Obsoletus Complex Research Articles

Phenology and attraction of potential Culicoides vectors of bluetongue virus in Basque Country (northern Spain)

Bluetongue virus is transmitted by Culicoides biting midges (Diptera: Ceratopogonidae). Culicoides associated with livestock were captured using CDC blacklight traps at three BTV-infected farms in Basque Country between November 2007 and December 2008. Twenty-seven and nineteen Culicoides species were collected in outdoor and indoor habitats respectively. Indoor insect community represented 86.1% of the whole captured individual biting midges. Culicoides obsoletus/Culicoides scoticus (two sibling species of the Obsoletus complex) were dominant throughout all months and sexes with maximum phenological peaks in November 2007 and June–July 2008. Culicoides lupicaris was the second most dominant species followed by Culicoides pulicaris (both species of the Pulicaris complex). Few specimens of Culicoides imicola, the principal Afro-Mediterranean vector of BTV, as well as four new species recorded for the Iberian Peninsula, were also collected. BTV was detected by RT-PCR from pools of C. obsoletus/C. scoticus, C. lupicaris and C. pulicaris parous females. dl-Lactic acid significantly attracted more C. obsoletus/C. scoticus females and males, C. lupicaris females, C. pulicaris females and Culicoides punctatus females and males; whereas acetone increased only the captures of Culicoides achrayi.

An outbreak of bluetongue virus serotype 9 in Southern Croatia

The aim of this study was to provide a description of the first epidemic of bluetongue and the first survey on midges of the genusCulicoidesin Croatia. Clinical signs were firstly observed on November 2001 in sheep in Konavle, Dubrovnik – Neretva County. During this epizootic the overall sheep morbidity and mortality were 5.2% (95% confidence interval (c.i.), 4.1-6.6%) and 2.29% (95% c.i., 1.6-3.3%), respectively. After the outbreak, 3,318 serum samples of ruminants from 53 villages of the Dubrovnik – Neretva County were examined for bluetongue virus (BTV) antibodies by competitive enzyme-linked immunosorbent assay (cELISA). In forty nine (92.45%, 95% c.i., 82.11-96.92%) of the 53 villages, animals with antibodies against bluetongue virus were found. In particular, a total of 178 cattle (49.86%, 95% c.i., 44.7-55.0%), 174 sheep (13.72%, 95% c.i., 11.9-15.7%) and 270 goats (15.95%, 95% c.i., 14.3-17.8%) were seropositive. Antibodies to bluetongue virus serotype 9 were detected in 212 positive sera by serum neutralization test. The percentage of positive animals decreased (P> 0.05) from the east to the west suggesting a possible east westward spreading of BTV infection. Fourteen light-trap midge collections from seven different sites were examined. Of the 4872Culicoidesspp. collected, 4,492 (92%, 95% c.i., 91.4-92.9%) of them belonged to the species ofObsoletus complex. This study showed for the first time that a pathogenic strain of BTV-9, probably from Montenegro, entered Croatia causing disease and death in local sheep and thatC. obsoletusandC. scoticuswere likely the major vectors of infection.

Contribution to the knowledge of Culicoides (Diptera: Ceratopogonidae) host preferences in France

Knowledge on host-feeding pattern of blood-sucking insects helps to understand the epidemiology of a vector-born disease. We determined blood meal origin from blood-fed Culicoides thanks to molecular techniques. A set of primers was used to selectively amplify segment of vertebrates' prepronociceptin gene from abdomen of engorged Culicoides (Diptera: Ceratopogonidae). Vertebrate DNA was successfully amplified in 91% of blood-fed Culicoides assayed. Direct sequencing and comparison of resultant sequences with sequences in GenBank, using BLAST, lead to the specific identification of the host in 100% of the cases. A total of 157 blood-fed females belonging to 13 different Culicoides' species were captured thanks to light traps in different areas of France between 2008 and 2009. Blood meal origin was determined for 143 blood-fed midges: 59 Culicoides obsoletus, 18 Culicoides dewulfi, 16 Culicoides scoticus, 11 Culicoides chiopterus, 10 Culicoides lupicaris, 1 Culicoides pulicaris, 8 Culicoides punctatus, 10 Culicoides pallidicornis, 3 Culicoides achrayi, 2 Culicoides furcillatus, 3 Culicoides brunnicans, 1 Culicoides picturatus and 1 Culicoides poperinghensis. The predominant species in our study belong to the C. obsoletus complex; they are considered as putative vectors of Bluetongue virus in the north of Europe. C. chiopterus sampled fed only on cattle, while blood meal origin of C. dewulfi, C. obsoletus and C. scoticus was diversified. In our sampling, we found that midges were fed mainly on cattle (54%), rabbits (20%), horses (17%), sheep (4%), pigs or wild boars (4%) and human (1%). Cattle DNA was found in at least 11 different species of Culicoides assayed.

