Circulation of Dirofilaria immitis DNA in mosquitos of Emilia-Romagna region (Italy): An alternative way to complete the epidemiological data set of heartworm parasite diffusion in our country

Dirofilaria immitis, the agent of canine and feline heartworm disease, is a widespread mosquito-borne helminth. The distribution of D. immitis in Europe is expanding from traditionally endemic areas to areas that were, until recently, considered free of infection (Fuehrer et al., 2021). Essential prerequisites for heartworm transmission include the presence of competent mosquito vectors, a climate that provides adequate temperature and humidity to support the mosquito population and that also allow maturation of ingested microfilariae into infective, third-stage larvae (L3) within the vector. The present study is aimed at updating current knowledge of the composition of competent vector species in the northern region of Emilia Romagna, a traditionally endemic area for D. immitis and D. repens. Mosquitoes were collected in 2022 by the "Istituto Zooprofilattico della Lombardia ed Emilia-Romagna (IZSLER)" as part of the regional surveillance plan for West Nile Virus (WNV). The capture zones included peri- urban and rural areas and mosquitos were captured with CDC-CO2 traps. Mosquitoes were stored at -20°C and then sorted by capture zone and species identified following the dichotomous keys (Becker et al. 2020). DNA from female mosquitoes was extracted and analyzed for the presence of D. immitis and D. repens (Sulesco et al. 2016). From the positive pools, RNA was also extracted and analyzed for the presence of WNV (Eiden et al., 2010). The majority of captured mosquitos belonged to Culex pipiens species followed by Aedes caspius, Aedes vexans, Aedes albopictus. Of these 8248 and 15746 female mosquitos (about 30% of each species) have been randomly selected and used to create pool for DNA extraction. A total of 140 pools (~ 20 mosquitos/each) in 2022 and 133 in 2023 have been analyzed. DNA of D. immitis was identified in 14 pools of 2022 captures and in 15 pools of 2023. None of the pools was positive for D. repens neither for WNV. In 2022, about 85% of the positive pools belonged to Ae. caspius species (11/13) and the other three pools to Ae. vexans. In 2023, 73% of the positive pools belonged to Ae. caspius, followed by Ae. vexans and Ae. albopictus (both 13.3%). A significant overlap emerged from the same traps positioned in Ferrara and Bologna province, which tested positive for D. immitis in both 2022 and 2023. These data highlight how, despite the abundance of C. pipiens captured, the most receptive species for D. immitis appear to be Ae. caspius and Ae. vexans. Furthermore, the geographical data highlights how the areas of the province of Ferrara and Bologna are the main geographical reservoirs of the parasite. Further in- depth studies should be carried out by using other types of traps and also focusing on urban areas. Becker et al., 2020, Springer Eiden et al., 2010 J Vet Diagnostic Invest 22(5):748-53 Fuehrer et al., 2021 Pathogens 10(10):1268 Sulesco et al., 2016 Parasit Vectors 9:1–10