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Abstract: 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. [Copyright &y& Elsevier]

Culicoides truuskae Labuschagne and Meiswinkel sp. n. is described and illustrated in both sexes from material collected in South Africa and Namibia. It is restricted to the xeric western margin of the subcontinent, occurring in Fynbos, Nama-Karoo and Succulent Karoo ecoregions in South Africa and Desert and Savanna ecoregions in Namibia experiencing 600 mm of rainfall annually. Culicoides truuskae sp. n. is part of the Afrotropical 'plain-wing' Culicoides in which the wing lacks a distinguishing pattern of light and dark spots; the diagnostic dark smudge that traverses wing cell r3 may result in C. truuskae sp. n. being misidentified as the sympatric but phyletically unrelated Culicoides herero (Enderlein) - (of the Similis group, subgenus Oecacta Poey). Additionally, this study is the first description of the male of C. herero. C. truuskae sp. n. and Culicoides coarctatus Clastrier and Wirth share similar characters in the male genitalia, although the two species are separable on wing pattern and female flagellum sensilla coeloconica (SCo) distribution. The breeding habitat and adult female blood-feeding preferences of C. truuskae sp. n. are not known. A maximum likelihood phylogenetic tree, using mitochondrial cytochrome c oxidase I (COI) sequence data, is provided to further clarify the relationship between C. truuskae sp. n., C. coarctatus and C. herero. Extensive light trap data, collected over 30 years, are used to map the distribution ranges of C. truuskae sp. n., C. coarctatus and C. herero in Southern Africa.Contribution: The description of this new species and the description of the male of C. herero increases our understanding of the diversity and distribution of Culicoides species in southern Africa.

As Bluetongue virus (BTV) spread in Italy following its first incursion in 2000, it soon became apparent that, besides Culicoides imicola, additional species of the subgenus Avaritia were involved as vectors, namely one or more of the species that belong to the so-called 'Culicoides obsoletus group', which comprises C. dewulfi, C. chiopterus, C. obsoletus sensu stricto, C. scoticus and C. montanus; the three last named species are considered generally as forming the Obsoletus complex. This study presents the findings made over the last decade and more, within the Italian entomological surveillance program for Bluetongue. It describes the integrated morphological and molecular approach used to identify the species of the 'C. obsoletus group', maps in detail their relative abundances and geographic ranges in Italy, clarifies the hitherto unknown comparative seasonal abundances of C. obsoletus s.s. and C. scoticus in a site in Central Italy, and provides further details on the potential vector status of five species of the 'C. obsoletus group', with emphasis on C. obsoletus s.s., C. scoticus and C. montanus. Unlike the situation in Northern Europe, Culicoides dewulfi and C. chiopterus are uncommon to rare in Italy. In contrast, the Obsoletus complex occurs abundantly throughout Italy, with C. obsoletus s.s. being the most prevalent and ecologically adaptive of the three species making up the complex. A longitudinal study conducted at a site in Central Italy revealed that: (i) species of the Obsoletus complex prefer horses to sheep; (ii) their parity rates range from 10% (March) to 56% (November); (iii) throughout the year C. scoticus is consistently more abundant than C. obsoletus s.s.; (iv) abundances in both, C. obsoletus s.s. and C. scoticus, peak in May-June, with the peak of the latter species being more evident. Bluetongue virus was first isolated from wild caught midges of the Obsoletus complex in 2002. Thereafter, pools of selected parous midges collected across Italy, and during multiple outbreaks of BT, have been found consistently PCR-positive for the virus. More recently, viral RNA has been detected in field specimens of C. dewulfi, C. obsoletus s.s., C. scoticus and C. montanus.

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.

