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In the Old World, African horse sickness (AHS) and equine encephalosis are transmitted to equids by Culicoides midges. AHS has a case-fatality-rate of 95% in horses. Though endemic to sub-Saharan Africa, AHS virus is able to incur northwards and to disseminate widely within Mediterranean countries. For example, >1,000 equines died during the AHS-epidemics affecting Spain, Portugal and Morocco (1987-1991). The virus was isolated not only from Culicoides imicola, but also from species endemic to Europe: C.

Culicoides (Diptera: Ceratopogonidae) host preferences and attack rates were quantified in early summer at a dairy farm in the Netherlands using livestock tethered at pasture. Midges were aspirated hourly over seven consecutive hours (17:00–23:00) from a dairy cow, a Shetland pony, and a sheep and correspondingly yielded seventeen, thirteen, and nine species. Of the 14,181 midges obtained, approximately 95% belonged to the C. obsoletus complex, C. dewulfi, C. chiopterus, and C. punctatus that together include all proven or potential vectors for arboviral diseases in livestock in northwestern Europe. On average, 7.6 and 3.5 times more Culicoides were collected, respectively, from the cow and the Shetland pony than from the sheep. In descending order of abundance, the C. obsoletus complex, C. dewulfi, and C. chiopterus dominated attacks on all three hosts, whereas C. punctatus and C. pulicaris favored only the two larger hosts. Irrespective of the host species involved, the three body regions attracted the same component species, C. chiopterus favoring the legs, C. punctatus and C. achrayi the belly, and the C. obsoletus complex, C. dewulfi, and C. pulicaris the head, back, and flanks. That known and potential vectors for animal diseases feed indiscriminately on a broad range of mammal hosts means that all major livestock species, including equines, are rendered susceptible to one or more Culicoides-borne pathogens.

In studies on Culicoides attacking livestock in the Netherlands, we chanced upon a species of the Obsoletus complex that we do not recognize, but whose dark wing pattern is distinctive. Nine cytochrome c oxidase (CO1) sequences of our so-called ‘dark obsoletus’ support its status as a separate species, the sequences differing significantly from those representing Culicoides obsoletus (Meigen) (90–91% homology) and Culicoides scoticus Downes & Kettle (87–88% homology). In the last decade, several research groups in Europe have encountered ‘mystery species’ related to C. obsoletus and in some instances have made their sequences for various genetic loci available in GenBank. These include a CO1 series submitted from Sweden in 2012 (annotated as ‘obsoletus 01, 02, or 03 MA-2012′) and of which some share a 99% identity with our sequences for ‘dark obsoletus’. Without doubt, the series from the Netherlands, along with a portion of the Swedish submissions, together represent a single species (‘dark obsoletus’). Whether this species is referable to the Russian Culicoides gornostaevae Mirzaeva recorded recently from Norway, Sweden and Poland, and based solely upon the external morphology of the male, is not clear. The presence in Western Europe of multiple undescribed species related to C. obsoletus means that the taxonomy of this important vector complex is not fully resolved; consequently, we know little about these cryptic species with regard to seasonality, geographic range and host preference. This is undesirable given that Culicoides-borne arboviruses causing disease in livestock are moving more regularly out of the tropics and spreading north into temperate latitudes.

Palaearctic Culicoides midges (Diptera: Ceratopogonidae) represent a vital link in the northward advance of certain arboviral pathogens of livestock such as that caused by bluetongue virus. The effects of relevant ecological factors on weekly Culicoides vector abundances during the bluetongue virus serotype 8 epidemics in the Netherlands in 2007 and 2008 were quantified within a hurdle modelling framework. The relative role of meteorological parameters showed a broadly consistent association across species, with larger catches linked to temperature-related variables and lower wind speed. Moreover, vector abundance was found to be influenced by edaphic factors, likely related to species-specific breeding habitat preferences that differed markedly amongst some species. This is the first study on Culicoides vector species in the Netherlands identified during an entomological surveillance programme, in which an attempt is made to pinpoint the factors that influence midge abundance levels. In addition to providing key inputs into risk-mitigating tools for midge-borne pathogens and disease transmission models, the adoption of methods that explicitly address certain features of abundance datasets (frequent zero-count observations and over-dispersion) helped enhance the robustness of the ecological analysis.

BACKGROUND: Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are biological vectors of internationally important arboviruses. To understand the role of Culicoides in the transmission of these viruses, it is essential to correctly identify the species involved. Within the western Palaearctic region, the main suspected vector species, C. obsoletus, C. scoticus, C. dewulfi and C. chiopterus, have similar wing patterns, which makes it difficult to separate and identify them correctly. METHODS: In this study, designed as an inter-laboratory ring trial with twelve partners from Europe and North Africa, we assess four PCR-based assays which are used routinely to differentiate the four species of Culicoides listed above. The assays based on mitochondrial or ribosomal DNA or microarray hybridisation were tested using aliquots of Culicoides DNA (extracted using commercial kits), crude lysates of ground specimens and whole Culicoides (265 individuals), and non-Culicoides Ceratopogonidae (13 individuals) collected from across Europe. RESULTS: A total of 800 molecular assays were implemented. The in-house assays functioned effectively, although specificity and sensitivity varied according to the molecular marker and DNA extraction method used. The Obsoletus group specificity was overall high (95-99%) while the sensitivity varied greatly (59.6-100%). DNA extraction methods impacted the sensitivity of the assays as well as the type of sample used as template for the DNA extraction. CONCLUSIONS: The results are discussed in terms of current use of species diagnostic assays and the future development of molecular tools for the rapid differentiation of cryptic Culicoides species.

