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.

A spatiotemporal model to assess the introduction risk of African horse sickness by import of animals and vectors in France.

BACKGROUND: African horse sickness (AHS) is a major, Culicoides-borne viral disease in equines whose introduction into Europe could have dramatic consequences. The disease is considered to be endemic in sub-Saharan Africa. Recent introductions of other Culicoides-borne viruses (bluetongue and Schmallenberg) into northern Europe have highlighted the risk that AHS may arrive in Europe as well. The aim of our study was to provide a spatiotemporal quantitative risk model of AHS introduction into France. The study focused on two pathways of introduction: the arrival of an infectious host (PW-host) and the arrival of an infectious Culicoides midge via the livestock trade (PW-vector). The risk of introduction was calculated by determining the probability of an infectious animal or vector entering the country and the probability of the virus then becoming established: i.e., the virus's arrival in France resulting in at least one local equine host being infected by one local vector. This risk was assessed using data from three consecutive years (2010 to 2012) for 22 regions in France. RESULTS: The results of the model indicate that the annual risk of AHS being introduced to France is very low but that major spatiotemporal differences exist. For both introduction pathways, risk is higher from July to October and peaks in July. In general, regions with warmer climates are more at risk, as are colder regions with larger equine populations; however, regional variation in animal importation patterns (number and species) also play a major role in determining risk. Despite the low probability that AHSV is present in the EU, intra-EU trade of equines contributes most to the risk of AHSV introduction to France because it involves a large number of horse movements. CONCLUSION: It is important to address spatiotemporal differences when assessing the risk of ASH introduction and thus also when implementing efficient surveillance efforts. The methods and results of this study may help develop surveillance techniques and other risk reduction measures that will prevent the introduction of AHS or minimize AHS' potential impact once introduced, both in France and the rest of Europe.

Assessment of the abundance of Culicoides chiopterus and Culicoides dewulfi in bovine dung: a comparison of larvae extraction techniques and emergence traps.

We compared two larvae extraction methods involving sugar-flotation and an adapted Berlese funnel-extraction with emergence traps. This was done in order to analyse the colonisation of cowpats by Culicoides chiopterus (Meigen 1830) and Culicoides dewulfi (Goetghebuer 1936) (Diptera: Ceratopogonidae) and to gain information on the comparability and efficacy of these three methods. With all three methods, a considerable number of individuals was obtained. Significantly more individuals were obtained via sugar-flotation and Berlese funnel-extraction compared to the emergence traps. These differences, likely due to natural mortality and sample processing are discussed. We recommend Berlese as an efficacious method for extracting Culicoides larvae from bovine dung. It produces data rapidly and extracted larvae are viable. In comparison with Berlese, slightly more larvae were obtained by sugar-flotation, but this method was very labour intensive.

Assessment of survival and body size variation of Culicoides imicola (Diptera: Ceratopogonidae) as functions of "Candidatus Cardinium" (Bacteroidetes) infection status.

"Candidatus Cardinium hertigii" (Bacteroidetes) is a maternally inherited endosymbiont known from several arthropods. Its mechanisms for persistence in host populations are mostly reproductive manipulation, though it has been occasionally reported to improve fitness parameters in several hosts. In Culicoides (Diptera: Ceratopogonidae) biting midges, the prevalence of "Candidatus Cardinium" infection was documented as moderate, with no detectable sex bias. We therefore investigated whether "Candidatus Cardinium" affects important fitness parameters, such as survival and body size, in Culicoides imicola, a dominant vector species. Field-collected midges were trapped and analyzed for survival under different environmental conditions and antibiotic treatment, taking into account "Candidatus Cardinium" infection status and parity status (i.e., parous or nulliparous). Additionally, wing lengths were measured as a proxy parameter for body size and analyzed together with "Candidatus Cardinium" infection data. The findings revealed no difference in survival of Culicoides infected with "Candidatus Cardinium" and that of uninfected midges in both parity states and under all tested conditions: optimal, starvation, heat, and antibiotic treatment. Beyond survival, no wing length difference was found for "Candidatus Cardinium"-infected versus uninfected midges. In aggregate, these findings support our conclusion that "Candidatus Cardinium" does not have an overt effect on the survival and size of adult C. imicola midges. "Candidatus Cardinium" may affect immature stages or may alter adult reproductive performance.

Bluetongue disease risk assessment based on observed and projected Culicoides obsoletus spp. vector densities.

