The vector potential of British Culicoides species for bluetongue virus

Two species of British Culicoides, C. nubeculosus and C. impunctatus were found to support bluetongue virus (BTV) multiplication after ingestion of the virus. Both species were infected by membrane feeding and C. nubeculosus also became infected after feeding on viraemic sheep. This species was shown to transfer the virus across a membrane after 8 days incubation at 25 degrees C and could therefore presumably act as a BTV vector. Six other British species of Culicoides supported BTV multiplication after intrathoracic inoculation of the virus.     

Culicoides vectors of bluetongue virus in Chester Zoo

On four nights in June 2008, light traps were operated for Culicoides biting midges, the vector species for bluetongue virus (BTV), at five sites in Chester Zoo in north-west England. Over 35,000 Culicoides midges, of 25 species, were captured, including high densities inside animal enclosures. Over 94 per cent of all the Culicoides trapped were females of the Obsoletus group, which is implicated as the vector of BTV serotype 8 in northern Europe. The mean catch of this group per trap per night was over 1500, suggesting a potential risk of BTV transmission if the virus is introduced to Chester Zoo in the animals or midges in the summer.     

Evaluation of Culicoides insignis (Diptera: Ceratopogonidae) as a vector of bluetongue virus.

Based upon epidemiological evidence, Culicoides insignis Lutz is a probable biological vector of bluetongue viruses (BTV) in South Florida, the Caribbean Region and Central America. The vector potential of this species for BTV was evaluated in the laboratory in a series of experiments using insects caught in the field. Although there was great variation in the percentage of flies that fed from any one catch, it was demonstrated that C. insignis became infected after membrane feeding on a mixture of blood and virus. The infection rates ranged from 20 to 62.5%. Following intrathoracic inoculation, BTV replicated to high titres in C. insignis. Such flies were also shown to be capable of transmitting BTV to susceptible sheep and embryonated chicken eggs. This series of experiments provides the first conclusive evidence that C. insignis is a biological vector of bluetongue virus. This is the first proven vector of BTV in the neotropics.     

Rapid diagnostic PCR assays for members of the Culicoides obsoletus and Culicoides pulicaris species complexes, implicated vectors of bluetongue virus in Europe

Biting midges of the Culicoides obsoletus Meigen and Culicoides pulicaris L. species complexes (Diptera: Ceratopogonidae) are increasingly implicated as vectors of bluetongue virus in Palearctic regions. However, predicting epidemiological risk and the spread of disease is hampered because whilst vector competence of Culicoides is expressed only in adult females, morphological identification of constituent species is only readily applicable to adult males and some species distinguishing traits have overlapping character states. Furthermore, adult males are typically rare in field collections, making characterisation of Culicoides communities impossible. Here we highlight the utility of mitochondrial cytochrome oxidase subunit I (COI) DNA sequences for taxonomic resolution and species identification of all species within C. obsoletus and C. pulicarus complexes. Culicoides were collected from 18 sites in the UK and Continental Europe, and identified to species level, or species complex level, based on morphological characters. The sample comprised four species from the C. obsoletus complex (n = 88) and five species from the C. pulicaris complex (n = 39). The DNA sequence of the 5' end of the COI gene was obtained from all individuals. Each member species formed a well-supported reciprocally monophyletic clade in a maximum likelihood phylogeny. Levels of DNA sequence divergence were sufficiently high between species to allow the design of species-specific PCR primers that can be used in PCR for identification of members of the C. pulicaris complex or in a multiplex PCR to identify members of the C. obsoletus complex. This approach provides a valuable diagnostic tool for monitoring species composition in mixed field collections of Culicoides.     

Serial cyclic transmission of bluetongue virus in sheep and Culicoides variipennis.

Bluetongue virus (strain 62-45S) was transmitted from sheep to sheep throughout a year by vector bites. A colonized population (SONORA strain, 000 line) of the biological vector Culicoides variipennis (Coquilllett) was used. Fifteen serial cyclic transmissions covered a period of 13 months from October through November of the following year. The mean infection rate of the biting gnats was 37 percent. The clinical response to bluetongue virus was significantly more severe in sheep infected by vector bites than in those inoculated with the virus at the same sheep-serial passage level. A second corroborative serial transmission was conducted for 7 months from June through December. The mean infection rate of the vector was 27%.     

