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Culicoides biting midges (Diptera: Ceratopogonidae) are vectors of a variety of pathogens including African horse sickness virus (AHSV), a member of the family Reoviridae, genus Orbivirus. AHSV causes African horse sickness (AHS), an endemic disease of equids with an extremely high mortality rate in horses in sub-Saharan Africa. Culicoides (Avaritia) imicola Kieffer is considered to be the principal vector of AHSV and is the dominant Culicoides species in South Africa. Due to the global distribution of Culicoides vectors, there is a potential risk of AHS spreading from endemic areas to areas traditionally free of the disease, which could have a severe economical impact on the affected equine industry. As part of any risk assessment it is essential to monitor known vectors as well as potential vector species. In the present study, sampling of Culicoides insects was compared using overnight collections in the conventional Onderstepoort light trap and mechanical aspiration of midges at sunset from bait horses. Culicoides imicola was confirmed as the predominant species using both trapping methods. Other species, mainly Culicoides (Avaritia) bolitinos Meiswinkel and Culicoides (Avaritia) gulbenkiani Caeiro, were highly underrepresented in the light trap collections, but made a significant contribution to the mechanical aspiration catches. The time for optimal collection differed between the trapping methods, leading to the conclusion that mechanical aspiration is a useful addition to conventional light trap collection and possibly the better choice when investigating insect vectors. An infection rate of 1.14% was calculated for the midge population based on real-time quantitative reverse-transcription polymerase chain reaction (RT-qPCR) assays of collected Culicoides midges, which exceeds previous estimates. This is probably due to the increased sensitivity of the RT-qPCR assay used in this study as compared to the virus isolation assays used in previous studies. RT-qPCR-positive midges were present in midge pools obtained from both light trap and mechanical aspiration. Seven of the positive pools consisted of C. imicola only, four contained mixed species and one pool contained no C. imicola, suggesting the presence of AHSV in midges of other species.