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.

The nematode Trichinella pseudospiralis is a cosmopolitan parasite capable of infecting both birds and mammals including humans. T. pseudospiralis has a limited zoonotic importance in comparison to that of the other Trichinella species. However, it has been recognized as the etiological agent of two outbreaks of trichinellosis due to the consumption of wild boar meat. The role played by birds in the epidemiology of T. pseudospiralis is still unclear and needs to be deepened. The aim of our work was to show the results of an extensive wild bird surveillance carried out in the Emilia-Romagna (E-R) region, northern Italy, over the last 16 years. As part of the regional wildlife surveillance program, 14,933 raptors and carrion-eating birds’ carcasses were necropsied from 2006 to 2021 and tested for the presence of Trichinella spp. larvae with only one positive result, an adult female of western marsh harrier. The larvae load (LPG) was evaluated on breast (100 LPG), wings (3.6 LPG), shoulder (2 LPG), head (4.5 LPG), thighs (8 LPG), lower legs (2 LPG) and tongue (0.77 LPG). The results of the present study confirm that also in northern Italy T. pseudospiralis has a low prevalence comparing to that of other Trichinella species. However, this study demonstrates that T. pseudospiralis can reach a high parasitic load in infested birds. The large distribution range, probably facilitated by its ability to infest birds, suggests the need for a continuous monitoring program.

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.

Background: Hepatozoon martis is an apicomplexan parasite infecting mustelids, but its geographical distribution and pathological relevance remain incompletely understood. To date, its presence in Italy has not been documented. This study aimed to investigate the occurrence of H. martis in martens from northern and central Italy using an integrated pathological and molecular approach. Methods: Between 2023 and 2025, carcasses of 17 martens ( Martes spp.) collected within wildlife health surveillance programs were examined. Tissue samples were screened for apicomplexan DNA by qPCR, and conventional PCR targeting the 18S rDNA, followed by sequencing and phylogenetic analyses, were applied for species characterization. Histopathological examinations were performed on tissues from freshly deceased animals. Results: Eight of 17 martens (47%), 16 identified as Martes foina and one Martes martes , tested positive for Hepatozoon infection. Sequencing confirmed H. martis in multiple tissues, including spleen, heart, lymph nodes, and muscle. Histopathological analysis revealed multifocal granulomatous myocarditis and myositis, with intracellular zoites and type II meronts observed within inflammatory lesions. Phylogenetic analyses of short and long 18S rDNA fragments showed that Italian isolates clustered with H. martis sequences from other European countries, displaying low genetic variability. Hepatozoon DNA was also detected in one Ixodes sp. collected from an infected animal. Conclusions: This study provides the first evidence to our knowledge of H. martis infection in Italy and highlights its pathogenic potential in stone martens. The low genetic diversity observed among European isolates suggests widespread circulation and gene flow across regions. Further investigations are required to elucidate transmission pathways, identify competent vectors, and assess the pathological significance of H. martis in wildlife.