Circulation of Dirofilaria immitis and Dirofilaria repens in the Danube Delta Biosphere Reserve, Romania

Background: Dirofilariosis is an emerging vector-borne parasitic disease in Europe. Monitoring of wild and domestic carnivores demonstrated circulation of Dirofilaria spp. in Romania in the past. For the implementation of control measures, knowledge on the native mosquito community responsible for Dirofilaria spp. transmission is required. Methods: Mosquito samples originated from a longitudinal study previously performed in the Danube Delta Biosphere Reserve. Mosquito pools were screened for Dirofilaria immitis and Dirofilaria repens. The samples comprised 240,572 female mosquito specimens collected every ten days between April and September in 2014 at four different trapping sites. In addition, blood samples of 36 randomly selected dogs were collected in 2016 in each of the four mosquito sampling sites. A duplex real-time assay was used to detect the presence of one or both Dirofilaria species for each sample. This assay targets the cytochrome c oxidase subunit 1 and the 16S rRNA gene fragments to differentiate both parasites. Results: Dirofilaria immitis and D. repens were detected in mosquito pools at all four trapping sites. In the 2118 mosquito pools tested, D. immitis was identified for eight and D. repens for six of the 14 screened mosquito taxa, with a higher prevalence of D. immitis (4.53% of analysed pools) compared to D. repens (1.09%). Dirofilaria spp. were also identified in dogs from the same sampling sites with a prevalence of 30.56%. For both Dirofilaria species, the highest estimated infection rates (EIRs) were found in Anopheles maculipennis (s.l.) (D. immitis: EIR = 0.206 per 100 specimens, D. repens: EIR = 0.066 per 100 specimens). In contrast, Coquillettidia richiardii and Anopheles hyrcanus as the most frequent taxa had infection rates which were significantly lower: Cq. richiardii (D. immitis: EIR=0.021; D. repens: EIR = 0.004); An. hyrcanus (D. immitis: EIR = 0.028; D. repens: EIR = 0.006). The number of positive pools per calendar week was positively correlated with the number of screened pools per calendar week, suggesting constant Dirofilaria spp. transmission during the observation period. Conclusions: This study further confirms significant circulation of Dirofilaria spp. in eastern Europe, with high parasite prevalence in domestic canids and mosquitoes. Therefore, systematic monitoring studies are required to better understand the environmental risk factors for Dirofilaria transmission, allowing the implementation of effective surveillance and control measures.