Gert J. Venter

Oral susceptibility to bluetongue virus of Culicoides (Diptera: Ceratopogonidae) from the United Kingdom.

Abstract Oral susceptibility to infection with bluetongue virus (family Resviridae, genus Orbivirus, BTV) serotype 9 was characterized in three Palaearctic species of Culicoides (Diptera: Ceratopogonidae). Variation in susceptibility to infection by using a recently described feeding technique was shown to occur between populations of Culicoides obsoletus Meigen complex midges from different geographic regions of the United Kingdom with virus infection rates varying from 0.4 to 7.4% of those tested. Susceptibility to infection was consistent on an annual basis at selected sites. Prevalence of infection in the most susceptible populations of both the C. obsoletus and Culicoides pulicaris L. complexes was comparable with that of Culicoides imicola Kieffer, the major vector of BTV in southern Europe and throughout Africa, when using the same feeding method and virus. These results are discussed with reference to the potential threat of the virus to susceptible livestock in northern Europe.

Comparison of the efficiency of five suction light traps under field conditions in South Africa for the collection of Culicoides species.

Culicoides biting midges (Diptera: Ceratopogonidae) are involved in the transmission of a variety of pathogens, the economically most important being the orbiviruses that cause bluetongue and African horse sickness; both of which have been shown to be multi-vector diseases. The identification of all potential vectors will be crucial for the implementation of integrated control measures and disease risk analysis. The primary monitoring tools used for the collection of Culicoides midges are various models of suction light traps. In order to facilitate comparison of data between laboratories the efficiency of five traps (Onderstepoort, Rieb, mini-CDC, Pirbright, BG-sentinel), used at present and in the past in Europe, was compared in the field in South Africa. Comparisons were done either in three replicates of a 4x4 or two replicates of a 5x5 randomized Latin square design. The Onderstepoort trap collected significantly more Culicoides midges than the other traps. Relatively small but statistically significant differences were found in the species composition, parous rates, sex ratios as well as the ratio of Culicoides midges to other insects, as determined by the different traps. It will be important to determine the significance and underlying causes for these differences.

Temperature Dependence of the Extrinsic Incubation Period of Orbiviruses in Culicoides Biting Midges

Background The rate at which viruses replicate and disseminate in competent arthropod vectors is limited by the temperature of their environment, and this can be an important determinant of geographical and seasonal limits to their transmission by arthropods in temperate regions. Methodology/Principal Findings Here, we present a novel statistical methodology for estimating the relationship between temperature and the extrinsic incubation period (EIP) and apply it to both published and novel data on virus replication for three internationally important orbiviruses (African horse sickness virus (AHSV), bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV)) in their Culicoides vectors. Our analyses show that there can be differences in vector competence for different orbiviruses in the same vector species and for the same orbivirus in different vector species. Both the rate of virus replication (approximately 0.017-0.021 per degree-day) and the minimum temperature required for replication (11-13°C), however, were generally consistent for different orbiviruses and across different Culicoides vector species. The estimates obtained in the present study suggest that previous publications have underestimated the replication rate and threshold temperature because the statistical methods they used included an implicit assumption that all negative vectors were infected. Conclusions/Significance Robust estimates of the temperature dependence of arbovirus replication are essential for building accurate models of transmission and for informing policy decisions about seasonal relaxations to movement restrictions. The methodology developed in this study provides the required robustness and is superior to methods used previously. Importantly, the methods are generic and can readily be applied to other arbovirus-vector systems, as long as the assumptions described in the text are valid.

Bionomics of temperate and tropical Culicoides midges: knowledge gaps and consequences for transmission of Culicoides-borne viruses.

Culicoides midges are abundant hematophagous flies that vector arboviruses of veterinary and medical importance. Dramatic changes in the epidemiology of Culicoides-borne arboviruses have occurred since 1998, including the emergence of exotic viruses in northern temperate regions, increases in global disease incidence, and enhanced virus diversity in tropical zones. Drivers may include changes in climate, land use, trade, and animal husbandry. New Culicoides species and new wild reservoir hosts have been implicated in transmission, highlighting the dynamic nature of pathogen-vector-host interactions. Focusing on potential vector species worldwide and key elements of vectorial capacity, we review the sensitivity of Culicoides life cycles to abiotic and biotic factors. We consider implications for designing control measures and understanding impacts of environmental change in different ecological contexts. Critical geographical, biological, and taxonomic knowledge gaps are prioritized. Recent developments in genomics and mathematical modeling may enhance ecological understanding of these complex arbovirus systems.

Life-history parameters of Culicoides (Avaritia) imicola Kieffer in the laboratory at different rearing temperatures.

This laboratory study investigates the sub-adult developmental cycle of field collected Culicoides (Avaritia) imicola Kieffer (Diptera; Ceratopogonidae). The period required from blood-feeding field-collected females to the production of progeny adults occupied 34-56 days at 20 degrees C, 15-21 days at 25 degrees C and 11-16 days at 28 degrees C, demonstrating clear temperature dependence. When reared at 28 degrees C, C. imicola demonstrated higher variability in fecundity (between 2.4 and 20.6 eggs/female) and lower hatching rates (50.0-62.2%), although larval survival rates to pupation were low at all temperatures (20-30%). Similarly, the mean emergence rate from pupae was the highest at lower temperatures. These results highlight the difficulty in establishing and maintaining a laboratory colony of this species from field-collected material and results are discussed in reference to future research directions that may aid this process.

