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.     

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.     

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.     

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.     

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.     

Real Time RT-PCR Assays for Detection and Typing of African Horse Sickness Virus

Although African horse sickness (AHS) can cause up to 95% mortality in horses, naïve animals can be protected by vaccination against the homologous AHSV serotype. Genome segment 2 (Seg-2) encodes outer capsid protein VP2, the most variable of the AHSV proteins. VP2 is also a primary target for AHSV specific neutralising antibodies, and consequently determines the identity of the nine AHSV serotypes. In contrast VP1 (the viral polymerase) and VP3 (the sub-core shell protein), encoded by Seg-1 and Seg-3 respectively, are highly conserved, representing virus species/orbivirus-serogroup-specific antigens. We report development and evaluation of real-time RT-PCR assays targeting AHSV Seg-1 or Seg-3, that can detect any AHSV type (virus species/serogroup-specific assays), as well as type-specific assays targeting Seg-2 of the nine AHSV serotypes. These assays were evaluated using isolates of different AHSV serotypes and other closely related orbiviruses, from the ‘Orbivirus Reference Collection’ (ORC) at The Pirbright Institute. The assays were shown to be AHSV virus-species-specific, or type-specific (as designed) and can be used for rapid, sensitive and reliable detection and identification (typing) of AHSV RNA in infected blood, tissue samples, homogenised Culicoides, or tissue culture supernatant. None of the assays amplified cDNAs from closely related heterologous orbiviruses, or from uninfected host animals or cell cultures.     

Immunogenicity of plant‐produced African horse sickness virus‐like particles: implications for a novel vaccine

African horse sickness (AHS) is a debilitating and often fatal viral disease affecting horses in much of Africa, caused by the dsRNA orbivirus African horse sickness virus (AHSV). Vaccination remains the single most effective weapon in combatting AHS, as there is no treatment for the disease apart from good animal husbandry. However, the only commercially available vaccine is a live‐attenuated version of the virus (LAV). The threat of outbreaks of the disease outside its endemic region and the fact that the LAV is not licensed for use elsewhere in the world, have spurred attempts to develop an alternative safer, yet cost‐effective recombinant vaccine. Here, we report the plant‐based production of a virus‐like particle (VLP) AHSV serotype five candidate vaccine by Agrobacterium tumefaciens‐mediated transient expression of all four capsid proteins in Nicotiana benthamiana using the cowpea mosaic virus‐based HyperTrans (CPMV‐HT) and associated pEAQ plant expression vector system. The production process is fast and simple, scalable, economically viable, and most importantly, guinea pig antiserum raised against the vaccine was shown to neutralize live virus in cell‐based assays. To our knowledge, this is the first report of AHSV VLPs produced in plants, which has important implications for the containment of, and fight against the spread of, this deadly disease.     

Bloodmeal analysis in Culicoides midges collected near horses,donkeys and zebras in the Eastern Cape,South Africa

An upsurge in African horse sickness (AHS) in the Eastern Cape, South Africa, from 2006 led to an epidemiological reassessment of the disease there. Light trapping surveys carried out near horses, donkeys and zebras in 2014–2016 collected 39 species of Culicoides midge (Diptera: Ceratopogonidae) that are potential vectors of AHS. To establish if these midges fed on equids, DNA sequences were obtained from the gut contents of 52 female midges (35 freshly blood‐fed, 13 gravid and four parous), representing 11 species collected across 11 sites. Culicoides leucostictus fed on all three equids. Culicoides bolitinos, Culicoides imicola and Culicoides magnus fed on both horses and donkeys. Culicoides onderstepoortensis fed on donkeys, and Culicoides similis and Culicoides pycnostictus fed on zebras. Bloodmeals from cows, pigs, warthogs, impalas and a domestic dog were also identified in various species, but none of the midges tested had fed on birds. These results contribute to knowledge of the vectorial capacity of several species of Culicoides with regard to AHS in the Eastern Cape and point to potential reservoir hosts, of which donkeys, zebras and domestic dogs have previously been found to harbour AHS. Blood‐fed midges were also obtained throughout winter, indicating the potential for endemic AHS in the province.     

