Vector competence of Culicoides biting midges from Switzerland for African horse sickness virus and epizootic haemorrhagic disease virus.

INTRODUCTION: Culicoides biting midges unexpectedly arose in Europe as highly efficient vectors of bluetongue virus in the epidemics that started in the Netherlands in 2006. They are known vectors of other orbiviruses, such as African horse sickness (AHSV) and epizootic haemorrhagic disease viruses (EHDV), which are not endemic to Europe. We investigated whether Culicoides occurring in Switzerland at two altitudes (Swiss Plateau, 650 meters above sea level [masl]; and pre-alpine, 2,130 masl) can act as vectors for AHSV and EHDV (two strains each). Biting midges were collected from farms, allowed to feed on virus-spiked blood meals through an artificial membrane in the laboratory and incubated for eight days under two temperature regimes (22 ± 6 °C or 26 ± 6 °C) reflecting a summer day or a hot spell on the Swiss Plateau. Vector competence was assessed from head homogenates by RT-qPCR and virus isolation. Overall, over 15,000 biting midges were exposed to any one of the four viruses. Fully disseminated infections were identified for all four virus strains in 14 individuals (6 C. obsoletus, 8 C. scoticus, as identified by MALDI-TOF mass spectrometry), all originating from the Swiss Plateau, by RT-qPCR. Viable virus could be isolated from 8 of these specimens. Dissemination rates ranged from 1-5%. No viral dissemination was observed in biting midges from the high altitude, predominantly belonging to the species C. grisescens, which were only investigated at the high temperature regime. However, a multivariable logistic regression model revealed no statistical difference in the dissemination rates based on the origin of midges (altitude), virus strain or temperature regime. Thus, AHDV and EHDV transmission is feasible on the Swiss Plateau but unlikely in the pre-alpine area by considering vector abundance. Ways of potential virus introduction include illegal animal movement but also long-distance wind-dispersal of infectious Culicoides.

INTRODUCTION: Les moucherons culicoïdes sont apparus de manière inattendue en Europe en tant que vecteurs très efficaces du virus de la fièvre catarrhale du mouton lors des épidémies qui ont commencé aux Pays-Bas en 2006. Ils sont des vecteurs connus d'autres orbivirus, tels que la peste équine (AHSV) et la maladie à virus hémorragique épizootique (EHDV), qui ne sont pas endémiques en Europe. Nous avons cherché à savoir si les culicoïdes présents en Suisse à deux altitudes (Plateau suisse, 650 mètres au-dessus du niveau de la mer et Préalpes, 2130 mètres au-dessus du niveau de la mer) peuvent agir comme vecteurs pour l'AHSV et l'EHDV (deux souches chacune). Des moucherons piqueurs ont été collectés dans des élevages, laissés se nourrir de repas de sang contaminé par le virus à travers une membrane artificielle en laboratoire et incubés pendant huit jours sous deux régimes de température (22 ± 6 °C ou 26 ± 6 °C) reflétant une journée d'été ou une vague de chaleur sur le plateau suisse. La compétence vectorielle a été évaluée à partir d'homogénats de tête par RT-qPCR et isolement du virus. Dans l'ensemble, plus de 15 000 moucherons piqueurs ont été exposés à l'un des quatre virus. Des infections entièrement disséminées ont été identifiées pour les quatre souches virales chez 14 individus (6 C. obsoletus, 8 C. scoticus, identifiés par spectrométrie de masse MALDI-TOF), tous originaires du plateau suisse, par RT-qPCR. Le virus viable a pu être isolé à partir de 8 de ces échantillons. Les taux de diffusion allaient de 1 à 5 %. Aucune dissémination virale n'a été observée chez les moucherons piqueurs de haute altitude, appartenant majoritairement à l'espèce C. grisescens, qui n'ont été étudiées qu'au régime de haute température. Cependant, un modèle de régression logistique multivariable n'a révélé aucune différence statistique dans les taux de dissémination en fonction de l'origine des moucherons (altitude), de la souche virale ou du régime de température. Ainsi, la transmission de l'AHDV et de l'EHDV est possible sur le plateau suisse mais peu probable dans la zone préalpine en considérant l'abondance des vecteurs. Les voies d'introduction potentielle du virus comprennent les déplacements illégaux d'animaux, mais aussi la dispersion par le vent sur de longues distances de culicoïdes infectieux.

