A comparison of the susceptibility of the biting midge Culicoides imicola to infection with recent and historical isolates of African horse sickness virus

The susceptibility of Culicoides (Avaritia) imicola Kiefer (Diptera: Ceratopogonidae) to 21 isolates representing all nine known serotypes of African horse sickness virus (AHSV), recovered from clinical cases of the disease in South Africa during 1998–2004, was compared with its susceptibility to approximately 40‐year‐old isolates stored at the Agricultural Research Council‐Onderstepoort Veterinary Institute. Field‐collected C. imicola were fed through a chicken skin membrane on sheep blood spiked with one of the virus isolates to a concentration in the range of 5.6–7.5log ₁₀TCID₅₀/mL. After 10 days incubation at 23.5 °C, five of the nine historical serotypes (AHSV‐1, ‐2, ‐3, ‐7 and ‐9) could not be isolated from C. imicola. All nine serotypes were recovered for the 21 recent isolates, for 16 of which the virus recovery rates were higher than for the corresponding historical isolates. These results emphasize the need to assess the oral susceptibility of local Culicoides populations to viruses in circulation during outbreaks in order to estimate their vector potential.     

The oral susceptibility of South African field populations of Culicoides to African horse sickness virus

Twenty‐two isolates of African horse sickness virus (AHSV), representing its distinct serotypes, geographical and historical origins, were fed to three populations of South African livestock‐associated Culicoides spp. (Diptera, Ceratopogonidae). Infective blood meals included 12 recent isolates, nine historical reference strains and one live attenuated vaccine strain serotype 7 (AHSV‐7) of the virus. Field‐collected midges were fed through a chicken‐skin membrane on sheep blood spiked with one of the viruses, which concentrations ranged from 5.4 to 8.8 log₁₀TCID₅₀/mL of blood. After 10 days incubation at 23.5°C, AHSV was isolated from 11 Culicoides species. Standard in vitro passaging of AHSV‐7, used for the preparation of live attenuated vaccine, did not reduce its ability to infect Culicoides species. Virus recovery rates in orally infected Culicoides midges differed significantly between species and populations, serotypes, isolates and seasons. Significant variations in oral susceptibility recorded in this study emphasize a complex inter‐relationship between virus and vector, which is further influenced by multiple intrinsic and extrinsic factors. As it is not possible to standardize all these factors under laboratory conditions, conclusive assessment of the role of field‐collected Culicoides midges in the transmission of orbiviruses remains problematic. Nevertheless, results of this study suggest the potential for multi‐vector transmission of AHSV virus in South Africa.     

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.     

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.     

Oral susceptibility of South African Culicoides species to live-attenuated serotype-specific vaccine strains of African horse sickness virus (AHSV)

Abstract. The oral susceptibility of livestock‐associated South African Culicoides midges (Diptera: Ceratopogonidae) to infection with the tissue culture‐attenuated vaccine strains of African horse sickness virus (AHSV) currently in use is reported. Field‐collected Culicoides were fed on horse blood‐virus mixtures each containing one of the seven serotype‐specific vaccine strains of AHSV, namely serotypes 1, 2, 3, 4, 6, 7 and 8. The mean titres of virus in the bloodmeals for the seven vaccine strains were between 6.8 and 7.6 log₁₀TCID50/mL. All females (n = 3262) that survived 10 days extrinsic incubation (10 dEI) at 23.5°C were individually assayed in microplate BHK‐21 cell cultures. In midges tested immediately after feeding, AHSV was detected in 96.1% individuals; mean virus titre was 2.0 log₁₀TCID50/midge. After 10 dEI virus recovery rates varied in Culicoides (Avaritia) imicola Kieffer from 1% (AHSV‐2) to 11% (AHSV‐7) and in Culicoides (A.) bolitinos Meiswinkel from 0% (AHSV‐3) to 14.6% (AHSV‐2). Although our results indicate that two major field vectors C. imicola and C. bolitinos are susceptible to oral infection with vaccine strains of AHSV, the level of viral replication for most of the vaccine strains tested was below the postulated threshold (=2.5 log₁₀TCID50/midge) for fully disseminated orbivirus infection. In this study, for the first time AHSV has been recovered after 10 dEI from six non‐Avaritia livestock‐associated Old World species: C. engubandei de Meillon (AHSV‐4), C. magnus Colaço (AHSV‐3, ‐4), C. zuluensis de Meillon (AHSV‐2, ‐4), C. pycnostictus Ingram & Macfie (AHSV‐2), C. bedfordi Ingram & Macfie (AHSV‐7), and C. dutoiti de Meillon (AHSV‐7). As little is known about the virogenesis of AHSV in the southern African species of Culicoides, the epidemiological significance of our findings in relation to the potential for transmission of current AHSV vaccine strains by Culicoides requires further assessment.     

