Salmonella in Wild Boar Meat: Prevalence and Risk Assessment in Central Italy (Umbria and Marche Region).

A survey was conducted from 2018 to 2023 to assess the presence of Salmonella in 280 hunted wild boar (carcasses after evisceration and skinning, N = 226; liver, N = 258; and fecal samples, N = 174). The overall prevalence was 2.86% (confidence interval 95%, 1.45-5.45%) with five positive samples detected in carcasses, three in the liver, and one in a fecal sample. This prevalence was in line with those found in nearby areas denoting a low number of positive samples. Positive animals were over 24 months of age and weighed, before skinning, 59.00 ± 9.11 Kg and no difference was detected in microbial loads between samples positive and negative for Salmonella (aerobic colony count of 4.59 and 4.66 log CFU/400 cm2, and Enterobacteriaceae count of 2.89 and 2.73 log CFU/400 cm2 (mean values) in positive and negative subjects, respectively). Salmonella Stanleyville was the most frequently isolated serotype. A semiquantitative risk assessment was conducted for the first time in game meat considering two products, meat cuts intended for cooking and fermented dry sausages. Only proper cooking can reduce the risk of ingestion of Salmonella to the minimum for consumers, whereas ready-to-eat dry sausages constitute risk products in terms of foodborne Salmonellosis (risk score of 64 out of 100).

Tapeworms detected in wolf populations in Central Italy (Umbria and Marche regions): A long-term study.

Tapeworms are trophically-transmitted and multi-host parasites with a complex indirect life cycle, strictly depending on predator-prey interactions. Their presence in a free-living population, mainly definitive hosts, is arduous to study due to the complexity of collecting fecal samples. However, epidemiological studies on their frequency are crucial from a public health perspective, providing information on food habits and prey selection of predators. The present study aims to update the frequency of tapeworms detected in stool samples by molecular analysis in Italian wolf populations of Umbria and Marche regions collected from 2014 to 2022. Tapeworm's total frequency was 43.2%. In detail, Taenia serialis was detected in 27 samples (21.6%), T. hydatigena in 22 (17.6%), and Mesocestoides corti (syn. M. vogae) in 2 (1.6%). Three samples were identified as M. litteratus and E. granulosus s.s. (G3) and T. pisiformis, with a proportion of 0.8%, respectively. The low frequency of E. granulosus in a hyperendemic area is discussed. The results show for the first time a high frequency of Taenia serialis not comparable to other Italian studies conducted on wild Carnivora; thus, a new ecological niche is conceivable. These findings suggest a plausible wolf-roe deer cycle for T. serialisin the investigated area.

Brominated flame retardants (PBDEs and HBCDs) and perfluoroalkyl substances (PFASs) in wild boars (Sus scrofa) from Central Italy.

Twenty-six samples of wild boar liver and muscle from the Central Apennine Mountain (Italy) were analysed for 19 perfluoro-alkyl substances (PFASs), 10 polybrominated diphenylethers (PBDEs) and 3 hexabromocyclododecanes (HBCDs). All samples were analysed by gas chromatography-tandem mass spectrometry for PBDEs and liquid chromatography-tandem mass spectrometry for PFASs and HBCDs, using an in-house developed analytical procedure. The brominated flame retardants (BFR) levels in livers were negligible: Σ10PBDEs reached a maximum value of 0.079 µg/kg, whereas HBCDs were not quantified in almost all of the samples analysed. BFR concentrations in muscles were higher, but not significantly therefore, for Σ10PBDEs lower bound, a mean value of 0.045 µg/kg (0.005-0.155 µg/kg range) was measured, while α-HBCD was quantified with a maximum of 0.084 µg/kg in 9 of the samples. Only two muscles contained all 3 HBCD isomers at concentrations of approximately 0.200 µg/kg. Σ19PFAS in the 26 wild boar livers was in the range 31.9-228 µg/kg, with a mean value of 87.7 µg/kg, reaching levels significantly higher than in muscles, which exhibited a mean concentration of 3.08 µg/kg (0.59-9.12 µg/kg range). Perfluorooctanesulfonic acid (PFOS) was the most prevalent compound in all liver samples, accounting for more than half of the total PFASs contamination, confirming that the liver is the primary target organ for PFOS exposure Perfluorotridecanoic acid (PFTrDA), which accounts for 25-30-% of the total contamination, was the most abundant compound in the muscle, followed by PFOS. The estimated daily intake (EDIs) of BFRs remained below the estimated chronic human daily dietary intake (Dr,h) defined from European Food Safety Authority (EFSA). Furthermore, the exposure to PFASs in muscle was 7.7 times lower than the EFSA's tolerable daily intake (TDI). In contrast, exposure due to liver consumption was significant: the EDI exceeded the EFSA's 2020 TDI by approximately 7 times.

