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.     

Detection of African horse sickness virus in Culicoides imicola pools using RT‐qPCR

African horse sickness (AHS) is an infectious, non‐contagious arthropod‐borne disease of equids, caused by the African horse sickness virus (AHSV), an orbivirus of the Reoviridae family. It is endemic in sub‐Saharan Africa and thought to be the most lethal viral disease of horses. This study focused on detection of AHSV in Culicoides imicola (Diptera: Ceratopogonidae) pools by the application of a RT‐qPCR. Midges were fed on AHSV‐infected blood. A single blood‐engorged female was allocated to pools of unfed nulliparous female midges. Pool sizes varied from 1 to 200. RNA was extracted and prepared for RT‐qPCR. The virus was successfully detected and the optimal pool size for the limit of detection of the virus was determined at a range between 1 to 25. Results from this investigation highlight the need for a standardized protocol for AHSV investigation in Culicoides midges especially for comparison among different studies and for the determination of infection rate.     

Comparing Culicoides biting rates in horses and cattle in The Netherlands: potential relevance to African horse sickness epidemiology

To determine relative host preference rates and to establish whether a pair of animals in close proximity (between–host interaction) modified Culicoides attack and abundance profiles, compared to those tethered in isolation (host independence), Culicoides midges were pooted hourly from two sets of experimental animals: (1) a heifer cow and Fjord horse tethered close together (5 m apart), and (2) a heifer and Fjord tethered in isolation (45 m apart). Over 12 days, 570 3‐min observations yielded 23 090 midges, representing 24 species. Approximately 95% belonged to the Culicoides obsoletus (Meigen) complex (two species), Culicoides dewulfi Goetghebuer, Culicoides chiopterus (Meigen), Culicoides punctatus (Meigen), Culicoides pulicaris (L.), and Culicoides achrayi Kettle & Lawson (Diptera: Ceratopogonidae) combined. There was no evidence for between‐host interaction. Mean Culicoides species‐specific attack rates did not differ between animal species, except that C. chiopterus was 7× more abundant on the legs of the heifer compared to the horse, and C. dewulfi twice as abundant on the upper half of the horse compared to the heifer. By contrast, mean species–specific biting rates of the C. obsoletus complex, C. chiopterus, C. dewulfi, C. punctatus, and C. pulicaris midges were 5×, 100×, 1.7×, 2×, and 2.5× lower in the horses compared to the heifers, respectively. It is not clear why high Culicoides attack rates observed in the horses do not convert into high biting rates as seen in cattle; this should be a subject for future research. In light of its apparent predilection for equids, the ability of C. dewulfi to replicate African horse sickness virus (AHSV) extrinsically should be investigated in the laboratory.     

Diel activity and preferred landing sites in Culicoides biting midges attacking Fjord horses

In the summer of 2014, in the central part of The Netherlands, Culicoides spp. (Diptera: Ceratopogonidae) attack rates, biting rates, and preferred landing sites were determined for a pair of Fjord horses maintained permanently at pasture in an area devoid of cattle. Eleven body regions of the horses were screened for midges, each region sampled randomly for 5 min using a handheld mouth aspirator (pooter). Observations were confined to the hour immediately before and after sunset. Culicoides spp. were obtained from every body region, of which the four most abundant species – Culicoides chiopterus (Meigen), Culicoides punctatus (Meigen), the species complex Culicoides obsoletus (Meigen), and Culicoides dewulfi Goetghebuer – all were proven or potential vectors for arboviral diseases in livestock. Culicoides spp. activity was distinctly bimodal across the day, surging at sunset and 1 h after sunrise. Midges were inactive between 11:00 and 16:00 hours, these hours marking the time of day when horses can be pastured most safely but, thereafter, to avoid escalating attacks, would have to be stabled protectively. Around sunset, the mean attack rate of the four most abundant species ranged from 3.0 to 11.7 midges per min; of these, C. dewulfi and C. chiopterus were reared out of the dung of experimental horses. The Netherlands is home to the world's densest horse population (11 per km²), of which half are estimated to stay outdoors permanently with no access to protective housing. In the absence of a preventive vaccination policy, it is difficult to envisage how horses in northern Europe will be protected from infection during an outbreak of a Culicoides‐transmitted disease like African horse sickness.     

