Surveillance of biting midges (Culicoides spp.) in Northern Ireland: influence of seasonality,surrounding habitat and livestock housing

Biting midges, Culicoides spp. (Diptera: Ceratopogonidae), are important vectors of viral pathogens. Following the outbreak of bluetongue serotype 8 in Europe between 2006 and 2009, many Culicoides surveillance programmes were initiated to identify vector‐active periods, in accordance with European Commission regulation 2007/1266/EC. This study utilized surveillance data from 4 years of continuous light‐trapping at 14 sites in Northern Ireland. The number of captured Culicoides varied from none during the vector‐free period (December–April) to more than 36 000 per night during peak activity in the summer. The Obsoletus group represented 75% of Culicoides collected and the Pulicaris group represented 21%. A total of 91% of Culicoides were female, of which 42% were parous. Abundance data, sex ratios and parous rates suggested that both the Obsoletus and Pulicaris groups underwent three generations/year. The Obsoletus group was associated with cattle‐rearing habitats and woodland, the Impunctatus group was found in habitats related to sheep rearing and the Pulicaris group were associated with both cattle and sheep. Housing did not reduce incursion of female Obsoletus group Culicoides but it did for males and for the Pulicaris group Culicoides. The influence of housing was strongly affected by time of year, probably reflecting the presence of livestock indoors/outdoors.     

Latitudinal diversity of biting midge species within the Obsoletus group across three habitats in Europe

Culicoides species from the Obsoletus group are important vectors of bluetongue and Schmallenberg virus. This group consists of several species that cannot easily be identified using morphological characteristics. Therefore, limited information is available about their distribution and habitat preferences. In this study, we aimed to elucidate the species composition of the Obsoletus group in three habitat types at climatically different latitudes across Europe. Traps were placed in three habitat types in three countries at different latitudes. After DNA extraction, biting midges were identified using PCR and gel electrophoresis. Extraction of DNA using Chelex proved to be a cost and time efficient method for species identification. A latitudinal effect on the relative abundance of species from the Obsoletus group was found. Species composition was unique for most country‐habitat combinations. The majority of biting midges were either C. obsoletus s.s. or C. scoticus, and both species were found at all latitudes and habitats. Their wide distribution and their high abundance at livestock farms make these species likely candidates for rapid farm‐to‐farm transmission of pathogens throughout Europe. Our results emphasize the need to differentiate Obsoletus group species to better understand their ecology and contribution to pathogen transmission.     

Farms,pastures and woodlands: the fine‐scale distribution of Palearctic Culicoides spp. biting midges along an agro‐ecological gradient

The spatial epidemiology of Bluetongue virus (BTV) at the landscape level relates to the fine‐scale distribution and dispersal capacities of its vectors, midges belonging to the genus Culicoides Latreille (Diptera: Ceratopogonidae). Although many previous researches have carried out Culicoides sampling on farms, little is known of the fine‐scale distribution of Culicoides in the landscape immediately surrounding farms. The aim of this study was to gain a better understanding of Culicoides populations at increasing distances from typical dairy farms in north‐west Europe, through the use of eight Onderstepoort‐type black‐light traps positioned along linear transects departing from farms, going through pastures and entering woodlands. A total of 16 902 Culicoides were collected in autumn 2008 and spring 2009. The majority were females, of which more than 97% were recognized as potential vectors. In pastures, we found decreasing numbers of female Culicoides as a function of the distance to the farm. This pattern was modelled by leptokurtic models, with parameters depending on season and species. By contrast, the low number of male Culicoides caught were homogeneously distributed along the transects. When transects entered woodlands, we found a higher abundance of Culicoides than expected considering the distance of the sampling sites to the farm, although this varied according to species.     

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.     

