Rene Bødker

Culicoids as Vectors of Schmallenberg Virus

To the Editor: In autumn 2011, an unidentified disease of livestock was reported on both sides of the Dutch–Germany border. By using metagenomics, the etiologic agent of this disease was identified as a novel orthobunyavirus and named Schmallenberg virus (SBV) (1). Other members of the genus Orthobunyavirus (e.g., Akabane virus) are widespread in Africa and Asia; biting midges (Culicoides spp.) and mosquitoes are responsible for transmitting these viruses. Hence, we reasonably assumed that European culicoids might be responsible for transmitting SBV within Europe. We present evidence that culicoids captured October 2011 in Denmark contained SBV RNA and most likely are vectors for this agent. In autumn 2011, culicoids were collected from several sites within Denmark. One site, a chicken farm in Hokkerup (Figure A1), was selected for study because of its location close (6 km) to the German border and proximity (<10 km) to an SBV-infected sheep farm in Germany, as reported on March 9, 2012, by the Friedrich Loeffler Institute surveillance website (www.fli.bund.de). The culicoids were collected during October 14–16 by using a Mosquito Magnet Independence trap (Mosquito Magnet, Lititz, PA, USA) baited with carbon dioxide and octenol. Midges were sorted manually into 91 specimens of the C. obsoletus group (comprising C. obsoletus, C. chiopterus, C. dewulfi, and C. scoticus) and 17 of the C. punctatus sensu stricto group, then stored at −20°C. Pools of culicoids were homogenized in water (100 µL) by using a 3-mm stainless steel bead (Dejay Distribution Ltd., Launceston, UK) in a TissueLyser II (QIAGEN, Hilden, Germany) for 1 min at 25 Hz (2). After homogenization, additional water (100 µL) was added to the samples, and then the mixture was centrifuged at 3,000 × g for 5 min. Nucleic acids were extracted from the supernatant (100 µL) by using a MagNA pure LC Total Nucleic Acid Isolation Kit on a MagNA pure LC (Roche Diagnostics, Basel, Switzerland) and eluted in water (50 µL). Two separate 1-step reverse transcription quantitative PCRs (RT-qPCRs), targeting the L segment and the S segment of SBV RNA, were performed according to protocols provided by the Friedrich Loeffler Institute in Germany (1) on the extracted nucleic acids by using a Mx3005p qPCR system (Agilent Technologies, Palo Alto, CA, USA). Another RT-qPCR targeting ruminant β-actin mRNA was performed as an internal endogenous control (3). Two of 22 pools tested strongly positive for the large (L) and small (S) segments of SBV RNA. Each positive sample was derived from 5 midges of the C. obsoletus group. One pool produced cycle threshold (Ct) values of 26.4 and 24.5 (in the L segment– and S segment–specific assays, respectively), whereas the second positive pool gave Ct values of 28.8 (L segment) and 27.6 (S segment). These pools were negative for the internal endogenous control that targeted the bovine/ovine β-actin mRNA. This result makes it unlikely that the detection of SBV RNA within the midges resulted from recent blood meals from infected animals remaining within the culicoids and suggests the virus has replicated within the midges. The PCR amplicons (145 bp; Figure) from the L segment–specific RT-qPCR were sequenced by using BigDye 1.1 chemistry on an ABI 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The sequences of 80 bp from the amplicons, excluding the primer sequences, had 100% identity with the expected region of the SBV segment L (1). Figure RNA extracted from pools of Culicoides obsoletus group midges was tested in 1-step reverse transcription quantitative PCRs (RT-qPCRs) for the Schmallenberg virus large segment, and the products were analyzed by agarose gel electrophoresis. Lanes 1–8, ... Reported Ct values generated by using the same assays from blood of naturally infected cattle were 24–35 (1). Usually, ≈100 µL of bovine/ovine blood is used for virus detection, whereas <1 µL of blood remains in a midge after a blood meal. This uptake of blood should therefore lead to a Ct value that is at least 6–7 units higher (≈100-fold lower level of RNA) when a single midge is tested by RT-qPCR (4). Thus, even if all 5 culicoids in a pool had recently taken a blood meal from a viremic animal, the Ct values observed here strongly suggest replication of SBV within the C. obsoletus group midges. However, in principle, other hosts of SBV could have a much higher level of viremia than cattle and could provide the levels of SBV RNA detected. C. punctatus s.s. midges cannot be ruled out as a possible vector of SBV because of the limited number of insects tested. Our study demonstrates the presence of SBV RNA in C. obsoletus group midges caught in Denmark during October 2011. The low Ct values (i.e., high SBV RNA levels) and the absence of ruminant β-actin mRNA in these samples strongly suggest that SBV replicates in these midges and hence that the C. obsoletus group midges are natural vectors for this virus.

