Vector competence of selected South African Culicoides species for the Bryanston serotype of equine encephalosis virus

Equine encephalosis virus (EEV) was recognized and described in the Republic of South Africa in 1967 and subsequent serological studies have shown this orbivirus to be both widespread and prevalent in southern Africa. In the present study it was shown that wild-caught Culicoides (Avaritia) imicola Kieffer (Diptera: Ceratopogonidae) can become infected with and permit the replication of the Bryanston serotype of EEV following membrane-feeding on infective blood containing 5.0 log10 plaque-forming-units (PFU)/ml. The mean prevalence of Bryanston virus infection in C. imicola after 10 days extrinsic incubation at 23.5 degrees C was 22.3% (23/103). The mean infectivity of Bryanston virus in the infected C. imicola increased from 1.3 log10 PFU/midge, in insects assayed immediately after feeding on the blood-virus mixture, to 2.6 log10 PFU/midge in insects assayed after incubation. The virus concentration in individual C. imicola infected with the Bryanston serotype of EEV ranged from 0.7 to 3.6 log10 PFU/midge. Bryanston virus titres higher than 2.5 log10 TCID50, found in individual C. imicola, suggest that this species may be able to transmit this virus to susceptible hosts. Prevalence of virus infection in C. imicola was determined by PFU and microtitration assays on both BHK and Vero cells and confirmation of the Bryanston serotype of EEV was determined by plaque inhibition. No virus replication could be demonstrated in 102 C. nivosus tested after the incubation period, suggesting that not all Culicoides species are equally susceptible to Bryanston virus infection. Other Culicoides species that survived the incubation period and that were negative for the presence of Bryanston virus were C. pycnostictus (42), C. leucostictus (7), C. magnus (2), C. bolitinos (1) and C. bedfordi (1).     

Oral susceptibility of South African stock-associated Culicoides species to bluetongue virus

Field-collected South African Culicoides species (Diptera, Ceratopogonidae) were fed on sheep blood containing bluetongue virus (BTV) represented by 13 low-passage reference serotypes: -1, -2, -4, -6, -7, -8, -9, -10, -11, -12, -13, -16 and -19. After 10 days of extrinsic incubation at 23.5 degrees C, of the 13 serotypes used, seven were recovered from C. (Avaritia) imicola Kieffer and 11 from C. (A.) bolitinos Meiswinkel. Virus recovery rates and the mean titres for most serotypes were significantly higher in C. bolitinos than in C. imicola. In addition, BTV was recovered from three non-Avaritia Culicoides species, namely C. (Remmia) enderleini Cornet & Brunhes (BTV-9), C. (Hoffmania) milnei Austen (BTV-4) and C. (H.) zuluensis de Meillon (BTV-16). No virus could be recovered from 316 individuals representing a further 14 Culicoides species. In Culicoides species fed on blood containing similar or identical virus titres of distinct BTV serotypes, significant differences were found in virus recovery rates. The results of this study confirm the higher vector competence of C. bolitinos compared with C. imicola.     

The susceptibility of Culicoides imicola and other South African livestock-associated Culicoides species to infection with bluetongue virus serotype 8

In 2006, a strain of bluetongue virus serotype 8 (BTV-8) of sub-Saharan origin was responsible for the first outbreaks in recorded history of clinical bluetongue disease (BT) in northern Europe. In this study, we examine the oral susceptibility of Culicoides (Avaritia) imicola Kieffer (Diptera: Ceratopogonidae) and other livestock-associated Culicoides species from southern Africa to infection with several strains of BTV-8. Following feeding using an artificial membrane-based method and incubation, virus was found in <1% of C. imicola individuals tested. Higher rates of susceptibility were found, however, for a variety of other South African species, including Culicoides (Avaritia) bolitinos Meiswinkel. Although these results do not preclude the role of C. imicola as a vector of BTV-8, its low susceptibility to BTV indicates that other less abundant Culicoides species may have the potential to play decisive roles in the epidemiology of this virus and should not be excluded from risk assessment studies.     