Potential SARS-CoV-2 Susceptibility of Cetaceans Stranded along the Italian Coastline.

Due to marine mammals' demonstrated susceptibility to SARS-CoV-2, based upon the homology level of their angiotensin-converting enzyme 2 (ACE2) viral receptor with the human one, alongside the global SARS-CoV-2 occurrence and fecal contamination of the river and marine ecosystems, SARS-CoV-2 infection may be plausibly expected to occur also in cetaceans, with special emphasis on inshore species like bottlenose dolphins (Tursiops truncatus). Moreover, based on immune and inflammatory responses to SARS-CoV-2 infection in humans, macrophages could also play an important role in antiviral defense mechanisms. In order to provide a more in-depth insight into SARS-CoV-2 susceptibility in marine mammals, we evaluated the presence of SARS-CoV-2 and the expression of ACE2 and the pan-macrophage marker CD68. Aliquots of tissue samples, belonging to cetaceans stranded along the Italian coastline during 2020-2021, were collected for SARS-CoV-2 analysis by real-time PCR (RT-PCRT) (N = 43) and Immunohistochemistry (IHC) (N = 59); thirty-two aliquots of pulmonary tissue sample (N = 17 Tursiops truncatus, N = 15 Stenella coeruleoalba) available at the Mediterranean Marine Mammal Tissue Bank (MMMTB) of the University of Padua (Legnaro, Padua, Italy) were analyzed to investigate ACE2 expression by IHC. In addition, ACE2 and CD68 were also investigated by Double-Labeling Immunofluorescence (IF) Confocal Laser Microscopy. No SARS-CoV-2 positivity was found in samples analyzed for the survey while ACE2 protein was detected in the lower respiratory tract albeit heterogeneously for age, gender/sex, and species, suggesting that ACE2 expression can vary between different lung regions and among individuals. Finally, double IF analysis showed elevated colocalization of ACE2 and CD68 in macrophages only when an evident inflammatory reaction was present, such as in human SARS-CoV-2 infection.

Detection of Leishmania spp. in Chronic Dermatitis: Retrospective Study in Exposed Horse Populations.

Leishmania infantum is a protozoan causing human zoonotic visceral leishmaniasis (ZVL) and visceral-cutaneous canine leishmaniosis (CanL) in the Mediterranean Basin. L. infantum is able to infect a large number of wild and domestic species, including cats, dogs, and horses. Since the 1990s, clinical cases of equine leishmaniasis (EL), typically characterized by cutaneous forms, have been increasingly diagnosed worldwide. The aim of the present study was to evaluate the presence of clinical forms of EL in CanL-endemic areas in Italy, where exposure of equine populations was ascertained from recent serological surveys. For this purpose, formalin-fixed and paraffin-embedded skin biopsies of 47 horses presenting chronic dermatitis compatible with EL were retrospectively selected for the study and subjected to conventional and q-PCR. A singular positivity for L. infantum was found; BLAST analysis of sequence amplicons revealed a 99-100% homology with L. infantum sequences. The histological examination revealed a nodular lymphoplasmacytic and histiocytic infiltrate; immunohistochemistry showed rare macrophages containing numerous positive amastigotes. The present retrospective study reports, for the first time, a case of a cutaneous lesion by L. infantum occurring in an Italian horse. Pathological and healthy skin samples should be investigated on a larger scale to provide information on the potential clinical impact of EL in the practice, and to define the role of horses in epidemiological ZVL and CanL scenarios.

