Comparison of the efficiency of the Onderstepoort- and Centres for Disease Control ultraviolet light traps for the collection of livestock associated Culicoides species in South Africa.

Comparative monitoring of the abundance and distribution of Culicoides biting midges (Diptera: Ceratopogonidae), the biological vectors of the causative agents of several diseases of global veterinary importance, will be crucial in determining the risk of disease outbreak and spread. Ultraviolet (UV) suction traps have become the most frequent method used for the monitoring of Culicoides diversity and abundance. The current study compared the trapping efficiency of the two most used UV suction light traps, i.e., the Onderstepoort (OP)- and the Centres for Disease Control trap, for the collection of livestock associated Culicoides species in South Africa. The study confirmed the superiority of the OP trap and indicated a correlation in species composition and age grading results as determine with the two trap types. Substantial variations in the comparative trap efficiency, as found between areas and sites within an area, suggest that a universal conversion factor between the two trap types may not be advisable as it is unclear to what extent species composition and environmental factors may influence the conversion factor. Light traps, independent of trap model, can be considered acceptable for determining the serial comparison of population numbers for seasonal fluctuation and species abundance in distribution surveys.

Evaluation of light emitting diode suction traps for the collection of livestock-associated Culicoides species in South Africa.

Risk analysis of pathogens transmitted by Culicoides (Diptera; Ceratopogonidae) depends on the ability to detect all potential vectors attacking livestock in an area. Onderstepoort 220-V ultraviolet (UV) down-draught light traps are considered the gold standard for this purpose. To improve the flexibility of this trap in the field, in the absence of 220-V power, the possibility of using low-energy light emitting diodes (LEDs) was assessed. The efficiency of a standard 220-V Onderstepoort trap (30 cm 8 W fluorescent UV light tube) was compared to that of 220-V Onderstepoort traps fitted with either two, four or eight individual white LEDs. The Onderstepoort 220-V trap was also compared to a 12-V Onderstepoort trap fitted with an 8 W fluorescent UV light tube, a 12-V Onderstepoort trap with 12 individual white LEDs and 12-V and 220-V Onderstepoort traps fitted with 12 individual UV LEDs. Higher numbers of Culicoides as well as species diversity were collected with a brighter light source. The use of UV LEDs in both the 12-V and 220-V combinations was comparable to the Onderstepoort 220-V light trap with ration to species diversity collected. The Onderstepoort 220-V light trap is recommended if large numbers of Culicoides need to be collected.

Assessment of the Hemotek® system for the in vitro feeding of field-collected Culicoides imicola (Diptera: Ceratopogonidae) in South Africa.

The optimising and standardisation of in vitro blood feeding protocols for field-collected Culicoides species (Diptera: Ceratopogonidae) will be of essence for the comparison of the vector competencies of various populations of viruses of veterinary importance and the establishment of laboratory colonies of putative vector species. A custom-made feeding chamber to accommodate the small size of Culicoides imicola Kieffer was designed for the commercially available Hemotek® system and compared to existing membrane and cotton pledge feeding methods. High feeding rates coupled to higher mean blood meal volume than that of the existing OVI device indicated that the Hemotek system will be suitable for the feeding of field-collected Culicoides. The Hemotek system was subsequently used to identify factors that may affect feeding success in the laboratory. Evaluated factors were the source (host) and temperature of the blood meal, time of the day of feeding, the position of the blood reservoir in relation to the midges and exposure time to the blood. While only feeding orientation and the temperature of the blood source seems to significantly affect the feeding rate, all the factors did influence the volume of blood consumed.

The applicability of spectrophotometry for the assessment of blood meal volume inartificially fed Culicoides imicola in South Africa.

