First evidence of widespread positivity to anticoagulant rodenticides in grey wolves (Canis lupus).

Second-generation Anticoagulant Rodenticides (ARs) can be critical for carnivores, due to their widespread use and impacts. However, although many studies explored the impacts of ARs on small and mesocarnivores, none assessed the extent to which they could contaminate large carnivores in anthropized landscapes. We filled this gap by exploring spatiotemporal trends in grey wolf (Canis lupus) exposure to ARs in central and northern Italy, by subjecting a large sample of dead wolves (n = 186) to the LC-MS/MS method. Most wolves (n = 115/186, 61.8 %) tested positive for ARs (1 compound, n = 36; 2 compounds, n = 47; 3 compounds, n = 16; 4 or more compounds, n = 16). Bromadiolone, brodifacoum and difenacoum, were the most common compounds, with brodifacoum and bromadiolone being the ARs that co-occurred the most (n = 61). Both the probability of testing positive for multiple ARs and the concentration of brodifacoum, and bromadiolone in the liver, systematically increased in wolves that were found at more anthropized sites. Moreover, wolves became more likely to test positive for ARs through time, particularly after 2020. Our results underline that rodent control, based on ARs, increases the risks of unintentional poisoning of non-target wildlife. However, this risk does not only involve small and mesocarnivores, but also large carnivores at the top of the food chain, such as wolves. Therefore, rodent control is adding one further conservation threat to endangered large carnivores in anthropized landscapes of Europe, whose severity could increase over time and be far higher than previously thought. Large-scale monitoring schemes for ARs in European large carnivores should be devised as soon as possible.

Eleven Years of Health Monitoring in Wild Boars (Sus scrofa) in the Emilia-Romagna Region (Italy).

In recent years, the growth of wild ungulates has increased the focus on their health monitoring. In particular, the health status of wild boars is relevant for the economic impact on the pig industry. The Emilia-Romagna region activated a wildlife monitoring plan to better evaluate the health status of the wild boar population. Between 2011 and 2021, samples of found dead and hunted wild boar have been examined for trichinellosis, tuberculosis, brucellosis, african swine fever, classical swine fever, Aujeszky's disease, swine vesicular disease, and swine influenza A. Trichinella britovi was identified in 0.001% of the examined wild boars; neither M. bovis nor M. tuberculosis were found in M. tuberculosis complex positive samples; 2.3% were positive for Brucella suis; 29.4% of the sera were positive for Aujeszky's disease virus; and 0.9% of the samples were positive for swine influenza A virus. With an uncertain population estimate, the number of animals tested, the number of positives, and the sampling method do not allow us to make many inferences but suggest the need to implement and strengthen the existing surveillance activity, as it seems to be the only viable alternative for safeguarding animal and human health.

Antimicrobial Resistance and Virulence Factors Assessment in Escherichia coli Isolated from Swine in Italy from 2017 to 2021

Prudent antibiotic use in pigs is critical to ensuring animal health and preventing the development of critical resistance. We evaluated the antimicrobial resistance (AMR) pattern in commensal and enterotoxigenic Escherichia coli (ETEC) isolates obtained in 2017−2021 from pigs suffering from enteric disorders. Overall, the selected 826 E. coli isolates showed the highest level of resistance to ampicillin (95.9%), tetracycline (89.7%), cefazolin (79.3%), and trimethoprim/sulfamethoxazole (74.8%). The resistance rates of the isolates to ampicillin increased (p < 0.05), reaching 99.2% of resistant strains in 2021. Regarding isolates harboring virulence genes, ETEC F18+ were significantly more resistant to florfenicol, gentamicin, kanamycin, and trimethoprim/sulfamethoxazole than ETEC F4+ strains. E. coli lacking virulence factor genes were more resistant to amoxicillin with clavulanic acid and cefazolin, but less resistant to gentamicin (p < 0.01) than isolates harboring virulence factors. Throughout the study period, a significant number of ETEC F18+ isolates developed resistance to florfenicol, gentamicin, and kanamycin. Finally, ETEC 18+ significantly (p < 0.05) increased resistance to all the tested antibiotics. In conclusion, AMR varied for E. coli over time and showed high levels for molecules widely administered in the swine industry, emphasizing the need for continuous surveillance. The observed differences in AMR between commensal and ETEC isolates may lead to the hypothesis that plasmids carrying virulence genes are also responsible for AMR in E. coli, suggesting more research on genetic variation between pathogenic and nonpathogenic E. coli.