Replication of live-attenuated vaccine strains of bluetongue virus in orally infected South African Culicoides species

Field-collected South African Culicoides (Diptera, Ceratopogonidae) were fed on sheep blood containing 16 live-attenuated vaccine strains of bluetongue virus (BTV) comprising serotypes -1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -13, -14, -16 and -19. After 10 days extrinsic incubation at 23.5 degrees C, 11 and seven of the 16 BTV serotypes used were recovered from Culicoides (Avaritia) imicola Kieffer and Culicoides (A.) bolitinos Meiswinkel, respectively. One serotype was also recovered from Culicoides (Remmia) enderleini Cornet & Brunhes. Bluetongue virus recovery rates and the mean titres for most serotypes were significantly higher in C. bolitinos than in C. imicola. Significant differences were found in virus recovery rates from Culicoides species fed on blood containing similar or identical virus titres of different BTV serotypes. In addition, we demonstrated that a single passage of live-attenuated BTV-1, -2, -4, -9 and -16 through the insect vector, followed by passaging in insect cells, did not alter its infectivity for C. imicola and that the oral susceptibility of C. imicola to the attenuated vaccine strains of BTV-1, -4, -9 and -16 remained similar for at least three consecutive seasons.     

Phylogenetic analysis of Culicoides species from France based on nuclear ITS1-rDNA sequences

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) play important roles in the transmission of viral diseases affecting wild and domestic ruminants and horses, including Bluetongue (BT) and African horse sickness (AHS) respectively. In southern Europe, BT has been largely transmitted by the classical Afro-Asian vector Culicoides imicola Kieffer. However, other species such as C. obsoletus Meigen, C. scoticus Downs & Kettle and C. pulicaris Linné may also be involved in BTV transmission. As a consequence of the discovery of C. imicola followed by BTV-2 outbreaks on the island of Corsica in October 2000, further studies on these biting midges have been carried out. To better characterize the evolution and phylogenetic relations of Culicoides, molecular analysis in parallel with a morphology-based taxonomic approach were performed. Phylogenetic analyses of French Culicoides species were undertaken using the ribosomal DNA (rDNA) internal transcribed spacer 1 (ITS1) as a molecular target. This region was shown to be useful in understanding evolutionary and genetic relationships between species. Construction of several trees showed that molecular phylogeny within the genus Culicoides correlates not only with morphological-based taxonomy but also with ecological patterns.     

African horse sickness epidemiology: vector competence of south african Culicoides species for virus serotypes 3, 5 and 8

The oral susceptibilities of 17 Culicoides species to infection with African horse sickness virus (AHSV) serotypes 3, 5 and 8 were determined by feeding field-collected midges on AHSV infected horse blood. The mean titres of virus in the bloodmeals for the three serotypes of AHSV were between 5.7 and 6.5 log10 TCID50/ml. Virus was detected, after 10 days incubation at 23.5 degrees C, in the Culicoides imicola Kieffer (Diptera: Ceratopogonidae) that had fed on blood containing AHSV 5 (8.5%) and 8 (26.8%), and in the Culicoides bolitinos Meiswinkel that had fed on AHSV 3 (3.8%), 5 (20.6%) and 8 (1.7%). Although 44.4% of the C. imicola were shown to have ingested AHSV 3 immediately after feeding, no virus was detected in 96 C. imicola after incubation. The relatively high titres of virus recorded in individual midges of both species after 10 days incubation suggested a fully disseminated infection. Previously, C. imicola was considered to be the only field vector of AHSV in Africa. Identifying C. bolitinos as a potential vector for AHSV is an important finding, which if proven will have a significant impact on our understanding of the epidemiology of AHS. No AHSVs could be detected in the other 15 species of Culicoides assayed, which suggests that some of the southern African Culicoides species are refractory to AHSV infection. However, further work with larger numbers of each species will be necessary to confirm this observation.     

