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.

Identifying potential candidate Culicoides spp. for the study of interactions with Candidatus Cardinium hertigii.

Culicoides biting midges (Diptera: Ceratopogonidae) are vectors responsible for the transmission of several viruses of veterinary importance. Previous screens of Culicoides have described the presence of the endosymbiont Candidatus Cardinium hertigii (Bacteroidetes). However, any impacts of this microbe on vectorial capacity, akin to those conferred by Wolbachia in mosquitoes, are yet to be uncovered and await a suitable system to study Cardinium-midge interactions. To identify potential candidate species to investigate these interactions, accurate knowledge of the distribution of the endosymbiont within Culicoides populations is needed. We used conventional and nested PCR assays to screen Cardinium infection in 337 individuals of 25 Culicoides species from both Palearctic and Afrotropical regions. Infections were observed in several vector species including C. imicola and the Pulicaris complex (C. pulicaris, C. bysta, C. newsteadi and C. punctatus) with varying prevalence. Phylogenetic analysis based on the Gyrase B gene grouped all new isolates within 'group C' of the genus, a clade that has to date been exclusively described in Culicoides. Through a comparison of our results with previous screens, we suggest C. imicola and C. sonorensis represent good candidates for onward study of Cardinium-midge interactions.

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.

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.

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.

Colonization of the Mediterranean basin by the vector biting midge species Culicoides imicola: an old story.

Understanding the demographic history and genetic make-up of colonizing species is critical for inferring population sources and colonization routes. This is of main interest for designing accurate control measures in areas newly colonized by vector species of economically important pathogens. The biting midge Culicoides imicola is a major vector of orbiviruses to livestock. Historically, the distribution of this species was limited to the Afrotropical region. Entomological surveys first revealed the presence of C. imicola in the south of the Mediterranean basin by the 1970s. Following recurrent reports of massive bluetongue outbreaks since the 1990s, the presence of the species was confirmed in northern areas. In this study, we addressed the chronology and processes of C. imicola colonization in the Mediterranean basin. We characterized the genetic structure of its populations across Mediterranean and African regions using both mitochondrial and nuclear markers, and combined phylogeographical analyses with population genetics and approximate Bayesian computation. We found a west/east genetic differentiation between populations, occurring both within Africa and within the Mediterranean basin. We demonstrated that three of these groups had experienced demographic expansions in the Pleistocene, probably because of climate changes during this period. Finally, we showed that C. imicola could have colonized the Mediterranean basin in the Late Pleistocene or Early Holocene through a single event of introduction; however, we cannot exclude the hypothesis involving two routes of colonization. Thus, the recent bluetongue outbreaks are not linked to C. imicola colonization event, but rather to biological changes in the vector or the virus.

Vector competence of Culicoides for arboviruses: three major periods of research, their influence on current studies and future directions.

The spectacular and unprecedented outbreaks of bluetongue virus (BTV) that have occurred in Europe since 1998 have led to increased interest in those factors that determine competence of Culicoides biting midges (Diptera: Ceratopogonidae) for arboviruses. In this review the authors critically examine three major periods of research into the biological transmission by Culicoides of two economically important arboviruses ofthefamily Reoviridae: African horse sicknessvirus (AHSV) and BTV. First they examine early studies, largely conducted in southern Africa, that played a key role in initially implicating Culicoides as agents of AHSV and BTV transmission. Then they examine advances in understanding made following the establishment of colonies of the BTV vector species Culicoides sonorensis, which have largely shaped our current understanding of BTV and AHSV transmission. They then consider attempts in recent years to implicate vectors of BTV in the European Union during what has become the most economically damaging series of outbreaks in recorded history. In some cases the origin of these outbreaks was uncertain and unexpected, particularly in northern Europe, where BTV had not previously occurred. Limitations imposed on studies of vector competence by the biology of Culicoides are then discussed, along with advances in the technologies now available and the logistics of working upon agents requiring biosecure containment outside their endemic range. Finally, the authors suggest areas that have either been poorly addressed to date or entirely ignored and ways in which studies could be conducted to provide standardised data for comparison worldwide.

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.

Degenerative trends of the palmaris longus muscle in a South African population.

The literature reports that the palmaris longus muscle (PL) is only found in mammals in which the forelimbs are weight-bearing extremities. It is suggested that the function of this muscle has been taken over by the other flexors in the forearm. Terms used in the literature to describe the diminishing of this muscle include retrogressive or phylogenetic degenerative trends. The aims of this study were to determine the prevalence of PL in a South African population and whether a phylogenetic degenerative trend for the PL exists. To determine the prevalence of the PL, five groups, representing different age intervals (Years 0-20, 21-40, 41-60, 61-80, and 81-99) were used. A sample of 706 participants of various ages was randomly selected. Statistical analysis included comparisons of the prevalence of the muscle between males and females and left and right sides, using a student t-test. A Chi-squared test was used to determine a possible phylogenetic degenerative trend of PL within the five groups. The sample yielded a bilateral absence of the PL in 11.9% of the cases. The muscle was unilaterally absent on the left side in 7.65% and 6.94% on the right side. The Chi-squared tests revealed a P-value of 0.27 for the left arm and 0.39 for the right arm. No obvious trend could be established for the phylogenetic degeneration of the PL in this study. It would appear that the PL muscle should not be considered as a phylogenetically degenerating muscle in a South African population.

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 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.

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.

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 log(10)TCID(50)/mL of blood. After 10 days incubation at 23.5 degrees 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.

Duration of repellency of N,N-diethyl-3-methylbenzamide, citronella oil and cypermethrin against Culicoides species when applied to polyester mesh.

The repellent efficacy of 15% N,N-diethyl-3-methylbenzamide (DEET), 0.6% citronella oil, and 0.3% alpha-cyano-cypermethrin against Culicoides species was compared in three 5x5 Latin squares (15 replicates) under South African field conditions. DEET, citronella oil or alpha-cyano-cypermethrin were applied to polyester meshes that were fitted to down-draught suction 220V UV light traps which were operated overnight. No significant repellent effect against Culicoides was found for the citronella oil or the alpha-cyano-cypermethrin. DEET had a significant repellent effect against Culicoides species and C. imicola for all catches made from after sunset to before sunrise.

Description and comparison of the pupae of a further two Culicoides (Avaritia) species from the dung of large herbivores in South Africa (Diptera:Ceratopogonidae).

In 2007 Nevill, Venter, Meiswinkel & Nevill demonstrated that the pupae of five Culicoides species belonging to the Imicola complex of the subgenus Avaritia could readily be differentiated from one another using various morphological characters. Three of the described species, Culicoides bolitinos Meiswinkel 1989; Culicoides loxodontis Meiswinkel 1992 and Culicoides sp. # 107 (= C. kwagga, Meiswinkel, unpublished thesis 1995), were reared from the dung of large herbivores, which included buffaloes, elephants, white and black rhinoceroses and zebras. However, during that study a further two Avaritia species, neither of which belonged to the Imicola complex, were reared from dung and these are the subject of the present study. For the past 20 years the adults of these two new closely related species have been known as Culicoides sp. # 54 pale form (p.f.) Meiswinkel and Culicoides sp. # 54 dark form (d.f.) Meiswinkel. The taxonomic description and formal naming of the adults of these two species has yet to be done. The present description and comparison of their pupae show that they are two clearly distinct species; that there is no group of morphological characters that can be used to differentiate these two species from the previously described five species of the Imicola complex; and finally that there was no difference between the pupae of C. sp. # 54 d.f. nor C. sp. # 54 p.f. reared from the dung of different host animals.