Genetic structure and population dynamics of the biting midgesCulicoides obsoletusandCulicoides scoticus: implications for the transmission and maintenance of bluetongue

Culicoides species belonging to the Obsoletus complex (Diptera: Ceratopogonidae) have been indicated as primary bluetongue (BT) vectors in many European countries and their possible involvement in the maintenance and overwintering of BT viruses has been suggested, even in regions where Culicoides imicola Keiffer is the main vector. The Obsoletus complex includes two predominant taxa, Culicoides obsoletus (Meigen) and Culicoides scoticus Downes & Kettle. However, the role played by each species in the epidemiology of BT is still unknown. Taxonomic identification is mainly based on the morphology of male genitalia and the lack of other reliable diagnostic features makes the screening of trap-collected vector populations, mainly females, particularly difficult. Although molecular markers have facilitated species identification, little information is yet available on the biology, abundance and population dynamics of the two taxa. The aim of this work was to investigate the genetic profile and temporal distribution of C. obsoletus and C. scoticus by using isozyme electrophoresis applied to adult midges, collected weekly at two selected farms in southern Sardinia. A total of nine enzyme loci were analysed and five of them provided diagnostic allozyme markers (Hk, Mdh, Pgi, Idh-1 and Idh-2). Nei's genetic distance between the two taxa was in the range of other well-separated taxa (D = 1.792), supporting their status as true species. Culicoides scoticus represented almost 61% of the 562 specimens analysed; its genetic structure was characterized by a very low level of intra-population variation (mean heterozygosity H(e) = 0.019) and higher genetic divergence between populations (F(ST) = 0.0016) than in C. obsoletus. The latter species had significantly more heterozygotes (H(e) = 0.123), a higher percentage of polymorphic loci, and no inter-population differentiation (F(ST) ≅ 0). We suggest that different biological and ecological constraints, such as breeding habitat requirements, may contribute to shaping the genetic profiles of C. scoticus and C. obsoletus. However, enough gene flow was maintained between populations of each species as no spatial and temporal structuring was sustained by Fisher's exact probability test (P > 0.5). The seasonal distributions of C. scoticus and C. obsoletus only partially overlapped: both species were mainly found early in the year, when the main vector, C. imicola, was present in low numbers, and peaked in abundance in April and May. Culicoides scoticus was predominant until May, decreased rapidly in the following months and increased again in winter, whereas C. obsoletus decreased more slowly and was still present in early summer. Consequently, C. scoticus may be a good candidate for playing a role in the transmission and maintenance of BT virus in Sardinia, as well as in other Mediterranean countries, during the months of late winter and early spring when the seroconversion of sentinel animals is still occurring in the absence of the main vector.

Bluetongue Virus Detection by Real-Time RT-PCR inCulicoidesCaptured During the 2006 Epizootic in Belgium and Development of an Internal Control

