Revealing potential bridge vectors for BTV and SBV: a study on Culicoides blood feeding preferences in natural ecosystems in Spain

Several species of Culicoides (Diptera: Ceratopogonidae) are vectors of pathogens, such as the bluetongue (BTV) and Schmallenberg (SBV) viruses, which cause important diseases in domestic and wild ruminants. As wild ruminants can contribute to overwintering and epizootics of both diseases, knowledge of the host‐feeding behaviour of Culicoides in natural ecosystems is important to better understand their epidemiology. Blood‐engorged Culicoides females trapped in natural areas inhabited by different wild ruminant species were genetically analysed to identify host species. The origin of bloodmeals was identified in 114 females of 14 species of Culicoides. A total of 104 (91.1%) Culicoides fed on mammals and 10 (8.9%) on birds. The most abundant host identified was red deer (66.7%), followed by humans (13%) and fallow deer (6.1%). Eleven of the 14 species of Culicoides fed exclusively on mammalian hosts. Among them, five are mammalophilic species considered to be important BTV and/or SBV vectors. The results of the present study confirm that Culicoides imicola, Culicoides obsoletus, Culicoides scoticus, Culicoides pulicaris and Culicoides punctatus fed on wild ruminants, and therefore support the hypothesis that these species can act as bridge vectors by facilitating the circulation of pathogens between wild and domestic ruminant communities.     

Experimental infection of Aedes (Stegomyia) albopictus and Culex pipiens mosquitoes with Bluetongue virus

Bluetongue disease (BT), caused by Bluetongue virus (BTV), infects wild and domestic ruminants, causing severe economic damage in the cattle and sheep industry. Proven vectors of BTV are biting midges belonging to the Culicoides genus, but other arthropods are considered potential vectors, such as ticks, mosquitoes, wingless flies, and sand flies. The present study represents the first attempt to evaluate the vectorial capacity of Culex pipiens and Aedes albopictus for BTV. Mosquitoes were artificially fed with blood containing BTV serotype 1. Infection, dissemination and transmission rates were evaluated at 0, 3, 7, 14 and 21 days after an infected blood meal. Viral RNA was only detected up to 3 days post infection in the bodies of both species. This study indicates that the two Italian populations of Cx. pipiens and Ae. albopictus are not susceptible to BTV infection.     

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.     

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.     

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.     

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.     

A Spatial Simulation Model for the Dispersal of the Bluetongue Vector Culicoides brevitarsis in Australia

Background

The spread of Bluetongue virus (BTV) among ruminants is caused by movement of infected host animals or by movement of infected Culicoides midges, the vector of BTV. Biologically plausible models of Culicoides dispersal are necessary for predicting the spread of BTV and are important for planning control and eradication strategies.

Methods

A spatially-explicit simulation model which captures the two underlying population mechanisms, population dynamics and movement, was developed using extensive data from a trapping program for C. brevitarsis on the east coast of Australia. A realistic midge flight sub-model was developed and the annual incursion and population establishment of C. brevitarsis was simulated. Data from the literature was used to parameterise the model.

Results

The model was shown to reproduce the spread of C. brevitarsis southwards along the east Australian coastline in spring, from an endemic population to the north. Such incursions were shown to be reliant on wind-dispersal; Culicoides midge active flight on its own was not capable of achieving known rates of southern spread, nor was re-emergence of southern populations due to overwintering larvae. Data from midge trapping programmes were used to qualitatively validate the resulting simulation model.

Conclusions

The model described in this paper is intended to form the vector component of an extended model that will also include BTV transmission. A model of midge movement and population dynamics has been developed in sufficient detail such that the extended model may be used to evaluate the timing and extent of BTV outbreaks. This extended model could then be used as a platform for addressing the effectiveness of spatially targeted vaccination strategies or animal movement bans as BTV spread mitigation measures, or the impact of climate change on the risk and extent of outbreaks. These questions involving incursive Culicoides spread cannot be simply addressed with non-spatial models.     