Summer seasonal prevalence of Culicoides species from pre-alpine areas in Switzerland.

Biting midges (Diptera: Ceratopogonidae) are arthropods of veterinary importance since they can transmit pathogens and cause severe allergic dermatitis in horses. Very little is known about the species at higher altitudes and their seasonal dynamics. In this work, adult Culicoides were collected with Onderstepoort UV-light suction traps (OVI) from June to September 2016 at two areas situated at around 1600 m asl (pre-alpine area I, 2 farms) and 2030 m asl (pre-alpine area II, 1 farm) in the Canton of Grisons (south-east Switzerland). Overall, 17 049 Culicoides were collected, including 871 parous females. A total of 50 individuals/trap/night (n = 1050) were identified to species (17 species) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) or by polymerase chain reaction (PCR) and sequencing. The remaining 15 128 Culicoides were classified to species groups' level. Culicoides obsoletus (Meigen, 1818), a multivoltine species, was mainly present at 1600 m asl, whereas at high altitudes (2030 m asl), C. grisescens Edwards, 1939 I&II were the most abundant species. In particular, C. grisescens II, which seems to be univoltine, occurred later in the season but significantly increasing over time. Species diversity was higher at pre-alpine I area (n = 16 species) compared to pre-alpine II (n = 10 species).

Vector competence of field populations of the mosquito species Aedes japonicus japonicus and Culex pipiens from Switzerland for two West Nile virus strains.

Aedes (Hulecoeteomyia) japonicus japonicus (Diptera: Culicidae) (Theobald) is an invasive mosquito species in Central Europe, where it has colonized several areas. In this study, field-collected specimens of Ae. japonicus and Culex pipiens (Diptera: Culicidae) (Linnaeus) from Zürich (Switzerland) were orally exposed to two strains (NY99 and FIN) of the avian zoonotic pathogen West Nile virus (WNV) (family Flaviviridae, genus Flavivirus). Dissemination and transmission of the viruses after incubation for 12-15 days under a fluctuating Central European midsummer temperature regime (24 ± 7 °C) was investigated by detection of viral RNA in homogenates of pools of both head/thorax and saliva by reverse transcription real-time polymerase chain reaction (PCR). Culex pipiens was susceptible to WNV NY99 only, whereas both virus strains could be detected in Ae. japonicus, with the additional isolation of WNV NY99 in Vero cell culture from one saliva pool. Given the high abundances of Ae. japonicus in many newly colonized areas, its recently demonstrated broad host range, including mammalian and avian blood hosts, and its vector competence, this species is a potential key bridge vector of WNV in Central Europe.

Sterilization of Hulecoeteomyia japonica japonica (= Aedes japonicus japonicus) (Theobald, 1901) by high-energy photon irradiation: implications for a sterile insect technique approach in Europe.

Hulecoeteomyia japonica japonica (= Aedes japonicus japonicus) (Diptera: Culicidae) (Theobald 1901), a container-breeding invasive species in North America and Europe, is attracting particular attention for its high local abundances and possible roles in the transmission of human and animal pathogens. The preferential habitats of this species are forested and bushy areas, which renders control measures extremely inefficient. Use of the sterile insect technique (SIT) may contribute to the implementation of area-wide integrated pest management strategies, as has been successfully proven with other aedine mosquito species. The present study investigates the effects of irradiation at a dose of 40 Gy on fitness parameters in H. j. japonica. Irradiation was performed on 16-24-h-old pupae from a colonized strain (PA) using a TrueBeam linear accelerator. Males from the PA strain were crossed with females of the same colony or with field-collected females. Irradiation induced a slight increase in mortality in male pupae, but did not alter the survival and mating abilities of emerging adult males. Rates of blood feeding and fertility were lower when PA strain males were kept with field-collected females rather than PA females. Irradiated males induced reductions in fertility (residual fertility: 2.6%) and fecundity in mated females. The data indicate that the SIT is a suitable technique to enhance the control of this species.

Field evaluation of baited traps for surveillance of Aedes japonicus japonicus in Switzerland.

