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.     

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.     

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.     

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.     

Modelling the distributions and spatial coincidence of bluetongue vectors Culicoides imicola and the Culicoides obsoletus group throughout the Iberian peninsula

Data obtained by a Spanish national surveillance programme in 2005 were used to develop climatic models for predictions of the distribution of the bluetongue virus (BTV) vectors Culicoides imicola Kieffer (Diptera: Ceratopogonidae) and the Culicoides obsoletus group Meigen throughout the Iberian peninsula. Models were generated using logistic regression to predict the probability of species occurrence at an 8-km spatial resolution. Predictor variables included the annual mean values and seasonalities of a remotely sensed normalized difference vegetation index (NDVI), a sun index, interpolated precipitation and temperature. Using an information-theoretic paradigm based on Akaike's criterion, a set of best models accounting for 95% of model selection certainty were selected and used to generate an average predictive model for each vector. The predictive performances (i.e. the discrimination capacity and calibration) of the average models were evaluated by both internal and external validation. External validation was achieved by comparing average model predictions with surveillance programme data obtained in 2004 and 2006. The discriminatory capacity of both models was found to be reasonably high. The estimated areas under the receiver operating characteristic (ROC) curve (AUC) were 0.78 and 0.70 for the C. imicola and C. obsoletus group models, respectively, in external validation, and 0.81 and 0.75, respectively, in internal validation. The predictions of both models were in close agreement with the observed distribution patterns of both vectors. Both models, however, showed a systematic bias in their predicted probability of occurrence: observed occurrence was systematically overestimated for C. imicola and underestimated for the C. obsoletus group. Average models were used to determine the areas of spatial coincidence of the two vectors. Although their spatial distributions were highly complementary, areas of spatial coincidence were identified, mainly in Portugal and in the southwest of peninsular Spain. In a hypothetical scenario in which both Culicoides members had similar vectorial capacity for a BTV strain, these areas should be considered of special epidemiological concern because any epizootic event could be intensified by consecutive vector activity developed for both species during the year; consequently, the probability of BTV spreading to remaining areas occupied by both vectors might also be higher.     

Impacts of climate, host and landscape factors on Culicoides species in Scotland

Culicoides biting midges (Diptera: Ceratopogonidae) vector a wide variety of internationally important arboviral pathogens of livestock and represent a widespread biting nuisance. This study investigated the influence of landscape, host and remotely-sensed climate factors on local abundance of livestock-associated species in Scotland, within a hierarchical generalized linear model framework. The Culicoides obsoletus group and the Culicoides pulicaris group accounted for 56% and 41%, respectively, of adult females trapped. Culicoides impunctatus Goetghebuer and C. pulicaris s.s. Linnaeus were the most abundant and widespread species in the C. pulicaris group (accounting for 29% and 10%, respectively, of females trapped). Abundance models performed well for C. impunctatus, Culicoides deltus Edwards and Culicoides punctatus Meigen (adjusted R(2) : 0.59-0.70), but not for C. pulicaris s.s. (adjusted R(2) : 0.36) and the C. obsoletus group (adjusted R(2) : 0.08). Local-scale abundance patterns were best explained by models combining host, landscape and climate factors. The abundance of C. impunctatus was negatively associated with cattle density, but positively associated with pasture cover, consistent with this species' preference in the larval stage for lightly grazed, wet rush pasture. Predicted abundances of this species varied widely among farms even over short distances (less than a few km). Modelling approaches that may facilitate the more accurate prediction of local abundance patterns for a wider range of Culicoides species are discussed.     

Phylogeny of the subgenus Culicoides and related species in Italy, inferred from internal transcribed spacer 2 ribosomal DNA sequences

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) include vectors for the economically important animal diseases, bluetongue (BT) and African horse sickness (AHS). In the Mediterranean Basin, these diseases are transmitted by four species of Culicoides: the first three belong in the subgenus Avaritia Fox and are Culicoides imicola Kieffer, Culicoides obsoletus (Meigen) and Culicoides scoticus Downes and Kettle; the fourth is Culicoides pulicaris (Linnaeus) in the subgenus Culicoides Latreille. In the Palaearctic Region, this subgenus (usually referred to as the C. pulicaris group) now includes a loose miscellany of some 50 taxa. The lack of clarity surrounding its taxonomy stimulated the present morphological and molecular study of 11 species collected in Italy. Phylogenetic analysis of nuclear ribosomal DNA internal transcribed spacer 2 (ITS2) sequence variation demonstrated a high degree of divergence. These results, combined with those from a parallel morphological study, disclosed: (1) that some previously described taxa should be resurrected from synonymy; (2) that there are new species to be described; (3) that the subgenus Culicoides (as currently employed) is a polyphyletic assemblage of four lineages - the subgenus Culicoides sensu stricto, the subgenus Silvicola Mirzaeva and Isaev, the subgenus Hoffmania Fox and the hitherto unrecognized Fagineus species complex. Each is discussed briefly (but not defined) and its constituent Palaearctic taxa listed. Strong congruence between morphological and molecular data holds promise for resolving many of the difficult taxonomic issues plaguing the accurate identification of vector Culicoides around the world.     