Bluetongue (BT) is an orbiviral disease of ruminant livestock. In Europe, the insect vectors implicated in BT virus transmission are Culicoides imicola, C. obsoletus, C. scoticus, C. pulicaris and C. dewulfi. C. imicola - an Afro-Asiatic species - is believed to account for approximately 90% of BT virus transmission; the latter four species are endemic to the Palaearctic region and are becoming increasingly important as the zone of BT virus infection in Europe moves northwards. As Culicoides distribution and ecology is critical in the spread of BT, it is essential to map their geographic ranges and to determine the ecological and climatic factors influencing their occurrence. Since 2000, more than 50 000 light trap collections have been made across Italy and the presence/absence and abundance of C. imicola ascertained; 3000 of these collections were also screened for two species (C. obsoletus, C. scoticus) of the Obsoletus Complex. To gain further insights into vector breeding habitats, spatial analysis techniques and statistical methodologies were used to investigate the association between the trap collection (municipality [polygons] and geographic point level) data and environmental and climatic variables. For the first dataset, 100 municipalities with the largest collections of C. imicola and 100 of the Obsoletus Complex, were selected and the average values of six independent climatic/environmental variables were calculated for each polygon. Factor analysis identified two principal factors explaining 84% of the total variability in climatic and environmental variables; 87.5% of trap collections were correctly classified by a discriminant analysis model. C. imicola was found to predominate in more sparsely vegetated habitats. Obsoletus Complex species were associated with a more densely vegetated habitat. For the second dataset, the presence/ absence of C. imicola at 172 sites in the region of Calabria (southern Italy) was investigated using 13 climatic, environmental and pedological variables. Factor analysis identified four factors; 89.5% of trap collections were correctly classified by a discriminant analysis model. Proximity to water-holding, clay soils was the most important factor explaining the markedly disjoint distribution of C. imicola in the 'toe' of Italy. Soil type may explain the patchy distribution of C. imicola elsewhere in Mediterranean Europe.

During the recent severe outbreaks of bluetongue (BT) in the Mediterranean Basin, the BT virus (BTV) spread beyond its historical limits into the Balkan region. One of the primary impacts of BT is the cessation in livestock trade which can have severe economic and social consequences. The authors briefly describe the development of the collaborative East-BTnet programme which aims to assist all affected and at-risk Balkan states and adjoining countries in the management of BT, and in the development of individual national surveillance systems. The beneficiary countries involved, and led by the World organisation for animal health (Office International des Epizooties) Collaborating Centre for veterinary training, epidemiology, food safety and animal welfare of the Istituto Zooprofilattico dell'Abruzzo e del Molise 'G. Caporale' in collaboration with the Institute for the Protection and the Security of the Citizen, the European Commission Joint Research Centre (IPSC-JRC), were Albania, Bosnia-Herzegovina, Bulgaria, Croatia, Cyprus, the Former Yugoslavia Republic of Macedonia, Kosovo, Malta, Romania, Serbia and Montenegro, Slovenia and Turkey. A regional web-based surveillance network is a valuable tool for controlling and managing transboundary animal diseases such as BT. Its implementation in the Balkan region and in adjoining areas of south-eastern Europe is described and discussed.

A survey for Culicoides Latreille, 1809, was made on Malta in 2002 to establish whether Culicoides imicola Kieffer, 1913, the principal vector of bluetongue virus (BTV) in the Mediterranean Basin, or any other suspected vector species, was present. The collections and analyses were performed in accordance with the protocols of the National Reference Centre for Exotic Diseases (CESME Centro Studi Malattie Esotiche) in Teramo, Italy. Eighty-four catches were made between May and October at six permanent sites, namely: Mellieha, Rabat, San Gwann, Zejtun (Malta), Gharb and Sannat (Gozo island). The traps were placed near cattle (four farms), cattle and sheep (one farm: Rabat) and sheep and goats (one farm: Mellieha). Culicoides midges were found in 91.66% (77/84) of the catches and the highest number of midges per catch was 1 726. Culicoides imicola was confirmed on Malta for the first time in October 2002 and was found at four sites (San Gwann, Sannat, Gharb and Mellieha) but at very low abundance levels (<0.1% of the total Culicoides collected). Culicoides paolae Boorman, 1996 was the most widespread and abundant species (more than 80% of total Culicoides). Midges of the Obsoletus Complex were rare, with less than 10 individuals captured. Other species of Culicoides identified in the collections were: C. submaritimus Dzhafarov, 1962, C. cataneii Clastrier, 1957, C. circumscriptus Kieffer, 1918, C. jumineri Callot and Kremer, 1969, C. kingi Austen, 1912, C. maritimus Kieffer, 1924 and C. newsteadi Austen, 1921.

Between 2000 and 2003, thousands of light-trap collections for Culicoides were made throughout Italy and a detailed distribution map of the primary vector of bluetongue (BT) virus (BTV), C. imicola compiled. In some areas, however, where clinical BT occurred and C. imicola could not be captured, the virus was isolated from biting midges belonging to the Obsoletus and/or the Pulicaris Complexes. Thus, the distribution and abundance of these two species complexes in Italy, as determined from about 3,000 collections, are reported here also and compared to that of C. imicola (from about 24,000 collections). The probable spread of the main vector of BT, C. imicola, into the northern third of Italy, and the widespread prevalence of additional vectors of the Obsoletus and Pulicaris Complexes, indicate nearly all regions of Italy to be at some risk to incursions of BTV. However, these complexes comprise at least six and twelve species, respectively, so precisely which species are able to transmit BTV remains incompletely known.