Spatial and Temporal Distribution of Culicoides Species in Mainland Portugal (2005–2010). Results of the Portuguese Entomological Surveillance Programme

Bluetongue virus (BTV) is transmitted by Culicoides biting midges and causes an infectious, non-contagious disease of ruminants. It has been rapidly emerging in southern Europe since 1998. In mainland Portugal, strains of BTV belonging to three serotypes have been detected: BTV-10 (1956-1960), BTV-4 (2004-2006 and 2013) and BTV-1 (2007-2012). This paper describes the design, implementation and results of the Entomological Surveillance Programme covering mainland Portugal, between 2005 and 2010, including 5,650 caches. Culicoides imicola Kieffer was mostly found in central and southern regions of Portugal, although it was sporadically detected in northern latitudes. Its peak activity occurred in the autumn and it was active during the winter months in limited areas of the country. Obsoletus group was present at the highest densities in the north although they were found throughout the country in substantial numbers. Culicoides activity occurred all year round but peaked in the spring. A generalized linear mixed model was developed for the analysis of the environmental factors associated with activity of the species of Culicoides suspected vectors of BTV in the country. For C. imicola Kieffer, the most important variables were month, diurnal temperature range (DTR), the number of frost days (FRS) and median monthly temperature (TMP). For the Obsoletus group, the most important factors were month, diurnal temperature range (DTR), and linear and quadratic terms for median monthly temperature (TMP). The results reported can improve our understanding of climatic factors in Culicoides activity influencing their distribution and seasonal pattern.     

Culicoides Species Communities Associated with Wild Ruminant Ecosystems in Spain: Tracking the Way to Determine Potential Bridge Vectors for Arboviruses

The genus Culicoides Latreille 1809 is a well-known vector for protozoa, filarial worms and, above all, numerous viruses. The Bluetongue virus (BTV) and the recently emerged Schmallenberg virus (SBV) are responsible for important infectious, non-contagious, insect-borne viral diseases found in domestic ruminants and transmitted by Culicoides spp. Both of these diseases have been detected in wild ruminants, but their role as reservoirs during the vector-free season still remains relatively unknown. In fact, we tend to ignore the possibility of wild ruminants acting as a source of disease (BTV, SBV) and permitting its reintroduction to domestic ruminants during the following vector season. In this context, a knowledge of the composition of the Culicoides species communities that inhabit areas where there are wild ruminants is of major importance as the presence of a vector species is a prerequisite for disease transmission. In this study, samplings were conducted in areas inhabited by different wild ruminant species; samples were taken in both 2009 and 2010, on a monthly basis, during the peak season for midge activity (in summer and autumn). A total of 102,693 specimens of 40 different species of the genus Culicoides were trapped; these included major BTV and SBV vector species. The most abundant vector species were C. imicola and species of the Obsoletus group, which represented 15% and 11% of total numbers of specimens, respectively. At the local scale, the presence of major BTV and SBV vector species in areas with wild ruminants coincided with that of the nearest sentinel farms included in the Spanish Bluetongue Entomological Surveillance Programme, although their relative abundance varied. The data suggest that such species do not exhibit strong host specificity towards either domestic or wild ruminants and that they could consequently play a prominent role as bridge vectors for different pathogens between both types of ruminants. This finding would support the hypothesis that wild ruminants could act as reservoirs for such pathogens, and subsequently be involved in the reintroduction of disease to livestock on neighbouring farms.     

Sugar‐feeding behaviour and longevity of European Culicoides biting midges

Most haematophagous insect vectors can also use sugar as an energy source; thus their sugar‐feeding behaviour influences their longevity and blood‐feeding rate and hence their vectorial capacity. Scant information is available on the sugar‐feeding behaviour of Culicoides Latreille biting midges (Diptera: Ceratopogonidae), which are vectors of bluetongue and Schmallenberg viruses. The longevity of laboratory‐reared Culicoides nubeculosus (Meigen) under fluctuating temperatures (16 and 28 °C) and with access to water or water and blood was on average 6.4 days and 8.9 days, respectively, which was around one third of the lifespan of siblings with access to sugar or sugar and blood (22.2 days and 27.1 days, respectively). Access to honeydew significantly increased the midge's longevity, whereas the provision of extrafloral nectaries had no impact. Females with access to sugar produced a significantly higher number of eggs (65.5 ± 5.2) than their starved sisters (45.4 ± 8.4). More than 80% of field‐caught female Culicoides from the two most abundant European groups, Obsoletus (n = 2243) and Pulicaris (n = 805), were fructose‐positive. Fructose‐positivity was high in all physiological stages and no seasonal variability was noted. The high rate of natural sugar feeding of Culicoides offers opportunities for the development of novel control strategies using toxic sugar baits and for the monitoring of vector‐borne diseases using sugar‐treated FTA (nucleic acid preservation) cards in the field.     