Bluetongue epidemiology in wild ruminants from Southern Spain

Serum samples from 210 wild ruminants collected between 2006 and 2007 in southern Spain were tested for antibodies against bluetongue virus (BTV) by means of a competitive ELISA assay. Eighty-seven of the 210 wild ruminants analysed (41%) showed antibodies against BTV. Statistically significant differences were found in the seroprevalence among species: 66% (65 of 98) for red deer (Cervus elaphus), 50% (ten of 20) for fallow deer (Dama dama), 33% (three of nine) for mouflon (Ovis aries musimon) and 11% (nine of 83) for Spanish ibex (Capra pyrenaica). Overall, the sites where seropositive wild ruminants were found coincide with the areas where BTV had been detected in livestock, but in eastern Sierra Morena, the virus circulated in wild ruminants, although it had not been detected in domestic ruminants in the same areas. Wild ruminants over 1-year of age (sub-adults and adults) had significantly higher seroprevalences than juvenile animals. Statistically significant differences were also observed between BTV seroprevalence and management (free-ranging vs. captivity) with higher prevalence in free-ranging animals. The high seroprevalences obtained suggest that BTV is widespread in wild ruminants in southern Spain. This factor could have an important influence on the evolution of the infection in domestic livestock and indicates the need to include wild ruminant species in BTV surveillance or control programs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Type I Interferon Limits the Capacity of Bluetongue Virus To Infect Hematopoietic Precursors and Dendritic Cells In Vitro and In Vivo

Hematopoietic stem cells (HSCs) give rise to progenitors with potential to produce multiple cell types, including dendritic cells (DCs). DCs are the principal antigen-presenting cells and represent the crucial link between innate and adaptive immune responses. Bluetongue virus (BTV), an economically important Orbivirus of the Reoviridae family, causes a hemorrhagic disease mainly in sheep and occasionally in other species of ruminants. BTV is transmitted between its mammalian hosts by certain species of biting midges (Culicoides spp.) and is a potent alpha interferon (IFN-α) inducer. In the present report, we show that BTV infects cells of hematopoietic origin but not HSCs in immunocompetent sheep. However, BTV infects HSCs in the absence of type I IFN (IFN-I) signaling in vitro and in vivo. Infection of HSCs in vitro results in cellular death by apoptosis. Furthermore, BTV infects bone marrow-derived DCs (BM-DCs), interfering with their development to mature DCs in the absence of type I IFN signaling. Costimulatory molecules CD80 and CD86 and costimulatory molecules CD40 and major histocompatibility complex class II (MHC-II) are affected by BTV infection, suggesting that BTV interferes with DC antigen-presenting capacity. In vivo, different DC populations are also affected during the course of infection, probably as a result of a direct effect of BTV replication in DCs and the production of infectious virus. These new findings suggest that BTV infection of HSCs and DCs can impair the immune response, leading to persistence or animal death, and that this relies on IFN-I.     

Culicoides Midge Bites Modulate the Host Response and Impact on Bluetongue Virus Infection in Sheep

Many haematophagous insects produce factors that help their blood meal and coincidently favor pathogen transmission. However nothing is known about the ability of Culicoides midges to interfere with the infectivity of the viruses they transmit. Among these, Bluetongue Virus (BTV) induces a hemorrhagic fever- type disease and its recent emergence in Europe had a major economical impact. We observed that needle inoculation of BTV8 in the site of uninfected C. nubeculosus feeding reduced viraemia and clinical disease intensity compared to plain needle inoculation. The sheep that developed the highest local inflammatory reaction had the lowest viral load, suggesting that the inflammatory response to midge bites may participate in the individual sensitivity to BTV viraemia development. Conversely compared to needle inoculation, inoculation of BTV8 by infected C. nubeculosus bites promoted viraemia and clinical symptom expression, in association with delayed IFN- induced gene expression and retarded neutralizing antibody responses. The effects of uninfected and infected midge bites on BTV viraemia and on the host response indicate that BTV transmission by infected midges is the most reliable experimental method to study the physio-pathological events relevant to a natural infection and to pertinent vaccine evaluation in the target species. It also leads the way to identify the promoting viral infectivity factors of infected Culicoides in order to possibly develop new control strategies against BTV and other Culicoides transmitted viruses.     

