Bluetongue Virus Transmission by Culicoides

At peak viremia, pots of approximately 250 adult C. sonorensis were exposed to the inner thigh of each sheep. Once returned to the laboratory, blood-fed females were selected under light CO₂ anesthesia and incubated for 8 days at 25 °C. Those surviving this period were selected under CO₂ anesthesia and placed individually in racked 96 sample polypropylene tubes (Qiagen, Crawley, UK). A selected number of blood-fed females were also processed immediately following blood feeding without incubation to obtain BTV RNA values of uptake only (D0 midges). Individual Culicoides were then homogenized using a 3-mm stainless steel ball bearing (Dejay Distribution Ltd., UK) in 100 µL of Schneider’s media (Sigma Aldrich, Poole, UK), before the addition of a further 900 µL of Schneider’s media. Pools of 400 µL were then created in polypropylene tubes using 50 µL of homogenate from each of 8 individuals and screened using the detection rtRT-PCR assay [37 (link)]. Where BTV was detected, individual samples from that pool were retested using the detection rtRT-PCR assay and the BTV-1/BTV-8 typing rtRT-PCR assays, as for sheep blood [38 (link)].