Saliva proteins of vector Culicoides modify structure and infectivity of bluetongue virus particles

Bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) are related orbiviruses, transmitted between their ruminant hosts primarily by certain haematophagous midge vectors (Culicoides spp.). The larger of the BTV outer-capsid proteins, 'VP2', can be cleaved by proteases (including trypsin or chymotrypsin), forming infectious subviral particles (ISVP) which have enhanced infectivity for adult Culicoides, or KC cells (a cell-line derived from C. sonorensis). We demonstrate that VP2 present on purified virus particles from 3 different BTV strains can also be cleaved by treatment with saliva from adult Culicoides. The saliva proteins from C. sonorensis (a competent BTV vector), cleaved BTV-VP2 more efficiently than those from C. nubeculosus (a less competent/non-vector species). Electrophoresis and mass spectrometry identified a trypsin-like protease in C. sonorensis saliva, which was significantly reduced or absent from C. nubeculosus saliva. Incubating purified BTV-1 with C. sonorensis saliva proteins also increased their infectivity for KC cells ～10 fold, while infectivity for BHK cells was reduced by 2-6 fold. Treatment of an 'eastern' strain of EHDV-2 with saliva proteins of either C. sonorensis or C. nubeculosus cleaved VP2, but a 'western' strain of EHDV-2 remained unmodified. These results indicate that temperature, strain of virus and protein composition of Culicoides saliva (particularly its protease content which is dependent upon vector species), can all play a significant role in the efficiency of VP2 cleavage, influencing virus infectivity. Saliva of several other arthropod species has previously been shown to increase transmission, infectivity and virulence of certain arboviruses, by modulating and/or suppressing the mammalian immune response. The findings presented here, however, demonstrate a novel mechanism by which proteases in Culicoides saliva can also directly modify the orbivirus particle structure, leading to increased infectivity specifically for Culicoides cells and, in turn, efficiency of transmission to the insect vector.     

Virus recovery rates for wild-type and live-attenuated vaccine strains of African horse sickness virus serotype 7 in orally infected South African Culicoides species

Previously reported virus recovery rates from Culicoides (Avaritia) imicola Kieffer and Culicoides (Avaritia) bolitinos Meiswinkel (Diptera, Ceratopogonidae) orally infected with vaccine strain of African horse sickness virus serotype 7 (AHSV-7) were compared with results obtained from concurrently conducted oral infections with five recent AHSV-7 isolates from naturally infected horses from various localities in South Africa. Culicoides were fed sheep bloods spiked with 10(7.6) TCID(50)/mL of a live-attenuated vaccine strain AHSV-7, and with five field isolates in which virus titre in the bloodmeals ranged from 10(7.1) to 10(8.2) TCID(50)/mL). After an extrinsic incubation of 10 days at 23.5 degrees C, virus recovery rates were significantly higher in C. imicola (13.3%) and C. bolitinos (4.2%) infected with the live-attenuated virus than in midges infected with any of the field isolates. The virus recovery rates for the latter groups ranged from 0% to 9.5% for C. imicola and from 0% to 1.5% for C. bolitinos. The C. imicola population at Onderstepoort was significantly more susceptible to infection with AHSV-7 isolated at Onderstepoort than to the virus strains isolated from other localities. Results of this study suggest that tissue culture attenuation of AHSV-7 does not reduce its ability to orally infect competent Culicoides species and may even lead to enhanced replication in the vector. Furthermore, oral susceptibility in a midge population appears to vary for geographically distinct isolates of AHSV-7.     