A comparison of the susceptibility of Culicoides imicola and C. bolitinos to oral infection with eight serotypes of epizootic haemorrhagic disease virus

Abstract. The mechanisms involved in introduction, maintenance and perpetuation of epizootic haemorrhagic disease virus (EHDV) in South Africa are not fully understood. This paper reports on the susceptibility of South African livestock associated Culicoides (Diptera: Ceratopogonidae) species to oral infection with eight EHDV serotypes. Virus was recovered from eight of 17 field-collected Culicoides species 10 days after oral feeding on blood/virus mixtures. Six EHDV serotypes were recovered from C. (Avaritia) imicola Kieffer, and seven serotypes were recovered from C. (A.) bolitinos Meiswinkel. Virus recovery rates in C. imicola ranged from 0.4% for EHDV 2 to 14.4% for EHDV 7, and in C. bolitinos from 0.6% for EHDV 6 to 12.3% for EHDV 2. There was a significant difference in virus recovery rates between serotypes in both species. Other Culicoides species that yielded EHDV after 10 days extrinsic incubation included C. (Meijerehelea) leucostictus Kieffer, C. (Culicoides) magnus Colaço, C. (Beltranmyia) nivosus de Meillon, C. (A.) gulbenkiani Caeiro, C. (Hoffmania) zuluensis de Meillon and C. onderstepoortensis Fiedler. Culicoides midges shown in this study to be susceptible to oral infection with EHDV are widely distributed in South Africa but differ considerably in their abundance, host preference and breeding sites.     

Susceptibility of Culicoides species biting midges to deltamethrin‐treated nets as determined under laboratory and field conditions in the Balearic Islands,Spain

Culicoides Latreille (Diptera: Ceratopogonidae) are vectors of several arboviruses, including bluetongue virus (BTV) and African horse sickness virus (AHSV), which cause diseases in, respectively, sheep and cattle, and horses, and have economic repercussions mainly as a result of trade restrictions. Insecticides can be used to reduce vector populations and hence the spread of disease. Despite the economic importance of these diseases, relatively few studies have evaluated the efficacy of commercially available insecticides and the effectiveness of treated nets against Culicoides species. The aim of the present study was to evaluate the insecticidal effect of commercially available polyethylene nets (ZeroVector^®) treated with deltamethrin (4.4 g/kg ± 15%) on Culicoides species. Laboratory and field trials were conducted in Culicoides populations collected in Majorca in the Balearic Islands, Spain. The present study shows that deltamethrin‐treated nets provoke high and rapid mortality (90–100%) in Culicoides midges under laboratory conditions and increase mortality by 13% when deployed in the field.     

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.     

A comparison of the vector competence of the biting midges,Culicoides (Avaritia) bolitinos and C. (A.) imicola,for the Bryanston serotype of equine encephalosis virus

Abstract Equine encephalosis virus (EEV) is widespread and prevalent in southern Africa. In this study, the oral susceptibility of Culicoides (Avaritia) imicola Kieffer (Diptera: Ceratopogonidae) to EEV was confirmed. In addition, C. (A.) bolitinos Meiswinkel, collected in the high‐lying eastern Free State, South Africa, was systemically infected with the Bryanston serotype of EEV after feeding through a membrane on artificially infected equine blood containing 4.7 log₁₀ PFU/mL of EEV. The mean infectivity of Bryanston virus in C. bolitinos increased from 1.2 log₁₀ PFU/midge, in midges assayed for virus immediately after feeding on the blood‐virus mixture, to 3.1 log₁₀ PFU/midge in midges assayed after 10 days' incubation at 23.5°C. Elevated virus infectivity titres, found in individual infected C. bolitinos, suggested that this Culicoides species is a vector of EEV. This bovine dung‐breeding Culicoides species may play an important role in transmitting EEV in the cooler parts of southern Africa, where it can be the most abundant Culicoides species collected near livestock. In the present study the prevalence of infection obtained for C. bolitinos (2.2%) with the Bryanston serotype of EEV was significantly lower than that of C. imicola (18.4%). After incubation, the Bryanston serotype of EEV was also isolated from one of 110 C. onderstepoortensis Fiedler assayed. However, the virus titre in this midge was 1.2 log₁₀ PFU/midge, which is not different from the titre that would be expected immediately after feeding on the blood‐virus mixture. Culicoides species that survived the incubation period and that were negative for the presence of Bryanston virus were C. magnus Colaço (96), C. bedfordi Ingram & Macfie (95) and C. pycnostictus Ingram & Macfie (45).     

Development and evaluation of real‐time PCR assays for bloodmeal identification in Culicoides midges

Culicoides (Diptera: Ceratopogonidae) midges are the biological vectors of a number of arboviruses of veterinary importance. However, knowledge relating to the basic biology of some species, including their host‐feeding preferences, is limited. Identification of host‐feeding preferences in haematophagous insects can help to elucidate the transmission dynamics of the arboviruses they may transmit. In this study, a series of semi‐quantitative real‐time polymerase chain reaction (qPCR) assays to identify the vertebrate host sources of bloodmeals of Culicoides midges was developed. Two pan‐reactive species group and seven species‐specific qPCR assays were developed and evaluated. The assays are quick to perform and less expensive than nucleic acid sequencing of bloodmeals. Using these assays, it was possible to rapidly test nearly 700 blood‐fed midges of various species from several geographic locations in Australia.     