Bluetongue: a historical and epidemiological perspective with the emphasis on South Africa

ABSTRACT: Bluetongue (BT) is a non-contagious, infectious, arthropod transmitted viral disease of domestic and wild ruminants that is caused by the bluetongue virus (BTV), the prototype member of the Orbivirus genus in the family Reoviridae. Bluetongue was first described in South Africa, where it has probably been endemic in wild ruminants since antiquity. Since its discovery BT has had a major impact on sheep breeders in the country and has therefore been a key focus of research at the Onderstepoort Veterinary Research Institute in Pretoria. Several key discoveries were made at this Institute, including the demonstration that the aetiological agent of BT was a dsRNA virus that is transmitted by Culicoides midges and that multiple BTV serotypes circulate in nature. It is currently recognized that BT is endemic throughout most of South Africa and 22 of the 26 known serotypes have been detected in the region. Multiple serotypes circulate each vector season with the occurrence of different serotypes depending largely on herd -immunity. Indigenous sheep breeds, cattle and wild ruminants are frequently infected but rarely demonstrate clinical signs, whereas improved European sheep breeds are most susceptible. The immunization of susceptible sheep remains the most effective and practical control measure against BT. In order to protect sheep against multiple circulating serotypes, three pentavalent attenuated vaccines have been developed. Despite the proven efficacy of these vaccines in protecting sheep against the disease, several disadvantages are associated with their use in the field.     

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.     

Bovine virus diarrhea and the vector-borne diseases Anaplasmosis and Bluetongue: a sero-surveillance in free-ranging red deer (<Emphasis Type="Italic">Cervus elaphus</Emphasis>) in selected areas of Switzerland

Due to climate changes, diseases emerging from southerly adjacent areas (Mediterranean countries) are likely to spread northward. Expanded migration of red deer harbors the risk of introducing new pathogens into a naive population of either wild or domestic animals. Little is known about the importance of red deer as a potential reservoir for diseases of domestic ruminants in Switzerland. Deer is susceptible for all three agents that were selected in this study: bovine virus diarrhea virus (BVDV), Anaplasma marginale (AM), and Bluetongue virus (BTV). The goal of this project was to establish the serological status of red deer in Switzerland concerning BVDV, AM, and BTV, and to assess the possible impact of disease dynamics with a focus on potential transmission of these diseases from red deer to cattle or vice versa. Sampling areas were selected according the following criteria: abundance of red deer, potential insect vector distribution due to climatic conditions, and traditional alpine pasture husbandry along with known migration routes of red deer. Blood samples were collected during the regular hunting season 2004 and 2005 by hunters and gamekeepers. There was no serological evidence for the presence of the vector-borne diseases AM and BT in red deer in Switzerland. Four out of 234 sera showed a positive result for BVD, corresponding to a sero-prevalence of 1.7% (95% CI 0.46–4.38). Facing the fact of the high sero-prevalence for BVD in Swiss cattle (60–80%) disease transmission from red deer to cattle in these areas under investigation is rather unlikely.     

Expression and purification of the outer shell proteinVP2 of the 4th serotype Bluetongue virus,and preparation of monoclonal antibodies against this protein

Bluetongue (BT) is an arbovirus transmitted disease by bites of the genus Culicoides and infects wild and domestic ruminants particularly in sheep. As an important outer shell protein which defines BTV serotypes, VP2 has been shown to be an ideal target antigen for identification of different BTV serotypes. In order to prepare a monoclonal antibody (mAb) against the VP2 protein of BTV-4, the corresponding encoding gene L2 was divided into three segments and then cloned into pET-28a (+) and pMAL-c5X vectors to generate recombinant plasmids, which were expressed in Escherichia coli BL21 (DE3) as histidine (His)-tagged (His-4A/4B/4C) and maltose-binding protein (MBP)-tagged (MBP-4A/4B/4C) fusion proteins. After affinity purification of His-4A/4B/4C with Ni-NTA agarose and MBP-4A/4B/4C with amylose resin, His-4A/4B/4C were used to immunize BALB/mice and MBP-4A/4B/4C were used to screen for mAb-secreting hybridomas. Five hybridoma cell lines stably secreting mAbs against different VP2 segments were obtained, in which 4A-1G7 and 4B-1B6 could recognize BTV-4 and also cross-react with other BTV serotypes. With the joint action of the two mAbs, BTV-4 and BTV-20 infection would be distinguished from other BTV serotypes. The successful preparation of recombinant VP2 segments and mAbs provides valuable materials that can be used in serological diagnosis of BTV-4.     