Culicoides species as potential vectors of African horse sickness virus in the southern regions of South Africa

African horse sickness (AHS), a disease of equids caused by the AHS virus, is of major concern in South Africa. With mortality reaching up to 95% in susceptible horses and the apparent reoccurrence of cases in regions deemed non‐endemic, most particularly the Eastern Cape, epidemiological research into factors contributing to the increase in the range of this economically important virus became imperative. The vectors, Culicoides (Diptera: Ceratopogonidae), are considered unable to proliferate during the unfavourable climatic conditions experienced in winter in the province, although the annual occurrence of AHS suggests that the virus has become established and that vector activity continues throughout the year. Surveillance of Culicoides within the province is sparse and little was known of the diversity of vector species or the abundance of known vectors, Culicoides imicola and Culicoides bolitinos. Surveillance was performed using light trapping methods at selected sites with varying equid species over two winter and two outbreak seasons, aiming to determine diversity, abundance and vector epidemiology of Culicoides within the province. The research provided an updated checklist of Culicoides species within the Eastern Cape, contributing to an increase in the knowledge of AHS vector epidemiology, as well as prevention and control in southern Africa.     

Effect of temperature on the transmission of orbiviruses by the biting midge,Culicoides sonorensis

The influence of temperature on the likelihood of Culicoides sonorensis Wirth & Jones (Diptera: Ceratopogonidae) transmitting African horse sickness virus (AHSV) serotypes 4 and 6, bluetongue virus (BTV) serotypes 10 and 16 and epizootic haemorrhagic disease of deer virus (EHDV) serotype 1 was investigated. Extrinsic incubation periods (EIP), vector competence and vector survival were determined at 15, 20, 25 and 30 degrees C. The effect of humidity on vector survival was also investigated by maintaining adult C. sonorensis at 40, 75 and 85% r.h. at each temperature. Higher temperatures were associated with a shorter EIP for all virus serotypes except AHSV6, to which C. sonorensis was orally refractory, increased vector competence for AHSV4 and EHDV1, but not for BTV10 or BTV16, and a reduction in vector survival. Humidity interacted with temperature in influencing vector survival, such that at low temperatures, lower humidity (40 and 75% r.h.) was detrimental for survival (up to 18% reduction in longevity), whereas at high temperatures, high humidity (85% r.h.) was detrimental (up to 36% reduction in longevity). In general, the transmission potential of C. sonorensis for AHSV4, EHDV1, BTV10 and BTV16 was greater at higher temperatures, because although vector survival was reduced, this was more than compensated for by the accompanying decrease in duration of the EIP.     

Host preferences of Palaearctic Culicoides biting midges: implications for transmission of orbiviruses

Feeding success depends on host availability, host defensive reactions and host preferences. Host choice is a critical determinant of the intensity at which pathogens are transmitted. The aim of the current study was to describe host preferences of Palaearctic Culicoides species (Diptera: Ceratopogonidae) Latreille using traps baited with the five different host species of poultry, horse, cattle, sheep and goat. Collections were carried out nightly in July and August 2009 in western France with three replicates of a 5 × 5 randomized Latin square (five sites, five hosts). Moreover, an ultraviolet (UV) light/suction trap was operated during host‐baited collections to correlate Culicoides biting rates and UV light/suction trap catches. A total of 660 Culicoides belonging to 12 species, but comprised mainly of Culicoides scoticus Downes and Kettle, Culicoides dewulfi Goetghebuer and Culicoides obsoletus Meigen, were collected on animal baits. Abundance was highest for the horse, which accounted for 95% of all Culicoides caught, representing 10 species. The horse, the largest bait, was the most attractive host, even when abundance data were corrected by weight, body surface or Kleiber's scaling factor. Culicoides obsoletus was the only dominant species attracted by birds. Both C. scoticus and C. dewulfi were collected mainly from the upper body of the horse. Finally, the quantification of host preferences allows for discussion of implications for the transmission of Culicoides‐borne pathogens such as bluetongue virus.     

Phylogenetic analysis of the mitochondrial cytochrome oxidase subunit I gene of five species of the Culicoides imicola species complex

The phylogenetic status of members of the Culicoides imicola Kieffer (Diptera: Ceratopogonidae) species complex of haematophagous midges is unknown, and simple means to identify the members using all life stages are unavailable. In this study, the status of three confirmed (C. imicola s.s., C. bolitinos Meiswinkel and C. loxodontis Meiswinkel) and two provisional (C. tuttifrutti Meiswinkel and C. kwagga Meiswinkel) members of the complex from South Africa was assessed using phylogenetic analysis of partial DNA and amino acid sequences of the mitochondrial cytochrome oxidase subunit I (COI) gene. The four or five individuals of each species analysed contained one or two haplotypes each. Interspecific divergence was significant and characterized by strong A <--> T transversion bias. Phylogenetic trees constructed using neighbour-joining, parsimony and maximum likelihood showed each species to be distinct. Combinations of sites for two restriction enzymes in the COI sequences were species-specific and could form the basis of a diagnostic PCR assay.     

