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.     

Vector competence of South African Culicoides species for bluetongue virus serotype 1 (BTV-1) with special reference to the effect of temperature on the rate of virus replication in C. imicola and C. bolitinos

Abstract. The oral susceptibility of 22 South African livestock associated Culicoides species to infection with bluetongue virus serotype 1 (BTV‐1) and its replication rate in C. imicola Kieffer and C. bolitinos Meiswinkel (Diptera: Ceratopogonidae) over a range of different incubation periods and temperatures are reported. Field‐collected Culicoides were fed on sheep blood containing 7.5 log₁₀TCID₅₀/mL of BTV‐1, and then held at constant different temperatures. Virus replication was measured over time by assaying individual flies in BHK‐21 cells using a microtitration procedure. Regardless of the incubation temperatures (10, 15, 18, 23.5 and 30°C) the mean virus titre/midge, infection rates (IR) and the proportion of infected females with transmission potential (TP = virus titre/midge ≥ 3 log₁₀ TCID₅₀) were found to be significantly higher in C. bolitinos than in C. imicola. Results from days 4–10 post‐infection (dpi), at 15–30°C, shows that the mean IR and TP values in C. bolitinos ranged from 36.7 to 87.8%, and from 8.4 to 87.7%, respectively; in C. imicola the respective values were 11.0–13.7% and 0–46.8%. In both species the highest IR was recorded at 25°C and the highest TP at 30°C. The time required for the development of TP in C. bolitinos ranged from 2 dpi at 25°C to 8 dpi at 15°C. In C. imicola it ranged from 4 dpi at 30°C to 10 dpi at 23.5°C; no individuals with TP were detected at 15°C. There was no evidence of virus replication in flies held at 10°C. When, at various points of incubation, individual flies were transferred from 10°C to 23.5°C and then assayed 4–10 days later, virus was recovered from both species. The mean virus titres/midge, and proportion of individuals with TP and IR, were again significantly higher in C. bolitinos than in C. imicola. Also the infection prevalence in C. magnus Colaço was higher than in C. imicola. Low infection prevalences were found in C. bedfordi Ingram & Macfie, C. leucostictus Kieffer, C. pycnostictus Ingram & Macfie, C. gulbenkiani Caeiro and C. milnei Austen. BTV‐1 was not detected in 14 other Culicoides species tested; however, some of these were tested in limited numbers. The present study indicates a multivector potential for BTV transmission in South Africa. In C. imicola and C. bolitinos the replication rates are distinct and are significantly influenced by temperature. These findings are discussed in relation to the epidemiology of bluetongue in South Africa.     

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.     

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 degrees 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).     

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.     

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.     

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.     

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.     

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.     

Protection of livestock against bluetongue virus vector Culicoides imicola using insecticide‐treated netting in open areas

The protection of livestock against Culicoides species (Diptera: Ceratopogonidae) using physical barriers or chemically treated barriers is difficult owing to the small size of these biting midges and animal welfare concerns associated with the reduction of air flow. Culicoides imicola Kieffer is the main bluetongue virus vector in the Mediterranean basin, including the southern Iberian peninsula, where livestock is mainly housed in open pens or sheds which offer no physical protection against C. imicola. In this study we assessed the efficacy of surrounding yearling ewe pens with a canvas barrier or a cypermethrin‐treated canvas barrier in reducing the entry of Culicoides spp. and C. imicola. Analyses were based on comparisons of Culicoides catches in traps in pens with and without barriers, and in traps located outside pens. Although there was no clear reduction in the abundance of Culicoides other than C. imicola in pens with either barrier, the C. imicola presence was markedly reduced by the insecticide‐treated barrier compared with the untreated barrier; the latter did not reduce the abundance of this species in pens. Estimates of the protection conferred against C. imicola by the treated barrier differed depending on whether catch comparisons were based on outside traps or on traps located inside no‐barrier pens. The results suggest that the use of insecticide‐treated barriers may reduce contact between livestock and C. imicola in open areas or sheds. More research is necessary to assess the degree of protection as a function of barrier height, C. imicola abundance, and the size of the area to be protected.     

Principal climatic and edaphic determinants of Culicoides biting midge abundance during the 2007–2008 bluetongue epidemic in the Netherlands,based on OVI light trap data

Palaearctic Culicoides midges (Diptera: Ceratopogonidae) represent a vital link in the northward advance of certain arboviral pathogens of livestock such as that caused by bluetongue virus. The effects of relevant ecological factors on weekly Culicoides vector abundances during the bluetongue virus serotype 8 epidemics in the Netherlands in 2007 and 2008 were quantified within a hurdle modelling framework. The relative role of meteorological parameters showed a broadly consistent association across species, with larger catches linked to temperature‐related variables and lower wind speed. Moreover, vector abundance was found to be influenced by edaphic factors, likely related to species‐specific breeding habitat preferences that differed markedly amongst some species. This is the first study on Culicoides vector species in the Netherlands identified during an entomological surveillance programme, in which an attempt is made to pinpoint the factors that influence midge abundance levels. In addition to providing key inputs into risk‐mitigating tools for midge‐borne pathogens and disease transmission models, the adoption of methods that explicitly address certain features of abundance datasets (frequent zero‐count observations and over‐dispersion) helped enhance the robustness of the ecological analysis.     