High-throughput screening of tick-borne pathogens in Europe

Due to increased travel, climatic, and environmental changes, the incidence of tick-borne disease in both humans and animals is increasing throughout Europe. Therefore, extended surveillance tools are desirable. To accurately screen tick-borne pathogens (TBPs), a large scale epidemiological study was conducted on 7050 Ixodes ricinus nymphs collected from France, Denmark, and the Netherlands using a powerful new high-throughput approach. This advanced methodology permitted the simultaneous detection of 25 bacterial, and 12 parasitic species (including; Borrelia, Anaplasma, Ehrlichia, Rickettsia, Bartonella, Candidatus Neoehrlichia, Coxiella, Francisella, Babesia, and Theileria genus) across 94 samples. We successfully determined the prevalence of expected (Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Rickettsia helvetica, Candidatus Neoehrlichia mikurensis, Babesia divergens, Babesia venatorum), unexpected (Borrelia miyamotoi), and rare (Bartonella henselae) pathogens in the three European countries. Moreover we detected Borrelia spielmanii, Borrelia miyamotoi, Babesia divergens, and Babesia venatorum for the first time in Danish ticks. This surveillance method represents a major improvement in epidemiological studies, able to facilitate comprehensive testing of TBPs, and which can also be customized to monitor emerging diseases.

Multiple detection of pathogens in ticks: development of a high throughput real time PCR chip used as a new epidemiologic investigative tool

Worldwide, ticks transmit more pathogens than other arthropods. Around 60 bacteria, 30 parasites and 100 viruses have been registered as tick-borne pathogens; a third of these pathogens are responsible for zoonoses. Usually, detection of tick-borne pathogens depends on the tick species collected: assays are performed for a restricted number of pathogens that are known to be transmitted by a particular tick species collected at a particular site. To better understand the epidemiology of tick-borne pathogens, it will be important to detect for each sample (one tick or one pool of ticks) most of the diseases they potentially transmit, regardless of the tick species. The aim is therefore to develop a new epidemiologic investigative tool which could detect high number of tick-borne pathogens by real time PCR. We developed a chip (BioMark™ dynamic arrays, Fluidigm Corporation) targeting pathogens of worldwide distribution transmitted by ticks. The designed epidemiologic arrays may detect 48 pathogens in 48 samples corresponding to 2304 qPCR reactions on the same time. Specific primers and probe have been designed for each pathogen and their specificity have been tested in silico with Blast. To begin, we targeted: (i) 37 pathogens whose Francisella tularensis, Coxiella burnetii, Neoehrlichia mikurensis, 5 species of Anaplasma, 3 species of Ehrlichia, 8 species of Borrelia, 2 species of Bartonella, 4 species of Rickettsia, 10 species of Babesia and 2 species of Theileria, (ii) 5 species of ticks whose 3 species of Ixodes and 2 species of Dermacentor. Sensitivity of primers and probe has been tested on a dilution range of reference DNAs of the targeted pathogens on a Lightcycler 480. Specificity then has been tested on a Biomark™dynamic array. The chip was secondly evaluated on field samples corresponding to 47 pools of 25 nymphs collected in two sites in France, the Netherlands and Denmark (corresponding to 7050 nymphs in total). We succesfully detected and determined the prevalence of Anaplasma phagocytophilum, Neoehrlichia mikurensis, Rickettsia helvetica, Bartonella henselae, five different genospecies of Borrelia burgdorferi s.l., the recently identified pathogen Borrelia miyamotoi, and two parasite species Babesia divergens and Babesia venatorum. This fast and low-cost tool allows comprehensive testing of tick-borne pathogens and can be customized to fit regional demands or to accommodate new or emerging pathogens. The tool represents a major improvement for surveillance and future epidemiological studies.

Prevalence, risk factors and spatial analysis of liver fluke infections in Danish cattle herds