A geographic information system (GIS) based on grids was developed by the National Reference Center for Veterinary Epidemiology at the Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise 'G. Caporale' (IZS) in Teramo to identify potential infectious sites for bluetongue (BT) disease in Italy. Geographical and climatic variables were used to build a spatial process model (SPM); the different layers were combined by sequential addition. The final grids (with a cell size of 0.0387 decimal degrees) were generated for each season of the year, and the suitability of each cell for the presence of C. imicola given a value ranking from 0 to 10. While this model more accurately predicts the presence of C. imicola in the Basilicata and Sicily regions, it still over-predicted its presence in the Puglia region. This could be due to the occurrence of calcareous soils which dominate the Puglia landscape. The present SPM is an additive model that assigns an equal weight to each variable. However, the results suggest the existence of hitherto unconsidered variables that significantly influence the prevalence of C. imicola. To reflect their importance, these variables should be assigned a higher weighting in future models. However, the decision in regard to precisely what this weighting should be depends on a very thorough knowledge of the ecology of C. imicola.

Thirty-six subgenera comprise the biting midge genus Culicoides Latreille, 1809. One of these, the relatively small subgenus Avaritia Fox, 1955, is the most important as it contains nearly half of the 30 world species of Culicoides known to play a greater or lesser role in the transmission of orbiviral diseases to livestock pantropically. These diseases include bluetongue (BT), African horse sickness (AHS) and epizootic haemorrhagic disease of deer (EHD). The subgenus Avaritia is distributed globally and the 70 species described have been subdivided into seven species groups and four subgroups. These 11 informal categories, variously labelled as either 'subgroups', 'groups' or 'complexes', are reduced here to seven in number (six species complexes and one subgroup) and although they appear natural, they are nearly all poorly defined. In this study two of these, namely the Imicola and Orientalis species complexes, are re-evaluated to determine which morphological characters define them more precisely, and so may help to establish their monophyly in the future. The two complexes are separable on eight discrete adult characters (two in the female, six in the male); these characters, and three secondary ones, are discussed and illustrated. The Imicola and Orientalis Complexes together embrace 23 species; these species are assigned to their respective complexes and according to recent nomenclatural adjustments. The taxonomy of vector Culicoides worldwide remains superficial; to improve the situation it is recommended that the 'traditional' morphological method be integrated with the 'modern' molecular approach.

The thirty species of Culicoides biting midges that play a greater or lesser role in the transmission of bluetongue (BT) disease in the pantropical regions of the world are listed. Where known, each species is assigned to its correct subgenus and species complex. In the Mediterranean region there are four species of Culicoides involved in the transmission of BT and belong in the subgenera Avaritia Fox, 1955 (three species) and Culicoides Latreille, 1809 (one species). Using both morphological and molecular second internal transcribed spacer (ITS2) sequence data, the authors reappraise the taxonomy of these four species and their congeners. A total of 56 populations of Culicoides collected from across Italy and representing 17 species (18 including the outgroup taxon C. imicola Kieffer, 1913) were analysed. The findings revealed the following: C. imicola is the only species of the Imicola Complex (subgenus Avaritia) to occur in the Mediterranean region. In Europe the subgenera Avaritia and Culicoides (usually, but not quite correctly, equated with the C. obsoletus and C. pulicaris groups, respectively) are both polyphyletic, each comprising three or more species complexes (including a hitherto unknown complex). About half the species studied could not be identified with certainty; furthermore, the results indicate that at least three previously described species of Palaearctic Culicoides should be resurrected from synonymy. Finally, a high level of taxonomic congruence occurred between the morphological and the molecular data. One of the 'new' vector species, C. pulicaris, was described by the father of taxonomy, Carl Linnaeus, in 1758, but today, almost 250 years later, no monograph has appeared that treats the Culicoides fauna of the northern hemisphere as a whole. At a time when such economically important livestock diseases as BT are affecting ever larger areas of Europe, it would seem appropriate to commence the production of such a monograph to aid in the field identification of vector Culicoides. This 'unfinished business' might best be achieved through a collaborative network embracing all ceratopogonid specialists currently active in both the Palaearctic and Nearctic faunal realms.