Culicoides in relation to transmission of African Horse Sickness virus in The Gambia

Twelve light trap collections made near overnight shelters of horses and donkeys in four villages in the Central River Division of The Gambia captured fourteen species of biting midge of the genus Culicoides . Five species new to The Gambia were identified. This brought the number of recognized species of Culicoides (after a revision of C. schultzei ) to twenty-nine in The Gambia. Species known or suspected as vectors of African horse sickness virus (AHSV) and bluetongue virus (BTV) comprised 83% of female captures, 65% of captures being C. imicola or its sibling species , C. miombo . Captures of female Culicoides in the late dry season were almost as large as in the early dry season, despite the extreme heat and dryness at this time of the year.
Tests on batches of formalin-preserved female midges, using AHSV or BTV antigen capture ELISAs, did not show the presence of any virus amongst 2286 females in 240 aliquots. Nearly all Gambian equines are reportedly seropositive to AHSV and these results suggest that virus challenge from Culicoides vectors may be a factor in the health of Gambian horses and donkeys.     

Phylogenetic status and matrilineal structure of the biting midge, Culicoides imicola, in Portugal, Rhodes and Israel

The biting midge Culicoides imicola Kieffer (Diptera: Ceratopogonidae) is the most important Old World vector of African horse sickness (AHS) and bluetongue (BT). Recent increases of BT incidence in the Mediterranean basin are attributed to its increased abundance and distribution. The phylogenetic status and genetic structure of C. imicola in this region are unknown, despite the importance of these aspects for BT epidemiology in the North American BT vector. In this study, analyses of partial mitochondrial cytochrome oxidase subunit I gene (COI) sequences were used to infer phylogenetic relationships among 50 C. imicola from Portugal, Rhodes, Israel, and South Africa and four other species of the Imicola Complex from southern Africa, and to estimate levels of matrilineal subdivision in C. imicola between Portugal and Israel. Eleven haplotypes were detected in C. imicola, and these formed one well-supported clade in maximum likelihood and Bayesian trees implying that the C. imicola samples comprise one phylogenetic species. Molecular variance was distributed mainly between Portugal and Israel, with no haplotypes shared between these countries, suggesting that female-mediated gene flow at this scale has been either limited or non-existent. Our results provide phylogenetic evidence that C. imicola in the study areas are potentially competent AHS and BT vectors. The geographical structure of the C. imicola COI haplotypes was concordant with that of BT virus serotypes in recent BT outbreaks in the Mediterranean basin, suggesting that population subdivision in its vector can impose spatial constraints on BT virus transmission.     

Determination of the oral susceptibility of South African livestock-associated biting midges,Culicoides species,to bovine ephemeral fever virus

A total of 10 607 Culicoides midges (Diptera: Ceratopogonidae) were fed on either sheep or horse blood containing not less than 6.5 log10 TCID50/ml of bovine ephemeral fever virus (BEFV). Insects were collected during two consecutive summers from two distinct climatic areas. Two seed viruses, originating from either South Africa or Australia, were used separately in the feeding trials. Blood-engorged females were incubated at 23.5 degrees C for 10 days and then individually assayed in microplate BHK-21 cell cultures. Of the 4110 Culicoides that survived, 43% were C. (Avaritia) imicola Kieffer and 27% were C. (A.) bolitinos Meiswinkel. The remainder represented 18 other livestock-associated Culicoides species. Although BEFV was detected in 18.9% of midges assayed immediately after feeding, no virus could be detected after incubation. The absence of evidence of either virus maintenance or measurable replication suggests that most of the abundant livestock-associated Culicoides species found in South Africa are refractory to oral infection with BEFV. Future studies should be carried out using species of mosquitoes that are associated with cattle in the BEF endemic areas.     