After the emergence of bluetongue (BT) in Belgium in 2006, two types of entomological surveys were initiated, the one to identify the local vector species, and the other to study their population dynamics. In the vector study, Culicoides were captured near farms with recently infected cattle or sheep; in the population study Culicoides were captured in two meadows situated in the BT-affected region. A total of 130 pools of parous, non-blood engorged female midges (with a mean of 7.5 midges per pool) were analysed with real-time reverse transcription PCR (RT-qPCR) targeting bluetongue virus (BTV) segment 5. To ensure the RNA integrity of the samples, all pools were also tested in a second RT-qPCR targeting Culicoides 18S rRNA, which served as an internal control. Seventeen pools with negative results for both 18S and BTV were excluded, most of which originated from the population survey. In the vector survey near outbreak sites, female midges of the obsoletus complex, including C. obsoletus, C. scoticus, C. dewulfi and C. chiopterus, dominated the black-light trap collections with 19 of 89 pools being BTV-positive. Moreover, all the collections from the vector survey included at least one positive pool of the obsoletus complex compared with only 20% collections (C. obsoletus/C. scoticus) in the population survey. The current study also revealed the presence of BTV RNA in one of five pools of C. pulicaris females captured near recent BT outbreaks, suggesting that this species might have played a role in transmission. Finally, the use of RT-qPCR for the recognition of new potential BTV vector species and the impact of an appropriate monitoring method and internal control are discussed.

Monitoring von Gnitzen (Culicoides spp.), den potentiellen Vektoren des Blauzungenkrankheitsvirus, in den 12 Klimaregionen der Schweiz

Midges of the genus Culicoides are the only known biological vectors of the bluetongue disease virus (BTV). Their occurrence at altitudes below 900 meters above sea level (m a.s.l.) is monitored in Switzerland by the Swiss Federal Veterinary Office, to determine vector-free periods around winter. In this study, data about the number of midges caught at stations representing the 12 climatic regions of Switzerland are shown. The tiny midges of 1-3 mm in size were caught once per week with UV light traps and grouped under the stereomicroscope into Obsoletus complex, Pulicaris complex and other Culicoides spp. Midges were caught at all stations, albeit in very different numbers. The highest monthly average was 10,000 midges per night (Dittingen/BL); the third highest average of all 12 stations was recorded for the highest-located station (Juf/GR, 2130 m a.s.l.). At stations below 1500 m a.s.l., midges of the Obsoletus complex (98% in Dittingen), which in Central Europe are most likely considered to be responsible for the transmission of BTV, were predominant. With increasing altitude, midges of the Pulicaris complex prevailed (91% in Juf). Hence, there are no regions of the populated areas in Switzerland which are free of midges, but the vector competence regarding BTV of the various midges needs to be urgently clarified.

Monitoring of Culicoides at 20 locations in northwest Germany

Entomological monitoring was carried out from April 2007 to May 2008 at 20 locations in the areas of Lower Saxony, Schleswig-Holstein, Hamburg, and Bremen. A total number of 26 Culicoides species were sampled by light traps during the first week of every month. Culicoides diversity was highest in summer, achieving more than 20 species and genera per month. Numbers of Culicoides were highest in spring and summer with a maximum of 325,000 individuals in May 2008 at a single location. During the winter, the number of individuals decreased considerably, but few individuals of Culicoides were present even during the coldest months in January and February with Culicoides obsoletus remaining the only species complex. The total number of Ceratopogonidae and the number of individuals from C. obsoletus complex and Culicoides pulicaris complex were significantly correlated with temperature almost at any date and location.

Changes of the abundance of Culicoides obsoletus s.s. and Culicoides scoticus in Southwest Germany identified by a PCR-based differentiation

The outbreak of bluetongue disease in Central Europe necessitates new approaches in the identification of vectors to follow-up changes of populations of species and not of complexes. Since females of species of the complex of Culicoides obsoletus are difficult to be identified according to morphological criteria, we applied a polymerase chain reaction (PCR)-based strategy targeting the mitochondrial cytochrome oxidase subunit I to differentiate between the species Culicoides obsoletus s.s. and Culicoides scoticus. Catches of culicoids obtained from May to November 2007 in an ultraviolet lamp trap at a cattle farm in Rhineland-Palatinate, Southern Germany were surveyed for changes of the abundance of both species. Only in May 2007, the samples contained similar proportions of both species. Afterwards, C. scoticus dominated with up to 88%. Calculating the number of specimens of both species within the total catches of culicoids, the numbers of C. obsoletus s.s. slightly decreased from May to July and increased to a little maximum in August. C. scoticus seemed to have three maxima in this period of time, the strongest one in August, presumably due to different generations and not to climatic conditions. These results indicate that the applied PCR strategy can be used for a detailed analysis of culicoids as basis for the estimation of the transmission risk of the bluetongue virus by different species of the Obsoletus complex.