BackgroundFasciola hepatica, a trematode parasite (liver fluke), infects a wide range of host species causing fasciolosis. The disease is prevalent world-wide and causes considerable economic losses to the livestock industry. Fasciolosis is regarded as an emerging food-borne zoonosis. To promote awareness among farmers and to implement strategies to control the infection, this study examined the prevalence, spatial distribution and risk factors for F. hepatica infection in Danish cattle herds.MethodsA retrospective population based study was performed using meat inspection data of approximately 1.5 million cattle slaughtered in the period 2011 to 2013. Annual cumulative prevalence of recorded liver fluke findings was calculated for each year. Global and local spatial cluster analysis was used to identify and map spatial patterns of F. hepatica positive and negative herds to explore environmental indicators of infection. Herd level, trade and environmental risk factors were evaluated for association with infection using logistic regression. Herd infection status as predicted from the final risk factor model was compared with the observed status using heat maps to assess how well the model fitted the observed spatial pattern.ResultsDuring the investigated period (2011–2013), an increase in annual herd prevalence was noted (2011–25.6%; 2012–28.4%; 2013–29.3%). The spatial analysis suggested significant clustering of positive and negative herds. Presence of streams, wetlands and pastures on farms showed a significant association with the presence of infection in cattle herds. Buying animals from positive herds was a risk factor on conventional farms. Additionally, risk of being infected with F. hepatica was higher in non-dairy herds of medium size (≥30 and < 100) when compared to dairy and large (≥100) cattle herds. The observed spatial pattern could be reproduced by predictions of the risk factor model.ConclusionsThis study showed an increase in annual herd level prevalence (2011 to 2013) indicating that an increasing proportion of herds are infected with F. hepatica infection every year in Denmark. Fasciolosis was found to be associated with both herd and environmental factors where the infection was influenced by local factors that clustered geographically.

Quantifying Dispersal of European Culicoides (Diptera: Ceratopogonidae) Vectors between Farms Using a Novel Mark-Release-Recapture Technique

Studying the dispersal of small flying insects such as Culicoides constitutes a great challenge due to huge population sizes and lack of a method to efficiently mark and objectively detect many specimens at a time. We here describe a novel mark-release-recapture method for Culicoides in the field using fluorescein isothiocyanate (FITC) as marking agent without anaesthesia. Using a plate scanner, this detection technique can be used to analyse thousands of individual Culicoides specimens per day at a reasonable cost. We marked and released an estimated 853 specimens of the Pulicaris group and 607 specimens of the Obsoletus group on a cattle farm in Denmark. An estimated 9,090 (8,918–9,260) Obsoletus group specimens and 14,272 (14,194–14,448) Pulicaris group specimens were captured in the surroundings and subsequently analysed. Two (0.3%) Obsoletus group specimens and 28 (4.6%) Pulicaris group specimens were recaptured. The two recaptured Obsoletus group specimens were caught at the release point on the night following release. Eight (29%) of the recaptured Pulicaris group specimens were caught at a pig farm 1,750 m upwind from the release point. Five of these were recaptured on the night following release and the three other were recaptured on the second night after release. This is the first time that movement of Culicoides vectors between farms in Europe has been directly quantified. The findings suggest an extensive and rapid exchange of disease vectors between farms. Rapid movement of vectors between neighboring farms may explain the the high rate of spatial spread of Schmallenberg and bluetongue virus (BTV) in northern Europe.

The range of attraction for light traps catching Culicoides biting midges (Diptera: Ceratopogonidae)

BackgroundCulicoides are vectors of e.g. bluetongue virus and Schmallenberg virus in northern Europe. Light trapping is an important tool for detecting the presence and quantifying the abundance of vectors in the field. Until now, few studies have investigated the range of attraction of light traps.MethodsHere we test a previously described mathematical model (Model I) and two novel models for the attraction of vectors to light traps (Model II and III). In Model I, Culicoides fly to the nearest trap from within a fixed range of attraction. In Model II Culicoides fly towards areas with greater light intensity, and in Model III Culicoides evaluate light sources in the field of view and fly towards the strongest. Model II and III incorporated the directionally dependent light field created around light traps with fluorescent light tubes. All three models were fitted to light trap collections obtained from two novel experimental setups in the field where traps were placed in different configurations.ResultsResults showed that overlapping ranges of attraction of neighboring traps extended the shared range of attraction. Model I did not fit data from any of the experimental setups. Model II could only fit data from one of the setups, while Model III fitted data from both experimental setups.ConclusionsThe model with the best fit, Model III, indicates that Culicoides continuously evaluate the light source direction and intensity. The maximum range of attraction of a single 4W CDC light trap was estimated to be approximately 15.25 meters. The attraction towards light traps is different from the attraction to host animals and thus light trap catches may not represent the vector species and numbers attracted to hosts.

Spatial abundance and clustering of Culicoides (Diptera: Ceratopogonidae) on a local scale

