Mosquito-borne viruses like West Nile virus (WNV) and Usutu virus (USUV) present growing public health concerns, especially with climate change and expanding vector ranges. This study describes the development and validation of a duplex Real-Time RT-PCR assay targeting β-actin (ACTB) mRNA as an endogenous control and a conserved 92 bp region shared by WNV and USUV genomes. Degenerate primers for ACTB ensure RNA extraction quality and PCR performance while enabling simultaneous detection of both viruses. A total of 1002 mosquito pools collected in Piedmont, Italy, during the 2024 vector season under the National Surveillance Plan for Arboviruses (PNA), were tested. The assay showed 100% accuracy—ACTB mRNA was detected in all pools, and six pools tested positive for WNV or USUV (three each). Diagnostic specificity was confirmed on 40 horse and bovine serum samples. Sanger sequencing confirmed ACTB identity across multiple mosquito species. The assay also demonstrated reproducibility across different operators and thermocyclers. The limit of detection (LOD) evaluation showed that the assay is capable of detecting viral RNA at very low concentrations, confirming its high analytical sensitivity. The duplex RT-PCR here developed is a reliable, sensitive, and specific tool for arbovirus surveillance, combining pathogen detection with internal quality control of RNA extraction and amplification, thus improving early warning and rapid response to mosquito-borne disease threats.

Complete animal welfare evaluation in intensive farming is challenging. With this study, we investigate new biomarkers for animal physical and mental health by comparing plasma expression of biochemical indicators in dairy cows reared in three different systems: (A) semi-intensive free-stall, (B) non-intensive tie-stall, and (C) intensive free-stall. Additionally, protein levels of mature brain-derived neurotrophic factor (mBDNF) and its precursor form (proBDNF) and indoleamine 2,3-dioxygenase (IDO1) specific activity were evaluated in brain samples collected from 12 cattle culled between 73 and 138 months of age. Alterations in plasma lipid composition and in the kynurenine pathway of tryptophan metabolism were observed in the tie-stall-reared animals. The total plasma BDNF concentration was higher in tie-stall group compared to the two free-housing groups. Brain analysis of the tie-stall animals revealed a different mBDNF/proBDNF ratio, with a higher level of proBDNF (p < 0.001). Our data are similar to previous studies on animal models of depression, which reported that inhibition of the conversion of proBDNF in its mature form and/or elevated peripheral kynurenine pathway activation may underlie cerebral biochemical changes and induce depressive-like state behavior in animals.

Aedes japonicus is an invasive Asian mosquito species, and to date it is widespread in many European countries. In Italy, it was first recorded in 2015 at the Austrian border and it then spread throughout the Northeast of the country. In 2019, it was also identified in Piedmont region, near the Swiss border. In the framework of the Italian program for prevention, surveillance, and response to Arboviruses, from June to November 2021, biweekly entomological surveillance was performed in the Liguria region (Northwest Italy). The collected mosquitoes were morphologically and genetically identified and molecularly analysed for the detection of West Nile and Usutu viruses. Six female mosquitoes, trapped on the 6th of July 2021 using a gravid trap in Albenga (Savona province), were morphologically identified as Ae. japonicus and the identification was genetically confirmed. The pool tested was negative for the presence of West Nile and Usutu viruses. The detection of Ae. japonicus was performed in a coastal area characterized by the presence of many floriculture activities. Considering the distance from the established Ae. japonicus mosquito populations in Italy and other European countries, this could represent an independent introduction in this country.

Background Tick-borne diseases are common throughout Europe. Ticks transmit pathogens to the host while feeding and together with mosquitoes, they are major vectors of infectious agents worldwide. In recent years, there has been a marked increase in the incidence of tick-bite events and tick-borne disease in northwest Italy, but information on the prevalence of tick-borne pathogens in ticks removed from humans remains scarce. To fill this gap, we report here the prevalence of tick bites and tick-borne pathogens documented for humans in Piedmont, northwest Italy, in the 3-year period 2017–2019. Methods Ticks attached to humans during 2017–2019 were collected from residents of urban and rural area by physicians and veterinarians working with local veterinary agencies. All ticks ( n = 1290) were morphologically identified to the species level. A subset of ticks removed from children (age 0–18 years) and the elderly (> 70 years), both age groups considered to be at-risk populations, was screened by biomolecular analysis to detect pathogens (e.g. Rickettsia spp., Borrelia spp., Anaplasma spp.). Pathogen identity was confirmed by Sanger sequencing. Results Ticks were taxonomically assigned to ten species of six genera ( Amblyomma , Dermacentor , Haemaphysalis , Hyalomma , Ixodes and Rhipicephalus ). Most belonged to the genus Ixodes : 1009 ticks (78.22%) were classified as Ixodes ricinus . A subset of 500 ticks collected from the two at-risk populations were subjected to PCR assay to determine the presence of Rickettsia spp., Borrelia spp., and Anaplasma spp. The overall prevalence of infection was 22.8% ( n = 114; 95% confidence interval [CI]: 19.19–26.73%), meaning that at least one pathogen was detected: Rickettsia spp. (prevalence 15%, n = 76; 95% CI 12.17–18.65%); Borrelia spp. (prevalence 6.4%, n = 32; 95% CI 4.42–8.92%); and Anaplasma spp. (prevalence 1.2%, n = 6; 95% CI 0.44–2.6%). Conclusions Our data underline the importance of surveillance in the epidemiology of tick-borne diseases and the implementation of strategies to control tick infestation and associated pathogens. Graphical Abstract

Background. Tick-borne diseases are widespread in many European Countries and high incidence has been reported in the past few years. Ticks are able to transmit several pathogens to the host while feeding and are considered the most important vectors of infectious agents worldwide together with mosquitos. As tick-bite events has remarkably increased in North-Western Italy and information about the prevalence of tick-borne pathogens in ticks removed from humans in Italy are scarce, this study aimed to identify the species of ticks biting humans and the tick-borne pathogens they can transmit.Methods. Ticks collected from bitten humans during 2017-2019 were morphologically identified and screened by biomolecular essays. A total of 1,290 ticks were analysed. Most of the ticks belonged to the Ixodes genus: 1,009 ticks (78.0%) were classified as Ixodes ricinus. Some Ixodes ticks (n = 158; 12.3%) were identified only at genus level due to lack of morphological features. Overall, 500 ticks were PCR tested for the presence of Rickettsia spp., Borrelia spp., and Anaplasma spp. in at-risk population (elderly and children). Pathogen identity was confirmed by Sanger sequencing. Results. The overall prevalence was 22.8%; (n = 114; 95%CI 19.19-26.73%), meaning that at least one pathogen was detected. Molecular analysis was carried out on a subset of 500 ticks showing the presence of R. helvetica (n=31), R. monacensis (n=35), R. slovaca (n=3), R. aeshlimannii (n=1), Rickettsia spp. (n=6); B. afzelii (n=11), B. burgdorferi sensu stricto (n=1), B. garinii (n=3), B. lusitaniae (n=4), and B. valaisiana (n=2), Borrellia spp. (n=11); Anaplasmaphagocytophilum(n=6).Conclusions. These data highlight the importance of surveillance to assess the epidemiology of TBDs that pose a threat to human health by the implementation of control strategies for both tick infestations and their associated pathogens.