Spatial distribution of bluetongue virus and its Culicoides vectors in Sicily

During the recent Mediterranean epizootic of bluetongue, an extensive programme of serological and vector (Culicoides biting midges (Diptera: Ceratopogonidae)) surveillance was carried out across Sicily. This paper presents the analysis of 911 light trap catches collected at the times of peak Culicoides abundance (summer to autumn 2000-2002) in 269 sites, in order to produce detailed maps of the spatial distribution of the main European vector, Culicoides imicola Kieffer and that of potential novel vectors. Whereas C. imicola was found at only 12% of sites, potential novel vectors, Culicoides obsoletus group Meigen, Culicoides pulicaris Linnaeus and Culicoides newsteadi Austen were present at over 50% of sites. However, the spatial distribution of C. imicola showed the closest correspondence to that of the 2000 and 2001 bluetongue (BT) outbreaks and its presence and abundance were significant predictors of the probability of an outbreak, suggesting that it was the main vector during these years. Although C. imicola may have played a role in transmission in several sites near Paternó, it was absent from the majority of sites at which outbreaks occurred in 2002 and from all sites in the province of Messina. All three potential novel vectors were widespread across sites at which outbreaks occurred during 2002. Of these, C. newsteadi was an unlikely candidate, as it was significantly less prevalent in outbreak vs. non-outbreak sites in Messina. It is hypothesized that the yearly distribution and intensity of outbreaks is directly attributable to the distribution and abundance of the vectors involved in transmission during each year. When C. imicola operated as the main vector in 2000 and 2001, outbreaks were few in number and were restricted to coastal regions due to low abundance and prevalence of this species. In 2002, it is hypothesized that BTV transmission was handed over to more prevalent and abundant novel vector species, leading to numerous and widespread outbreaks and probably to overwintering of the virus between 2001 and 2002. Based on catch ranges in outbreak vs. non-outbreak sites, it is tentatively suggested that nightly catches of 400 or more C. obsoletus and 150 or more C. pulicaris allow BTV transmission at a site, and provide a strategy for a fuller examination of the relationship between BTV transmission and the abundance and distribution of different vector species.     

Geographical and seasonal distribution of the bluetongue virus vector, Culicoides imicola, in central Italy

Following the first incursion of bluetongue virus (BTV) into Italy, the geographical and seasonal distribution of the biting midge Culicoides imicola Kieffer (Diptera: Ceratopogonidae), the main vector of BTV and African horse sickness virus, was investigated in two regions of central Italy (Lazio and Tuscany). Surveillance of Culicoides was carried out between July 2001 and December 2002 using light traps: 1917 collections were made in 381 trap sites, well distributed across both regions. During the survey, bluetongue outbreaks were recorded in both regions. Culicoides imicola was found in 89 (23%) trap sites, distributed fairly continuously along the whole western coastline, between 41.2697 degrees N and 44.05724 degrees N. It was found only occasionally inland and usually in low abundance, with catches of more than 1000 specimens per night found in only two sample sites and 74% of catches numbering fewer than 10 specimens. Adults were caught from March to mid December, with peaks ranging from the end of August to mid November. The coastal distribution and the presence of only few sites with year-round records of adult vectors suggests that colonization may have occurred recently, by passive wind-dispersal from external source areas (Sardinia and Corsica). Alternatively, the species may occur in established, previously undetected, autochthonous populations that are limited from extension inland and northern-ward within Lazio and Tuscany by cool winter temperatures.     

Spatial and temporal distribution of bluetongue and its Culicoides vectors in Bulgaria

