Animals, including wildlife, are part of One-Health concept since many infectious diseases can affect both humans and animals. In this study, 126 red foxes (Vulpes vulpes) from Northern Italy in 2022–2023 were tested by molecular assays for Protoparvovirus carnivoran 1 (PPVC-1), Canine adenovirus type 1 and 2 (CAdV-1 and CAdV-2), Circovirus canine (CanineCV), Canine distemper virus (CDV), and Leptospira spp. A total of 39 of 126 (30.9%) red foxes were infected with at least one pathogen and five of these were coinfected: 20/126 (15.9%) red foxes tested positive for PPVC-1, 3/126 (2.4%) for CAdV, 20/126 (15.9%) for CanineCV, and 2/126 (1.6%) for Leptospira spp. DNA. No foxes tested positive for CDV RNA. The pathogens identified were genetically analysed. New findings were reported such as a fox with multiple feline panleukopenia virus (FPV) and canine parvovirus type 2b (CPV-2b) infection associated with quasispecies dynamics, typical genetic characteristics of the identified CanineCV, and the first detection in red foxes of Leptospira ST198 related to L. interrogans serogroup Australis. Further studies are necessary to investigate the transmission between domestic animals and wildlife and to understand the role of red foxes in the maintenance of these pathogens not only in the wild but also in urban and peri-urban environments.

Cases of sand fly-borne diseases in the Emilia-Romagna region, such as meningitis caused by Toscana virus and human leishmaniasis, are reported annually through dedicated surveillance systems. Sand flies are abundant in the hilly part of the region, while the lowland is unsuitable habitat for sand flies, which are found in lower numbers in this environment with respect to the hilly areas. In this study, we retrieved sand flies collected during entomological surveillance of the West Nile virus (from 2018 to 2021) to assess their abundance and screen them for the presence of pathogens. Over the four-year period, we collected 3022 sand flies, more than half in 2021. The most abundant sand fly species was Phlebotomus (Ph.) perfiliewi, followed by Ph. perniciosus; while more rarely sampled species were Ph. papatasi, Ph. mascittii and Sergentomyia minuta. Sand flies were collected from the end of May to the end of September. The pattern of distribution of the species is characterized by an abundant number of Ph. perfiliewi in the eastern part of the region, which then falls to almost none in the western part of the region, while Ph. perniciosus seems more uniformly distributed throughout. We tested more than 1500 female sand flies in 54 pools to detect phleboviruses and Leishmania species using different PCR protocols. Toscana virus and Leishmania infantum, both human pathogens, were detected in 5 pools and 7 pools, respectively. We also detected Fermo virus, a phlebovirus uncharacterized in terms of relevance to public health, in 4 pools. We recorded different sand fly abundance in different seasons in Emilia-Romagna. During the season more favorable for sand flies, we also detected pathogens transmitted by these insects. This finding implies a health risk linked to sand fly-borne pathogens in the surveyed area in lowland, despite being considered a less suitable habitat for sand flies with respect to the hilly areas.

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has recently emerged as a promising method for arthropods identification. A total of 125 ticks collected from 2013 until 2023 in Italy either stored frozen at -20 °C (n = 75) or preserved in 70% ethanol (n = 50) were identified using morphological keys before being analyzed by MALDI-TOF MS and, when necessary, by molecular biology in Marseille, France. Of the 125 samples analyzed by MALDI-TOF MS, 106 (84.8%) yielded good quality MS spectra (i.e., intensity>3000 arbitrary units with low background noise). The spectra of 21 specimens from eight tick species, including Dermacentor marginatus, Haemaphysalis parva, Haemaphysalis punctata, Ixodes hexagonus, Ixodes ricinus, Rhipicephalus pusillus, Rhipicephalus sanguineus s.s., and Rhipicephalus turanicus whose morphological identities were confirmed by molecular methods, were used to update an in-house MALDI-TOF MS arthropod spectra database. Blind testing of the remaining 85 good quality spectra against the updated database produced the log score values (LSVs) ranging from 1.56 to 2.57. Overall, identification by MALDI-TOF MS matched morphological identification for 59/85 (69.4%) spectra, whereas 26/85 (30.6%) spectra were discordant with morphological identification. Molecular identification using 16S rRNA gene sequencing was available for 17/26 discordant samples, and 16/17 (94.1%) supported the MALDI-TOF MS result. Considering only samples confirmed by both MALDI-TOF MS and molecular methods, 71/72 (98.6%) were correctly identified by MALDI-TOF MS, with 68/72 (94.4%) showing LSVs > 1.8, a value previously reported as relevant for arthropod identification. This study confirms the effectiveness of MALDI-TOF MS for tick identification and highlights its potential as a new tool for tick identification in Italy.

Trichinella spp. are zoonotic nematodes with a global distribution, primarily maintained through wildlife reservoirs which complicates eradication efforts. In Europe, four species—T. spiralis, T. britovi, T. nativa, and T. pseudospiralis—circulate in wild carnivores and omnivores, with T. britovi and T. spiralis being the most prevalent in wild and domestic hosts. This study aimed to assess the presence and species distribution of Trichinella spp. in wild mammals. This study presents the results of five years of wildlife surveillance carried out within the long-standing monitoring program implemented in the Emilia-Romagna region, northern Italy, which has been in place since 2006. Between 2020 and 2024, a total of 104,338 wild mammals, including red foxes, wolves, and wild boar, were tested using the magnetic stirrer digestion method in accordance with EU regulations. A total of 12 animals (0.011%) were found to be infected with Trichinella larvae. Molecular identification of positive samples, performed at the European Union Reference Laboratory for Parasites, showed the presence of T. britovi in 11 animals, while one isolate remained unidentified. T. britovi was detected primarily in wolves and red foxes. No infections were identified in wild boar during the study period. Overall, the low prevalence observed confirms that Trichinella spp. infections in wildlife in Emilia-Romagna are sporadic but persistently present. Only T. britovi was detected, supporting its role as the predominant species in this area. These findings highlight the importance of long-term surveillance for early detection and risk assessment within a One Health framework.

Purpose The aim of this study was to describe the occurrence and phylogenetic diversity of Hepatozoon spp. in small mammals from the Emilia-Romagna region, northern Italy, and to assess their genetic relationships with previously described lineages. Methods Tissue samples (spleen and ear) from wild animals belonging to eight small mammal species were examined. Molecular screening was performed using PCR targeting the 18 S rRNA gene of Hepatozoon spp. Positive amplicons were sequenced and analyzed using phylogenetic methods to determine their taxonomic placement, and one positive fresh sample was additionally subjected to histopathological characterization. Results Hepatozoon DNA was detected in two rodent species, the brown rat (Rattus norvegicus) and the European edible dormouse (Glis glis). Sequences showed high identity (98.9–100%) with Hepatozoon sp. previously reported in rodents from Spain and Tanzania, and 98.32–98.9% identity with Hepatozoon ophisauri. Phylogenetic analysis placed the sequences from R. norvegicus within a rodent-associated clade, whereas those from G. glis formed a distinct lineage related to Hepatozoon detected in a great gerbil. Histopathological analysis in a European edible dormouse revealed multifocal pulmonary granulomatous inflammatory lesions associated with intracytoplasmic parasitic zoites and developmental stages consistent with type I and type II meronts. Conclusion This study provides molecular evidence of distinct Hepatozoon lineages in European rodents, potentially representing undescribed taxa, and highlights the need for integrative studies combining molecular, morphological, and ecological data.