Wildlife Hosts of Leishmania infantum in a Re-Emerging Focus of Human Leishmaniasis, in Emilia-Romagna, Northeast Italy.

In the last decade, an upsurge of human leishmaniasis has been reported in the Emilia-Romagna region, Northeast Italy. Epidemiologic data have raised doubts about the role of dogs as the main reservoirs for Leishmania infantum. In the present study, a total of 1,077 wild animals were screened for L. infantum DNA in earlobe and spleen samples from 2019 to 2022. The lymph nodes were tested only in 23 animals already positive in the earlobe and/or spleen. A total of 71 (6.6%) animals resulted positive in at least one of the sampled tissues, including 3/18 (16.7%) wolves, 6/39 (15.4%) European hares, 38/309 (12.3%) roe deer, 1/11 (9.1%) red deer, 8/146 (4.9%) wild boars, 13/319 (4.1%) red foxes, 1/54 (1.9%) porcupine, and 1/59 (1.7%) European badger. Most of the infected animals (62/71) tested positive only in the earlobe tissue, only four animals (two roe deer and two wild boars) tested positive only in the spleen, and five animals (three roe deer and two red foxes) resulted positive for both tissues. L. infantum DNA was detected in the lymph nodes of 6/23 animals. L. infantum detection occurred in all seasons associated with low real-time PCR Ct values. Further research is needed in order to clarify the role of wildlife in the re-emerging focus of leishmaniasis in Northeast Italy.

Influenza A in Wild Boars: Viral Circulation in the Emilia-Romagna Region (Northern Italy) between 2017 and 2022.

A systematic surveillance against influenza A viruses (IAVs) in the Suidae population is essential, considering their role as IAV mixing vessels. However, the viral circulation in wild Sus scrofa species is poorly investigated in comparison to the knowledge of IAV infection dynamics in domestic pigs. This study investigated the circulation and the genetic diversity of wild boars' IAVs detected in the Emilia-Romagna region (2017-2022). A total of 4605 lung samples were screened via an M gene real-time RT-PCR for SwIAV; positive samples were subtyped by multiplex RT-PCR, and viral isolation was attempted. Isolated strains (3 out of the 17 positives) were fully sequenced to evaluate viral genotypic diversity. H1N1 was the most frequently detected subtype, with identification of H1pdm09N1 and H1avN1. Whole-genome phylogenetic analysis revealed SwIAVs belonging to different genotypes, with different genetic combinations, and highlighted the simultaneous circulation of the same genotypes in both pigs and wild boars, supporting the hypothesis of SwIAV spillover events at the wildlife-livestock interface. This study represents an update on the wild boar SwIAV Italian situation, and the strains' complete genome analysis showed an evolving and interesting situation that deserves further investigation.

Integrated Use of Molecular Techniques to Detect and Genetically Characterise DNA Viruses in Italian Wolves (Canis lupus italicus).

In this study, internal organs (tongue, intestine, and spleen) of 23 free-ranging Italian wolves (Canis lupus italicus) found dead between 2017 and 2019 were tested for Carnivore protoparvovirus 1, Canine adenovirus (CAdV), and Canine circovirus (CanineCV) using real-time PCR assays. Genetic characterisation of the identified viruses was carried out by amplification, sequencing, and analysis of the complete viral genome or informative viral genes. All the wolves tested positive for at least one of the DNA viruses screened, and 11/23 were coinfected. Carnivore protoparvoviruses were the most frequently detected viruses (21/23), followed by CanineCV (11/23) and CAdV (4/23). From the analysis of the partial VP2 gene of 13 carnivore protoparvoviruses, 12 were canine parvovirus type 2b, closely related to the strains detected in dogs and wild carnivores from Italy, and one was a feline panleukopenia-like virus. Of the four CAdV identified, two were CAdV-1 and two were CAdV-2. The complete genome of seven CanineCVs was sequenced and related to the CanineCV identified in dogs, wolves, and foxes worldwide. Close correlations emerged between the viruses identified in wolves and those circulating in domestic dogs. Further studies are needed to investigate if these pathogens may be potentially cross-transmitted between the two species.

Illegal fishing with electrofishing devices in the Po river basin, Emilia Romagna, Italy.