Répartition et abondance des moucherons piqueurs, vecteurs potentiels de la fièvre catarrhale ovine, en Suisse

Indigenous biting midges proved to be highly efficient vectors for the recently introduced bluetongue virus serotype 8 (BTV-8). Since its introduction in Northern Europe, the virus has rapidly spread, reaching Switzerland in 2008. The aims of this project are to determine the distribution, abundance, and activity pat­terns of biting midges occurring in Switzerland. Insects were caught with Onderstepoort ultraviolet light traps once weekly at stations representing the 12 climatic regions of Switzerland throughout the whole year. In addition, catches were carried out at five stations in an Alpine region of Switzerland at alti­tudes between 1300 and 2000 metres above sea level from the end of June to the end of October 2008. Midges were grouped under the stereomicroscope into Obsoletus complex, Pulicaris complex or other Culicoides spp. Midges were caught at all stations, albeit in very different numbers. The highest monthly average was 10,000 midges per night (Dittingen); the third high­est average was recorded at the highest station (Juf, 2130 m). At stations below 1500 m, midges of the Obsoletus complex (98% in Dittingen) were predominant. In Central Europe, they are considered the most likely vectors responsible for BTV trans­mission. With increasing altitude, midges of the Pulicaris com­plex prevailed (91% in Juf). Catches in two neighbouring Alpine mountains of similar altitude (approximately 2000 m) varied considerably. It is most likely that there are no midge-free zones in the agricultural areas (including Alpine summer pastures) of Switzerland, but the vector competence of the various midges with regard to BTV needs to be urgently clarified.

Standardisation des méthodes de barcode moléculaire pour identifier les espèces de <em>Culicoides</em> en Europe

The design of efficient control strategies for Culicoides midges lies in correct species identification. From the approximately 1340 described Culicoides species, only 30 have been incriminated as vectors of bluetongue disease virus. However, the actual number of vector species could be different considering that relatively few species have been studied. In Europe, the most important vec­tor was assumed to be Culicoides imicola, an Afro-Asian vector. However, during the last two years other species such as C. puli­caris (Pulicaris complex), C. dewulfi, C. chiopterus and species of the Obsoletus complex have increased in importance, as their populations have been found abundant in outbreak areas, outside the geographical range of C. imicola. Whereas C. imicola is the most easily identified, C. obsoletus sensu stricto and C. scoticus, from the Obsoletus complex, are extremely difficult to identify by using traditional methods such as morphological examina­tion. The deoxyribonucleic acid (DNA)-barcoding regions is located in the cytochrome oxidase 1 gene (CO1). Recent stud­ies have shown that this region is valuable in the identification of Culicoides species; however, the CO1 fragment to be used must be selected with great care. We propose to standardize the methodology and the selection of the fragment to be amplified to homogenize results obtained by different groups.

Evaluation de la stabulation comme moyen de protection des bovins des piqûres de <em>Culicoides</em>, vecteurs de la fièvre catarrhale ovine

Light-trap catches of Culicoides biting midges were compared inside and outside animal housing, in the presence and in the absence of cattle. A three-time replicated 4x4 Latin square design was used at four farms in Bala, North Wales, over 12 nights in May and June; the experiment was repeated in October. In the two studies, over 70,000 and over 4500 Culicoides were trapped of which 93% and 86%, respectively, belonged to C. Obsoletus complex. Across the four farms, in May and June, the presence of cattle increased catches of C. obsoletus by 2.3 times, and outside traps caught them 6.5 times more than inside traps. Similar patterns were apparent in October, but the difference between inside and outside catches was reduced. Catches were strongly correlated with minimum temperature and maximum wind speed, and these two variables explained a large amount of night-to-night variation in catch. Outside catches were reduced to a greater extent than inside catches by colder minimum tem­peratures and stronger maximum wind speeds. These conditions occurred more frequently in October than in May and June, thereby suppressing the outside catches more than the inside catches, and reducing the apparent degree of exophily of C. obsoletus in autumn. The results suggest that the risk of animals receiving bites from C. obsoletus is reduced by housing at both times of year, but the benefit would be greatest on warm, still nights when outside catches are at their greatest.

