First Evidence of Bovine Leukemia Virus in Culicoides Biting Midges and the Stable Fly Stomoxys calcitrans Collected on Cattle Farms in Poland.

Bovine leukemia virus (BLV) is a cell-associated retrovirus of cattle transmitted mainly through the transfer of infected lymphocytes, but the possible role of hematophagous insects in its epidemiology remains incompletely understood. This study investigated BLV DNA in field-collected Culicoides biting midges and Stomoxys calcitrans from cattle farms in Poland. Insects were collected on four farms in northeastern Poland. Culicoides spp. were identified morphologically, classified by gonotrophic status, and pooled by species and status, whereas S. calcitrans were analyzed individually. DNA quality was verified by PCR targeting insect markers. Bovine host DNA was detected by PCR targeting mitochondrial cytochrome b in Culicoides and prepronociceptin (PNOC) in S. calcitrans. BLV proviral DNA was detected by TaqMan real-time PCR targeting the pol gene, followed by nested PCR and sequencing of a 400 bp fragment of the env gene. BLV DNA was detected in 20 of 138 Culicoides pools (14.5%). BLV copy numbers ranged from 2 to 19,000 copies per 500 ng of total DNA. All BLV-positive Culicoides pools were also positive for bovine cytochrome b DNA. Among the pol-positive pools, 18 were positive in nested env PCR, and 15 yielded high-quality sequences. One S. calcitrans specimen was also positive in both BLV assays. Sequences obtained from insects were identical to those from infected cattle, and phylogenetic analysis assigned them to genotypes G4 and G7, the predominant BLV genotypes circulating in Poland. These findings provide field-based molecular evidence that Culicoides biting midges and S. calcitrans can carry bovine blood and BLV DNA under natural farm conditions, supporting their possible role in the mechanical transfer of BLV between cattle.