Distribution of Culicoides in Greece

Culicoides biting midges (Diptera: Ceratopogonidae) were trapped between 1999 and 2004 at 122 locations in mainland Greece and on most of the larger Aegean and Ionian islands, using OVI light traps, in order to determine the distribution and seasonal activity of bluetongue virus vectors and other Culicoides species. Thirty‐nine Culicoides species were identified, six of which (C. furcillatus, C. impunctatus, C. paolae, C. pictipennis, C. riethi, and C. scoticus) were identified for the first time in Greece. Two of these (C. impunctatus and C. scoticus) may be of veterinary importance due to their role as vectors of bluetongue virus and related orbiviruses. In addition, C. imicola was detected for the first time in mainland Greece.     

Phylogenetic relationships of the subgenus AvaritiaFox, 1955 including Culicoides obsoletus (Diptera, Ceratopogonidae) in Italy based on internal transcribed spacer 2 ribosomal DNA sequences

Abstract. Biting midges of the genus Culicoides include vector species for orbiviral diseases, such as bluetongue and African horse sickness. Although the Afro-Asiatic species C. imicola is the major vector of bluetongue in the Mediterranean basin, recent outbreaks in regions where C. imicola is absent has incriminated other Culicoides, including those belonging to the Obsoletus Complex of the subgenus Avaritia Fox, 1955. The classical taxonomy of this species complex is unclear and this stimulated the molecular analysis of twenty Culicoides populations sampled from eighteen localities across Italy. Ribosomal internal transcribed spacer 2 sequences were used to characterize the intra- and interspecific variation between Italian members of the Obsoletus Complex and related species, by means of an analysis of molecular variance and phylogenetic analyses. Although morphological differentiation is often extremely difficult, the molecular analysis clearly demonstrated a high degree of divergence between most species. The study showed that at least seven species of the subgenus Avaritia occur in Italy; these are C. obsoletus, C. scoticus, C. montanus, C. dewulfi, C. imicola and two species that could not be identified with certainty, but one of which is similar to C. chiopterus. Finally, a simple polymerase chain reaction assay was developed that rapidly discriminates between four members of the Obsoletus Complex in Italy, a prerequisite for vector identification.     

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.     

The Mondrian matrix: Culicoides biting midge abundance and seasonal incidence during the 2006–2008 epidemic of bluetongue in the Netherlands

During the northern Europe epidemic of bluetongue (BT), Onderstepoort‐type blacklight traps were used to capture Culicoides Latreille (Diptera: Ceratopogonidae) biting midges weekly between November 2006 and December 2008 on 21 livestock farms in the Netherlands. Proven and potential vectors for the bluetongue virus (BTV) comprised almost 80% of the midges collected: the Obsoletus complex, constituting C. obsoletus (Meigen) and C. scoticus Downes & Kettle (44.2%), C. dewulfi Goetghebuer (16.4%), C. chiopterus (Meigen) (16.3%) and C. pulicaris (Linnaeus) (0.1%). Half of the 24 commonest species of Culicoides captured completed only one (univoltine) or two (bivoltine) generations annually, whereas multivoltine species (including all BTV vectors) cycled through five to six generations (exceeding the one to four generations calculated in earlier decades). Whether this increment signals a change in the phenology of northern Europe Culicoides or simply is an adaptive response that manifests during warmer episodes, thus heightening periodically the incursive potential of midge‐borne arboviruses, remains to be clarified. Culicoides duddingstoni Kettle & Lawson, C. grisescens Edwards, C. maritimus Kieffer, C. pallidicornis Kieffer and C. riethi Kieffer are new records for the biting midge fauna of the Netherlands. It is suggested that C. punctatus (Meigen) be added to the European list of vector Culicoides.     

Comparaison des populations de Culicoides Latreille 1809 (Diptera : Ceratopogonidae) présentes au sein d’une bergerie belge et d’une prairie ovine associée

Culicoides biting midges (Diptera: Ceratopogonidae) serve as biological vectors for several pathogens, including the Bluetongue virus and the recently described Schmallenberg virus in northern Europe. These diseases have caused considerable direct and indirect economic losses to the sheep and cattle industries. This study undertaken between August and December 2007 on a sheep farm in the Namur province (Belgium) aims to evaluate Culicoides populations present inside a partially opened sheepfold and in a nearby sheep meadow, using light traps. The comparative analysis of insects trapped at 18 dates at regular intervals showed that Culicoides were most abundant inside this livestock building (17,450 midges) than in surrounding meadow (1,121 midges); this meadow had however a greater species diversity. The two species C. obsoletus and C. scoticus constituting the Obsoletus complex predominated for all trappings and females were much more numerous than males. Important capture of engorged females of the Obsoletus complex inside the sheepfold seems to reflect the possibility of an opportunistic endophagous behavior. Maintaining sheep inside livestock buildings in order to reduce the risk of Culicoides bites – and thus of pathogens transmission – however requires to limit biting midge populations which are likely to enter or to develop inside these buildings. Implementation of effective sanitation and hygiene measures against midges present inside farms, as well as establishing of measures to protect livestock against intrusion and improvement of “midge-proofing” of animal housing are therefore highly recommended.     