Bluetongue is an arboviral disease of ruminants causing significant economic losses. Our risk assessment is based on the epidemiological key parameter, the basic reproduction number. It is defined as the number of secondary cases caused by one primary case in a fully susceptible host population, in which values greater than one indicate the possibility, i.e., the risk, for a major disease outbreak. In the course of the Bluetongue virus serotype 8 (BTV-8) outbreak in Europe in 2006 we developed such a risk assessment for the University of Veterinary Medicine Vienna, Austria. Basic reproduction numbers were calculated using a well-known formula for vector-borne diseases considering the population densities of hosts (cattle and small ruminants) and vectors (biting midges of the Culicoides obsoletus spp.) as well as temperature dependent rates. The latter comprise the biting and mortality rate of midges as well as the reciprocal of the extrinsic incubation period. Most important, but generally unknown, is the spatio-temporal distribution of the vector density. Therefore, we established a continuously operating daily monitoring to quantify the seasonal cycle of the vector population by a statistical model. We used cross-correlation maps and Poisson regression to describe vector densities by environmental temperature and precipitation. Our results comprise time series of observed and simulated Culicoides obsoletus spp. counts as well as basic reproduction numbers for the period 2009-2011. For a spatio-temporal risk assessment we projected our results from the location of Vienna to the entire region of Austria. We compiled both daily maps of vector densities and the basic reproduction numbers, respectively. Basic reproduction numbers above one were generally found between June and August except in the mountainous regions of the Alps. The highest values coincide with the locations of confirmed BTV cases.

A spatial assessment of Culicoides spp. distribution and bluetongue disease risk areas in Austria.

Bluetongue disease (BT) was introduced into Central Europe in the summer of 2006 and has since affected most European countries. In this study we analysed the distribution of the biting midge vector Culicoides spp. in Austria and modelled Bluetongue disease risk zones. Culicoides spp. abundance data was collected from weekly catches of 14 months from 54 trapping locations. The corresponding weather data mean temperature (p < 0.001), wind (p < 0.001), relative humidity (p = 0.019) and altitude (p = 0.059) were identified as predictors on Culicoides spp. distribution in a regression model (R 2.8.0). The majority of catches were detected at temperatures above 10 degrees C and at relative humidities between 65-80%. The point data of these parameters originating from 186 meteorological stations were interpolated using the Geostatistical Analyst Kriging tool (ESRI ArcGIS 9.3). To create seasonal risk maps we overlaid regions with optimal temperature and humidity conditions with domestic ruminants density data. Our results show that the summer season holds the greatest risk of a BT epidemic with 25.9% of the analysed area providing optimal conditions for vector abundance and 12.4% showing contact risk with ruminant hosts. This project (1) provides fundamental data on the Culicoides spp. distribution in Austria, (2) determines limiting climatic parameters on vector abundance and (3) identifies risk areas by including areas of possible host-parasite-interactions. These high-risk areas can subsequently be given special attention for precautionary monitoring and surveillance measures.

Assessment of vector/host contact: comparison of animal-baited traps and UV-light/suction trap for collecting Culicoides biting midges (Diptera: Ceratopogonidae), vectors of Orbiviruses.

BACKGROUND: The emergence and massive spread of bluetongue in Western Europe during 2006-2008 had disastrous consequences for sheep and cattle production and confirmed the ability of Palaearctic Culicoides (Diptera: Ceratopogonidae) to transmit the virus. Some aspects of Culicoides ecology, especially host-seeking and feeding behaviors, remain insufficiently described due to the difficulty of collecting them directly on a bait animal, the most reliable method to evaluate biting rates.Our aim was to compare typical animal-baited traps (drop trap and direct aspiration) to both a new sticky cover trap and a UV-light/suction trap (the most commonly used method to collect Culicoides). METHODS/RESULTS: Collections were made from 1.45 hours before sunset to 1.45 hours after sunset in June/July 2009 at an experimental sheep farm (INRA, Nouzilly, Western France), with 3 replicates of a 4 sites×4 traps randomized Latin square using one sheep per site. Collected Culicoides individuals were sorted morphologically to species, sex and physiological stages for females. Sibling species were identified using a molecular assay. A total of 534 Culicoides belonging to 17 species was collected. Abundance was maximal in the drop trap (232 females and 4 males from 10 species) whereas the diversity was the highest in the UV-light/suction trap (136 females and 5 males from 15 species). Significant between-trap differences abundance and parity rates were observed. CONCLUSIONS: Only the direct aspiration collected exclusively host-seeking females, despite a concern that human manipulation may influence estimation of the biting rate. The sticky cover trap assessed accurately the biting rate of abundant species even if it might act as an interception trap. The drop trap collected the highest abundance of Culicoides and may have caught individuals not attracted by sheep but by its structure. Finally, abundances obtained using the UV-light/suction trap did not estimate accurately Culicoides biting rate.

Changes in waste stabilisation pond performance resulting from the retrofit of activated sludge treatment upstream: part II--Management and operating issues.

Bolivar Wastewater Treatment Plant (WWTP) was originally commissioned with trickling filter secondary treatment, followed by waste stabilisation pond (WSP) treatment and marine discharge. In 1999, a dissolved air flotation/filtration (DAFF) plant was commissioned to treat a portion of the WSP effluent for horticultural reuse. In 2001, the trickling filters were replaced with activated sludge treatment. A shift in WSP ecology became evident soon after this time, characterised by a statistically significant reduction in algal counts in the pond effluent, and increased variability in algal counts and occasional population crashes in the ponds. While the photosynthetic capacity of the WSPs has been reduced, the concomitant reduction in organic loading has meant that the WSPs have not become overloaded. As a result of the improvement in water quality leaving the ponds, significant cost savings and improved product water quality have been realised in the subsequent DAFF treatment stage. A number of operating issues have arisen from the change, however, including the re-emergence of a midge fly nuisance at the site. Control of midge flies using chemical spraying has negated the cost savings realised in the DAFF treatment stage. While biomanipulation of the WSP may provide a less aggressive method of midge control, this case demonstrates the difficulty of predicting in advance all ramifications of a retrospective process change.