Ornithodoros coriaceus (pajaroello tick) as a vector of bluetongue virus

Preliminary studies demonstrated that the argasid tick, Ornithodoros coriaceus Koch, could become infected with bluetongue virus (BTV). Ticks became infected after feeding through artificial membranes on BTV-infected suspensions of cell cultures, chicken embryos, and sheep blood. Ticks also became infected after natural feeding on viremic sheep (BTV serotype 17) and cattle (BTV serotype 11). Virus was recovered from the hemolymph and salivary glands of ticks which had ingested BTV either through an artificial membrane or by natural feeding on a host animal. Ticks infected with BTV serotype 13 were capable of transmitting the virus to a susceptible cow at 42 days after ingestion of virus-infected cultures, thus demonstrating the potential of the tick to serve as a biological vector of BTV.     

Simultaneous quantification of the relative abundance of species complex members: application to Culicoides obsoletus and Culicoides scoticus (Diptera: Ceratopogonidae), potential vectors of bluetongue virus

The two sympatric sibling species Culicoides obsoletus (Meigen) and Culicoides scoticus Downes and Kettle (Diptera: Ceratopogonidae), are known to be competent vectors for bluetongue virus in the Palaearctic region. However, morphological identification of constituent species is only readily applicable to adult males and these two species distinguishing traits have overlapping character states. As their vector competence may differ in space and time, the correct identification and quantification of specimens of both species are essential for understanding bluetongue epidemiology. However, no molecular tools are available for high-throughput identification of the two species. We therefore developed a quantitative duplex real-time PCR assay to determine the relative abundance of each sibling species in a sample using TaqMan probes. For each species, standard curves were constructed from serial dilutions of purified plasmid DNA containing ITS1-5.8S-ITS2 (rDNA) in the range of 10(-1) to 10(-5)ng/μL. Standard curves were used to quantify samples of mixed C. obsoletus/C. scoticus specimens. Specificity was evaluated with 5156 specimens representing 62 species. Based on the DNA quantities detected according to the standard curves, a quadratic model developed on 1100 males and validated on 555 females was able to predict the relative abundance of each species simultaneously in a one-shot reaction (Pearson coefficient of 0.999). Our assay showed a requirement of two specimens or less for 95% of the predictions, making it highly applicable to field collections. Extensive use of this real-time PCR assay will provide a better understanding of geographical distribution, dynamics, and bionomics on a species level, which is essential for risk assessment. This approach is an important contribution to medical entomology for investigating the vector role of arthropod sibling species.     

The Culicoides 'snapshot': a novel approach used to assess vector densities widely and rapidly during the 2006 outbreak of bluetongue (BT) in The Netherlands

A novel method was developed and implemented during the recent outbreak of bluetongue (BT) in sheep and cattle in The Netherlands to obtain rapidly a 'snapshot' of Culicoides vector densities at the national level. The country was divided into 110 raster cells, each measuring 20kmx20km; within 106 of these cells, a farm was selected with a minimum of 10 cattle and sampled for Culicoides for one night only using the Onderstepoort-type blacklight trap. Prior to deployment of the light traps in the field, local veterinarians were trained in their use and in the preservation of captured Culicoides. The collections were despatched daily by courier to a field laboratory where the Culicoides were counted and identified. The 'snapshot' commenced on 12 September 2006 and was completed on 28 September coinciding with the 5-7 weeks of BT virus (BTV) activity in The Netherlands and when the number of weekly cases of disease was on the rise. Analysis of the 106 collections was completed on 5 October. The number of grid cells in which a taxon occurred is represented by the index 20(2)gFR (=20kmx20km grid Frequency Rate); this index essentially reflects the percentage of examined raster cells found to contain the potential vector in question. The 'snapshot' results can be summarised as follows: The northward advance of BT in Europe compels the competent authorities in affected and in neighbouring territories to acquire rapidly baseline information around which to plan sound vector surveillance and livestock movement strategies. The Culicoides 'snapshot' is a tool well suited to this purpose. It is stressed that a vector surveillance program must be built upon a firm taxonomic base because misidentifications will flaw the mapped seasonal and geographic distribution patterns upon which veterinary authorities depend.     

Genetic variation of bluetongue virus serotype 11 isolated from host (sheep) and vector (Culicoides variipennis) at the same site

Genetic relatedness of 2 strains of bluetongue virus (BTV) serotype 11 that were isolated from the same geographic site--one from host (sheep) and the other from the vector Culicoides variipennis during an enzootic of bluetongue at Bruneau, Idaho, in August 1973--was determined by comparing the oligonucleotide fingerprint analyses of the individual double-stranded RNA segments of the genomes. It was observed that the 2 strains of BTV-11 exhibit considerable differences in their genotypes, the percentage of diversity being different for each of the corresponding RNA species of the 2 strains of BTV-11. These results indicate that more than one genotype of BTV can circulate in juxtaposition in a given geographic site. The observed genotypic diversity might be due to the accumulation of point mutations on specific RNA species or antecedent reassortment of RNA segments between different BTV in nature or both.