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 and seasonal distribution of Culicoides imicola in Iberia in relation to the transmission of African horse sickness virus

Abstract. Collections of biting midges were made over 24 months from sixty sites spread across Iberia. Information on the distribution of the vector of African horse sickness virus, Culicoides imicola , from these 3119 samples showed that this species was annually present across south-western Spain as far as 3^o53'W and throughout most of Portugal, up to 41^o5'N. C.imicola was found in all areas where African horse sickness epizootics had occurred in 1987-90 and also in areas outside the epizootic zones.
Seasonal patterns of capture success of C.imicola , from seventeen frequently sampled sites where the vector was present, usually showed a late summer-early autumn peak. At the sites furthest south there was a discrete peak, mostly in September or October, before and after which the numbers captured increased or decreased steadily. At higher latitudes peak abundances occurred as early as May or as late as November, population build up was less uniform and numbers often declined rapidly after the peak was reached. Both the distribution and seasonal abundance patterns closely matched transmission patterns of African horse sickness virus, which rose during late summer and caused most cases during the autumn months.     

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.     

Latitudinal diversity of biting midge species within the Obsoletus group across three habitats in Europe

Culicoides species from the Obsoletus group are important vectors of bluetongue and Schmallenberg virus. This group consists of several species that cannot easily be identified using morphological characteristics. Therefore, limited information is available about their distribution and habitat preferences. In this study, we aimed to elucidate the species composition of the Obsoletus group in three habitat types at climatically different latitudes across Europe. Traps were placed in three habitat types in three countries at different latitudes. After DNA extraction, biting midges were identified using PCR and gel electrophoresis. Extraction of DNA using Chelex proved to be a cost and time efficient method for species identification. A latitudinal effect on the relative abundance of species from the Obsoletus group was found. Species composition was unique for most country‐habitat combinations. The majority of biting midges were either C. obsoletus s.s. or C. scoticus, and both species were found at all latitudes and habitats. Their wide distribution and their high abundance at livestock farms make these species likely candidates for rapid farm‐to‐farm transmission of pathogens throughout Europe. Our results emphasize the need to differentiate Obsoletus group species to better understand their ecology and contribution to pathogen transmission.     

Preliminary note on the distribution and ecology of Culicoides imicola in Portugal

Abstract. Data on Culicoides imicola were obtained during studies carried out during the recent outbreak of African horse sickness in Portugal. The previous most northerly published record of C.imicola in Portugal was 38^o40'N (Pégöes). In the present work the geographical distribution of this species is extended to the parallel of 41^o17'N. We have also confirmed the continuous presence of adult C.imicola in Southern Portugal (Alentejo and Algarve) throughout the year. In the laboratory we obtained this species from a sample of cattle faeces and from another of soil contaminated with animal excreta. In relation to host association 57.37% of C.imicola were trapped in the vicinity of pigsties. Finally, we collected 11 ,463 Culicoides of which 12.47% were C. imicola.     

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.     

Control techniques for Culicoides biting midges and their application in the U.K. and northwestern Palaearctic

The recent emergence of bluetongue virus (Reoviridae: Orbivirus) (BTV) in northern Europe, for the first time in recorded history, has led to an urgent need for methods to control the disease caused by this virus and the midges that spread it. This paper reviews various methods of vector control that have been employed elsewhere and assesses their likely efficacy for controlling vectors of BTV in northern Europe. Methods of controlling Culicoides spp. (Diptera: Ceratopogonidae) have included: (a) application of insecticides and pathogens to habitats where larvae develop; (b) environmental interventions to remove larval breeding sites; (c) controlling adult midges by treating either resting sites, such as animal housing, or host animals with insecticides; (d) housing livestock in screened buildings, and (e) using repellents or host kairomones to lure and kill adult midges. The major vectors of BTV in northern Europe are species from the Culicoides obsoletus (Meigen) and Culicoides pulicaris (L.) groups, for which there are scant data on breeding habits, resting behaviour and host-oriented responses. Consequently, there is little information on which to base a rational strategy for controlling midges or for predicting the likely impact of interventions. However, data extrapolated from the results of vector control operations conducted elsewhere, combined with some assessment of how acceptable or not different methods may be within northern Europe, indicate that the treatment of livestock and animal housing with pyrethroids, the use of midge-proofed stabling for viraemic or high-value animals and the promotion of good farm practice to at least partially eliminate local breeding sites are the best options currently available. Research to assess and improve the efficacy of these methods is required and, in the longer term, efforts should be made to develop better bait systems for monitoring and, possibly, controlling midges. All these studies will need better methods of analysing the ecology and behaviour of midges in the field than are currently in use. The paucity of control options and basic knowledge serve to warn us that we must be better prepared for the possible emergence of other midge-borne diseases, particularly African horse sickness.     

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.