Passé et futur : études de morphologie et de biologie moléculaire des <em>Culicoides</em>

Culicoides are the biological vectors of bluetongue virus, an arbovirus transmitted by females after the ingestion of blood from viraemic animals. Conventional microscopy is usually used for taxonomic characterization and recently molecular charac­terization became a very useful tool to differentiate species with similar morphological characteristics, such as those belonging to Culicoides Obsoletus complex and C. dewulfi. On the other hand, scanning electronic microscopy (SEM) allows the visuali­zation of very small details, which can also be used in Culicoides species identification as a complement of other methods. In this work we performed both techniques in order to detect simulta­neously subtle features. Female and male specimens of Culicoides obsoletus sensu stricto, C. scoticus and C. dewulfi were washed in ethylic alcohol in gradients until 100%, followed by immersion in acetone. The insects were dried using the point drying method, mounted in stubs and coated with gold palladium. Specimens were observed under electronic microscope. Deoxyribonucleic acid (DNA) was extracted individually from 360 specimens of the C. Obsoletus complex (C. obsoletus s.s., C. scoticus) and 18 specimens of C. dewulfi, then analysed by PCR Multiplex. Several images of heads including details of the eyes (contiguous and bare), sensilla, flagellomeres, absence of teeth in the cibarial arch and pharynx, maxillary palps and mouth parts, thorax, abdomen and legs (in both sexes), genitalia, hind tibial comb, and fifth tarsi with empodium and claws are presented. PCR Multiplex results are shown. In this work, the SEM technique was used to show specific morphological details not obtained by optical microscopy of C. obsoletus. The SEM technique may be helpful to differentiate members of Culicoides species complexes and can be comple­mentary to other classification methods of Culicoides. Samples of Culicoides analysed by PCR Multiplex and examined in parallel with SEM might improve the taxonomic classification of these insects.

<em>Culicoides imicola</em>, vecteur du virus de sérotype 8 de la fièvre catarrhale ovine

Based on its abundance near livestock and wide geographical distribution, one of the most important Culicoides virus vector species of orbiviruses is the Afro-Asiatic species C. (Avaritia) imi­cola. It is considered a proven vector of bluetongue virus (BTV) and is one of the world’s most widely distributed Culicoides spe­cies. Strong correlations were found between the presence of C. imicola and outbreaks of BT and African horse sickness (AHS) in Africa and BT in Southern Europe. In 2006, BT serotype 8 (BTV- 8) was responsible for the first outbreaks ever of BT recorded in Northern Europe. Outbreaks first appeared in the Netherlands and Belgium, and subsequently spread to Germany, France, and Luxemburg. The virus apparently overwintered in Northern Europe and, during 2007-2008, spread across the whole of Europe. The outbreaks in Northern Europe happened in the absence of C. imicola. Members of the Culicoides (Culicoides) pulicaris and C. (A.) Obsoletus complexes have been implicated in BTV transmission. In the present study, the oral susceptibil­ity of C. imicola and other South African livestock associated Culicoides species to various isolates of BTV-8 were determined by artificial feeding. During summer 2008, a Belgium and a recent (2004) South African field isolate of BTV-8 were fed to Culicoides collected at two localities in South Africa. Adult Culicoides midges were collected at the Agricultural Research Council; Onderstepoort Veterinary Institute (ARC-OVI) and at Clarens, in a cooler mountainous area of South Africa. At ARC-OVI the dominant species was C. imicola and at Clarens it was C. (A.) bolitinos. The South African field isolate was fed at con­centrations of 5.8 and 7.8 log10TCID50/mL in the blood meal and the Belgium isolate at 6.8 log10 TCID50/mL. The following summer (2009) the same South African (6.8 log10 TCID50/mL) and Belgium (6.8 log10 TCID50/mL), a Dutch (4.8 log10 TCID50/ mL) and a Spanish (6.1 log10 TCID50/mL) isolates of BTV-8 were fed to Culicoides species collected at ARC-OVI. In the same season, the South African and Belgium isolates were also fed to midges collected at Clarens. Virus recovery, after an incubation period of 10 days at 23.5°C in C. imicola, was low for all iso­lates for both years. The South African isolate (5.8 log10 TCID50/ mL) and the Spanish isolate of BTV-8 could not be recovered from 420 and 492 midges tested after incubation. The recov­ery rate for the other isolates ranged from 2/403 (0.5%) (2008: SA isolate) to 1/683 (0.1%) (2009: SA isolate). These relatively low recovery rates make direct comparison between isolates problematic. Virus recovery from C. bolitinos fed on the South African isolate (7.8 log10 TCID50/mL) was significantly higher than any of the other isolates. During 2008 the Belgium iso­late could be recovered from 4/229 (1.7%) C. bolitinos tested. Other Culicoides species from which BTV-8 could be isolated included C. (Meijerehelea) leucostictus, C. (A.) gulbenkiani and C. (unplaced) angolensis. Viral concentrations in infected C. imi­cola ranged from 0.7 to 2.4 and from 0.7 to 3.4 log10 TCID50/ mL in C. bolitinos. These results are in agreement with previous studies indicating a similarly low susceptibly in C. imicola for the reference strain of BTV-8. The relatively low oral susceptibility to near refractory status of C. imicola as determined for some of the isolates in this study will easily be compensated for by the high abundance of C. imicola in South Africa. Cumulative laboratory oral susceptibility results from South Africa indicate a consistently higher susceptibility in C. bolitinos for BTV as well as a multi-vector potential for BTV as well as AHSV. The unique biology of potential vector competent Culicoides species emphasizes the complex epidemiology of these diseases.