A novel intermediate host for Taenia serialis (Gervais, 1847): The European roe deer (Capreolus capreolus L. 1758) from the Monti Sibillini National Park (MSNP), Italy.

Taeniids are multi-host parasites with an indirect life cycle that strictly depends on a predator-prey relationship. Parasites with a complex life cycle may exhibit different degrees of host-specificity at each life stage. Knowing the host breadth is a fundamental concept of the biology and epidemiology of these multi-host parasites. Morphological identification of tapeworms is challenging and occasionally may produce misdiagnosis. Thus, molecular investigations were carried out for the identification of parasitic cysts detected from muscle tissues in a male roe deer necropsied at the Istituto Zooprofilattico Sperimentale dell'Umbria e delle Marche "Togo Rosati" (Central Italy). Sanger sequencing showed 99% query cover, 2e-109 e-value, and 100% identity with Taenia serialis. The exact definitive host was not revealed in this report, but red foxes and Italian wolves may play a significant role as being widespread within the area. Wildlife surveillance is crucial to monitor for human and animal health since global distribution and flexibility in intermediate hosts of many and even more critical taeniids species may enlarge their host range.

First Isolation of Brucella canis from a breeding kennel in Italy.

Brucella canis has been isolated for the first time in Italy in a commercial breeding kennel. It was diagnosed after a deep investigation related to the onset of reproductive disorders. Animals were tested with direct and indirect techniques. The agent was first detected in two Chihuahua aborted foetuses by direct culture. Further, it was also isolated from blood samples of dogs hosted in the kennel, which also showed reaction to conventional serological tests (microplate serum agglutination test). The isolates were identified as B. canis by standard microbiological methods and a Bruce­ladder multiplex PCR. To investigate the genomic diversity, whole genome sequencing was used, applying the core genome Multilocus Sequence Typing (cgMLST ). In a first round of serological testing performed on 598 animals, 269 (46.1%) tested positive. In the second round of laboratory testing carried out 4­5 weeks apart, the number of serologically positive dogs was 241 out of 683 tested (35.3%), while the number of dogs positive to isolation was 68 out of 683 tested (10.0%). The PCR showed a lack of sensitivity when compared to direct isolation. The epidemiological investigation did not identify the source of the infection, given the time elapsed from the onset of abortions to the definitive diagnosis of B. canis infection in the kennel. The genomic analyses featured the strains as ST21 and, according to the cgMLST, revealed the presence of a tight cluster with a maximum diversity of four allelic differences. The observed limited genomic variation, largely within the known outbreak cut­offs, suggests that the outbreak herein described was likely caused by a single introduction. Moreover, in a broader scale comparison using the public available genomes, we found that the closest genome, isolated in China, differed by more than 50 alleles making not possible to find out the likely origin of the outbreak. The lack of updated data on B. canis genome sequences in the public databases, together with the limited information retrieved from the epidemiological investigations on the outbreak, hampered identification of the source of B. canis infection.

Systemic Bovine Tuberculosis in a Crested Porcupine (Hystrix cristata) in the Marche Region, Italy.

Tuberculosis is a worldwide zoonosis involving a wide range of hosts among domestic and wild animals. We describe tuberculosis caused by Mycobacterium bovis in a wild crested porcupine (Hystrix cristata) found dead in the district of Macerata, Marche Region, Italy in 2019.

One Health Approach to Rickettsiosis: A Five-Year Study on Spotted Fever Group Rickettsiae in Ticks Collected from Humans, Animals and Environment.