To elucidate the epidemiology of vector-borne diseases that can affect livestock in the Mediterranean Basin and elsewhere, it is essential to obtain a clear understanding of the life-cycle and habits of the vector insects involved. One purpose of such investigations is to provide data for an epidemiological surveillance system. As this depends heavily upon the collection of specimens in the field, it is necessary to establish the kinds of information required, and how it can be obtained. This requires, in turn, that the method (and instrument) of capture be standardised, so that all data are as complete as possible, are comparable, and are informative at many levels. Within the surveillance system for bluetongue (BT) in Italy, the National Reference Centre for Exotic Diseases (CESME: Centro Studi Malattie Esotiche) is leading an intensive and countrywide survey for Culicoides (Diptera: Ceratopogonidae) using standardised methods and protocols developed in collaboration with the Onderstepoort Veterinary Institute in South Africa. These methods have now also been implemented outside Italy in Malta, Croatia, Albania and Romania. This system includes the field protocols developed for the collection of Culicoides, the laboratory protocols developed around the insect analyses and the computer-based recording of all field data. Finally, the authors provide an 'Easy key' for the rapid identification of the principal BT vector C. imicola, and for grouping species that belong to the Obsoletus and Pulicaris vector complexes, and to the Nubeculosus and Schultzei potential vector complexes.

Between July and September 2002, bluetongue (BT) virus (BTV) serotypes 2 and 9 caused mortalities amongst sheep in the communities of San Gregorio Magno (Salerno, Campania), Laviano (Salerno, Campania) and Carpino (Foggia, Puglia), central Italy. On three of the affected farms, approximately 10,000 specimens of Culicoides were captured, representing fifteen species. Not a single specimen of the classical Afro-Asiatic BT vector, C. imicola Kieffer, was found; species of the Obsoletus Complex dominated the light-trap collections (90%) and included C. obsoletus (Meigen), C. scoticus Downes and Kettle and C. dewulfi Goetghebuer. Fifty-eight pools of the Obsoletus Complex (excluding C. dewulfi), each numbering 100 individuals per pool, and containing only parous and gravid females, were assayed for virus. BTV serotype 2 (BTV-2) was isolated from three pools (San Gregorio and Carpino) and BTV-9 from one (Laviano). These results indicate clearly that a species other than C. imicola is involved in the current re-emergence of BT in the Mediterranean Basin, but whether this is only C. obsoletus sensu stricto, or only C. scoticus, or both together, has yet to be established.

The biting midge, Culicoides paolae Boorman, described from specimens collected in the extreme south of Italy in 1996, belongs in the subgenus Drymodesmyia. This subgenus was erected by Vargas in 1960 for the so-called Copiosus species group, an assemblage of 22 species endemic to the tropical regions of the New World and, where known, breed in vegetative materials including the decaying leaves (cladodes) and fruits of Central American cacti. The Mexican peoples have utilised these cacti for over 9,000 years; one of these, Opuntia ficus-indica Linnaeus, was brought to Europe by Christopher Columbus following his voyages of discovery. As a taxon C. paolae is very similar to the Central American C. jamaicensis Edwards, 1922 raising the possibility that it (or a closely related species of Drymodesmyia) was introduced into the Mediterranean Region at the time of Columbus, but was (perplexingly) discovered only 500 years later and named C. paolae. The comparison of Sardinian specimens of C. paolae with Panamanian material of C. jamaicensis (housed in the Natural History Museum in London) confirmed the two species to be very similar but unusual differences were noted around the precise distribution of the sensilla coeloconica on the female flagellum. Until it is understood whether these differences represent either intra- or interspecific variation, the question of the possible synonymy of C. paolae must be held in abeyance.

Blacklight traps were used to collect Culicoides biting midges weekly between September 1996 and August 1998 at 40 sites distributed equidistantly across South Africa. The seasonal and geographic prevalences of 86 species of Culicoides were elucidated simultaneously, and included C. imicola Kieffer and C. bolitinos Meiswinkel the principal vectors of bluetongue (BT) and African horse sickness (AHS) in the region. These two species were amongst the most prevalent Culicoides to be found and, together, comprised >50% of the more than three million biting midges captured. The data are presented as coloured matrices, and are transformed also into inverse distance weighting (IDW) interpolative maps. The data reveal that the prevalence of each vector is somewhat fractured and it is posited that this is (in part) due to significant differences in their respective breeding habitats. The results illustrate also that the presence of multiple vectors (in any region of the world) will complicate both the epidemiology of the orbiviral diseases they transmit and the formulation of rational livestock movement and disease control strategies. This is especially true for southern Europe where the recent devastating cycle of BT has been shown to involve at least three vectors. Finally, the influence that man has on the development of large foci of vector Culicoides around livestock may be less important than previously suggested but must be investigated further.