Programme national bulgare de surveillance du virus de la fièvre catarrhale : études de terrain et évaluation du risque en 2009

Bluetongue is a non-contagious, arthropod-borne viral disease of both domestic and wild ruminants. Bluetongue virus (BTV) is endemic in some areas where cattle and wild ruminants serve as reservoirs for the virus. Some authors suggest that a fundamental change in the European ecosystem since 1998, mostly linked to climate change, may have influenced the introduction and sur­vival of BTV in Europe. Genetic analyses of bluetongue viruses isolated in Europe during this period showed that six virus types (1, 2, 4, 8, 9 and 16) have entered the region since that period. The aim of this presentation was to summarize the results of the National BTV Surveillance Programme, field studies, and risk assessment of BTV entry into Bulgaria in 2009. As part of the Surveillance Programme of the disease in 2009, seroprevalence was performed in 19 sentinel BTV-seronegative animal herds, 7 of which were located in the western part of the country and the other 12 in the southern part, about 10 to 20 kil­ometres from the border. Each sentinel herd consisted of 10 large ear-tagged ruminants. The sentinel animals were tested every 30 days for the presence of BTV antibodies. Competitive enzyme-linked immunosorbent assays (cELISAs) were used to determine the antibodies against the common group antigen. Fourteen Onderstepoort black light traps were set up for Culicoides surveil­lance and monitoring in southern and western border districts of the country after April 1, 2009. Catches of Culicoides were sorted by traditional identification keys. The risk of BTV invasion was assessed according to the World Organisation for Animal Health standards. The hazard identification, possibility of BT outbreaks, and assessment of the epidemiological situation in neighbour­ing countries were made based on other authors’ reports on the density of small and large ruminants, and economical and trade relations between countries. In addition, the BTV situation on a Greek Aegean Sea Island was closely monitored in 2008-09. During the 2009 serum surveillance period, 3340 serum sam­ples from different species of ruminants were investigated, all with negative results for BTV antibodies; C. imicola was never detected in more than 12 years of Culicoides surveillance in Bulgaria. Analysis of 274 midges caught during 2009 showed that dominating Culicoides species were of the Obsoletus and Pulicaris complexes, well-known Palaearctic midges in Europe. The negative results from the Surveillance Programme supported the evidence of absence of active BTV circulation on the whole territory of Bulgaria during 2009. During the 2008-09 season, Greek Authorities reported several circulating BTV serotypes (1, 8, 16) on Lesvos Island. As the epidemiological situation on Greek islands in the Aegean Sea is complicated and not very clear, the risk of BTV entering Bulgaria ranges from high (in spring and summer) to moderate (in winter and autumn).