Comparison of two trapping methods for Culicoides biting midges and determination of African horse sickness virus prevalence in midge populations at Onderstepoort, South Africa

Culicoides biting midges (Diptera: Ceratopogonidae) are vectors of a variety of pathogens including African horse sickness virus (AHSV), a member of the family Reoviridae, genus Orbivirus. AHSV causes African horse sickness (AHS), an endemic disease of equids with an extremely high mortality rate in horses in sub-Saharan Africa. Culicoides (Avaritia) imicola Kieffer is considered to be the principal vector of AHSV and is the dominant Culicoides species in South Africa. Due to the global distribution of Culicoides vectors, there is a potential risk of AHS spreading from endemic areas to areas traditionally free of the disease, which could have a severe economical impact on the affected equine industry. As part of any risk assessment it is essential to monitor known vectors as well as potential vector species. In the present study, sampling of Culicoides insects was compared using overnight collections in the conventional Onderstepoort light trap and mechanical aspiration of midges at sunset from bait horses. Culicoides imicola was confirmed as the predominant species using both trapping methods. Other species, mainly Culicoides (Avaritia) bolitinos Meiswinkel and Culicoides (Avaritia) gulbenkiani Caeiro, were highly underrepresented in the light trap collections, but made a significant contribution to the mechanical aspiration catches. The time for optimal collection differed between the trapping methods, leading to the conclusion that mechanical aspiration is a useful addition to conventional light trap collection and possibly the better choice when investigating insect vectors. An infection rate of 1.14% was calculated for the midge population based on real-time quantitative reverse-transcription polymerase chain reaction (RT-qPCR) assays of collected Culicoides midges, which exceeds previous estimates. This is probably due to the increased sensitivity of the RT-qPCR assay used in this study as compared to the virus isolation assays used in previous studies. RT-qPCR-positive midges were present in midge pools obtained from both light trap and mechanical aspiration. Seven of the positive pools consisted of C. imicola only, four contained mixed species and one pool contained no C. imicola, suggesting the presence of AHSV in midges of other species.

Vector competence of selected South African Culicoides species for the Bryanston serotype of equine encephalosis virus.

Equine encephalosis virus (EEV) was recognized and described in the Republic of South Africa in 1967 and subsequent serological studies have shown this orbivirus to be both widespread and prevalent in southern Africa. In the present study it was shown that wild‐caught Culicoides (Avaritia) imicola Kieffer (Diptera: Ceratopogonidae) can become infected with and permit the replication of the Bryanston serotype of EEV following membrane‐feeding on infective blood containing 5.0 log10 plaque‐forming‐units (PFU)/ml. The mean prevalence of Bryanston virus infection in C. imicola after 10 days extrinsic incubation at 23.5°C was 22.3% (23/103). The mean infectivity of Bryanston virus in the infected C. imicola increased from 1.3 log10 PFU/midge, in insects assayed immediately after feeding on the blood‐virus mixture, to 2.6 log10 PFU/midge in insects assayed after incubation. The virus concentration in individual C. imicola infected with the Bryanston serotype of EEV ranged from 0.7 to 3.6 log10 PFU/midge. Bryanston virus titres higher than 2.5 log10 TCID50, found in individual C. imicola, suggest that this species may be able to transmit this virus to susceptible hosts. Prevalence of virus infection in C. imicola was determined by PFU and microtitration assays on both BHK and Vero cells and confirmation of the Bryanston serotype of EEV was determined by plaque inhibition. No virus replication could be demonstrated in 102 C. nivosus tested after the incubation period, suggesting that not all Culicoides species are equally susceptible to Bryanston virus infection. Other Culicoides species that survived the incubation period and that were negative for the presence of Bryanston virus were C. pycnostictus (42), C. leucostictus (7), C. magnus (2), C. bolitinos (1) and C. bedfordi (1).

First record of Culicoides oxystoma Kieffer and diversity of species within the Schultzei group of Culicoides Latreille (Diptera: Ceratopogonidae) biting midges in Senegal

The Schultzei group of Culicoides Latreille (Diptera: Ceratopogonidae) is distributed throughout Africa to northern Asia and Australasia and includes several potential vector species of livestock pathogens. The taxonomy of the species belonging to this species group is confounded by the wide geographical distribution and morphological variation exhibited by many species. In this work, morphological and molecular approaches were combined to assess the taxonomic validity of the species and morphological variants of the Schultzei group found in Senegal by comparing their genetic diversity with that of specimens from other geographical regions. The species list for Senegal was updated with four species: Culicoides kingi, C. oxystoma, C. enderleini and C. nevilli being recorded. This is the first record of C. oxystoma from Africa south of Sahara, and its genetic relationship with samples from Israel, Japan and Australia is presented. This work provides a basis for ecological studies of the seasonal and spatial dynamics of species of this species group that will contribute to better understanding of the epidemiology of the viruses they transmit.

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.

The attraction range of the Onderstepoort 220 V light trap for Culicoides biting midges as determined under South African field conditions

Despite some limitations suction light traps are the primary tools used for the collection of Culicoides species (Diptera: Ceratopogonidae). The range of attraction of the Onderstepoort light trap is not known but an insight into the range of a trap will determine where the trap must be positioned relative to the hosts present, possible breeding sites and environmental structures in the trapping vicinity. It will therefore contribute to a more meaningful interpretation and comparison of results between trapping events. In the present study the number of Culicoides midges collected in a single trap was compared to those of traps made with an additional trap respectively 1m, 4m and 8.5m away from the first. Treatments between sites were rotated in three replicates of a 4×4 Latin square design. While interactions were found in traps 4m apart no statistically significant interactions were found when they were 8.5m apart. The range of attraction, indicated by the interaction between two traps, will be between 2m and 4m. In interpreting light trap results the limitations of this collection method needs to be taken into consideration.