The efficacy of Centers for Disease Control (CDC) miniature light traps and ovitraps was tested in the outskirts of the city of Zurich in Switzerland for their use in the surveillance of Aedes (Hulecoeteomyia) japonicus japonicus (Theobald) (Diptera: Culicidae), the invasive Asian bush mosquito. Sets of single CDC traps were run overnight (n = 18) in three different environments (forest, suburban and urban) in 3 × 3 Latin square experimental designs. Traps were baited with: (a) carbon dioxide (CO2 ); (b) CO2 plus light, or (c) CO2 plus lure blend [Combi FRC 3003 (iGu® )]. At the same locations, mosquito eggs were collected weekly using standard ovitraps baited with different infusions (oak, hay or tap water) and equipped with different oviposition substrates (a block of extruded polystyrene, a germination paper strip or a wooden stick). Data were analysed using Poisson and negative binomial general linear models. The use of light (P < 0.001) or lure (P < 0.001) significantly increased the attractiveness of CDC traps baited with CO2 . Oak and hay infusions did not increase the attractiveness of ovitraps compared with standing tap water (P > 0.05), and extruded polystyrene blocks were preferred as an oviposition substrate over wooden sticks (P < 0.05) and seed germination paper (P < 0.05). Carbon dioxide-baited CDC miniature light traps complemented with light or iGu® lure and ovitraps containing standing tap water and polystyrene oviposition blocks can be considered as efficient and simple tools for use in Ae. j. japonicus surveillance programmes.

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

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

Life-history parameters of Culicoides (Avaritia) imicola Kieffer in the laboratory at different rearing temperatures.

This laboratory study investigates the sub-adult developmental cycle of field collected Culicoides (Avaritia) imicola Kieffer (Diptera; Ceratopogonidae). The period required from blood-feeding field-collected females to the production of progeny adults occupied 34-56 days at 20 degrees C, 15-21 days at 25 degrees C and 11-16 days at 28 degrees C, demonstrating clear temperature dependence. When reared at 28 degrees C, C. imicola demonstrated higher variability in fecundity (between 2.4 and 20.6 eggs/female) and lower hatching rates (50.0-62.2%), although larval survival rates to pupation were low at all temperatures (20-30%). Similarly, the mean emergence rate from pupae was the highest at lower temperatures. These results highlight the difficulty in establishing and maintaining a laboratory colony of this species from field-collected material and results are discussed in reference to future research directions that may aid this process.

Clinical signs and pathology shown by British sheep and cattle infected with bluetongue virus serotype 8 derived from the 2006 outbreak in northern Europe.

Four poll Dorset sheep and four Holstein-Friesian cattle were infected with the northern European strain of bluetongue virus (BTV), BTV-8, to assess its pathogenicity in UK breeds. The time course of infection was monitored in both species by using real-time reverse transcriptase-PCR (RT-PCR), conventional RT-PCR and serology. Two of the sheep developed severe clinical signs that would have been fatal in the field; the other two were moderately and mildly ill, respectively. The cattle were clinically unaffected, but had high levels of viral RNA in their bloodstream. Real-time RT-PCR detected viral RNA as early as one day after infection in the cattle and three days after infection in the sheep. Antibodies against BTV were detected by six days after infection in the sheep and eight days after infection in the cattle. Postmortem examinations revealed pathology in the cattle that was more severe than suggested by the mild clinical signs, but the pathological and clinical findings in the sheep were more consistent.

Spatial and temporal distribution of bluetongue and its Culicoides vectors in Bulgaria.

Surveillance of Culicoides (Diptera: Ceratopogonidae) biting midges was carried out between 2001 and 2003, at 119 sites within a 50 x 50-km grid distributed across Bulgaria, using light trap collections around the time of peak adult midge abundance. Sentinel and ad hoc serum surveillance of hosts susceptible to bluetongue infection was carried out at around 300 sites between 1999 and 2003. Following the initial incursion of bluetongue virus 9 (BTV-9) into Bourgas province in 1999, affecting 85 villages along the southern border, a further 76 villages were affected along the western border in 2001, with outbreaks extending as far north as 43.6 degrees N. The BTV-9 strain in circulation was found to have a low pathogenicity for Bulgarian sheep populations, with less than 2% of susceptible individuals becoming sick and seroconversions detected up to 30 km from recorded outbreaks in the south. The major Old World vector Culicoides imicola Kieffer was not detected among over 70,000 Culicoides identified in summer collections, suggesting that BTV-9 transmission in Bulgaria was primarily carried out by indigenous European vectors. The most likely candidates, the Palaearctic species complexes - the Culicoides obsoletus Meigen and C. pulicaris L. complexes - were widespread and abundant across the whole country. The C. obsoletus complex represented 75% of all individuals trapped in summer and occurred in high catch sizes (up to 15,000 individuals per night) but was not found across all outbreak sites, indicating that both Palearctic complexes probably played a role in transmission. Within the C. pulicaris complex, only C. pulicaris s.s., C. punctatus Meigen and C. newsteadi Austen were sufficiently abundant and prevalent to have been widely involved in transmission, whilst within the C. obsoletus complex most trapped males were C. obsoletus s.s. Adult vectors were found to be largely absent from sites in west Bulgaria for a period of at least 3 months over winter, which, taken along with the spatiotemporal pattern of outbreaks in the region between years, indicates the virus may be overwintering here by an alternative mechanism - either by covert persistence in the vertebrate host or possibly by persistence in larval stages of the vector.

