Quantifying bluetongue virus in adult Culicoides biting midges (Diptera: Ceratopogonidae)

A TissueLyser system (QIAGEN) was used to rapidly and accurately estimate bluetongue virus "loads" in individual adult Culicoides sonorensis Wirth & Jones (Diptera: Ceratopogonidae). The optimized homogenization program that was developed, involved shaking insects for 1 min at 25 Hz with 2- or 3-mm stainless steel ball bearings. This program was used to measure the quantities of bluetongue virus present in insects that had either been inoculated or had ingested a viremic bloodmeal through an artificial membrane. The virus titers obtained using either infection technique and the optimized program did not differ significantly from those obtained using a polypropylene motor-driven pestle, a method that is currently in common use for studies of vector competence). The advantages of the new method, as a rapid means of detecting fully disseminated infections in individual field-caught flies, are discussed. Its use is compared with the processing of pools of Culicoides by conventional methods, where the extent of dissemination of the virus is unknown and could wrongly implicate species that are of low importance in transmission.