Répartition de la fièvre catarrhale ovine et des <em>Culicoides spp. de 2005 à 2008 au Portugal

The first outbreak of bluetongue (BT) in Portugal occurred in 1956 and was caused by BT virus serotype 10 (BTV-10). The dis­ease was declared eradicated in 1960. Forty-four years later, in November 2004, BT re-occurred in Portugal, caused by BTV-4, and again, in September 2007, by BTV-1. It has persisted in some regions of the country since then. To help control the disease, a BT entomological programme was implemented in Portugal in 2005 and some of the results are now reported, focusing on Culicoides species as BTV potential vectors. In the framework of the national entomological programme, the country was divided into 45 geographical units (GUs) (50 km side squares). Mini-CDC light traps were used to collect insects in selected ruminant herds in different GUs. During the period of September 2005 to May 2008, 2990 valid catches were made and ten Culicoides species were identified. C. imicola was the main species identified and accounted for 81.73% of the specimens collected, with a peak occurrence in September. C. Obsoletus complex accounted for 4.45% of the specimens, with peaks occurring from May to July. C. pulicaris accounted for 0.04% only of the collections. C. imicola was caught 18.3 times more and mean specimens were collected 8.9 times more per trap than C. obsoletus. Although C. imicola was collected in more northern latitudes than previously reported in Portugal, these catches were sporadic and with a low number of specimens. C. imicola predominantly occurred in the East-Central and South regions of Portugal and prevailed in the areas where BT outbreaks occurred. Its marked seasonal occur­rence coincided with the period of BT outbreaks in Portugal. C. imicola is thus considered the main vector of BTV in Portugal, and official sanitary prophylactic and control measures should be implemented accordingly to decrease the risk of transmission.

Puces à ADN comme outil d'identification moléculaire pour <em>Culicoides</em>

A DNA microarray test based on internal transcribed spacer 1 (ITS1) genotype expression was developed to identify Culicoides species of Northwestern Europe belonging to the Culicoides Obsoletus complex. The assay was designed so as to allow interpretation by the naked eye. False positive and false nega­tive results were eliminated. The need for expensive laboratory equipment and reagents was kept as low as possible, making the technique affordable and feasible for any diagnostic laboratory with polymerase chain reaction (PCR) facilities. The microarray test could be validated through the three ringtests organised by Medreonet. Use of this microarray can improve monitoring adult and immature Culicoides species.

Etudes interlaboratoires sur les espèces du complexe Obsoletus

Culicoides species are difficult to identify but, recently, molecu­lar identification tools based on cytochrome oxidase subunit 1 (CO1), internal transcribed spacer 1 or 2 (ITS-1 or ITS-2) have been developed for Palaearctic species including vector spe­cies. In the framework of Medreonet network, the workpackage on “Regional surveillance of vectors” includes guidelines on Palaearctic and Mediterranean Culicoides species identification using molecular tools. Three ring trials were carried out to test whether the methods and tools used in the main laboratories of Europe were adequate and able to identify species correctly. Moreover, a questionnaire was sent to each laboratory to record the protocols and tools used for molecular identification. The four species of the Obsoletus complex, C. obsoletus, C. scoticus, C. chiopterus, and C. dewulfi were selected for the ring trial. All specimens were first morphologically identified by experts. The first ring trial containing eight specimens (four males and four females) of the four species was sent on the 4th of August 2008 to 13 different participating laboratories from seven coun­tries. Two types of material were sent: extracted deoxyribonucleic acid (DNA) (four samples/species) and phosphate-buffered saline (PBS)-ground biting midges (34 samples/species). Eventually, two laboratories did not participate. Only two out of the 11 partici­pating laboratories correctly identified the species. The molecu­lar identification used was based on ITS-1 and ITS-2 markers. A second ring trial was sent in April 2009 with only extracted DNA samples to avoid the different extraction steps which could interfere with tool sensitivity. A total of 20 extracted DNA sam­ples were sent to 10 participating laboratories. Four out of the 10 correctly identified the species. Three laboratories used methods based on ITS-2 or ITS-1 markers, and one laboratory used CO1. Finally, a third ring trial with 20 PBS-ground samples was sent in September 2009 to 10 laboratories. Only one laboratory cor­rectly identified the species. Sensitivity and specificity of the molecular tools used are discussed.