Emergence of Culicoides obsoletus group species from farm‐associated habitats in Germany

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) may transmit several arboviruses to ruminant livestock. The species of the Obsoletus group are considered to be among the most important vectors of bluetongue virus (BTV) in northern Europe. As agricultural environments offer suitable habitats for the development of their immature stages, the emergence of adult Culicoides from potential breeding sites was investigated at 20 cattle farms throughout Germany in 2012 and 2013. In analyses of species‐specific habitat preferences and relationships between Culicoides abundance in breeding substrates and their physicochemical characteristics, dungheaps emerged as the most important substrate for the development of Culicoides obsoletus sensu stricto (s.s.) (Meigen), whereas Culicoides chiopterus (Meigen) and Culicoides dewulfi Goetghebuer were generally restricted to cowpats. A decreasing pH value was associated with a higher abundance or a higher probability of observing these three species. Furthermore, the abundance of C. obsoletus s.s. was positively related to increasing moisture. Dungheaps were very productive breeding sites for this species and are therefore suggested as a target for potential control measures.     

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.     

Are Bogs Reservoirs for Emerging Disease Vectors? Evaluation of Culicoides Populations in the Hautes Fagnes Nature Reserve (Belgium)

Several species of Culicoides (Diptera: Ceratopogonidae) biting midges serve as biological vectors for the bluetongue virus (BTV) and the recently described Schmallenberg virus (SBV) in northern Europe. Since their recent emergence in this part of the continent, these diseases have caused considerable economic losses to the sheep and cattle industries. Much data is now available that describe the distribution, population dynamics, and feeding habits of these insects. However, little is known regarding the presence of Culicoides in unusual habitats such as peaty marshes, nor their potential vector capacity. This study evaluated Culicoides biting midges present in the bogs of a Belgian nature reserve compared to those residing at a nearby cattle farm. Culicoides were trapped in 2011 at four different sites (broadleaved and coniferous forested areas, open environments, and at a scientific station) located in the Hautes Fagnes Nature Reserve (Belgium). An additional light trap was operated on a nearby cattle farm. Very high numbers of biting midges were captured in the marshy area and most of them (70 to 95%) were Culicoides impunctatus, a potential vector of BTV and other pathogens. In addition, fewer numbers of C. obsoletus/C. scoticus species, C. chiopterus, and C. dewulfi were observed in the bogs compared to the farm. The wet environment and oligotrophic nature of the soil were probably responsible for these changes in the respective populations. A total of 297,808 Culicoides midges belonging to 27 species were identified during this study and 3 of these species (C. sphagnumensis, C. clintoni and C. comosioculatus) were described in Belgium for the first time.     

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.     

Schmallenberg Virus Circulation in Culicoides in Belgium in 2012: Field Validation of a Real Time RT-PCR Approach to Assess Virus Replication and Dissemination in Midges