Revealing potential bridge vectors for BTV and SBV: a study on Culicoides blood feeding preferences in natural ecosystems in Spain

Several species of Culicoides (Diptera: Ceratopogonidae) are vectors of pathogens, such as the bluetongue (BTV) and Schmallenberg (SBV) viruses, which cause important diseases in domestic and wild ruminants. As wild ruminants can contribute to overwintering and epizootics of both diseases, knowledge of the host‐feeding behaviour of Culicoides in natural ecosystems is important to better understand their epidemiology. Blood‐engorged Culicoides females trapped in natural areas inhabited by different wild ruminant species were genetically analysed to identify host species. The origin of bloodmeals was identified in 114 females of 14 species of Culicoides. A total of 104 (91.1%) Culicoides fed on mammals and 10 (8.9%) on birds. The most abundant host identified was red deer (66.7%), followed by humans (13%) and fallow deer (6.1%). Eleven of the 14 species of Culicoides fed exclusively on mammalian hosts. Among them, five are mammalophilic species considered to be important BTV and/or SBV vectors. The results of the present study confirm that Culicoides imicola, Culicoides obsoletus, Culicoides scoticus, Culicoides pulicaris and Culicoides punctatus fed on wild ruminants, and therefore support the hypothesis that these species can act as bridge vectors by facilitating the circulation of pathogens between wild and domestic ruminant communities.     

Host use patterns of Culicoides spp. biting midges at a big game preserve in Florida,U.S.A., and implications for the transmission of orbiviruses

Culicoides spp. biting midges (Diptera: Ceratopogonidae) are vectors of pathogens that have a significant economic impact on the livestock industry. White‐tailed deer (Odocoileus virginianus), a farmed species in the U.S.A., are susceptible to two Culicoides spp. borne orbiviruses: bluetongue virus and epizootic haemorrhagic disease virus. Elucidating host–vector interactions is an integral step in studying disease transmission. This study investigated the host range of Culicoides spp. present on a big game preserve in Florida on which a variety of Cervidae and Bovidae freely roam. Culicoides were captured with Centers for Disease Control and Prevention (CDC) miniature light traps run twice weekly on the preserve for 18 consecutive months (July 2015–December 2016). Host preference was quantified through forage ratios, based upon PCR‐based bloodmeal analysis of Culicoides spp. and overall animal relative abundance on the preserve. Culicoides stellifer preferentially fed on Cervus spp. and fallow deer (Dama dama) and displayed a relative avoidance of Bovidae and white‐tailed deer. Culicoides debilipalpis preferred white‐tailed deer and avoided all Bovidae. Culicoides pallidicornis and Culicoides biguttatus showed preferences for white‐tailed deer and Père David's deer (Elaphurus davidianus), respectively. These results add to current knowledge of preferred hosts of Florida Culicoides spp. and have implications for the spread of orbiviruses. Copyright © 2018 John Wiley & Sons, Ltd.     

Influence of carbon dioxide on numbers of Culicoides midges collected with suction light traps in South Africa

To implement risk management against diseases transmitted by species of Culicoides Latreille, 1809 (Diptera: Ceratopogonidae), it is essential to identify all potential vectors. Light traps are the most commonly used tool for the collection of Culicoides midges. Given the indiscriminate artificial attraction of light, traps will collect all night‐flying insects rather than only livestock‐associated Culicoides midges. Factors that may increase the efficacy of traps, especially for livestock‐associated Culicoides midges, require investigation. In the present study, results obtained with Centers for Disease Control (CDC) and Onderstepoort light traps baited with carbon dioxide (CO₂) were compared with those of unbaited controls. Comparisons were made using two replicates of a 4 × 4 randomized Latin square design. With both trap types, the mean numbers of Culicoides midges collected in 16 baited traps were higher than those caught in 16 unbaited traps. Although exceptionally low numbers were collected with the CDC traps, the increases in the numbers and frequency of collection of Culicoides imicola Kieffer, 1913 were more pronounced in the CDC traps compared with the Onderstepoort traps. These results indicate that the addition of CO₂ may increase the efficiency of these traps for the collection of C. imicola and other livestock‐associated Culicoides species.     