Background: Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are biological vectors of internationally important arboviruses. To understand the role of Culicoides in the transmission of these viruses, it is essential to correctly identify the species involved. Within the western Palaearctic region, the main suspected vector species, C. obsoletus, C. scoticus, C. dewulfi and C. chiopterus, have similar wing patterns, which makes it difficult to separate and identify them correctly. Methods: In this study, designed as an inter-laboratory ring trial with twelve partners from Europe and North Africa, we assess four PCR-based assays which are used routinely to differentiate the four species of Culicoides listed above. The assays based on mitochondrial or ribosomal DNA or microarray hybridisation were tested using aliquots of Culicoides DNA (extracted using commercial kits), crude lysates of ground specimens and whole Culicoides (265 individuals), and non-Culicoides Ceratopogonidae (13 individuals) collected from across Europe. Results: A total of 800 molecular assays were implemented. The in-house assays functioned effectively, although specificity and sensitivity varied according to the molecular marker and DNA extraction method used. The Obsoletus group specificity was overall high (95-99%) while the sensitivity varied greatly (59.6-100%). DNA extraction methods impacted the sensitivity of the assays as well as the type of sample used as template for the DNA extraction. Conclusions: The results are discussed in terms of current use of species diagnostic assays and the future development of molecular tools for the rapid differentiation of cryptic Culicoides species.

To determine which species of Culicoides biting midges carry Schmallenberg virus (SBV), we assayed midges collected in the Netherlands during autumn 2011. SBV RNA was found in C. scoticus, C. obsoletus sensu stricto, and C. chiopterus. The high proportion of infected midges might explain the rapid spread of SBV throughout Europe.

To determine which species of Culicoides biting midges carry Schmallenberg virus (SBV), we assayed midges collected in the Netherlands during autumn 2011. SBV RNA was found in C. scoticus, C. obsoletus sensu stricto, and C. chiopterus. The high proportion of infected midges might explain the rapid spread of SBV throughout Europe. During early summer 2011, Schmallenberg virus (SBV), a novel orthobunyavirus of the Simbu serogroup, spread across much of northern Europe, infecting ruminant livestock. The Simbu serogroup (family Bunyaviridae, genus Bunyavirus) includes Shamonda virus, Akabane virus, Sathuperi virus, and Aino virus. These viruses cause teratologic effects in ruminants and are arthropod-borne, and most have been isolated in the Old World from mosquitoes and Culicoides spp. biting midges (1). Recent preliminary studies indicate that =1 species of Culicoides midges act as field vectors for SBV in Europe (2). To determine which Culicoides midge species harbor SBV, we analyzed midges collected from 3 livestock holdings in eastern and northeastern parts of the Netherlands.

Geographical maps indicating the value of the basic reproduction number, R 0 , can be used to identify areas of higher risk for an outbreak after an introduction. We develop a methodology to create R 0 maps for vector-borne diseases, using bluetongue virus as a case study. This method provides a tool for gauging the extent of environmental effects on disease emergence. The method involves integrating vector-abundance data with statistical approaches to predict abundance from satellite imagery and with the biologically mechanistic modelling that underlies R 0 . We illustrate the method with three applications for bluetongue virus in the Netherlands: 1) a simple R 0 map for the situation in September 2006, 2) species-specific R 0 maps based on satellite-data derived predictions, and 3) monthly R 0 maps throughout the year. These applications ought to be considered as a proof-of-principle and illustrations of the methods described, rather than as ready-to-use risk maps. Altogether, this is a first step towards an integrative method to predict risk of establishment of diseases based on mathematical modelling combined with a geographic information system that may comprise climatic variables, landscape features, land use, and other relevant factors determining the risk of establishment for bluetongue as well as of other emerging vector-borne diseases.

Geographical maps indicating the value of the basic reproduction number, R0, can be used to identify areas of higher risk for an outbreak after an introduction. We develop a methodology to create R0 maps for vector-borne diseases, using bluetongue virus as a case study. This method provides a tool for gauging the extent of environmental effects on disease emergence. The method involves integrating vector-abundance data with statistical approaches to predict abundance from satellite imagery and with the biologically mechanistic modelling that underlies R0. We illustrate the method with three applications for bluetongue virus in the Netherlands: 1) a simple R0 map for the situation in September 2006, 2) species-specific R0 maps based on satellite-data derived predictions, and 3) monthly R0 maps throughout the year. These applications ought to be considered as a proof-of-principle and illustrations of the methods described, rather than as ready-to-use risk maps. Altogether, this is a first step towards an integrative method to predict risk of establishment of diseases based on mathematical modelling combined with a geographic information system that may comprise climatic variables, landscape features, land use, and other relevant factors determining the risk of establishment for bluetongue as well as of other emerging vector-borne diseases.

Geographical maps indicating the value of the basic reproduction number, R0, can be used to identify areas of higher risk for an outbreak after an introduction. We develop a methodology to create R0 maps for vector-borne diseases, using bluetongue virus as a case study. This method provides a tool for gauging the extent of environmental effects on disease emergence. The method involves integrating vector-abundance data with statistical approaches to predict abundance from satellite imagery and with the biologically mechanistic modelling that underlies R0. We illustrate the method with three applications for bluetongue virus in the Netherlands: 1) a simple R0 map for the situation in September 2006, 2) species-specific R0 maps based on satellite-data derived predictions, and 3) monthly R0 maps throughout the year. These applications ought to be considered as a proof-of-principle and illustrations of the methods described, rather than as ready-to-use risk maps. Altogether, this is a first step towards an integrative method to predict risk of establishment of diseases based on mathematical modelling combined with a geographic information system that may comprise climatic variables, landscape features, land use, and other relevant factors determining the risk of establishment for bluetongue as well as of other emerging vector-borne diseases.