Use of climatic data and satellite imagery to model the abundance of Culicoides imicola, the vector of African horse sickness virus, in Morocco

African horse sickness (AHS) is a vector-borne, infectious disease of equids caused by African horse sickness virus (AHSV) . The only proven field vector of the virus is the biting midge Culicoides imicola. Following a recent epizootic (1989–91) of AHS in Morocco, light traps and automatic weather stations were operated for 2 years at twenty-two sites distributed over much of the country. The annually-averaged mean daily trap catch of C. imicola at these sites was negatively correlated with wind speed, and positively correlated with the average and mean annual minimum NDVI (Normalized Difference Vegetation Index, a remotely sensed measure of vegetation activity). There were no significant correlations between the mean daily trap catch and air temperature, soil temperature, relative humidity, saturation deficit, rainfall, altitude or the mean annual maximum or range of NDVI. The best two-variable model, which combined Windspeed_MnAvMn (the average daily minimum wind speed of the least windy month) and NDVI_min (the average annual minimum NDVI) as predictors, explained over 50% of the variance in the annually-averaged mean daily trap catch of C. imicola. There was a significant, positive correlation between minimum wind speed at night and the daily mortality rate of adult female C. imicola and it is suggested that the relationship between wind speed and the abundance of C. imicola arises from effects on adult mortality or dispersal. Considering several climatic variables, in North Africa NDVI_min was most significantly correlated with total annual rainfall. It is suggested that the relationship between NDVI_min and the abundance of C. imicola arises from the impact of soil moisture on both. It is proposed that areas of Morocco with higher levels of soil moisture in late summer or autumn provide more, larger and/or more enduring breeding sites for C. imicola, as well as supporting more photosynthetically active vegetation and hence having higher NDVI.     

Indoor and outdoor winter activity of Culicoides biting midges,vectors of bluetongue virus,in Italy

Indoor and outdoor winter activity of Culicoides spp. (Diptera: Ceratopogonidae) in central Italy was investigated in order to evaluate whether indoor activity might account for the overwintering of bluetongue virus, as has been hypothesized by some authors. Weekly Culicoides collections were performed at three farms over three consecutive winter seasons. At each farm, two black‐light traps were operated simultaneously, indoors and outdoors. Culicoides were identified using both morphological and molecular means. The Culicoides obsoletus group accounted for 98.2% of sampled specimens. Within this group, C. obsoletus s.s. accounted for 56.8% and Culicoides scoticus for 43.2% of samples. Nulliparous, parous and engorged females were caught throughout the entire winter, both indoors and outdoors. At times, indoor catch sizes outnumbered outdoor collections. A significant inverse correlation was found between minimum temperature and the proportion of indoor Culicoides of the total midge catch, thus indicating that lower outdoor temperatures drive Culicoides midges indoors. High rates of engorged females were recorded indoors, possibly as the result of the propensity of C. obsoletus females to feed indoors. Higher proportions of parous females were found in indoor than in outdoor catches, indicating higher survival rates indoors and, consequently, higher vectorial capacities of midges sheltering indoors compared with those remaining outdoors.     

Breeding sites and species association of the main Bluetongue and Schmallenberg virus vectors,the Culicoides species (Diptera: Ceratopogonidae), in northern Europe

Several species of Culicoides (Diptera: Ceratopogonidae) biting midges are biological vectors of bluetongue virus (BTV) and, as recently discovered, Schmallenberg virus (SBV) in northern Europe. Since their recent emergence in this part of the continent, these diseases that affect domestic and wild ruminants have caused considerable economic losses to the sheep and cattle industries. The substrates that are suitable for larval development of the main vector species are still relatively unknown. This study assessed all the substrates present in the immediate surroundings of a Belgian cattle farm and aimed to highlight the main breeding sites of these midge species. A total of 1639 immature Culicoides and 1320 adult specimens belonging to 13 species were found in 15 out of the 43 substrates studied: maize silage residues for C. obsoletus/C. scoticus, old overwintered cattle dung in the meadow for C. chiopterus and C. dewulfi, ground of a flooded meadow, green filamentous algae and underlying substrate, silt from a pond, and ground of hollows caused by the crossing of machines on a dirt track for C. festivipennis, silt from a pond for C. nubeculosus, and ground of a flooded meadow for C. lupicaris. Identification of these micro-habitats and the associations among the species they contain could allow their localization and the development of new strategies of vector control, while preventing the creation of new Culicoides larval micro-habitats. Finally, measures designed to reduce larval populations could improve efficacy of vaccination campaigns against BTV in Europe.