The volume of the blood meal of haematophagous insects will determine the number of infective particles taken up during feeding and may as such denote the minimum dose needed to infect a competent vector. Culicoides midges resort among the smallest of haematophagous vectors and determining and comparing their blood meal volumes may be challenging. Collected Culicoides imicola females were fed on defibrinated bovine blood through a Parafilm® membrane using a Hemotek® system. After feeding, the weight of pools of 10 engorged females was compared to that of 10 unfed females to determine the volume of blood imbibed. After weighing, the pools were homogenized and their absorbance read at 410 nm. Spectrophotometer readings were then converted to blood meal volumes using calibration curves, obtained by the dilution of known volumes of blood used for feeding. Although the mean blood meal volumes determined spectrophotometrically (0.06 µL), differed significantly (P < 0.01) from those obtained by weighing (0.07 µL), the range in blood meal volumes determined spectrophotometrically (0.03-0.08 µL) and by weighing (0.01-0.11 µL) was positively correlated (r = 0.7; P < 0.01). Both methods can be used to determine the blood meal volume.

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.

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.

The efficiency of light-emitting diode suction traps for the collection of South African livestock-associated Culicoides species.

Culicoides biting midges (Diptera: Ceratopogonidae) are vectors of a range of orbiviruses that cause important veterinary diseases such as bluetongue and African horse sickness. The effective monitoring of Culicoides species diversity and abundance, both at livestock and near potential wildlife hosts, is essential for risk management. The Onderstepoort 220-V ultraviolet (UV) light trap is extensively used for this purpose. Reducing its power requirements by fitting low-energy light-emitting diodes (LEDs) can lead to greater flexibility in monitoring. A comparison of the efficiency of the 220-V Onderstepoort trap (8-W fluorescent UV light) with the efficiency of the 220-V or 12-V Onderstepoort traps fitted with red, white, blue or green LEDs or a 12-V fluorescent Onderstepoort trap demonstrated the 220-V Onderstepoort trap to be the most efficient. All the results showed nulliparous Culicoides imicola Kieffer females to be the dominant grouping. Despite the lower numbers collected, 12-V traps can be used in field situations to determine the most abundant species.

Evidence of African horse sickness virus infection of Equus zebra hartmannae in the south-western Khomas Region, Namibia.

Equine mortalities suspected to be due to African horse sickness (AHS) were reported from the arid Khomas Region, Namibia, in 2008. The area was previously considered a localized AHS-free area. Hartmann's mountain zebra (Equus zebra hartmannae), a potential but unconfirmed reservoir host of African horse sickness virus (AHSV), occurs in the region. Between 2009 and 2010 serum, blood and tissue samples from 31 culled E. z. hartmannae were analysed by reverse transcription-polymerase chain reaction (RT-PCR) (n = 31) and enzyme-linked immunosorbent assay (ELISA) (n = 18) to determine the presence of AHSV and/or antibodies against AHSV. The presence of antibodies against AHSV was demonstrated in all 18 samples assayed, and AHSV double stranded RNA was detected in 26% of the animals. This is evidence that E. z. hartmannae can become infected with AHSV.

Evaluation of larviposition site selection of Glossina brevipalpis.

Tsetse species (Diptera: Glossinidae) are vectors of trypanosome parasites which cause disease in both humans and livestock. In South Africa Glossina austeni Newstead, 1912 and G. brevipalpis Newstead, 1911 are responsible for the cyclical transmission of animal trypanosomes causing African animal trypanosomiasis also referred to as nagana. Gravid tsetse females deposit a single larva in specific sites but little information is available on biotic and abiotic factors that govern site selection. This study therefore aimed to characterize some of the substrate conditions that may influence selection of larviposition sites. Colonised, gravid female G. brevipalpis were presented with a choice of four larviposition sites. Sites differed in qualities of pH (5, 7, 9), salinity (0, 1.3, 4g/L) and the presence of other tsetse pupae (G. brevipalpis or G. austeni). These trials indicated no significant selection by gravid females with regard to pH and salinity. Females selected significantly more often for sites with pupae (P<0.05), but also favored sites containing conspecific over heterospecific pupae (P<0.05). These results present the first indication of an aggregation effect of tsetse pupae in G. brevipalpis. This may imply that G. brevipalpis larvae produce a pheromone during pupation as seen in G. morsitans morsitans. Isolation of such semio-chemicals would allow the development of larviposition traps to attract gravid females.