Indigenous Culicoides biting midges are suggested to be putative vectors for the recently emerged Schmallenberg virus (SBV) based on SBV RNA detection in field-caught midges. Furthermore, SBV replication and dissemination has been evidenced in C. sonorensis under laboratory conditions. After SBV had been detected in Culicoides biting midges from Belgium in August 2011, it spread all over the country by the end of 2011, as evidenced by very high between-herd seroprevalence rates in sheep and cattle. This study investigated if a renewed SBV circulation in midges occurred in 2012 in the context of high seroprevalence in the animal host population and evaluated if a recently proposed realtime RT-PCR approach that is meant to allow assessing the vector competence of Culicoides for SBV and bluetongue virus under laboratory conditions was applicable to field-caught midges. Therefore midges caught with 12 OVI traps in four different regions in Belgium between May and November 2012, were morphologically identified, age graded, pooled and tested for the presence of SBV RNA by realtime RT-PCR. The results demonstrate that although no SBV could be detected in nulliparous midges caught in May 2012, a renewed but short lived circulation of SBV in parous midges belonging to the subgenus Avaritia occured in August 2012 at all four regions. The infection prevalence reached up to 2.86% in the south of Belgium, the region where a lower seroprevalence was found at the end of 2011 than in the rest of the country. Furthermore, a frequency analysis of the Ct values obtained for 31 SBV-S segment positive pools of Avaritia midges showed a clear bimodal distribution with peaks of Ct values between 21–24 and 33–36. This closely resembles the laboratory results obtained for SBV infection of C. sonorensis and implicates indigenous midges belonging to the subgenus Avaritia as competent vectors for SBV.     

A new tool for the molecular identification of Culicoides species of the Obsoletus group: the glass slide microarray approach

Culicoides species of the Obsoletus group (Diptera: Ceratopogonidae) are potential vectors of bluetongue virus serotype 8 (BTV 8), which was introduced into central Western Europe in 2006. Correct morphological species identification of Obsoletus group females is especially difficult and molecular identification is the method of choice. In this study we present a new molecular tool based on probe hybridization using a DNA microarray format to identify Culicoides species of the Obsoletus group. The internal transcribed spacer 1 (ITS1) gene sequences of 55 Culicoides belonging to 13 different species were determined and used, together with 19 Culicoides ITS1 sequences sourced from GenBank, to design species-specific probes for the microarray test. This test was evaluated using the amplified ITS1 sequences of another 85 Culicoides specimens, belonging to 11 species. The microarray test successfully identified all samples (100%) of the Obsoletus group, identifying each specimen to species level within the group. This test has several advantages over existing polymerase chain reaction (PCR)-based molecular tools, including possible capability for parallel analysis of many species, high sensitivity and specificity, and low background signal noise. Hand-spotting of the microarray slide and the use of detection chemistry make this alternative technique affordable and feasible for any diagnostic laboratory with PCR facilities.     

Measurement of the Infection and Dissemination of Bluetongue Virus in Culicoides Biting Midges Using a Semi-Quantitative RT-PCR Assay and Isolation of Infectious Virus

Background

Culicoides biting midges (Diptera: Ceratopogonidae) are the biological vectors of globally significant arboviruses of livestock including bluetongue virus (BTV), African horse sickness virus (AHSV) and the recently emerging Schmallenberg virus (SBV). From 2006–2009 outbreaks of BTV in northern Europe inflicted major disruption and economic losses to farmers and several attempts were made to implicate Palaearctic Culicoides species as vectors. Results from these studies were difficult to interpret as they used semi-quantitative RT-PCR (sqPCR) assays as the major diagnostic tool, a technique that had not been validated for use in this role. In this study we validate the use of these assays by carrying out time-series detection of BTV RNA in two colony species of Culicoides and compare the results with the more traditional isolation of infectious BTV on cell culture.

Methodology/Principal Findings

A BTV serotype 1 strain mixed with horse blood was fed to several hundred individuals of Culicoides sonorensis (Wirth & Jones) and C. nubeculosus (Mg.) using a membrane-based assay and replete individuals were then incubated at 25°C. At daily intervals 25 Culicoides of each species were removed from incubation, homogenised and BTV quantified in each individual using sqPCR (C_q values) and virus isolation on a KC-C. sonorensis embryonic cell line, followed by antigen enzyme-linked immunosorbent assay (ELISA). In addition, comparisons were also drawn between the results obtained with whole C. sonorensis and with individually dissected individuals to determine the level of BTV dissemination.

Conclusions/Significance

C_q values generated from time-series infection experiments in both C. sonorensis and C. nubeculosus confirmed previous studies that relied upon the isolation and detection of infectious BTV. Implications on the testing of field-collected Culicoides as potential virus vectors by PCR assays and the use of such assays as front-line tools for use in diagnostic laboratories in this role are discussed.     

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.     

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.     

