Winter activity and virus infection of Culicoides biting midges (Diptera: Ceratopogonidae) in German livestock housing before and during the 2024 BTV-3 outbreak

BACKGROUND: Despite its importance for Culicoides-borne disease risk assessment, the winter activity of species of this biting midge genus remains poorly characterized. Moreover, information on virus circulation in winter-active Culicoides vectors, both under endemic conditions and during outbreaks, is almost non-existent. METHODS: UV-light traps to collect Culicoides biting midges were operated once a week for 24 h in cattle, sheep, goat and horse stables at 12 to 55 locations during four consecutive winter periods (November 1 to March 31, 2021 to 2025) throughout Germany. Collected females of the genus Culicoides were morphologically assigned to Obsoletus Group or Pulicaris Complex. Temperature data were recorded to relate biting midge activity to temperatures. Screening for bluetongue virus (BTV), Schmallenberg virus and epizootic hemorrhagic disease virus was done by real-time RT-PCR on 1,581 pools (46,358 females) of both groups collected before (winter 2022/2023) and during the BTV serotype 3 (BTV-3) outbreak (winters 2023/2024 and 2024/2025). RESULTS: Culicoides collections displayed notable variation between sites and months. November and March accounted for 95% of captures, while mid-winter months (December to February) yielded only 5%, with January and February each contributing 1% of total captures. The March 2024 and 2025 activity peaks coincided with mild temperatures and the inclusion of new monitoring sites, suggesting that one or both factors contributed. Although blood-fed females were very rare during mid-winter, their abundance often exceeded the EU threshold for parous Culicoides to define a vector-free period of < 5 individuals per trap and night. However, only a single Culicoides pool from November 2024 tested positive for BTV-3-RNA. All other pools were negative for all three viruses. CONCLUSIONS: Potential Culicoides vectors are active and feed on blood in livestock housings during wintertime. The presence of blood-fed females, exceeding the EU threshold, challenges the concept of a 'vector-free period'. However, despite extensive monitoring over four years including outbreak conditions, only one virus-positive Culicoides pool was detected during winter. Combined with the very low number of biting midges and the temperatures not allowing virus replication in arthropods, virus circulation during wintertime appears to be hardly existent even under endemic or outbreak conditions. Considering both Culicoides activity and virus circulation, the findings suggest that the risk of BTV infection in ruminants via biting midges during the central European winter (December to March) is negligible. BTV overwintering in central Europe likely depends on either rare infection events in ruminants and vectors during winter that sustain viremia until spring, or on persistence mechanisms that are currently unknown.