Vector competence of Culicoides species and the seroprevalence of homologous neutralizing antibody in horses for six serotypes of equine encephalosis virus (EEV) in South Africa

Abstract. Field‐collected Culicoides species (Diptera: Ceratopogonidae) were fed on horse blood–virus mixtures containing one of the six serotypes of equine encephalosis virus (EEV1 to EEV6). The virus mean titres in the bloodmeals varied between 6.1 and 7.0 log₁₀TCID₅₀/mL. Of 19 Culicoides species assayed after 10 days extrinsic incubation at 23.5°C, five yielded the challenge virus, namely Culicoides (Avaritia) imicola Kieffer (EEV1–6), C. (A.) bolitinos Meiswinkel (EEV1, 2, 4, 6), C. (Meijerehelea) leucostictus Kiefer (EEV1, 2), C. (Culicoides) magnus Colaço (EEV1) and C. (Hoffmania) zuluensis de Meillon (EEV2). Virus recovery rates ranged from 0.5 to 13%. The mean levels of viral replication differed between serotypes and Culicoides species and ranged from 1.0 to 2.3 log₁₀TCID₅₀/midge. Culicoides midges shown in this study to be susceptible to oral infection with EEV are widely distributed in South Africa but differ considerably in their abundance, host preference and breeding sites. Of 1456 horses tested, 1144 (77%) had antibody to EEV. Homologous virus‐neutralizing antibodies to all six serotypes were detected in individual horses from all eight geographical provinces of South Africa. The distribution, prevalence, and the rate of exposure to individual serotypes varied significantly between regions. The potential for vectoring of EEV in the field by several Culicoides species with unique ecologies and lack of cross‐protection to re‐infection with multiple serotypes highlights some of the mechanisms that are likely to play a role in the virus' natural maintenance cycle and the highly efficient level of countrywide transmission amongst South African horses.     

Emergence of Culicoides obsoletus group species from farm‐associated habitats in Germany

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) may transmit several arboviruses to ruminant livestock. The species of the Obsoletus group are considered to be among the most important vectors of bluetongue virus (BTV) in northern Europe. As agricultural environments offer suitable habitats for the development of their immature stages, the emergence of adult Culicoides from potential breeding sites was investigated at 20 cattle farms throughout Germany in 2012 and 2013. In analyses of species‐specific habitat preferences and relationships between Culicoides abundance in breeding substrates and their physicochemical characteristics, dungheaps emerged as the most important substrate for the development of Culicoides obsoletus sensu stricto (s.s.) (Meigen), whereas Culicoides chiopterus (Meigen) and Culicoides dewulfi Goetghebuer were generally restricted to cowpats. A decreasing pH value was associated with a higher abundance or a higher probability of observing these three species. Furthermore, the abundance of C. obsoletus s.s. was positively related to increasing moisture. Dungheaps were very productive breeding sites for this species and are therefore suggested as a target for potential control measures.     

Abundance,diversity and temporal activity of adult Culicoides spp. associated with cattle in West Bengal,India

Relative abundance, species composition and temporal activity of Culicoides midges were studied for a period of 2 years (2012–2014) using suction ultra violet light traps at two sites located in the agriculture heartland of West Bengal, India. Surveillance in close proximity to cattle recorded predominance of five species with C oxystoma and C. peregrinus as the most dominant species followed by C. fulvus, C. innoxius and C. anophelis. The temporal activity of midges was investigated for seven consecutive nights at one site in August-September, 2012 and the predominant species was Culicoides oxystoma followed by Culicoides peregrinus. All of the species exhibited crepuscular activity with their flight activity increasing from dusk to dawn. Engorged adults constituted dominant age group in collections. Studies on population ecology of the adults midges are of considerable importance predicting for the epidemicity of midge-borne diseases in cattle.     