Compétence vectorielle des espèces de <em>Culicoides</em> britanniques pour le sérotype 8 du virus de la fièvre catarrhale ovine

The proportion of infected vectors which are able to transmit an arbovirus to a susceptible host has a significant impact on the epidemic potential of such a virus. Assessing vector competence is therefore crucial to evaluate accurately the risk posed by such a disease to any non-endemic region. The vector competence of various Culicoides species in Scotland for bluetongue virus serotype 8 (BTV-8) was assessed by a pad-feeding technique, and a high-throughput virus extraction and isolation procedure. This was coupled with a multiplex polymerase chain reaction (PCR) to identify members of the Culicoides Obsoletus complex to spe­cies level. These results are compared with vector competence results of further Culicoides Obsoletus in South-East England assessed by the same method. A very low level of competence for this strain was detected in all Culicoides species tested, simi­lar to that described for this strain in C. imicola originating from both Corsica and the Onderstepoort Veterinary Institute in South Africa. The implications of this are discussed in relation to future studies and also with regard to wider aspects of orbivirus trans­mission in the European Union.

Variabilité spatiale des captures de <em>Culicoides</em> dans des fermes de la zone de Bala au nord du Pays de Galles

The aim of this study was to investigate the spatial variability of Culicoides catches at a local scale using environmental data and farm characteristics. A thrice-replicated random sampling design was set up on a 1x1 km grid in a 6x6 km study site in Bala, North Wales in July 2008 over a period of 12 days using Onderstepoort black light traps to catch Culicoides. Overall, 132 catches were made on 35 farms, 32 of which were sampled three times and three of which were sampled daily. A total of 357,229 Culicoides encompassing 19 species (mean catch = 2706 per trap and per night; maximum catch = 65,763) were caught. The species of the Obsoletus complex accounted for 62% of the catches. Environmental factors (such as land cover, land use, soil type, altitude and weather) and farm characteristics (animals, insecticide use, dung management, proximity to water and to potential breeding sites, and openness of the landscape) will be used to model the abundance of Culicoides species. The influ­ence of Culicoides abundance in neighbouring farms will also be assessed and accounted for. This will help better understand spa­tial distribution and variability of Culicoides densities at a local scale and provide elements for discussion on how to approach mapping and modelling of vector abundance at coarser scales.

Comparaison de l'efficacité de quatre modèles de pièges lumineux pour la collecte de <em>Culicoides</em> piqueurs en Allemagne

Biting midges of the genus Culicoides are vectors of bluetongue disease, an OIE-listed animal disease which has recently led to severe economic losses in susceptible livestock species in Europe. Therefore, several affected countries conduct monitor­ing programmes for Culicoides spp. To compare the number and species composition of Culicoides obtained, the efficacy of various traps used to collect biting midges needs to be assessed. Four aspiration black light traps were thus compared: the Onderstepoort, John W. Hock, Riebt, and Biogents. One of the traps was operated from the 1st of April 2009 to determine the onset of increased Culicoides activity and thus the best time to start the study. The comparison was carried out from May 16th until September 26th. The traps were set up at four different sites on pastures of the Friedrich-Loeffler-Institut where cattle, sheep and goats were present. The comparison was carried out according to the Medreonet protocol in a 4×4 randomized Latin square design with one night of activity for each trap at each site. The highest number of Culicoides specimens was captured by the Onderstepoort (1245), followed by the Biogents (365), the Hock (30) and the Riebt (4) traps. The majority of midges caught was classified as belonging to the Obsoletus complex (1399), including C. obsoletus s.s., C. scoticus and C. chiopterus. Other encountered species were C. punctatus s.s. (87), C. albicans (78) and C. riethi (50). Most biting midges were collected in May (594) and June (575). In September only one Culicoides speci­men was caught. The results show that the trap model has a great impact on the number of Culicoides caught. Culicoides monitor­ing programmes should be harmonised by using the same type of trap everywhere or by adjusting the numbers of collected biting midges using a trap factor. The trap model should be taken into consideration in the decision to declare the seasonally-determined vector-free periods.