Surveillance of Culicoides (Diptera: Ceratopogonidae) biting midges was carried out between 2001 and 2003, at 119 sites within a 50 x 50-km grid distributed across Bulgaria, using light trap collections around the time of peak adult midge abundance. Sentinel and ad hoc serum surveillance of hosts susceptible to bluetongue infection was carried out at around 300 sites between 1999 and 2003. Following the initial incursion of bluetongue virus 9 (BTV-9) into Bourgas province in 1999, affecting 85 villages along the southern border, a further 76 villages were affected along the western border in 2001, with outbreaks extending as far north as 43.6 degrees N. The BTV-9 strain in circulation was found to have a low pathogenicity for Bulgarian sheep populations, with less than 2% of susceptible individuals becoming sick and seroconversions detected up to 30 km from recorded outbreaks in the south. The major Old World vector Culicoides imicola Kieffer was not detected among over 70,000 Culicoides identified in summer collections, suggesting that BTV-9 transmission in Bulgaria was primarily carried out by indigenous European vectors. The most likely candidates, the Palaearctic species complexes - the Culicoides obsoletus Meigen and C. pulicaris L. complexes - were widespread and abundant across the whole country. The C. obsoletus complex represented 75% of all individuals trapped in summer and occurred in high catch sizes (up to 15,000 individuals per night) but was not found across all outbreak sites, indicating that both Palearctic complexes probably played a role in transmission. Within the C. pulicaris complex, only C. pulicaris s.s., C. punctatus Meigen and C. newsteadi Austen were sufficiently abundant and prevalent to have been widely involved in transmission, whilst within the C. obsoletus complex most trapped males were C. obsoletus s.s. Adult vectors were found to be largely absent from sites in west Bulgaria for a period of at least 3 months over winter, which, taken along with the spatiotemporal pattern of outbreaks in the region between years, indicates the virus may be overwintering here by an alternative mechanism - either by covert persistence in the vertebrate host or possibly by persistence in larval stages of the vector.     

Bluetongue vector species of Culicoides in Switzerland

Switzerland is historically recognized by the Office Internationale des Epizooties as free from bluetongue disease (BT) because of its latitude and climate. With bluetongue virus (BTV) moving north from the Mediterranean, an entomological survey was conducted in Switzerland in 2003 to assess the potential of the BTV vectors present. A total of 39 cattle farms located in three geographical regions, the Ticino region, the Western region and the region of the Grisons, were monitored during the vector season. Farms were located in areas at high risk of vector introduction and establishment based on the following characteristics: annual average temperature > 12.5 degrees C, average annual humidity >or= 60%, cattle farm. Onderstepoort black light traps were operated at the cattle farms generally for one night in July and one night in September. A total of 56 collections of Culicoides (Diptera: Ceratopogonidae) were identified morphologically. Only one single individual of Culicoides (Avaritia) imicola, the major Old World vector of BTV, was found in July 2003 in the Ticino region, one of the southernmost regions of Switzerland. In the absence of further specimens of C. imicola from Switzerland it is suggested that this individual may be a vagrant transported by wind from regions to the south of the country where populations of this species are known to occur. Alternative potential BTV vectors of the Culicoides (Culicoides) pulicaris and Culicoides (Avaritia) obsoletus complexes were abundant in all sampled regions with individual catches exceeding 70 000 midges per trap night.     

Modelling the distributions of Culicoides bluetongue virus vectors in Sicily in relation to satellite-derived climate variables

Surveillance data from 268 sites in Sicily are used to develop climatic models for prediction of the distribution of the main European bluetongue virus (BTV) vector Culicoides imicola Kieffer (Diptera: Ceratopogonidae) and of potential novel vectors, Culicoides pulicaris Linnaeus, Culicoides obsoletus group Meigen and Culicoides newsteadi Austen. The models containing the 'best' climatic predictors of distribution for each species, were selected from combinations of 40 temporally Fourier-processed remotely sensed variables and altitude at a 1 km spatial resolution using discriminant analysis. Kappa values of around 0.6 for all species models indicated substantial levels of agreement between model predictions and observed data. Whilst the distributions of C. obsoletus group and C. newsteadi were predicted by temperature variables, those of C. pulicaris and C. imicola were determined mainly by normalized difference vegetation index (NDVI), a variable correlated with soil moisture and vegetation biomass and productivity. These models were used to predict species presence in unsampled pixels across Italy and for C. imicola across Europe and North Africa. The predicted continuous presence of C. pulicaris along the appenine mountains, from north to south Italy, suggests BTV transmission may be possible in a large proportion of this region and that seasonal transhumance (seasonal movement of livestock between upland and lowland pastures) even in C. imicola-free areas should not generally be considered safe. The predicted distribution of C. imicola distribution shows substantial agreement with observed surveillance data from Greece and Iberia (including the Balearics) and parts of mainland Italy (Lazio, Tuscany and areas of the Ionian coast) but is generally much more restricted than the observed distribution (in Sardinia, Corsica and Morocco). The low number of presence sites for C. imicola in Sicily meant that only a restricted range of potential C. imicola habitats were included in the training set and that predictions could only be made within this range. Future modelling exercises will use abundance data collected according to a standardized protocol across the Mediterranean and, for Sicily in particular, should include non-climatic environmental variables that may influence breeding site suitability such as soil type.     