BackgroundBiting midges, Culicoides, of the Obsoletus group and the Pulicaris group have been involved in recent outbreaks of bluetongue virus and the former was also involved in the Schmallenberg virus outbreak in northern Europe.MethodsFor the first time, here we investigate the local abundance pattern of these two species groups in the field by intensive sampling with a grid of light traps on 16 catch nights. Neighboring trap catches can be spatially dependent on each other, hence we developed a conditional autoregressive (CAR) model framework to test a number of spatial and non-spatial covariates expected to affect Culicoides abundance.ResultsThe distance to sheep penned in the corner of the study field significantly increased the abundance level up to 200 meters away from the sheep. Spatial clustering was found to be significant but could not be explained by any known factors, and cluster locations shifted between catch nights. No significant temporal autocorrelation was detected. CAR models for both species groups identified a significant positive impact of humidity and significant negative impacts of precipitation and wind turbulence. Temperature was also found to be significant with a peak at just below 16 degrees Celcius. Surprisingly, there was a significant positive impact of wind speed. The CAR model for the Pulicaris group also identified a significant attraction to the smaller groups of sheep placed in the field. Furthermore, a large number of spatial covariates which were incorrectly found to be significant in ordinary regression models were not significant in the CAR models. The 95% C.I. on the prediction estimates ranged from 20.4% to 304.8%, underlining the difficulties of predicting the abundance of Culicoides.ConclusionsWe found that significant spatial clusters of Culicoides moved around in a dynamic pattern varying between catch nights. This conforms with the modeling but was not explained by any of the tested covariates. The mean abundance within these clusters was up to 11 times higher for the Obsoletus group and 4 times higher for the Pulicaris group compared to the rest of the field.

Optimal vaccination strategies against vector-borne diseases.

Using a process oriented semi-agent based model, we simulated the spread of Bluetongue virus by Culicoides, biting midges, between cattle in Denmark. We evaluated the minimum vaccination cover and minimum cost for eight different preventive vaccination strategies in Denmark. The simulation model replicates both a passive and active flight of midges between cattle distributed on pastures and cattle farms in Denmark. A seasonal abundance of midges and temperature dependence of biological processes were included in the model. The eight vaccination strategies were investigated under four different grazing conditions. Furthermore, scenarios were tested with three different index locations stratified for cattle density. The cheapest way to vaccinate cattle with a medium risk profile (less than 1000 total affected cattle) was to vaccinate cattle on pasture. Regional vaccination displayed better results when index cases were in the vaccinated areas. However, given that the long-range spread of midge borne disease is still poorly quantified, more robust national vaccination schemes seem preferable.

Spatio-temporal optimization of sampling for bluetongue vectors (Culicoides) near grazing livestock

BackgroundEstimating the abundance of Culicoides using light traps is influenced by a large variation in abundance in time and place. This study investigates the optimal trapping strategy to estimate the abundance or presence/absence of Culicoides on a field with grazing animals. We used 45 light traps to sample specimens from the Culicoides obsoletus species complex on a 14 hectare field during 16 nights in 2009.FindingsThe large number of traps and catch nights enabled us to simulate a series of samples consisting of different numbers of traps (1-15) on each night. We also varied the number of catch nights when simulating the sampling, and sampled with increasing minimum distances between traps. We used resampling to generate a distribution of different mean and median abundance in each sample. Finally, we used the hypergeometric distribution to estimate the probability of falsely detecting absence of vectors on the field. The variation in the estimated abundance decreased steeply when using up to six traps, and was less pronounced when using more traps, although no clear cutoff was found.ConclusionsDespite spatial clustering in vector abundance, we found no effect of increasing the distance between traps. We found that 18 traps were generally required to reach 90% probability of a true positive catch when sampling just one night. But when sampling over two nights the same probability level was obtained with just three traps per night. The results are useful for the design of vector monitoring programmes on fields with grazing animals.

Spatial Scan Statistics to Assess Sampling Strategy of Antimicrobial Resistance Monitoring Program

The collection and analysis of data on antimicrobial resistance in human and animal populations are important for establishing a baseline of the occurrence of resistance and for determining trends over time. In animals, targeted monitoring with a stratified sampling plan is normally used. However, to our knowledge it has not previously been analyzed whether animals have a random chance of being sampled by these programs, regardless of their spatial distribution. In this study, we used spatial scan statistics, based on a Poisson model, as a tool to evaluate the geographical distribution of animals sampled by the Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (DANMAP), by identifying spatial clusters of samples and detecting areas with significantly high or low sampling rates. These analyses were performed for each year and for the total 5-year study period for all collected and susceptibility tested pig samples in Denmark between 2002 and 2006. For the yearly analysis, both high and low sampling rates areas were significant, with two clusters in 2002 (relative risk [RR]: 2.91, p < 0.01 and RR: 0.06, p < 0.01) and one in 2005 (RR: < 0.01, p < 0.01). For the 5-year analysis, one high sampling rate cluster was detected (RR: 2.56, p = 0.01). These findings allowed subsequent investigation to clarify the source of the sampling clusters. Overall, the detected clusters presented different spatial locations over the years and we can conclude that they were more associated to temporary sampling problems than to a failure in the sampling strategy adopted by the monitoring program. Spatial scan statistics proved to be a useful tool for assessment of the randomness of the sampling distribution, which is important when evaluating the validity of the results obtained by an antimicrobial monitoring program.