Investigating the Role of Stable Flies (Stomoxys calcitrans) and Biting Midges (Culicoides spp.) as Potential Mechanical Vectors of African Swine Fever Virus in Lithuania, Poland and Romania.

Background Since its emergence in Georgia in 2007, the seasonal pattern of African swine fever virus (ASFV) genotype II outbreaks in European pig populations has been evident. It is hypothesized that summer-related farming practices, along with the increased activity and abundance of arthropod vectors during warmer months, contribute to the increased incidence of these outbreaks during this period. This study investigated the potential role of stable flies ( Stomoxys calcitrans ) and biting midges ( Culicoides spp.) as mechanical vectors of ASFV. Methods From August 2021 to August 2023, vector surveillance was conducted as part of a case-control study on 42 outbreak farms and 70 control farms across Romania, Lithuania, and Poland. Collected insect specimens were pooled and tested for ASFV DNA using real-time PCR. Results A total of 8,604 biting midges’ specimens and 742 S. calcitrans flies were collected, with ASF DNA detected in 27 out of 1,219 insect pools. Positive pools were predominantly observed in Culicoides punctatus , C. newsteadi , and the Obsoletus complex, with most detections occurring in August. However, ASFV isolation was unsuccessful. Statistical analyses revealed no significant association between farm status (outbreak vs. control) and ASFV DNA detection in biting midges’ pools, likely due to limited sample size. Additionally, two ASFV-positive S. calcitrans pools were identified, supporting their potential role as mechanical vehicles for ASFV. The findings highlight the affinity of C. punctatus , C. newsteadi , and the Obsoletus complex and S. calcitrans for pig farms, which may increase the likelihood of ASFV acquisition and dissemination, assuming viral infectivity is maintained in these insects. Environmental factors, such as pig farm density and proximity to other hosts, likely influence this risk. Conclusions These results emphasize the importance of vector control strategies, including insect netting, to mitigate ASFV transmission risks. Further research is needed to understand the dynamics of ASFV infection in arthropods. Notably, this study also reports the first identification of Culicoides riethi and Culicoides salinarius in Lithuania.