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Background Recent climate and environmental changes have resulted in the geographical expansion of Mediterranean Leishmania infantum vectors towards northern latitudes and higher altitudes in different European countries, including Italy, where new foci of canine leishmaniasis have been observed in the northern part of the country. Northern Italy is also an endemic area for mosquito-borne diseases. During entomological surveillance for West Nile virus, mosquitoes and other hematophagous insects were collected, including Phlebotomine sand flies. In this study, we report the results of Phlebotomine sand fly identification during the entomological surveillance conducted from 2017 to 2019. Methods The northeastern plain of Italy was divided by a grid with a length of 15 km, and a CO 2 -CDC trap was placed in each geographical unit. The traps were placed ~ 15 km apart. For each sampling site, geographical coordinates were recorded. The traps were operated every two weeks, from May to November. Sand flies collected by CO 2 -CDC traps were identified by morphological and molecular analysis. Results From 2017 to 2019, a total of 303 sand flies belonging to the species Phlebotomus perniciosus ( n  = 273), Sergentomyia minuta ( n  = 5), P. mascittii ( n  = 2) and P. perfiliewi ( n  = 2) were collected, along with 21 unidentified specimens. The trend for P. perniciosus collected during the entomological surveillance showed two peaks, one in July and a smaller one in September. Sand flies were collected at different altitudes, from −2 m above sea level (a.s.l.) to 145 m a.s.l. No correlation was observed between altitude and sand fly abundance. Conclusions Four Phlebotomine sand fly species are reported for the first time from the northeastern plain of Italy. Except for S. minuta , the sand fly species are competent vectors of Leishmania parasites and other arboviruses in the Mediterranean Basin. These findings demonstrate the ability of sand flies to colonize new environments previously considered unsuitable for these insects. Even though the density of the Phlebotomine sand fly population in the plain areas is consistently lower than that observed in hilly and low mountainous areas, the presence of these vectors could herald the onset of epidemic outbreaks of leishmaniasis and other arthropod-borne diseases in areas previously considered non-endemic. Graphical Abstract

Early detection of pathogens in vectors is important in preventing the spread of arboviral diseases, providing a timely indicator of pathogen circulation before outbreaks occur. However, entomological surveillance may face logistical constraints, such as maintaining the cold chain, and resource limitations, such as the field and laboratory workload of mosquito processing. We propose an FTA card-based trapping system that aims to simplify both field and laboratory phases of arbovirus surveillance. We modified a BG-Sentinel trap to include a mosquito collection chamber and a sugar feeding source through an FTA card soaked in a long-lasting viscous solution of honey and hydroxy-cellulose hydrogel. The FTA card ensures environmental preservation of nucleic acids, allowing continuous collection and feeding activity of specimens for several days and reducing the effort required for viral detection. We tested the trap prototype during two field seasons (2019 and 2021) in North-eastern Italy and compared it to CDC-CO 2 trapping applied in West Nile and Usutu virus regional surveillance. Collections by the BG-FTA approach detected high species diversity, including Culex pipiens , Aedes albopictus , Culex modestus , Anopheles maculipennis sensu lato and Ochlerotatus caspius . When used for two-days sampling, the BG-FTA trap performed equally to CDC also for the WNV-major vector Cx. pipiens . The FTA cards detected both WNV and USUV, confirming the reliability of this novel approach to detect viral circulation in infectious mosquitoes. We recommend this surveillance approach as a particularly useful alternative in multi-target surveillance, for sampling in remote areas and in contexts characterized by high mosquito densities and diversity.

A 10-month-old intact female Cane Corso dog was referred to the Veterinary Teaching Hospital of the University of Perugia (PG-VTH) for severe hemolytic anemia and thrombocytopenia. The dog had never traveled abroad and was regularly treated with antiparasitic products. On physical examination, the dog showed lethargy, delayed growth, weight loss, pale mucous membranes, and abdominal pain. The temperature was normal, and on examination, no ectoparasites were observed on the animal’s body surface. The main laboratory findings were hemolytic anemia, thrombocytopenia, and elevated liver enzymes. Babesia infection was initially diagnosed by blood smear evaluation via May–Grünwald–Giemsa staining and then confirmed by real-time polymerase chain reaction analysis; further sequencing analysis attributed the infection to Babesia vulpes ( B. vulpes ). An initial treatment with imidocarb dipropionate was only partially effective, while resolution of the infection was reached afterward with a combination of Malarone ® and azithromycin therapy. To the authors’ knowledge, this report describes the first case of B. vulpes infection in a dog in Italy.

