. Nowadays, with the persistence of competent mosquito populations, the effect of climate change, and increased possibility of importing malaria parasites from endemic counties due to growing migration, a malaria resurgence in Italy has become more likely. Hence, enhancing the understanding of the current distribution of the Anopheles maculipennis complex and the factors that influence the presence of this malaria vector is crucial, especially in Northern Italy, characterised by a high density of both human population and livestock. Methods To assess the presence and abundance of malaria vectors, a 4-year field survey in the plain areas of Lombardy and Emilia-Romagna region in Italy was conducted. Every sampling point was characterised in space by the land use in a 500-m radius and in time considering meteorological data collected in the short and long time periods before sampling. We combined the results of a linear regression model with a random forest analysis to understand the relative importance of the investigated niche dimensions in determining Anopheles mosquito presence and abundance. Results The estimated normalised variable importance indicates that rice fields were the most important land use class explaining the presence of Anopheles, followed by transitional woodlands and shrubland. Farm buildings were the third variable in terms of importance, likely because of the presence of animal shelters, followed by urbanised land. The two most important meteorological variables influencing the abundance of Anopheles in our study area were mean temperature in the 24 h before the sampling date and the sum of degree-days with temperature between 18 °C and 30 °C in the 14 days before the sampling date. Conclusions The results obtained in this study could be helpful in predicting the risk of autochthonous malaria transmission, based on local information on land cover classes that might facilitate the presence of malaria vectors and presence of short- and medium-term meteorological conditions favourable to mosquito development and activity. The results can support the design of vector control measures through environmental management. Graphical Abstract

Functional size measures are often used as the basis for estimating development effort, because they are available in the early stages of software development. Several simplified measurement methods have also been proposed, both to decrease the cost of measurement and to make functional size measurement applicable when functional user requirements are not yet known in full detail. Lately, machine learning techniques have been successfully used for software development effort estimation, but the usage of machine learning techniques in combination with simplified functional size measures has not yet been empirically evaluated. This paper aims to fill this gap: it reports to what extent functional size measures can be simplified, without decreasing the accuracy of effort estimates obtained via machine learning techniques. The reported evaluation addresses separately the effort models concerning (i) new software developed from scratch, (ii) software extensions obtained by adding new functionality, and (iii) software modifications that required also changing and possibly removing functionalities. We carried out an empirical study, in which effort estimation models were built via multiple Machine Learning techniques, using both traditional full-fledged functional size measures and simplified measures. Our study shows that using simplified functional size measures in place of traditional functional size measures for effort estimation does not yield practically relevant differences in accuracy. Therefore, software project managers can consider analyzing only a small and specific part of functional user requirements to get measures that effectively support effort estimation.

Abstract 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.

Abstract Background Mucosal Melanomas (MM) are highly aggressive neoplasms arising from mucosal melanocytes. Current treatments offer a limited survival benefit for patients with advanced MM; moreover, the lack of pre-clinical cellular systems has significantly limited the understanding of their immunobiology. Methods Five novel cell lines were obtained from patient-derived biopsies of MM arising in the sino-nasal mucosa and designated as SN-MM1-5. The morphology, ultrastructure and melanocytic identity of SN-MM cell lines were validated by transmission electron microscopy and immunohistochemistry. Moreover, in vivo tumorigenicity of SN-MM1-5 was tested by subcutaneous injection in NOD/SCID mice. Molecular characterization of SN-MM cell lines was performed by a mass-spectrometry proteomic approach, and their sensitivity to PI3K chemical inhibitor LY294002 was validated by Akt activation, measured by pAkt(Ser473) and pAkt(Thr308) in immunoblots, and MTS assay. Results This study reports the validation and functional characterization of five newly generated SN-MM cell lines. Compared to the normal counterpart, the proteomic profile of SN-MM is consistent with transformed melanocytes showing a heterogeneous degree of melanocytic differentiation and activation of cancer-related pathways. All SN-MM cell lines resulted tumorigenic in vivo and display recurrent structural variants according to aCGH analysis. Of relevance, the microscopic analysis of the corresponding xenotransplants allowed the identification of clusters of MITF-/CDH1-/CDH2 + /ZEB1 + /CD271 + cells, supporting the existence of melanoma-initiating cells also in MM, as confirmed in clinical samples. In vitro, SN-MM cell lines were sensitive to cisplatin, but not to temozolomide. Moreover, the proteomic analysis of SN-MM cell lines revealed that RICTOR, a subunit of mTORC2 complex, is the most significantly activated upstream regulator, suggesting a relevant role for the PI3K-Akt-mTOR pathway in these neoplasms. Consistently, phosphorylation of NDRG1 and Akt activation was observed in SN-MM, the latter being constitutive and sustained by PTEN loss in SN-MM2 and SN-MM3. The cell viability impairment induced by LY294002 confirmed a functional role for the PI3K-Akt-mTOR pathway in SN-MM cell lines. Conclusions Overall, these novel and unique cellular systems represent relevant experimental tools for a better understanding of the biology of these neoplasms and, as an extension, to MM from other sites.