Electric fishing is an illegal hunting method, unfortunately widely used by poachers to paralyze fish and to catch many animals in a short time. In Italy, it is authorized only for scientific and conservative purposes. Between 2014 and 2018, the Ferrara section of the Experimental Zooprophylactic Institute of Lombardy and Emilia Romagna, Italy, received nine cases of potentially illegal electric fishing in Po river and its tributary rivers. Necropsies were performed following standard protocols and samples of different tissues were collected and examined using histochemical and immunohistochemical techniques. Gross lesions frequently observed were circulatory alteration phenomena (i.e. multi-organ hyperemia, hemorrhages and congestion, hemopericardium), also found histologically, in addition to multifocal degenerative and necrotic muscular processes that could be attributed to injuries from electric current, as already reported in literature. Immunohistochemical investigations confirmed degenerative and necrotic lesions with myoglobin depletion and a corresponding fibrinogen accumulation. Myoglobin globules were also detected in the renal parenchyma, as consequent of rhabdomyolysis. The results of this study allowed to correlate electric fishing to gross, histologic and immunohistochemical lesions, which together constitute a pathognomonic picture to be considered a reference standard in this type of illegal controversy.

Natural distemper infection in stone martens (Martes foina): From infection to neutralizing antibodies.

We report an outbreak of canine distemper virus (CDV) among stone martens (Martes foina) in Italy. After being rescued in Northern Italy between April and June 2018, six subjects were kept in a wildlife and exotic animal rescue center in Bologna province. Subjects have been monitored for 15 months in captivity. Within this time-lapse, two subjects died, while among the remaining four, only one showed clinical symptoms referable to distemper. Surviving subjects have been regularly tested for CDV by means of reverse transcriptase-PCR from conjunctival and oropharyngeal swabs for eleven months. The identified viruses belonged to the Wildlife-Europe CDV genetic subgroup. Neutralizing antibodies were detected at the end of the eleven months, when all subjects tested reverse transcriptase-PCR negative. Our findings confirm the circulation of the Wildlife-Europe CDV genetic subgroup (Europe 1/South America 1 lineage) within the Italian wildlife, and improve knowledge on viral infection in stone martens.

Carbapenemase IncF-borne blaNDM-5 gene in the E. coli ST167 high-risk clone from canine clinical infection, Italy.

The blaNDM-5-producing E. coli Sequence Type (ST)167 high-risk clone is emerging worldwide in human clinical cases, while its presence in companion animals is sporadic and has never been described in Italy. Using a combined Oxford Nanopore (ONT) long-reads and Illumina short-reads sequencing approach, an E. coli ST167 isolated from a hospitalized dog, was in-depth characterized by WGS and the plasmid containing blaNDM-5 was fully reconstructed. The complete sequence of the pMOL008 mosaic plasmid (F36:F31:A4:B1; pMOL008) harbouring blaNDM-5, was resolved and characterized. Moreover, a (pro)phage and IncFII, containing blaCMY-2 and ermB, and IncI2 plasmid types were also identified. pMOL008 was almost identical to blaNDM-5-containing plasmids from E. coli ST167 isolated from Italian human clinical cases and from a Swiss dog and colonized humans. blaNDM-5 was located in a class 1 integron together with aadA2, aac(3)-IIa, mph(A), sul1, tet(A) and dfrA12. The risk of spill-over and spill-back transmission of carbapenem-resistance genes, related plasmids and strains between humans and dogs, represents a Public Health threat and highlights the importance of the One Health approach for the AMR surveillance.

Effect of production process and high-pressure processing on viability of Salmonella spp. in traditional Italian dry-cured coppa.

The aim of the study was to investigate the combined effect of the manufacturing process followed by HPP treatment on the inactivation of Salmonella spp. in artificially contaminated coppa samples, in order to verify the ability of the combined processes to achieve the objective of a 5-log reduction of Salmonella spp. needed for exportation to the U.S. Fresh anatomical cuts intended for coppa production were supplied by four different delicatessen factories located in Northern Italy. Raw meat underwent experimental contamination with Salmonella spp. using a mixture of 3 strains. Surface contamination of the fresh anatomical cuts was carried out by immersion into inoculum containing Salmonella spp. The conditions of the HPP treatment were: pressure 593 MPa, time 290 seconds, water treatment temperature 14°C. Surface and deep samples were performed post contamination (T0), end of the cold phase (T1), end of process (Tend), and after HPP treatment (postHPP) and Salmonella spp. Enumerated. The results of this study show a significant reduction of Salmonella spp. all through the production process (P<0.01) for all companies, followed by an additional reduction of bacterial counts due to HPP treatment (P<0.01), both in superficial and deep contaminations (P<0.01). The superficial overall reduction resulted of 1.58 to 5.04 log CFU/g during the production process. HPP treatment resulted in a significant (P<0.01) superficial and deep decrease in Salmonella spp. enumeration varying from 0.61 to 4.01 log and from 1.49 to 4.13 log. According to the data presented in this study, only the combined approach of coppa manufacturing process followed by HPP treatment always led to a 5-log reduction of Salmonella spp. required by USDA/FSIS guidelines.