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.     

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.     

Application of an embryonated chicken egg model to assess the vector competence of Australian Culicoides midges for bluetongue viruses

Culicoides biting midges (Diptera: Ceratopogonidae) are vectors of a number of globally important arboviruses that affect livestock, including bluetongue virus (BTV), African horse sickness virus and the recently emerged Schmallenberg virus. In this study, a model using embryonated chicken eggs (ECEs) was utilized to undertake vector competence studies of Australian Culicoides spp. for 13 laboratory‐adapted or wild‐type virus strains of BTV. A total of 7393 Culicoides brevitarsis were reared from bovine dung, and 3364 Culicoides were induced to feed from ECEs infected with different strains of BTV. Of those, 911 (27%) survived the putative extrinsic incubation period of 9–12 days. In some trials, virus was also transmitted onward to uninfected ECEs, completing the transmission cycle. This model does not rely on the use of colonized midges and has the capacity to assess the vector competence of field‐collected insects with strains of virus that have not previously been passaged in laboratory culture systems. There is also potential for this model to be used in investigations of the competence of Culicoides spp. for other arboviruses.     

Measurement of the Infection and Dissemination of Bluetongue Virus in Culicoides Biting Midges Using a Semi-Quantitative RT-PCR Assay and Isolation of Infectious Virus

Background

Culicoides biting midges (Diptera: Ceratopogonidae) are the biological vectors of globally significant arboviruses of livestock including bluetongue virus (BTV), African horse sickness virus (AHSV) and the recently emerging Schmallenberg virus (SBV). From 2006–2009 outbreaks of BTV in northern Europe inflicted major disruption and economic losses to farmers and several attempts were made to implicate Palaearctic Culicoides species as vectors. Results from these studies were difficult to interpret as they used semi-quantitative RT-PCR (sqPCR) assays as the major diagnostic tool, a technique that had not been validated for use in this role. In this study we validate the use of these assays by carrying out time-series detection of BTV RNA in two colony species of Culicoides and compare the results with the more traditional isolation of infectious BTV on cell culture.

Methodology/Principal Findings

A BTV serotype 1 strain mixed with horse blood was fed to several hundred individuals of Culicoides sonorensis (Wirth & Jones) and C. nubeculosus (Mg.) using a membrane-based assay and replete individuals were then incubated at 25°C. At daily intervals 25 Culicoides of each species were removed from incubation, homogenised and BTV quantified in each individual using sqPCR (C_q values) and virus isolation on a KC-C. sonorensis embryonic cell line, followed by antigen enzyme-linked immunosorbent assay (ELISA). In addition, comparisons were also drawn between the results obtained with whole C. sonorensis and with individually dissected individuals to determine the level of BTV dissemination.

Conclusions/Significance

C_q values generated from time-series infection experiments in both C. sonorensis and C. nubeculosus confirmed previous studies that relied upon the isolation and detection of infectious BTV. Implications on the testing of field-collected Culicoides as potential virus vectors by PCR assays and the use of such assays as front-line tools for use in diagnostic laboratories in this role are discussed.     

Larval development and emergence sites of farm‐associated Culicoides in the United Kingdom

Culicoides biting midges (Diptera: Ceratopogonidae) are the biological vectors of internationally important arboviruses of livestock including bluetongue virus (BTV). Information on the habitats used by Culicoides for larval development is valuable for establishing targeted vector control strategies and for improving local scale models of vector abundance. This study combines emergence trap collections of adult Culicoides identified using molecular markers and physiochemical measurements of habitats to investigate larval development sites of Palaearctic Culicoides in South East England. The known range of larval habitats for several Culicoides species is extended and the potential BTV vector species C. obsoletus and C. scoticus are confirmed to co‐occur in many larval habitats. The presence of emerging C. obsoletus was favoured by increasing substrate moisture level [odds ratio (OR) 6.94 (2.30; 20.90)] and substrate pH [OR 4.80 (1.66; 13.90)] [bias‐corrected D_xy: 0.68; area under the curve (AUC): 0.86] rather than any particular larval habitat type, as expected for a species with relatively wide larval habitat preference. Of the newly emerged sub‐genus Avaritia individuals collected, 23% were observed to have a degree of abdominal pigmentation commonly inferred to indicate parity. If consistent across species and locations, this observation represents a potential source of error for age structure analyses of Culicoides populations.