库蠓源蓝舌病病毒1型的分离和初步鉴定

为监测云南边境地区虫媒库蠓蓝舌病病毒携带情况,本研究对2013年-2017年从云南6个口岸及周边地区采集到的约5 400只库蠓样本,分180组。采用荧光定量RT-PCR检测、鸡胚和细胞分离、目的基因克隆测序分析和间接免疫荧光试验等进行病毒分离与鉴定。结果显示:采集库蠓样本中有20组检出蓝舌病病毒核酸,检出率为11.11%(20/180);接种后有1份样本能导致鸡胚胚体充血出血和死亡以及BHK-21细胞呈现明显的细胞病变;RT-PCR能从感染细胞样本中扩增出蓝舌病病毒VP7基因特异性片段,且该片段序列与国外BTV-1毒株相应序列的相似性达95%~99%;间接免疫荧光试验显示分离病毒能与BTV-1抗体发生特异性结合。结果表明,云南边境地区库蠓携带有蓝舌病病毒,且为BTV-1,因此应加强对云南边境地区蓝舌病的预防与控制。     

Occurrence of Culicoides spp. (Diptera: Ceratopogonidae) in Tunisia, with emphasis on the bluetongue vector Culicoides imicola

Following the bluetongue (BT) outbreaks in Tunisia from 1999 to 2002, BTV (bluetongue virus) serotype 2 was isolated; however, no entomological investigation was performed. In the study presented here, we assessed the Culicoides species populations (particularly C. imicola) in proximity to the BT outbreaks locations, both as a retrospective analysis and to update the list of Culicoides species present in Tunisia. The insects were caught using light traps and the species identification was performed according to the standard entomological methods. This study reveaaled the presence of significant numbers of C. imicola in all the tested locations. In addition, we reported a new Culicoides species for the Tunisian fauna C. punctatus.     

Spatial distribution of Culicoides species in Portugal in relation to the transmission of African horse sickness and bluetongue viruses

Surveillance of Culicoides (Diptera: Ceratopogonidae) biting midge vectors was carried out at 87 sites within a 50 x 50 km grid distributed across Portugal, using light trap collections at the time of peak midge abundance. Culicoides imicola (Kieffer) made up 66% of the 55 937 Culicoides in these summer collections. It was highly abundant in the central eastern portion of Portugal, between 37 degrees 5' N and 41 degrees 5' N, and in a band across to the Lisbon peninsula (at around 38 degrees 5' N). Of all the complexes, its distribution was most consistent with that of previous outbreaks of Culicoides-borne disease, suggesting that it may remain the major vector in Portugal. Its distribution was also broadly consistent with that predicted by a recent climate-driven model validating the use of remote sensing datasets for modelling of Culicoides distribution. Adult C. imicola were found to have overwintered at 12 of 20 sites re-surveyed in winter but it did so in very low numbers. Culicoides obsoletus (Meigen) and Culicoides pulicaris (Linnaeus) complex midges were widespread despite their low summer abundance. The observed coincidence of high abundances of C. imicola and high abundances of C. pulicaris in summer lead us to suggest that C. imicola could bring African horse sickness virus or bluetongue virus into contact with C. pulicaris and the latter complex, together with C. obsoletus, could then transmit these viruses across much wider areas of Europe. The fact that adult C. pulicaris are present in high abundances in winter may provide a mechanism by which these viruses can overwinter in these areas.