Rabbit haemorrhagic disease virus type 2 (RHDV2) causes a severe systemic disease with hepatic necrosis. Differently from classic RHDV, which affects only European rabbits (Oryctolagus cuniculus), RHDV2 can affect many leporid species, including hares (Lepus spp.) and cottontail rabbits (Sylvilagus spp.). RHDV2 emerged in Europe in 2010 and spread worldwide. During the last 5 years, there have been multiple outbreaks in North America since the first known event in 2016 in Quebec, Canada, including several detections in British Columbia, Canada, between 2018 and 2019, Washington State and Ohio, USA, in 2018 and 2019, and New York, USA, in 2020. However, the most widespread outbreak commenced in March 2020 in the southwestern USA and Mexico. In California, RHDV2 spread widely across several southern counties between 2020 and 2021, and the aim of this study was to report and characterize these early events of viral incursion and circulation within the state. Domestic and wild lagomorphs (n = 81) collected between August 2020 and February 2021 in California with a suspicion of RHDV2 infection were tested by reverse transcription quantitative real-time PCR on the liver, and histology and immunohistochemistry for pan-lagovirus were performed on liver sections. In addition, whole genome sequencing from 12 cases was performed. During this period, 33/81 lagomorphs including 24/59 domestic rabbits (O. cuniculus), 3/16 desert cottontail rabbits (Sylvilagus audubonii), and 6/6 black-tailed jackrabbits (Lepus californicus) tested positive. All RHDV2-positive animals had hepatic necrosis typical of pathogenic lagovirus infection, and the antigen was detected in sections from individuals of the three species. The 12 California sequences were closely related (98.9%-99.95%) to each other, and also very similar (99.0%-99.4%) to sequences obtained in other southwestern states during the 2020-2021 outbreak; however, they were less similar to strains obtained in New York in 2020 (96.7%-96.9%) and Quebec in 2016 (92.4%-92.6%), suggesting that those events could be related to different viral incursions. The California sequences were more similar (98.6%-98.7%) to a strain collected in British Columbia in 2018, which suggests that that event could have been related to the 2020 outbreak in the southwestern USA.

The introduction of new technologies in current digestive surgical practice is progressively reshaping the operating room, defining the fourth surgical revolution. The implementation of black boxes and control towers aims at streamlining workflow and reducing surgical error by early identification and analysis, while augmented reality and artificial intelligence augment surgeons’ perceptual and technical skills by superimposing three-dimensional models to real-time surgical images. Moreover, the operating room architecture is transitioning toward an integrated digital environment to improve efficiency and, ultimately, patients’ outcomes. This narrative review describes the most recent evidence regarding the role of these technologies in transforming the current digestive surgical practice, underlining their potential benefits and drawbacks in terms of efficiency and patients’ outcomes, as an attempt to foresee the digestive surgical practice of tomorrow.

Background Recently, mesenchymal stromal cells (MSCs) have gained recognition for their clinical utility in transplantation to induce tolerance and to improve/replace pharmacological immunosuppression. Cord blood (CB)-derived MSCs are particularly attractive for their immunological naivety and peculiar anti-inflammatory and anti-apoptotic properties. Objectives: The objective of this study was to obtain an inventory of CB MSCs able to support large-scale advanced therapy medicinal product (ATMP)-based clinical trials. Study design: We isolated MSCs by plastic adherence in a GMP-compliant culture system. We established a well-characterized master cell bank and expanded a working cell bank to generate batches of finished MSC(CB) products certified for clinical use. The MSC(CB) produced by our facility was used in approved clinical trials or for therapeutic use, following single-patient authorization as an immune-suppressant agent. Results: We show the feasibility of a well-defined MSC manufacturing process and describe the main indications for which the MSCs were employed. We delve into a regulatory framework governing advanced therapy medicinal products (ATMPs), emphasizing the need of stringent quality control and safety assessments. From March 2012 to June 2023, 263 of our Good Manufacturing Practice (GMP)-certified MSC(CB) preparations were administered as ATMPs in 40 subjects affected by Graft-vs.-Host Disease, nephrotic syndrome, or bronco-pulmonary dysplasia of the newborn. There was no infusion-related adverse event. No patient experienced any grade toxicity. Encouraging preliminary outcome results were reported. Clinical response was registered in the majority of patients treated under therapeutic use authorization. Conclusions: Our 10 years of experience with MSC(CB) described here provides valuable insights into the use of this innovative cell product in immune-mediated diseases.

Shah and colleagues [...]