[Risk assessment of bluetongue disease incursion into Germany using geographic information system (GIS)]. / Risikoabschätzung zur Einschleppung der Bluetongue Disease nach Deutschland mit Hilfe eines Geographischen Informationssystems (GIS).

Using a geographic information system (GIS), by analysis of the relationship between the spatial distribution of cattle density and the risk factors temperature, altitude and rainfall, we defined geographical habitats enabling optimal development and competence of Culicoides spp. to transmit Bluetongue-Virus (BTV): Risk zones (low, high, highest risk) were identified mainly in Baden-Württemberg, Hessen and Rheinland-Pfalz if persistently infected ruminants are imported into these zones in summer (June to August mainly), based on the current climatic conditions, BTD outbreaks are considered a real possibility. Overwintering of the virus seems unlikely. However, global warming will lead to a steady increase of the size of the risk zones. In addition, the possibility of primary outbreaks increases. The reason for this is not only the expected northern shift of Culicoides imicola, but in addition an increasing vector competence of domestic Culicoides species. We therefore recommend the storage of vaccines as well as conducting ecological studies analysing the presence of Culicoides vectors. Using the data from these studies, it will be possible to produce updated quantitative risk assessment via GIS.

Nematocera (Ceratopogonidae, Psychodidae, Simuliidae and Culicidae) and control methods.

The biology, veterinary importance and control of certain Nematocera are described and discussed. Culicoides spp. (family Ceratopogonidae) transmit the arboviruses of bluetongue (BT), African horse sickness (AHS), bovine ephemeral fever (BEF) and Akabane. Some other arboviruses have been isolated from these species, while fowl pox has been transmitted experimentally by Culicoides. These insects are vectors of the parasitic protozoans Leucocytozoon caulleryi and Haemoproteus nettionis, and the parasitic nematodes Onchocerca gutturosa, O. gibsoni and O. cervicalis. They also cause recurrent summer hypersensitivity in horses, ponies, donkeys, cattle and sheep. Farm animals can die as a result of mass attack by Simulium spp., which are also vectors of Leucocytozoon simondi, L. smithi and the filariae O. gutturosa, O. linealis and O. ochengi. Venezuelan equine encephalomyelitis (VEE) and Rift Valley fever (RVF) have been isolated from simuliids, and vesicular stomatitis virus New Jersey strain has been replicated in Simulium vittatum. Simuliids are well known as vectors of O. volvulus, the cause of human onchocercosis (river blindness). The family Psychodidae includes the genera Phlebotomus and Lutzomyia (subfamily Phlebotominae), vectors of Leishmania spp. in humans, dogs and other mammals. Vesicular stomatitis virus Indiana strain has been regularly isolated from phlebotomine sandflies. Mass attack by mosquitoes can also prove fatal to farm animals. Mosquitoes are vectors of the viruses of Akabane, BEF, RVF, Japanese encephalitis, VEE, western equine encephalomyelitis, eastern equine encephalomyelitis and west Nile meningoencephalitis, secondary vectors of AHS and suspected vectors of Israel turkey meningoencephalitis. The viruses of hog cholera, fowl pox and reticuloendotheliosis, the rickettsiae Eperythrozoon ovis and E. suis, and the bacterium Borrelia anserina are mechanically transmitted by mosquitoes. These insects also induce allergic dermatitis in horses. They transmit several filarial worms of both animals and humans, and are of great medical importance as vectors of major human diseases, including malaria, yellow fever, dengue fever and many more diseases caused by arboviruses.

Activity of Culicoides furens larvae (Diptera: Ceratopogonidae) as related to light and nutritional state.

The diel activity of fourth-instar Culicoides furens (Poey) was studied in the laboratory by means of video recordings. Larvae in 0.5% agar under a photoperiod of 12:12 (L:D) h were observed under three nutritional states: starved 24 h before and during observation; fed (with nematodes) 24 h before, but starved during, observation; and fed both before and during observation. Starved larvae displayed considerably reduced activity compared with those that were better fed, both in terms of speed and type of movement (the proportion of time spent in continuous rather than intermittent motion). Under all nutritional states, larvae were more active during dark than light hours; 3-hourly observations of speed plotted for four periods of 24 h strongly indicated a diel cycle of activity, which may, in the field, be accompanied by vertical migration in the soil. Overall, our observations indicated that during development, C. furens larvae rove constantly through the substrate, at speeds of approximately 2 m/h (and up to 4 m/h), and that this movement is maintained even in the presence of abundant food.