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.

Efficacy of alphacypermethrin-treated high density polyethylene mesh applied to jet stalls housing horses against Culicoides biting midges in South Africa.

The efficacy of alphacypermethrin-treated high density polyethylene (HDPE) mesh applied to jet stalls against Culicoides biting midges (Diptera: Ceratopogonidae) was determined by mechanical aspiration of midges from horses and using Onderstepoort 220 V downdraught black light traps in four blocks of a 3 × 2 randomised design under South African field conditions. The alphacypermethrin-treated HDPE mesh applied to the stall significantly (P = 0.008) reduced the number of Culicoides midges, predominantly Culicoides (Avaritia) imicola Kieffer, mechanically aspirated from horses housed in the stall. The mesh reduced the Culicoides midge attack rate in the treated stall compared to the untreated stall and a sentinel horse by 6 times and 14 times, respectively. The number of Culicoides midges and C. imicola collected in light traps from the untreated and alphacypermethrin HDPE mesh-treated stalls did not differ significantly (P = 0.82). Alphacypermethrin-treated HDPE mesh could be used to reduce exposure of horses in jet stalls to Culicoides midges, specifically C. imicola, and the risk of midge-borne Orbivirus transmission.

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.

Laboratory and field evaluations of chemical and plant-derived potential repellents against Culicoides biting midges in northern Spain.

The efficacy of 23 compounds in repelling Culicoides biting midges (Diptera: Ceratopogonidae), particularly Culicoides obsoletus (Meigen) females, was determined by means of a Y-tube olfactometer. The 10 most effective compounds were further evaluated in landing bioassays. The six most promising compounds (including chemical and plant-derived repellents) were evaluated at 10% and 25% concentrations in field assays using Centers for Disease Control (CDC) light traps. At least three compounds showed promising results against Culicoides biting midges with the methodologies used. Whereas olfactometer assays indicated DEET at 1 µg/µL to be the most effective repellent, filter paper landing bioassays showed plant-derived oils to be better. Light traps fitted with polyester mesh impregnated with a mixture of octanoic, decanoic and nonanoic fatty acids at 10% and 25% concentrations collected 2.2 and 3.6 times fewer midges than control traps and were as effective as DEET, which is presently considered the reference standard insect repellent. The best plant-derived product was lemon eucalyptus oil. Although these have been reported as safe potential repellents, the present results indicate DEET and the mixture of organic fatty acids to be superior and longer lasting.

Field and in vitro insecticidal efficacy of alphacypermethrin-treated high density polyethylene mesh against Culicoides biting midges in South Africa.

The efficacy of untreated and alphacypermethrin-treated high density polyethylene (HDPE) mesh against Culicoides biting midges (Diptera: Ceratopogonidae) was determined using Onderstepoort downdraught black light traps and a contact bioassay. Three traps were operated overnight in four replicates of a 3×3 randomised Latin square design near horses under South African field conditions. Both the untreated and alphacypermethrin-treated HDPE mesh significantly (P<0.05) reduced the numbers of Culicoides midges, predominantly Culicoides (Avaritia) imicola Kieffer, collected in the light traps by 4.2 and 7.2 times, respectively. A repellent effect of the alphacypermethrin-treated mesh was not confirmed because the number of midges collected in the light traps with untreated and alphacypermethrin-treated HDPE mesh was not significantly different (P=0.656). Bioassay of the insecticidal contact efficacy indicated median C. imicola mortality of 100% from 30 and 10 min following exposure to the alphacypermethrin-treated HDPE mesh for 1 or 3 min, respectively. In the bioassay, mortality was significantly higher (P=0.016) at 5 min post exposure in the midges exposed to the alphacypermethrin-treated mesh for 3 min (74.8%) compared to the 1 min exposure group (59.5%). The HDPE mesh could be used to reduce exposure of housed animals to Culicoides midges, specifically C. imicola, and viruses transmitted by these midges. Mesh treated with alphacypermethrin had the additional benefit of a rapid insecticidal effect on C. imicola.