Background Feline vector-borne pathogens (FeVBPs) have been increasingly investigated for their impact on cat health and their zoonotic potential. The aim of the present study was to assess the prevalence of FeVBPs and haemoplasmas in cats across Italy and to identify potential risk factors linked to their occurrence. Methods Blood samples from 958 owned cats living in the North ( n  = 556), Centre ( n  = 173) and South ( n  = 229) of Italy were tested for Babesia spp., Hepatozoon spp., Ehrlichia spp., Anaplasma spp. and filarioids by conventional PCR (cPCR) and for haemoplasmas and Bartonella spp. by SYBR green real-time PCR. Cats included in the study represent a sub-sample from a larger number of animals enrolled in a previous study, which were selected based on the geographical origin. Data on cats’ positivity for Leishmania infantum , feline leukaemia virus (FeLV) and for feline immunodeficiency virus (FIV), available from the previous study, were included and examined. Potential risk factors for pathogen infection were assessed in relationship to categorical variables including sex, geographical origin, breed, neutering status and age of cats. Results Out of the 958 cats, 194 (20.2%) were positive for at least one of the tested pathogens, 89 (16%) from the North, 32 (18.5%) from the Centre and 73 (31.9%) from the South of Italy. A high prevalence of FeVBPs was detected in male cats ( n  = 125, 27.8%), living in the southern part of the country ( n  = 73, 31.9%), younger than 18 months of age ( n  = 24, 22.4%) and not neutered (n = 39; 27.5%). In particular, 24 cats (2.5%) tested PCR-positive for Bartonella spp., of which 1.6% for B. henselae and 0.9% for B. clarridgeiae . A total of 111 cats scored PCR-positive for haemoplasmas (11.6%), specifically “ Candidatus Mycoplasma haemominutum” ( n  = 95, 9.9%), M. haemofelis ( n  = 14, 1.5%) and “ Candidatus Mycoplasma turicensis” ( n  = 2, 0.2%). Moreover, 39, 31 and 8 cats were positive for FeLV (4.1%), L. infantum (3.2%) and FIV (0.8%), respectively. Co-infections were registered for 19 (9.8%) cats. Conclusions These results confirm the occurrence of haemoplasmas and FeVBPs throughout Italy. Preventive measures to protect both animal and human health should be carried out also for owned cats, even if no health status of animals has been assessed in this study.

Tick-borne encephalitis virus (TBEV) is the causative agent of tick-borne encephalitis (TBE), a severe zoonosis occurring in the Palearctic region mainly transmitted through Ixodes ticks. In Italy, TBEV is restricted to the north-eastern part of the country. This report describes for the first time a case of clinical TBE in a roe deer (Capreolus capreolus L.). The case occurred in the Belluno province, Veneto region, an area endemic for TBEV. The affected roe deer showed ataxia, staggering movements, muscle tremors, wide-base stance of the front limbs, repetitive movements of the head, persistent teeth grinding, hypersalivation and prolonged recumbency. An autopsy revealed no significant lesions to explain the neurological signs. TBEV RNA was detected in the brain by real-time RT-PCR, and the nearly complete viral genome (10,897 nucleotides) was sequenced. Phylogenetic analysis of the gene encoding the envelope protein revealed a close relationship to TBEV of the European subtype, and 100% similarity with a partial sequence (520 nucleotides) of a TBEV found in ticks in the bordering Trento province. The histological examination of the midbrain revealed lymphohistiocytic encephalitis, satellitosis and microgliosis, consistent with a viral etiology. Other viral etiologies were ruled out by metagenomic analysis of the brain. This report underlines, for the first time, the occurrence of clinical encephalitic manifestations due to TBEV in a roe deer, suggesting that this pathogen should be included in the frame of differential diagnoses in roe deer with neurologic disease.

West Nile virus (WNV) and Usutu virus (USUV) are neurotropic mosquito-borne orthoflaviviruses maintained in an enzootic cycle, in which birds are amplifying/reservoir hosts, while humans and equids are dead-end hosts. As northern Italy, especially the Veneto Region, is considered an endemic area for WNV and USUV circulation, a surveillance plan based on a One Health approach has been implemented since 2008. This work reports the results of entomological, veterinary and human surveillances for WNV and USUV in the Veneto Region in 2022 and 2023, through virological and/or serological examinations. In 2022, 531 human WNV infections were recorded, and 93,213 mosquitoes and 2193 birds were virologically tested, showing infection rates (IRs) of 4.85% and 8.30%, respectively. The surveillance effort in 2023 provided these results: 56 human WNV infections were confirmed, and 133,648 mosquitoes and 1812 birds were virologically tested, showing IRs of 1.78% and 4.69%, respectively. This work highlights the exceptional circulation of WNV in the Veneto Region, due to the new re-introduction of WNV lineage 1 and co-circulation with WNV lineage 2. This paper confirms the efficacy of integrated surveillance for early warning of viral circulation and gives new insights about avian hosts involved in the enzootic cycle of orthoflavivirus in the endemic region of Italy.

