Background: Echinococcus multilocularis is a small tapeworm affecting wild and domestic carnivores and voles in a typical prey-predator life cycle. In Italy, there has been a focus of E. multilocularis since 1997 in the northern Italian Alps, later confirmed in red foxes collected from 2001 to 2005. In this study, we report the results of seven years of monitoring on E. multilocularis and other cestodes in foxes and describe the changes that occurred over time and among areas (eco-regions) showing different environmental and ecological features on a large scale. Methods: Eggs of cestodes were isolated from feces of 2872 foxes with a sedimentation/filtration technique. The cestode species was determined through multiplex PCR, targeting and sequencing ND1 and 12S genes. Analyses were aimed to highlight variations among different eco-regions and trends in prevalence across the study years. Results: Out of 2872 foxes, 217 (7.55%) samples resulted positive for cestode eggs at coproscopy, with differences of prevalence according to year, sampling area and age class. Eight species of cestodes were identified, with Taenia crassiceps (2.65%), Taenia polyacantha (1.98%) and E. multilocularis (1.04%) as the most represented. The other species, Mesocestoides litteratus, Taenia krabbei, T. serialis, T. taeniaeformis and Dipylidium caninum, accounted for < 1% altogether. Echinococcus multilocularis was identified in foxes from two out of six eco-regions, in 30 fecal samples, accounting for 1.04% within the cestode positives at coproscopy. All E. multilocularis isolates came from Bolzano province. Prevalence of cestodes, both collectively and for each of the three most represented species (T. crassiceps, T. polyacantha and E. multilocularis), varied based on the sampling year, and for E. multilocularis an apparent increasing trend across the last few years was evidenced. Conclusions: Our study confirms the presence of a focus of E. multilocularis in red foxes of northeast Italy. Although this focus seems still spatially limited, given its persistence and apparent increasing prevalence through the years, we recommend research to be conducted in the future on the ecological factors that, on a smaller scale, allow this zoonotic species to persist. On the same scale, we recommend a health education campaign to inform on the measures to prevent this zoonosis, targeted at people living in the area, especially hunters, dog owners, forestry workers and other potentially exposed categories.

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.

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.

Toxocara cati is a common roundworm of cats and wild felids and, together with T. canis, it is the main causative agent of human toxocariasis. Humans may become infected by ingestion of embryonated eggs via contaminated soil, food, or water, or by ingestion of raw or undercooked meat of paratenic hosts that are infected by Toxocara larvae. In this study, we report the detection of T. cati larvae from meat samples of ostriches and wild boars. These samples were inspected by enzymatic digestion, as part of the trichinellosis surveillance. As ostrich meat is intended for “carpaccio” preparation, a traditional Italian raw meat preparation, there is the need to make the consumption of this meat safe. For this purpose, it is recommended to freeze the meat before preparation. Our findings confirmed that T. cati larvae can contaminate muscle tissues of paratenic hosts, increasing the risk of infection due to the consumption of raw or undercooked meat.

Background: Different trapping devices and attractants are used in the mosquito surveillance programs currently running in Europe. Most of these devices target vector species belonging to the genera Culex or Aedes, and no studies have yet evaluated the effectiveness of different trapping devices for the specific targeting of Anopheles mosquito species, which are potential vectors of malaria in Europe. This study aims to fill this gap in knowledge by comparing the performance of trapping methods that are commonly used in European mosquito surveillance programs for Culex and Aedes for the specific collection of adults of species of the Anopheles maculipennis complex. Methods: The following combinations of traps and attractants were used: (i) BG-Sentinel 2 (BG trap) baited with a BG-Lure cartridge (BG + lure), (ii) BG trap baited with a BG-Lure cartridge and CO₂ (BG + lure + CO₂), (iii) Centers for Disease Control and Prevention-like trap (CDC trap) baited with CO₂ (CDC + CO₂), (iv) CDC trap used with light and baited with BG-Lure and CO₂ (CDC light + lure + CO₂). These combinations were compared in the field using a 4 × 4 Latin square study design. The trial was conducted in two sites in northeastern Italy in 2019. Anopheles species were identified morphologically and a sub-sample of An. maculipennis complex specimens were identified to species level by molecular analysis. Results: Forty-eight collections were performed on 12 different trapping days at each site, and a total of 1721 An. maculipennis complex specimens were captured. The molecular analysis of a sub-sample comprising 254 specimens identified both Anopheles messeae/Anopheles daciae (n = 103) and Anopheles maculipennis sensu stricto (n = 8) at site 1, while at site 2 only An. messeae/An. daciae (n = 143) was found. The four trapping devices differed with respect to the number of An. messeae/An. daciae captured. More mosquitoes were caught by the BG trap when it was used with additional lures (i.e. BG + lure + CO₂) than without the attractant, CO₂ [ratio_{BG+lure vs BG+lure+CO2} = 0.206, 95% confidence interval (CI) 0.101–0.420, P < 0.0001], while no significant differences were observed between CDC + CO₂ and CDC light + lure + CO₂ (P = 0.321). The addition of CO₂ to BG + lure increased the ability of this combination to capture An. messeae/An. daciae by a factor of 4.85, and it also trapped more mosquitoes of other, non-target species (Culex pipiens, ratio_{BG+lure vs BG+lure+CO2} = 0.119, 95% CI 0.056–0.250, P < 0.0001; Ochlerotatus caspius, ratio_{BG+lure vs BG+lure+CO2} = 0.035, 95% CI 0.015–0.080, P < 0.0001). Conclusions: Our results show that both the BG-Sentinel and CDC trap can be used to effectively sample An. messeae/An. daciae, but that the combination of the BG-Sentinel trap with the BG-Lure and CO₂ was the most effective means of achieving this. BG + lure + CO₂ is considered the best combination for the routine monitoring of host-seeking An. maculipennis complex species such as An. messeae/An. daciae. The BG-Sentinel and CDC traps have value as alternative methods to human landing catches and manual aspiration for the standardized monitoring of Anopheles species in Europe. Graphical

Ixodes ricinus ticks are ubiquitous in Europe, including in North-East Italy. These ticks are important vectors of several zoonotic pathogens of public health relevance. In this study, the habitat suitability range of I. ricinus ticks infected with zoonotic pathogens was predicted in North-East Italy, and relevant spatial predictors were identified. In 2015–2021, ticks were collected at 26 sampling sites in the study area. The collected ticks were screened for the presence of pathogens using PCR assays. For Borrelia, Rickettsia and Anaplasma/Ehrlichia species, data allowed for ecological niche modelling using Maxent. Environmental determinants potentially related to tick habitat suitability were used as model inputs. Predicted suitable habitat distributions revealed hotspots of the probability of pathogen presence in I. ricinus ticks mainly in the central and upper parts of the study area. Key environmental predictors were temperature, rainfall and altitude, and vegetation index for specific pathogens (Rickettsia and Anaplasma/Ehrlichia species). Increased risk of exposure to tick-borne pathogens upon tick bites in the predicted hotspot areas can, therefore, be expected. This provides useful information for public health risk managers in this and other similar regions.