Live attenuated bluetongue vaccine viruses in Dorset Poll sheep, before and after passage in vector midges (Diptera: Ceratopogonidae).

The aim of this study was to address concerns associating with the use of BTV attenuated commercial vaccines in European sheep. These concerns include development of viraemia, possibility of transmission by vectors, reversion to virulence and re-assortment with wild-type viruses. The two vaccine viruses (BTV 2 and 9) replicated in two species of Culicoides subsequent to oral infection reaching titres suggesting transmission would occur. Viraemia in Dorset Poll sheep inoculated with either vaccine or insect passaged vaccine viruses persisted for up to 17 days, recording titres that ranged from 2.5 to 6.25 log(10)TCID(50)/ml, which is easily sufficient to infect vector Culicoides. Moderate to severe clinical signs of BT, albeit short lived, were observed in sheep following vaccination. However, to date there is no evidence of increasing virulence following two sequential passages through the vectors.

Health care expenditure, laboratory services and IVD market.

What has been written until now should not be misinterpreted: without doubt there is wastage in the Italian health care expenditure which must be rationalized. Moreover, the public deficit-now over two million billion lira-will probably prevent any reinvestment in health care of resources liberated through the above-mentioned rationalization process. In the near future, the attention of the authorities should be focused on how to reduce public spending, which probably also includes spending on health care. The message which needed to be passed on here does not refuse to recognize the possibility of rationalizing health care expenditure in Italy nor does it reject the need for this course of action. Instead it tries to give the following warning: in the light of the above circumstances-or the fact that even now the authorities cannot admit to spending "much" in the absolute sense, especially with regard to technology, or the fundamental role of IVDs in health care processes, or the difficulties in which the companies of the sector have been placed-it is possible to state that any blind, or worse still, ill-equipped, intervention in this field would have the undoubted effect of damaging the health care sector, or even place many companies on their knees, without receiving the expected benefits on the balance sheet. Benefits in the form of efficiency and saving can only be obtained from an effective reorganisation of the health structures, in line with the reforms provided for by legislative decree n. 502/92 (and subsequent modifications)--which, due to aspects too numerous to mention, is still a dead letter--and by taking steps towards valuing the laboratory services. As already stated, to talk of inefficiency in general terms means talking of unproductive expenditure: this occurs when utilizing factors whose cost is "too high" and/or productivity is "too low". It is with this distinction in mind that intervention must come; assessing factors not individually but rationally integrated in the production process of which they are part. Given the values at issue, any action not specifically aimed at the elimination of process inefficiency penalizes the already "healthy" elements, causes damage to the system and does not result in any significant benefits for expenditure. To summarize further, the need is for: a definition of what and how much to guarantee that can be taken from the National Budget; the realization of models which, surpassing the current macroscopic causes of inefficiency (which reside in structural and process type diseconomies), would make a correct economic assessment of investments and expenditure viable. In this respect, clinical pathology could play a fundamental role, in that the investments in this particular sector, if well programmed, would lead to significant savings for the entire health care system. The path followed until now must be abandoned at all costs; a path which starts by underestimating the needs and continues with the utilization of resources in an irrational manner, finishing with a proposal for the following year to allocate fewer resources to technology, almost as if it were on the same level as the potatoes (with or maybe even get hungry), at least this would resolve balance sheet problems without affecting the running of the services.