The Spread of Bluetongue Virus Serotype 8 in Great Britain and Its Control by Vaccination

Background

Bluetongue (BT) is a viral disease of ruminants transmitted by Culicoides biting midges and has the ability to spread rapidly over large distances. In the summer of 2006, BTV serotype 8 (BTV-8) emerged for the first time in northern Europe, resulting in over 2000 infected farms by the end of the year. The virus subsequently overwintered and has since spread across much of Europe, causing tens of thousands of livestock deaths. In August 2007, BTV-8 reached Great Britain (GB), threatening the large and valuable livestock industry. A voluntary vaccination scheme was launched in GB in May 2008 and, in contrast with elsewhere in Europe, there were no reported cases in GB during 2008.

Methodology/Principal Findings

Here, we use carefully parameterised mathematical models to investigate the spread of BTV in GB and its control by vaccination. In the absence of vaccination, the model predicted severe outbreaks of BTV, particularly for warmer temperatures. Vaccination was predicted to reduce the severity of epidemics, with the greatest reduction achieved for high levels (95%) of vaccine uptake. However, even at this level of uptake the model predicted some spread of BTV. The sensitivity of the predictions to vaccination parameters (time to full protection in cattle, vaccine efficacy), the shape of the transmission kernel and temperature dependence in the transmission of BTV between farms was assessed.

Conclusions/Significance

A combination of lower temperatures and high levels of vaccine uptake (>80%) in the previously-affected areas are likely to be the major contributing factors in the control achieved in England in 2008. However, low levels of vaccination against BTV-8 or the introduction of other serotypes could result in further, potentially severe outbreaks in future.     

Environmental Drivers of Culicoides Phenology: How Important Is Species-Specific Variation When Determining Disease Policy?

Since 2006, arboviruses transmitted by Culicoides biting midges (Diptera: Ceratopogonidae) have caused significant disruption to ruminant production in northern Europe. The most serious incursions involved strains of bluetongue virus (BTV), which cause bluetongue (BT) disease. To control spread of BTV, movement of susceptible livestock is restricted with economic and animal welfare impacts. The timing of BTV transmission in temperate regions is partly determined by the seasonal presence of adult Culicoides females. Legislative measures therefore allow for the relaxation of ruminant movement restrictions during winter, when nightly light-suction trap catches of Culicoides fall below a threshold (the ‘seasonally vector free period’: SVFP). We analysed five years of time-series surveillance data from light-suction trapping in the UK to investigate whether significant inter-specific and yearly variation in adult phenology exists, and whether the SVFP is predictable from environmental factors. Because female vector Culicoides are not easily morphologically separated, inter-specific comparisons in phenology were drawn from male populations. We demonstrate significant inter-specific differences in Culicoides adult phenology with the season of Culicoides scoticus approximately eight weeks shorter than Culicoides obsoletus. Species-specific differences in the length of the SVFP were related to host density and local variation in landscape habitat. When the Avaritia Culicoides females were modelled as a group (as utilised in the SFVP), we were unable to detect links between environmental drivers and phenological metrics. We conclude that the current treatment of Avaritia Culicoides as a single group inhibits understanding of environmentally-driven spatial variation in species phenology and hinders the development of models for predicting the SVFP from environmental factors. Culicoides surveillance methods should be adapted to focus on concentrated assessments of species-specific abundance during the start and end of seasonal activity in temperate regions to facilitate refinement of ruminant movement restrictions thereby reducing the impact of Culicoides-borne arboviruses.     

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.