The spotted fever group of Rickettsiae is a heterogeneous group of Rickettsiae transmitted by ticks, causing similar diseases in humans (spotted fever). Until recently, it was supposed that a single pathogenic tick-borne SFG Rickettsia circulated in each different geographic area and that R. conorii subsp. conorii was the SFG Rickettsiae circulating in Italy, but in the last decade, thanks to molecular diagnostic, several different Rickettsia species, previously not considered pathogenic for decades, have been isolated from ticks and definitively associated to human disease, also in Italy. The present survey was carried out with the aim of investigating the presence of different SFG Rickettsia species in a geographic area where no information was available. Ticks collected from animals submitted to necropsy, removed from humans in local hospitals and collected from the environment were identified and tested by PCR for Rickettsia spp. based on the gltA gene, and positive PCR products were sequenced. A total of 3286 ticks were collected. Fifteen tick species were recognized, the most represented (79.52%) species in the collection was Ixodes ricinus, followed by Rhipicephalus sanguineus (9.13%). The overall prevalence of Rickettsia infection was 7.58%. Eight species of Rickettsia were identified, the most frequent was R. monacensis (56%), followed by R. helvetica (25.50%). Noteworthy, is the detection in the present study of Rrhipicephali, detected only twice in Italy. These are the first data available on SFG Rickettsiae circulation in the study area and they can be considered as starting point to assess the possible risk for humans.

Exposure of Owned Dogs and Feeding Ticks to Spotted Fever Group Rickettsioses in Central Italy.

Dogs may be useful sentinels for public health monitoring of spotted fever group rickettsioses (SFGR). The aim of this study was to determine the exposure to SFGR among dogs and feeding ticks in central Italy. A total of 344 dogs and 607 adult ticks (395 Rhipicephalus sanguineus and 212 Ixodes ricinus specimens) collected from the coats of sampled animals were included in the study. Canine serum samples were analyzed by indirect fluorescent antibody technique (IFAT) for IgG antibodies against Rickettsia conorii and Rickettsia rickettsii. All the ticks and buffy coats were processed by a PCR targeting a fragment of gltA followed by sequencing. Overall, 56 dogs (16.3%) tested positive for one or both rickettsial antigens by IFAT with endpoint titers ranging from 1:64 to 1:2048; 38 (11%) serum samples reacted against R. conorii, 46 (13.4%) reacted against R. rickettsii, and 28 (8.1%) reacted simultaneously against both rickettsial agents. All buffy coats were PCR negative. Rickettsial DNA was revealed in 39 (18.4%) I. ricinus and in 10 (2.5%) R. sanguineus specimens. The amplicons sequencing showed three SFGR, that is, R. conorii detected in 10 R. sanguineus specimens and Rickettsia helvetica and Rickettsia monacensis detected in 7 and 32 I. ricinus ticks. Nine out of the 10 R. conorii isolates were obtained from ticks collected from seronegative dogs, and one specimen from a dog tested positive for both R. rickettsii and R. conorii by immunofluorescence assay. Among the seven ticks tested positive for R. helvetica, six were recovered from the coats of seronegative dogs and one from a dog having antibodies against R. conorii; the 32 isolates of R. monacensis were obtained from 28 seronegative and 4 R. conorii/R. rickettsii-positive dogs. The results highlight the non-negligible exposure of the canine population to SFGR in the sampled areas.

Molecular Survey on Rickettsia spp., Anaplasma phagocytophilum, Borrelia burgdorferi Sensu Lato, and Babesia spp. in Ixodes ricinus Ticks Infesting Dogs in Central Italy.