An investigation on the Culicoides species composition at seven sites in southern Switzerland

In the past decade, there have been regular outbreaks of bluetongue (BT) in many parts of Europe. Owing to the presence of BT disease and its vectors in countries adjacent to Switzerland, an initial entomological survey was conducted in 2003, which established the presence of several midges of the genus Culicoides (Diptera: Ceratopogonidae). Subsequently, a sentinel herd monitoring system was established with the primary entomological aim being the determination and further study of Culicoides population compositions. Insects were collected in 2005 and 2006 at seven sentinel herd sites in the south of Switzerland (canton of Ticino) near the border of Italy, using Onderstepoort-type light traps. This region is botanically and zoologically similar to the Mediterranean and is one of the warmest and most humid areas of the country, hence it is considered a potential access path for BT disease into Switzerland. Collections were made at four cattle farms, two equestrian centres and one goat farm. Sites were sampled four times per month from June to October. Traps were operated from dusk until dawn and samples were collected monthly for analysis through microscopy as well as a Culicoides imicola -specific PCR. Results confirmed the absence of C. imicola (Kieffer) and demonstrated that the potential BT virus vectors are highly abundant, notably: Culicoides obsoletus (Meigen), Culicoides scoticus (Downes & Kettle) and Culicoides dewulfi (Goetghebuer) subgenus Avaritia and Culicoides pulicaris (Linnaeus) subgenus Culicoides . These findings expand the current knowledge of Culicoides population composition in the southern part of the Switzerland. Culicoides cataneii (Clastrier), Culicoides flavipulicaris (Dzhafarov), Culicoides indistinctus (Khalaf), Culicoides nubeculosus (Meigen) and species of the Grisescens complex were reported for the first time in Switzerland.     

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.     

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.     

Recovery rates of bluetongue virus serotypes 1, 2, 4 and 8 Spanish strains from orally infected Culicoides imicola in South Africa

Bluetongue (BT) is an infectious disease of ruminants that has spread northwards in Europe during the last decade. The aetiological agent of the disease is an arbovirus [bluetongue virus (BTV)] that belongs to the genus Orbivirus (family Reoviridae). The virus is transmitted by certain species of biting midge within the genus Culicoides (Diptera: Ceratopogonidae). Information on the vector status of the Culicoides species in a specific area will be essential to predict the risk for BTV incursion. Field-collected Culicoides (Avaritia) imicola Kieffer from South Africa were fed on blood containing several Spanish isolates of BTV. Despite the high virus concentrations in the bloodmeal (5.1-6.4 log(10) TCID(50) /mL of blood), virus was recovered from <1% of midges assayed after incubation. Virus concentrations >2.5 log(10) TCID(50) /midge in individual infected C. imicola suggest virus replication with possible risk for transmission to susceptible vertebrate hosts in the field for at least two of the serotypes assayed (BTV-1 and BTV-2). A third serotype (BTV-4) was very close to the estimated threshold for transmission. The relatively low to near refractory status of C. imicola compared with other vector species such as Culicoides bolitinos supports previous results, indicating that Culicoides species other than C. imicola may play a more important role in the epidemiology of BTV.     

五种库蠓脂肪酸的研究

本文用气相色语法分析了不显库蠓(Culicoides obsoletus)、兴安库蠓(C, sinanoensis)、怒江库蠓(C.Nujiangensis)、灰黑库蠓(C.Pulicaris)和刺整库蠓(C.Punctatus)等5种库蠓雌虫脂 肪醛的组成和含量。 结果表明,5种库蠓均含有12-14种主要脂肪酸,并以棕榈油酸(C_16：1)、棕搁酸 (C_16：0)、油酸(C_18：1)、硬脂酸(C_18：0)含量最高,共占脂肪酸总量的70%以上。对主要脂肪酸的含量进行聚类分析,发现不显库蠓种团内任意两种库蠓间的欧氏距离小于不显库蠓种团种类与灰黑库蠓种团种类间的欧氏距离,说明脂肪酸的定性、定量分析结果对库蠓分类有一定的参考价值。     

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.     

Presence in the Balearic Islands (Spain) of the midges Culicoides imicola and Culicoides obsoletus group

An outbreak of the livestock viral disease bluetongue (BT) was detected during September and October 2000 in the Balearic Islands, Spain. Due to the lack of information about the species of Culicoides (Diptera: Ceratopogonidae) reported in the affected area, six farms in Majorca, four in Minorca and one in Ibiza were selected to carry out surveillance of Culicoides adults using light traps. Here, for the first time, we report the presence in the Balearic Islands of Culicoides imicola Keiffer, the main vector of BT, and the Culicoides obsoletus Meigen group.     

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.     

Evaluation of housing as a means to protect cattle from Culicoides biting midges, the vectors of bluetongue virus

The housing of animals at night was investigated as a possible means of protecting them from attack by Culicoides biting midges (Diptera: Ceratopogonidae), the vectors of bluetongue. Light-trap catches of Culicoides were compared inside and outside animal housing, in the presence and absence of cattle. A three-replicate, 4 × 4 Latin square design was used at four farms in Bala, north Wales, over 12 nights in May and June 2007, and the experiment repeated in October. In the two studies, respectively, >70 000 and >4500 Culicoides were trapped, of which 93% and 86%, respectively, were of the Culicoides obsoletus group. 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 6.5 times more insects 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 higher maximum wind speeds. These conditions occur more frequently in October than in May and June, thereby suppressing outside catches more than 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 and the benefit would be greatest on warm, still nights when outside catches are at their greatest.