In Europe, Ixodes ricinus is the main vector for tick-borne pathogens (TBPs), the most common tick species in Italy, particularly represented in pre-alpine and hilly northern areas. From 2011 to 2017, ticks were collected by dragging in Belluno province (northeast Italy) and analyzed by molecular techniques for TBP detection. Several species of Rickettsia spp. and Borrelia spp. Anaplaspa phagocitophilum, Neoerlichia mikurensis and Babesia venatorum, were found to be circulating in the study area carried by I. ricinus (n = 2668, all stages). Overall, 39.1% of screened pools were positive for at least one TBP, with a prevalence of 12.25% and 29.2% in immature stages and adults, respectively. Pathogens were detected in 85% of the monitored municipalities, moreover the presence of TBPs varied from one to seven different pathogens in the same year. The annual TBPs prevalence fluctuations observed in each municipality highlights the necessity of performing continuous tick surveillance. In conclusion, the observation of TBPs in ticks remains an efficient strategy for monitoring the circulation of tick-borne diseases (TBDs) in a specific area.

Dogs are proved to be competent reservoir hosts for several vector-borne pathogens. Their prevalence varies according to the geographical area. Many vector-borne pathogens may be transmitted by blood transfusion. The purpose of this study was to determine the serological and molecular prevalence of some vector-borne pathogens in dog blood donors, living in central Italy. Blood samples of 126 donors (19 breeds) included were tested for a broad serological and DNA-base tests panel. The differences in pathogen prevalence according to age, sex, and breeds were tested (chi-square test, Fisher’s exact test). Overall, 50 animals (39.7%) tested positive at PCR (polymerase chain reaction) and/or serology (IFAT, indirect fluorescent antibody test) for at least one pathogen. Three dogs were positive at both serology and PCR. A tendency of hemoplasmas to be more prevalent in older dogs (41.2%) compared to the younger ones (25.7%) was noted. We highlight the difficulties of selecting healthy blood donor dogs in an endemic area for vector-borne infections. It is important to choose the serological and biomolecular investigations panel that is most suited to the donor’s environment. Close collaboration between clinician and parasitologists is important in the interpretation of IFAT and PCR results. Finally, we underline the important role of blood donors as an epidemiological tool for active surveillance against canine vector-borne diseases.

Background The ability of tick-borne agents to survive in stored blood bags is a key factor for their transmissibility by blood transfusion. The aim of this study was to evaluate the survival and potential infectivity of Rickettsia conorii (RC) in artificially contaminated canine whole blood (WB) and in leukoreduced whole blood (LR-WB) during the storage period. Methods RC was cultured on L929 cells. We used a one-week 25-cm 2 flask with 70–80% of L929 infected cells to prepare the bacterial inoculum by pelleting cells and suspending the pellet in the donors’ serum. We infected five 100 ml WB units with RC within 2 h from the collection and maintained it at room temperature for 4 h prior to refrigeration. We filtered 50 ml of each WB bag to obtain leukoreduced WB (LR-WB) at day 1 post-infection (dpi). We checked WB and LR-WB bags at 1, 4, 7, 14, 21, 28, 35 dpi for RC presence and viability through real-time PCR (rPCR) for DNA and mRNA, respectively, and by isolation. Identification of isolates was confirmed by indirect immunofluorescence and rPCRs. Results RC survived for the entire storage period in both whole and leukoreduced blood. All bags contained viable bacteria until 7 dpi; RC viability generally decreased over time, particularly in LR-WB bags where the isolation time was longer than in WB. Viable bacteria were still isolated at 35 dpi in 3 WB and 3 LR-WB. Conclusions Leukoreduction reduced but did not eliminate RC in infected units. The survival and infectivity of RC in canine blood during the storage period may represent a threat for recipients.

Phlebotomine sand flies (Diptera: Phlebotominae) are the principal vectors of Leishmania spp. (Kinetoplastida: Trypanosomatidae). In Central Europe, Phlebotomus mascittii is the predominant species, but largely understudied. To better understand factors driving its current distribution, we infer patterns of genetic diversity by testing for signals of population expansion based on two mitochondrial genes and model current and past climate and habitat suitability for seven post-glacial maximum periods, taking 19 climatic variables into account. Consequently, we elucidate their connections by environmental-geographical network analysis. Most analyzed populations share a main haplotype tracing back to a single glacial maximum refuge area on the Mediterranean coasts of South France, which is supported by network analysis. The rapid range expansion of Ph. mascittii likely started in the early mid-Holocene epoch until today and its spread possibly followed two routes. The first one was through northern France to Germany and then Belgium, and the second across the Ligurian coast through present-day Slovenia to Austria, toward the northern Balkans. Here we present a combined approach to reveal glacial refugia and post-glacial spread of Ph. mascittii and observed discrepancies between the modelled and the current known distribution might reveal yet overlooked populations and potential further spread. An integrated approach using phylogeographic data, climate modelling, and network analysis unlocks insights into the post-glacial expansion of the sand fly Phlebotomus mascittii in Europe and suggests two potential (parallel) dispersal routes.