Rabbit haemorrhagic disease virus type 2 (RHDV2) causes a severe systemic disease with hepatic necrosis. Differently from classic RHDV, which affects only European rabbits (Oryctolagus cuniculus), RHDV2 can affect many leporid species, including hares (Lepus spp.) and cottontail rabbits (Sylvilagus spp.). RHDV2 emerged in Europe in 2010 and spread worldwide. During the last 5 years, there have been multiple outbreaks in North America since the first known event in 2016 in Quebec, Canada, including several detections in British Columbia, Canada, between 2018 and 2019, Washington State and Ohio, USA, in 2018 and 2019, and New York, USA, in 2020. However, the most widespread outbreak commenced in March 2020 in the southwestern USA and Mexico. In California, RHDV2 spread widely across several southern counties between 2020 and 2021, and the aim of this study was to report and characterize these early events of viral incursion and circulation within the state. Domestic and wild lagomorphs (n = 81) collected between August 2020 and February 2021 in California with a suspicion of RHDV2 infection were tested by reverse transcription quantitative real-time PCR on the liver, and histology and immunohistochemistry for pan-lagovirus were performed on liver sections. In addition, whole genome sequencing from 12 cases was performed. During this period, 33/81 lagomorphs including 24/59 domestic rabbits (O. cuniculus), 3/16 desert cottontail rabbits (Sylvilagus audubonii), and 6/6 black-tailed jackrabbits (Lepus californicus) tested positive. All RHDV2-positive animals had hepatic necrosis typical of pathogenic lagovirus infection, and the antigen was detected in sections from individuals of the three species. The 12 California sequences were closely related (98.9%-99.95%) to each other, and also very similar (99.0%-99.4%) to sequences obtained in other southwestern states during the 2020-2021 outbreak; however, they were less similar to strains obtained in New York in 2020 (96.7%-96.9%) and Quebec in 2016 (92.4%-92.6%), suggesting that those events could be related to different viral incursions. The California sequences were more similar (98.6%-98.7%) to a strain collected in British Columbia in 2018, which suggests that that event could have been related to the 2020 outbreak in the southwestern USA.

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.

ABSTRACT The bacterium Treponema paraluisleporidarum causes syphilis in lagomorphs. In a set of 1,095 samples from four species—European brown hare, mountain hare, Corsican hare, and European rabbit—we tested for infection and 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 that 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 whole genome data 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 widespread infection and a high genetic variation of the syphilis-causing pathogen in a higher number of positively PCR-tested European lagomorphs (n = 302/1,095). 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.

Abstract A fatal Encephalomyocarditis virus (EMCV) infection epidemic involving fifteen primates occurred between October 2006 and February 2007 at the Natura Viva Zoo. This large open-field zoo park located near Lake Garda in Northern Italy hosts one thousand animals belonging to one hundred and fifty different species, including various lemur species. This lemur collection is the most relevant and rich in Italy. A second outbreak between September and November 2008 involved three lemurs. In all cases, the clinical signs were sudden deaths generally without any evident symptoms or only with mild unspecific clinical signs. Gross pathologic changes were characterized by myocarditis (diffuse or focal pallor of the myocardium), pulmonary congestion, emphysema, oedema and thoracic fluid. The EMCV was isolated and recognized as the causative agent of both outbreaks. The first outbreak in particular was associated with a rodent plague, confirming that rats are an important risk factor for the occurrence of the EMCV infection.

Over the last few decades, several pestiviruses have been discovered in ruminants, pigs, and, more recently, in non-ungulate hosts. Consequently, the nomenclature and taxonomy of pestiviruses have been updated. The Tunisian sheep-like pestivirus (TSV, Pestivirus N) is an additional ovine pestivirus genetically closely related to classical swine fever virus (CSFV). In this study, during a survey of pestivirus infections in ovine farms in the Lombardy region of Northern Italy, we identified and isolated a pestivirus strain from a sheep that was found to belong to Pestivirus N species based on its genomic nucleotide identity. The sheep itself and its lamb were found to be persistently infected. We performed molecular characterization and phylogenetic analysis of three viral genomic regions (a fragment of 5′-UTR, partial Npro, and the whole E2 region). In conclusion, these results confirmed circulating TSV in Northern Italy after notification in Sicily, Italy, and France. Correlation with Italian, Tunisian, and French strains showed that detection might have resulted from the trading of live animals between countries, which supports the need for health control measures.

African swine fever (ASF) is a severe viral disease characterized by high lethality in suids and caused by the African Swine Fever Virus (ASFV). The ASF genotype I virus was introduced to Europe in 1957, marking the onset of the first European epidemic wave. In 2007, ASFV genotype II was detected in Georgia, affecting domestic pigs and wild boars before spreading to various European and extra-European countries, including Italy. The first case of ASFV in Italy was documented on 7 January 2022, in a wild boar in the Piedmont region. Since then, several ASFV-positive wild boar carcasses have been identified in the Piedmont and Liguria regions. By June 2023, ASFV had spread to Lombardy, one of the major pig-producing regions in northern Italy; the virus was first detected in early summer in wild boar carcasses. Two months later, it was diagnosed in a commercial pig farm as a consequence of the disease’s spread amongst wild boars and an increase in the viral environmental load. This report aims to describe the features of ASFV domestic pig outbreaks that occurred in the Zinasco municipality (Lombardy) and the joint efforts to mitigate potential direct and indirect economic impacts on the Italian and global pig industry. The epidemiological investigation and the measures implemented, which were all performed according to national and European regulations, as well as exceptional ad hoc measures aimed at protecting the pig industry, are described in order to provide a practical and effective approach to combating ASF.