Dogs are a common feeding hosts for Ixodes ricinus and may act as reservoir hosts for zoonotic tick-borne pathogens (TBPs) and as carriers of infected ticks into human settings. The aim of this work was to evaluate the presence of several selected TBPs of significant public health concern by molecular methods in I. ricinus recovered from dogs living in urban and suburban settings in central Italy. A total of 212 I. ricinus specimens were collected from the coat of domestic dogs. DNA was extracted from each specimen individually and tested for Rickettsia spp., Borrelia burgdorferi sensu lato, Babesia spp., and Anaplasma phagocytophilum, using real-time and conventional PCR protocols, followed by sequencing. Sixty-one ticks (28.8%) tested positive for TBPs; 57 samples were infected by one pathogen, while four showed coinfections. Rickettsia spp. was detected in 39 specimens (18.4%), of which 32 were identified as Rickettsia monacensis and seven as Rickettsia helvetica. Twenty-two samples (10.4%) tested positive for A. phagocytophilum; Borrelia lusitaniae and Borrelia afzelii were detected in two specimens and one specimen, respectively. One tick (0.5%) was found to be positive for Babesia venatorum (EU1). Our findings reveal the significant exposure of dogs to TBPs of public health concern and provide data on the role of dogs in the circulation of I. ricinus-borne pathogens in central Italy.

Serological Survey of Hantavirus and Flavivirus Among Wild Rodents in Central Italy.

Hantaviruses are a group of zoonotic viruses carried by rodents. Puumala virus (PUUV) and Dobrava virus (DOBV) are the causative agents of human hantavirus infections in Europe. Knowledge about hantavirus circulation in Italy is very scarce. West Nile virus (WNV) and Usutu virus (USUV) are emerging neuropathogenic flaviviruses, both endemic in most part of the Italian territories. To monitor the circulation of PUUV, DOBV, WNV, and USUV in natural environment in central Italy, we carried out serological surveillance in wild rodents. During this study, 90 animals were captured in forested areas of Abruzzo and Marche regions and tested with serological assays for the specific pathogens. Serological test provided no evidence of PUUV and DOBV circulation in the studied area. However, four rodents (Apodemus flavicollis) were found to be positive by WNV ELISA test. Two of them were confirmed as WNV by virus neutralization test.

Seasonal monitoring of Aedes albopictus: practical applications and outcomes.

The introduction of the Asian tiger mosquito Aedes (Stegomya) albopictus (Diptera: Culicidae) into temperate regions poses serious concerns for the risk of the spreading of arboviral epidemics, as confirmed by the Chikungunya fever outbreak in Italy. This article describes the implementation and the results of a strategy for the pest management implemented over 4 years in Pesaro (a city in the Marche region, Italy). The strategy used 60 integrated wide­sized ovitraps for monitoring purposes. Twenty-day larvicide-based treatment cycles were implemented for the manholes of the urban area and also the inhabitants were involved in pest control relating to their own properties. It was observed that the weekly median of eggs laid decreased consistently from 2008 to 2011, indicating the good performance of the vector control and a reduction in the related epidemics risk.

Ultrastructural morphometrical and immunocytochemical analyses of hepatocyte nuclei from mice fed on genetically modified soybean.

No direct evidence that genetically modified (GM) food may represent a possible danger for health has been reported so far; however, the scientific literature in this field is still quite poor. Therefore, we carried out an ultrastructural morphometrical and immunocytochemical study on hepatocytes from mice fed on GM soybean, in order to investigate eventual modifications of nuclear components of these cells involved in multiple metabolic pathways related to food processing. Our observations demonstrate significant modifications of some nuclear features in GM-fed mice. In particular, GM fed-mice show irregularly shaped nuclei, which generally represents an index of high metabolic rate, and a higher number of nuclear pores, suggestive of intense molecular trafficking. Moreover, the roundish nucleoli of control animals change in more irregular nucleoli with numerous small fibrillar centres and abundant dense fibrillar component in GM-fed mice, modifications typical of increased metabolic rate. Accordingly, nucleoplasmic (snRNPs and SC-35) and nucleolar (fibrillarin) splicing factors are more abundant in hepatocyte nuclei of GM-fed than in control mice. In conclusion, our data suggest that GM soybean intake can influence hepatocyte nuclear features in young and adult mice; however, the mechanisms responsible for such alterations remain unknown.