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Abstract 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.

Surveillance of Echinococcus multilocularis at the edge of its range is hindered by fragmented distributional patterns and low prevalence in definitive hosts. Thus, tests with adequate levels of sensitivity are especially important for discriminating between infected and non-infected areas. In this study we reassessed the prevalence of E. multilocularis at the southern border of its distribution in Province of Bolzano (Alto Adige, northeastern Alps, Italy), to improve surveillance in wildlife and provide more accurate estimates of exposure risk. We compared the diagnostic test currently implemented for surveillance based on coproscopy and multiplex PCR (CMPCR) to a real-time quantitative PCR (qPCR) in 235 fox faeces collected in 2019 and 2020. The performances of the two tests were estimated using a scraping technique (SFCT) applied to the small intestines of a subsample (n = 123) of the same foxes as the reference standard. True prevalence was calculated and the sample size required by each faecal test for the detection of the parasite was then estimated. True prevalence of E. multilocularis in foxes (14.3%) was markedly higher than reported in the last decade, which was never more than 5% from 2012 to 2018 in the same area. In addition, qPCR showed a much higher sensitivity (83%) compared to CMPCR (21%) and agreement with the reference standard was far higher for qPCR (0.816) than CMPCR (0.298) meaning that for the latter protocol, a smaller sample size would be required to detect the disease. Alto Adige should be considered a highly endemic area. Routine surveillance on definitive hosts at the edges of the E. multilocularis distribution should be applied to smaller geographic areas, and rapid, sensitive diagnostic tools using directly host faeces, such as qPCR, should be adopted.

The assessment of red fox population density is considered relevant to the surveillance of zoonotic agents vectored by this species. However, density is difficult to estimate reliably, since the ecological plasticity and elusive behavior of this carnivore hinder classic methods of inference. In this study, red fox population density was estimated using a non-invasive molecular spatial capture-recapture (SCR) approach in two study areas: one in a known hotspot of the zoonotic cestode Echinococcus multilocularis, and another naïve to the parasite. Parasitological investigations on collected samples confirmed the presence of the parasite exclusively in the former area; the SCR results indicated a higher fox population density in the control area than in the hotspot, suggesting either that the relationship between fox density and parasite prevalence is not linear and/or the existence of other latent factors supporting the parasitic cycle in the known focus. In addition, fox spotlight count data for the two study areas were used to estimate the index of kilometric abundance (IKA). Although this method is cheaper and less time-consuming than SCR, IKA values were the highest in the areas with the lower molecular SCR density estimates, confirming that IKA should be regarded as a relative index only.

Orf-virus (ORFV) is a parapoxvirus that infects small ruminants worldwide causing sporadic zoonotic infections, mainly transmitted by direct contact with sheep and goats. Following an ORFV case in a hunter of Alpine chamois (Rupicapra rupicapra), who did not report previous contact to domestic animals, a serological survey in Western Austria was conducted to assess the seroprevalence of ORFV in this species. In addition, this study also tested blood/tissue samples of chamois from different areas of the adjacent province of Bolzano/Northern Italy for antibodies against ORFV using immunofluorescence and ELISA. The observed seropositivity rates in the chamois tested on the Austrian and Italian side of the Alps were 23.5% and 9.5%, respectively, with a combined 95% confidence interval ranging from 0.0678 to 0.238. Although the prevalence was significantly lower than the one observed in Austrian sheep flocks, this study provided the first evidence that parapoxviruses have spilled over into chamois populations to a significant degree in the Tyrol regions of Austria and Italy.