Effect of production process and high-pressure processing on viability of Listeria innocua in traditional Italian dry-cured coppa.

In this study the effect of the application of High Pressure Treatment (HPP) combined with four different manufacturing processes on the inactivation of Listeria innocua, used as a surrogate for L. monocytogenes, in artificially contaminated coppa samples was evaluated in order to verify the most suitable strategy to meet the Listeria inactivation requirements needed for the exportation of dry-cured meat in the U.S. Fresh anatomical cuts intended for coppa production were supplied by four different delicatessen factories located in Northern Italy. Raw meat underwent experimental contamination with Listeria innocua using a mixture of 5 strains. Surface contamination of the fresh anatomical cuts was carried out by immersion into inoculum containing Listeria spp. The conditions of the HPP treatment were: pressure 593 MPa, time 290 seconds, water treatment temperature 14°C. Listeria innocua was enumerated on surface and deep samples post contamination, resting, ripening and HPP treatment. The results of this study show how the reduction of the microbial load on coppa during the production process did not vary among three companies (P>0.05) ranging from 3.73 to 4.30 log CFU/g, while it was significantly different (P<0.01) for the fourth company (0.92 log CFU/g). HPP treatment resulted in a significant (P<0.01) deep decrease of L. innocua count with values ranging between 1.63-3.54 log CFU/g with no significant differences between companies. Regarding superficial contamination, HPP treatment resulted significant (P<0.01) only in Coppa produced by two companies. The results highlight that there were processes less effective to inhibit the pathogen; in particular for company D an increase of L. innocua count was shown during processing and HPP alone cannot be able to in reaching the Listeria inactivation requirements needed for exportation of dry-cured meat in the U.S. According to the data reported in this paper, HPP treatment increases the ability of the manufacturing process of coppa in reducing Listeria count with the objective of a lethality treatment.

Semi-Quantitative Ultrasonographic Evaluation of NAFLD.

Nonalcoholic fatty liver disease (NAFLD) embraces histopathological entities ranging from the relatively benign simple steatosis to the progressive form nonalcoholic steatohepatitis (NASH), which is associated with fibrosis and an increased risk of progression to cirrhosis and hepatocellular carcinoma. NAFLD is the most common liver disease and is associated with extrahepatic comorbidities including a major cardiovascular disease burden. The non-invasive diagnosis of NAFLD and the identification of subjects at risk of progressive liver disease and cardio-metabolic complications are key in implementing personalized treatment schedules and follow-up strategies. In this review, we highlight the potential role of ultrasound semiquantitative scores for detecting and assessing steatosis severity, progression of NAFLD, and cardio-metabolic risk. Ultrasonographic scores of fatty liver severity act as sensors of cardio-metabolic health and may assist in selecting patients to submit to second-line non-invasive imaging techniques and/or liver biopsy.

Direct Oral Anticoagulants in Patients with Liver Disease in the Era of Non-Alcoholic Fatty Liver Disease Global Epidemic: A Narrative Review.

Atrial fibrillation (AF) and venous thromboembolism (VTE) are highly prevalent and relevant healthcare issues. Direct oral anticoagulants (DOACs) are now the first-choice for anticoagulant treatment of these conditions displaying a better efficacy/safety profile than vitamin-K antagonists, mainly due to significantly reduced risk of major bleeding, especially of intracranial haemorrhage. Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in developed countries showing a continuously growing prevalence. Nonalcoholic steatohepatitis (NASH), its evolutive form, will be the leading cause for liver transplantation by 2020. NAFLD is independently associated with an increased risk of abnormalities of cardiac structure and function, including cardiac rhythm disorders (mainly AF). Moreover, data suggest an increased risk of unprovoked VTE associated with NAFLD/NASH. Therefore, a growing number of patients with chronic liver disease (CLD) will be candidate for anticoagulant therapy in the near future. Cirrhosis of any etiology is characterized by an unstable thrombosis/bleeding haemostatic balance, making anticoagulant therapy particularly challenging in this condition. Given that patients with significant active liver disease and cirrhosis were excluded from all pivotal randomized controlled trials on DOACs, this comprehensive review aims at critically discussing real-world evidence, including the latest population studies, regarding the use of DOACs in patients with CLD/cirrhosis.