Barcoding of biting midges in the genus Culicoides: a tool for species determination

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are insect vectors of economically important veterinary diseases such as African horse sickness virus and bluetongue virus. However, the identification of Culicoides based on morphological features is difficult. The sequencing of mitochondrial cytochrome oxidase subunit I (COI), referred to as DNA barcoding, has been proposed as a tool for rapid identification to species. Hence, a study was undertaken to establish DNA barcodes for all morphologically determined Culicoides species in Swedish collections. In total, 237 specimens of Culicoides representing 37 morphologically distinct species were used. The barcoding generated 37 supported clusters, 31 of which were in agreement with the morphological determination. However, two pairs of closely related species could not be separated using the DNA barcode approach. Moreover, Culicoides obsoletus Meigen and Culicoides newsteadi Austen showed relatively deep intraspecific divergence (more than 10 times the average), which led to the creation of two cryptic species within each of C. obsoletus and C. newsteadi. The use of COI barcodes as a tool for the species identification of biting midges can differentiate 95% of species studied. Identification of some closely related species should employ a less conserved region, such as a ribosomal internal transcribed spacer.     

First molecular identification of the vertebrate hosts of Culicoides imicola in Europe and a review of its blood‐feeding patterns worldwide: implications for the transmission of bluetongue disease and African horse sickness

Culicoides (Diptera: Ceratopogonidae) are vectors of pathogens that affect wildlife, livestock and, occasionally, humans. Culicoides imicola (Kieffer, 1913) is considered to be the main vector of the pathogens that cause bluetongue disease (BT) and African horse sickness (AHS) in southern Europe. The study of blood‐feeding patterns in Culicoides is an essential step towards understanding the epidemiology of these pathogens. Molecular tools that increase the accuracy and sensitivity of traditional methods have been developed to identify the hosts of potential insect vectors. However, to the present group's knowledge, molecular studies that identify the hosts of C. imicola in Europe are lacking. The present study genetically characterizes the barcoding region of C. imicola trapped on farms in southern Spain and identifies its vertebrate hosts in the area. The report also reviews available information on the blood‐feeding patterns of C. imicola worldwide. Culicoides imicola from Spain feed on blood of six mammals that include species known to be hosts of the BT and AHS viruses. This study provides evidence of the importance of livestock as sources of bloodmeals for C. imicola and the relevance of this species in the transmission of BT and AHS viruses in Europe.     

Characterization of two cryptic species,Culicoides stigma and C.parroti (Diptera: Ceratopogonidae), based on barcode regions and morphology

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are insect vectors of economically important veterinary diseases such as African horse sickness, bluetongue, and Schmallenberg virus. The identification of Culicoides based on morphological features can be difficult. Three species of biting midges, Culicoides nubeculosus, C. stigma, and C. parroti have emerged in the laboratory from mud collected around watering troughs on a farm in northern France. Emerging Culicoides were characterized morphologically and molecularly using molecular markers. The closely related species C. stigma and C.parroti showed highly divergent sequences for both mitochondrial (cytochrome B and cytochrome oxidase I) and ribosomal DNA first internal transcribed spacer. A RFLP based on a single restriction using the same enzyme (HaeIII) for both cytochrome C oxidase I and cytochrome B is proposed to identify these species.     

First detection of endosymbiotic bacteria in biting midges Culicoides pulicaris and Culicoides punctatus,important Palaearctic vectors of bluetongue virus

Heritable bacteria have been highlighted as important components of vector biology, acting as required symbionts with an anabolic role, altering competence for disease transmission, and affecting patterns of gene flow by altering cross compatibility. In this paper, we tested eight U.K. species of Culicoides (Diptera: Ceratopogonidae) midge for the presence of five genera of endosymbiotic bacteria: Cardinium (Bacteroidales: Bacteroidaceae); Wolbachia (Rickettsiales: Rickettsiaceae); Spiroplasma (Entomoplasmatales: Spiroplasmataceae); Arsenophonus (Enterobacteriales: Enterobacteriaceae), and Rickettsia (Rickettsiales: Rickettsiaceae). Cardinium spp. were detected in both sexes of Culicoides pulicaris and Culicoides punctatus, two known vectors of bluetongue virus. Cardinium spp. were not detected in any other species, including the Culicoides obsoletus group, the main vector of bluetongue and Schmallenberg viruses in northern Europe. The other endosymbionts were not detected in any Culicoides species. The Cardinium strain detected in U.K. Culicoides species is very closely related to the Candidatus Cardinium hertigii group C, previously identified in Culicoides species in Asia. Further, we infer that the symbiont is not a sex ratio distorter and shows geographic variation in prevalence within a species. Despite its detection in several species of Culicoides that vector arboviruses worldwide, the absence of Cardinium in the C. obsoletus group suggests that infections of these symbionts may not be necessary to the arboviral vector competence of biting midges.     