Morphology and DNA barcoding reveal three species in one: description of Culicoides cryptipulicaris sp. nov. and Culicoides quasipulicaris sp. nov. in the subgenus Culicoides

Species of the genus Culicoides (Diptera: Ceratopogonidae) are well known for their importance in the field of medical and veterinary entomology. Culicoides spp. transmit a wide variety of pathogens, primarily viruses that affect animals and humans. In Europe, the most economically important disease transmitted by Culicoides is bluetongue (BT). Culicoides spp. have been recently involved as primary vectors for Schmallenberg disease. The taxonomy within the subgenus Culicoides has been historically difficult and reorganizations have been proposed regularly. The subgenus Culicoides includes species that are considered to be potential vectors for BT. High morphological intraspecific variability has been attributed to these species. This highlights the apparent presence of previously undetected cryptic species diversity in the subgenus. In the present study, a detailed morphological and molecular study of specimens belonging to Culicoides pulicaris s.l. and specimens resembling a cross between C. pulicaris and Culicoides punctatus revealed the presence of two new species: Culicoides cryptipulicaris and Culicoides quasipulicaris. Females of C. quasipulicaris and males of both species were morphologically distinguished from C. pulicaris (Linnaeus, 1758), whereas females of C. cryptipulicaris were identified using molecular techniques exclusively.     

Detection of Extended-Spectrum Beta-Lactamase (ESBL)-Producing Escherichia coli on Flies at Poultry Farms

In the Netherlands, extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli bacteria are highly prevalent in poultry, and chicken meat has been implicated as a source of ESBL-producing E. coli present in the human population. The current study describes the isolation of ESBL-producing E. coli from house flies and blow flies caught at two poultry farms, offering a potential alternative route of transmission of ESBL-producing E. coli from poultry to humans. Overall, 87 flies were analyzed in 19 pools. ESBL-producing E. coli bacteria were detected in two fly pools (10.5%): a pool of three blow flies from a broiler farm and a pool of eight house flies from a laying-hen farm. From each positive fly pool, six isolates were characterized and compared with isolates obtained from manure (n = 53) sampled at both farms and rinse water (n = 10) from the broiler farm. Among six fly isolates from the broiler farm, four different types were detected with respect to phylogenetic group, sequence type (ST), and ESBL genotype: A₀/ST3519/SHV-12, A₁/ST10/SHV-12, A₁/ST58/SHV-12, and B1/ST448/CTX-M-1. These types, as well as six additional types, were also present in manure and/or rinse water at the same farm. At the laying-hen farm, all fly and manure isolates were identical, carrying bla_TEM-52 in an A₁/ST48 genetic background. The data imply that flies acquire ESBL-producing E. coli at poultry farms, warranting further evaluation of the contribution of flies to dissemination of ESBL-producing E. coli in the community.     

Implicating Culicoides Biting Midges as Vectors of Schmallenberg Virus Using Semi-Quantitative RT-PCR

Background

The recent unprecedented emergence of arboviruses transmitted by Culicoides biting midges in northern Europe has necessitated the development of techniques to differentiate competent vector species. At present these techniques are entirely reliant upon interpretation of semi-quantitative RT-PCR (sqPCR) data in the form of C_q values used to infer the presence of viral RNA in samples.

Methodology/Principal Findings

This study investigates the advantages and limitations of sqPCR in this role by comparing infection and dissemination rates of Schmallenberg virus (SBV) in two colony lines of Culicoides. Through the use of these behaviorally malleable lines we provide tools for demarcating arbovirus infection and dissemination rates in Culicoides which to date have prevented clear implication of primary vector species in northern Europe. The study demonstrates biological transmission of SBV in an arthropod vector, supporting the conclusions from field-caught Culicoides and provides a general framework for future assessment of vector competence of Culicoides for arboviruses using sqPCR.

Conclusions/Significance

When adopting novel diagnostic technologies, correctly implicating vectors of arboviral pathogens requires a coherent laboratory framework to fully understand the implications of results produced in the field. This study illustrates these difficulties and provides a full examination of sqPCR in this role for the Culicoides-arbovirus system.