Veterinary forensic sciences to solve a fatal case of predation on flamingos (Phoenicopterus roseus).

The present case study concerns a case of predation of 4 individuals of captive pink flamingo in Emilia Romagna Region, Northeastern Italy. The pink flamingo (Phoenicopterus roseus) is a species included in the Red List of Threatened Species established by the International Union for Conservation of Nature (IUCN) which lists species in danger of extinction. During the Winter of 2013, 4 flamingos (2 in the Comacchio area, and 2 from Argenta and Codigoro oases - Ferrara province) were found dead some of them headless, with their bodies severely bitten. At first, a fox (Vulpes vulpes) was suspected to be the predator responsible for the killing and the birds were taken to the laboratory for further investigations. The investigations included: field observations, study of the predator behaviour, necropsy examinations, assessment of the intercanine distance, and genetic analysis on the predator's traces. The intercanine distance indicated that the predator could not have been a fox. The analysis of salivary DNA samples enabled us to establish that the predator was in fact a dog. This case highlights the importance of co-operation among the various branches of forensic sciences and the great usefulness of the roles filled by other veterinary forensic experts involved in solving crime.

Outbreak of type C botulism in birds and mammals in the Emilia Romagna region, northern Italy.

Over a 7-day period beginning 8 August 2011, a large number of wild birds of several species were found dead or with neurologic clinical signs along the shore of Crostolo stream, in the Emilia Romagna region, Italy. Twenty-eight Mallards (Anas platyrhynchos), two Hooded Crows (Corvus corone cornix), and three coypus (Myocastor coypus) were found moribund on the Crostolo stream bank, collected, and sent to Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Reggio Emilia Section. The cause of mortality was determined to be Clostridium botulinum type C toxin. The toxin was identified by a mouse bioassay for botulinum toxins and confirmed in bird sera and blowfly larvae (Lucilia caesar) collected from the stomachs of birds.

Development of a minor groove binding probe based real-time PCR for the diagnosis and quantification of Leishmania infantum in dog specimens.

A new quantitative real-time PCR (qPCR) assay based on Taqman® technology and minor groove binding (MGB) probe was developed for the diagnosis of leishmaniosis and quantification of Leishmania infantum DNA in infected dogs. This method was based on the amplification of a 122bp fragment of the highly conserved kDNA minicircles of L. infantum. The reaction was performed using the StepOnePlus™ system with StepOne software™. This assay was able to detect the presence of protozoan parasite DNA in amounts as low as 0.03 parasites per reaction. The standard curve designed for the quantification of parasites showed linearity over seven log DNA concentration range with a correlation coefficient >0.999 and both intra- and inter-assay variability demonstrated the high efficiency and reproducibility of the assay. The qPCR also proved to be successfully applicable to different clinical samples including blood, bone marrow, lymph node aspirates and conjunctival swabs.

First reports of Trichinella pseudospiralis in wild boars (Sus scrofa) of Italy.

Trichinella pseudospiralis is a non-encapsulated species infecting both mammals and birds. In Italy, this parasite was reported only in two night-birds of prey of Central Italy. In January 2010, Trichinella larvae were detected in three wild boars (Sus scrofa) of two regions of Northern Italy by enzymatic digestion. The parasites were identified as T. pseudospiralis by multiplex-PCR. The first infected wild boar was hunted in the Emilia Romagna region and the other two infected wild boars were bred outdoors in a small family farm of the Friuli Venezia Giulia region. These new epidemiological data reinforce the role of the wild boar as the main reservoir of T. pseudospiralis in Europe.

Diagnóstico pluridimensional dinâmico em psiquiatria / Dynamic pluridimensional diagnosis in psychiatry

Devemos a Kretschmer o conceito de diagnóstico pluridimensional em psiquiatria. Entre nós, um dos que mais estudou o assunto foi Leme Lopes. Sem alterar a idéia básica de diagnóstico pluridimensional dinâmico, propomos um novo esquema, inspirado nas séries complementares de Freud. Mostram a vantagem e a clareza do novo esquema para uma visão holística dos pacientes (AU)