A modified vaccinia Ankara virus (MVA) vaccine expressing African horse sickness virus (AHSV) VP2 protects against AHSV challenge in an IFNAR -/- mouse model

African horse sickness (AHS) is a lethal viral disease of equids, which is transmitted by Culicoides midges that become infected after biting a viraemic host. The use of live attenuated vaccines has been vital for the control of this disease in endemic regions. However, there are safety concerns over their use in non-endemic countries. Research efforts over the last two decades have therefore focused on developing alternative vaccines based on recombinant baculovirus or live viral vectors expressing structural components of the AHS virion. However, ethical and financial considerations, relating to the use of infected horses in high biosecurity installations, have made progress very slow. We have therefore assessed the potential of an experimental mouse-model for AHSV infection for vaccine and immunology research. We initially characterised AHSV infection in this model, then tested the protective efficacy of a recombinant vaccine based on modified vaccinia Ankara expressing AHS-4 VP2 (MVA-VP2).     

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.     

Farms,pastures and woodlands: the fine‐scale distribution of Palearctic Culicoides spp. biting midges along an agro‐ecological gradient

The spatial epidemiology of Bluetongue virus (BTV) at the landscape level relates to the fine‐scale distribution and dispersal capacities of its vectors, midges belonging to the genus Culicoides Latreille (Diptera: Ceratopogonidae). Although many previous researches have carried out Culicoides sampling on farms, little is known of the fine‐scale distribution of Culicoides in the landscape immediately surrounding farms. The aim of this study was to gain a better understanding of Culicoides populations at increasing distances from typical dairy farms in north‐west Europe, through the use of eight Onderstepoort‐type black‐light traps positioned along linear transects departing from farms, going through pastures and entering woodlands. A total of 16 902 Culicoides were collected in autumn 2008 and spring 2009. The majority were females, of which more than 97% were recognized as potential vectors. In pastures, we found decreasing numbers of female Culicoides as a function of the distance to the farm. This pattern was modelled by leptokurtic models, with parameters depending on season and species. By contrast, the low number of male Culicoides caught were homogeneously distributed along the transects. When transects entered woodlands, we found a higher abundance of Culicoides than expected considering the distance of the sampling sites to the farm, although this varied according to species.     

Seasonal abundance of livestock‐associated Culicoides species in northeastern Brazil

Culicoides biting midges (Diptera: Ceratopogonidae) are of great medical and veterinary importance because the haematophagous females of some species can transmit diseases to humans and animals. In order to determine the presence and seasonal abundance of the bluetongue virus (BTV) vector Culicoides insignis Lutz at domestic animal sheds in northeastern Brazil, insects were collected once a month between January and December 2010. Light traps were set from 18.00 to 06.00 hours at a pigsty, chicken coop and bovine corral. Culicoides insignis accounted for 81% of the 22 316 specimens collected. Other well‐represented species were: Culicoides paucienfuscatus Barbosa (3246 individuals), Culicoides diabolicus Hoffman (308), Culicoides leopoldoi Ortiz (224) and Culicoides duartei Tavares and Luna Dias (221). The remainder accounted for 4% of the total sample. Culicoides insignis occurred mostly at the cattle corral, 98.2% in the rainy season. This study confirms the presence and close association of C. insignis with cattle in Maranhão state, northeastern Brazil and emphasizes the risk of bluetongue infections spreading in the area.