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Background West Nile (WNV) and Usutu (USUV) are emerging vector-borne zoonotic flaviviruses. They are antigenically very similar, sharing the same life cycle with birds as amplification host, Culicidae as vector, and man/horse as dead-end host. They can co-circulate in an overlapping geographic range. In Europe, surveillance plans annually detect several outbreaks. Methods In Italy, a WNV/USUV surveillance plan is in place through passive and active surveillance. After a 2018 WNV outbreak, a reinforced integrated risk-based surveillance was performed in four municipalities through clinical and serological surveillance in horses, Culicidae catches, and testing on human blood-based products for transfusion. Results Eight WNV cases in eight equine holdings were detected. Twenty-three mosquitoe catches were performed and 2367 specimens of Culex pipiens caught; 17 pools were USUV positive. A total of 8889 human blood donations were tested, and two asymptomatic donors were USUV positive. Conclusions Different surveillance components simultaneously detected WNV only in horses and USUV only in humans and mosquitoes. While in endemic areas (i.e. northern Italy) entomological surveillance is successfully used as an early detection warning, this method in central Italy seems ineffective. To achieve a high level of sensitivity, the entomological trapping effort should probably exceed a reasonable balance between cost and performance. Besides, WNV/USUV early detection can be addressed by horses and birds. Further research is needed to adapt the surveillance components in different epidemiological contexts. Graphical Abstract

Coccidiosis due to Eimeria spp. are widespread parasitic infections in cattle and water buffaloes and may impair health, welfare, and production of these livestock species. The aims of this study were (i) to investigate the prevalence and seasonal dynamics of eimeriosis and (ii) to characterize the Eimeria species in large ruminants in a Mediterranean area, in order to plan effective control strategies. Parasitological data were obtained from a 10-year surveillance program (2010–2019) on 3,631 farms (2,089 buffalo and 1,542 cattle farms) sampled in central and southern Italy. Pooled fecal samples were analyzed using the FLOTAC technique with an analytic sensitivity of 2 oocysts per gram of feces (OPG) utilizing a saturated sodium chloride flotation solution (specific gravity = 1.200). Eimeria species identification was performed by morphometric analysis after a one week incubation of oocysts in a 2.5% potassium dichromate solution. The results showed high prevalence of Eimeria (up to 100%) in both cattle and buffaloes in the 10 years of surveillance, even if a slight reduction was reported in the last three years. The overall prevalence of eimeriosis was 91.7% (95% confidence interval, 95% CI = 90.2–93.1) in cattle farms and 81.5% (95% CI = 79.8–83.1) in water buffalo farms. The mean OPG value was 66.8 (min = 2; max = 8,065) in cattle and 55.9 (min = 2; max = 15,415) in water buffaloes, but this difference was not statistically significant (p > 0.05). In total, nine species of Eimeria were found in cattle the most prevalent being Eimeria bovis, E. ellipsoidalis, E. cylindrica, and E. zuernii, whereas in water buffaloes eight species of Eimeria were found, the most prevalent being E. ellipsoidalis, E. auburnensis, E. bovis, and E. zuernii. Mixed infections were common in both ruminant species. The seasonal pattern showed a higher prevalence of eimeriosis in cattle in spring (86.9%) whereas in buffalo farms the prevalence was higher in winter (82.3%) and summer (82.4%). In conclusion, the 10-year surveillance program indicates that eimeriosis is common in cattle and water buffaloes and therefore continuous effective control strategies are needed.

Background Angiostrongylus vasorum is a nematode residing in the heart and pulmonary vessels of dogs and wild carnivores. In Europe the red fox is its reservoir, while only three records from wolves have been published. Angiostrongylus vasorum has a worldwide distribution, and many pieces of evidence demonstrate that it is spreading from endemic areas to new ones. In Italy, A. vasorum was reported with increasing frequency in dogs and foxes in the last decades, and now it is considered endemic throughout the country. Angiostrongylus vasorum can be asymptomatic or cause respiratory and circulatory disorders, at times causing severe disseminated infections. Methods Between February 2012 and December 2016, 25 wolves found dead in central Italy were submitted to the Istituto Zooprofilattico del Lazio e della Toscana for post-mortem examination. Samples of lungs, heart, liver, spleen, kidneys, mediastinic lymph nodes and brain were collected from each animal for histological examination. When adult and larval nematodes were microscopically seen in lungs, the other organs were processed, and five histological sections for each organ were examined. To confirm parasite identification, lung samples were submitted to a PCR-sequencing protocol targeting the ITS2 region of A. vasorum. Results Seven wolves (28.0%) harboured nematode larvae in lung sections. In two of the positive wolves, adult nematodes were visible in pulmonary arteries, in four animals larvae were also detected in other organs. DNA sequencing reactions confirmed parasite identification as A. vasorum in all the cases. Conclusions As a result of the high prevalence of A. vasorum reported in wolves in the present study, a focus of high circulation could be hypothesised in central Italy. Nevertheless, the similarly high prevalence in foxes originating from the same areas were reported in previous papers. Histopathological evidence highlights the pathogenic potential of A. vasorum in the wolf, especially in juvenile animals.