The attraction range of the Onderstepoort 220V 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.

Recovery rates of bluetongue virus serotypes 1, 2, 4 and 8 Spanish strains from orally infected Culicoides imicola in South Africa.

Bluetongue (BT) is an infectious disease of ruminants that has spread northwards in Europe during the last decade. The aetiological agent of the disease is an arbovirus [bluetongue virus (BTV)] that belongs to the genus Orbivirus (family Reoviridae). The virus is transmitted by certain species of biting midge within the genus Culicoides (Diptera: Ceratopogonidae). Information on the vector status of the Culicoides species in a specific area will be essential to predict the risk for BTV incursion. Field-collected Culicoides (Avaritia) imicola Kieffer from South Africa were fed on blood containing several Spanish isolates of BTV. Despite the high virus concentrations in the bloodmeal (5.1-6.4 log(10) TCID(50) /mL of blood), virus was recovered from <1% of midges assayed after incubation. Virus concentrations >2.5 log(10) TCID(50) /midge in individual infected C. imicola suggest virus replication with possible risk for transmission to susceptible vertebrate hosts in the field for at least two of the serotypes assayed (BTV-1 and BTV-2). A third serotype (BTV-4) was very close to the estimated threshold for transmission. The relatively low to near refractory status of C. imicola compared with other vector species such as Culicoides bolitinos supports previous results, indicating that Culicoides species other than C. imicola may play a more important role in the epidemiology of BTV.

The repellent effect of organic fatty acids on Culicoides midges as determined with suction light traps in South Africa.

The efficacy of a 15% (w/w) mixture of octanoic, nonanoic and decanoic acids in light mineral oil to repel Culicoides biting midges (Diptera; Ceratopogonidae) was determined in three replicates of a 4 × 4 Latin square design under South African field conditions. The fatty acids were applied to ± 0.07 m(2) polyester meshes with a mesh size 2-3mm fitted to 220 V 8 W Onderstepoort downdraught light traps. To reduce the relatively strong attraction of the light trap, the black light tubes in the Onderstepoort trap were replaced with 8 W 23 cm white light tubes. The traps were operating overnight next to cattle. Two traps treated with the mixture of fatty acids collected 1.7 times fewer midges than two untreated traps. Although this mixture of fatty acids had shown a repellent effect against a number of blood-feeding insects this is the first indication that it also has a significant repellent effect against Culicoides species and especially Culicoides (Avaritia) imicola Kieffer when applied to polyester mesh.

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.

The effect of 1-octen-3-ol and 4-methylphenol on Culicoides midge numbers collected with suction light traps in South Africa.

Despite some shortcomings, suction light traps are the primary monitoring tool for the collection of Culicoides species (Diptera: Ceratopogonidae). Factors that may increase the efficiency of these traps need to be investigated. In the present study the numbers of Culicoides midges collected with two Onderstepoort black light traps baited with a mixture of 1-octen-3-ol and 4-methylphenol, as a potential olfactory cue, were compared to those of two unbaited traps. Comparisons were done in two and three replicates of a 4 × 4 randomized Latin square design in the presence and absence of cattle. The addition of 1-octen-3-ol and 4-methylphenol, released at 9.1 and 15.5mg/h, respectively, did not influence species richness, numbers collected, sex ratios or age-grading results. Comparisons of Culicoides numbers and especially the abundance of Culicoides imicola Kieffer in collections done in the presence and absence of cattle confirm previous findings that show that host animals will be the primary attraction for Culicoides midges and that light traps mostly sample midges already in the near vicinity of the host.

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 5.6-7.5 log (10)TCID(50)/mL. After 10 days incubation at 23.5 degrees 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.