Ixodes ricinus as potential vector for Usutu virus.

Usutu virus (USUV) is an emerging flavivirus that is maintained in an enzootic cycle with mosquitoes as vectors and birds as amplifying hosts. In Europe, the virus has caused mass mortality of wild birds, mainly among Common Blackbird (Turdus merula) populations. While mosquitoes are the primary vectors for USUV, Common Blackbirds and other avian species are exposed to other arthropod ectoparasites, such as ticks. It is unknown, however, if ticks can maintain and transmit USUV. We addressed this question using in vitro and in vivo experiments and field collected data. USUV replicated in IRE/CTVM19 Ixodes ricinus tick cells and in injected ticks. Moreover, I. ricinus nymphs acquired the virus via artificial membrane blood-feeding and maintained the virus for at least 70 days. Transstadial transmission of USUV from nymphs to adults was confirmed in 4.9% of the ticks. USUV disseminated from the midgut to the haemocoel, and was transmitted via the saliva of the tick during artificial membrane blood-feeding. We further explored the role of ticks by monitoring USUV in questing ticks and in ticks feeding on wild birds in the Netherlands between 2016 and 2019. In total, 622 wild birds and the Ixodes ticks they carried were tested for USUV RNA. Of these birds, 48 (7.7%) carried USUV-positive ticks. The presence of negative-sense USUV RNA in ticks, as confirmed via small RNA-sequencing, showed active virus replication. In contrast, we did not detect USUV in 15,381 questing ticks collected in 2017 and 2019. We conclude that I. ricinus can be infected with USUV and can transstadially and horizontally transmit USUV. However, in comparison to mosquito-borne transmission, the role of I. ricinus ticks in the epidemiology of USUV is expected to be minor.

Interactive effects of climate, land use and soil type on Culex pipiens/torrentium abundance.

The incidence and risk of mosquito-borne disease outbreaks in Northwestern Europe has increased over the last few decades. Understanding the underlying environmental drivers of mosquito population dynamics helps to adequately assess mosquito-borne disease risk. While previous studies have focussed primarily on the effects of climatic conditions (i.e., temperature and precipitation) and/or local environmental conditions individually, it remains unclear how climatic conditions interact with local environmental factors such as land use and soil type, and how these subsequently affect mosquito abundance. Here, we set out to study the interactive effects of land use, soil type and climatic conditions on the abundance of Culex pipiens/torrentium, highly abundant vectors of West Nile virus and Usutu virus. Mosquitoes were sampled at 14 sites throughout the Netherlands. At each site, weekly mosquito collections were carried out between early July and mid-October 2020 and 2021. To assess the effect of the aforementioned environmental factors, we performed a series of generalized linear mixed models and non-parametric statistical tests. Our results show that mosquito abundance and species richness consistently differ among land use- and soil types, with peri-urban areas with peat/clay soils having the highest Cx. pipiens/torrentium abundance and sandy rural areas having the lowest. Furthermore, we observed differences in precipitation-mediated effects on Cx. pipiens/torrentium abundance between (peri-)urban and other land uses and soil types. In contrast, effects of temperature on Cx. pipiens/torrentium abundance remain similar between different land use and soil types. Our study highlights the importance of both land use and soil type in conjunction with climatic conditions for understanding mosquito abundances. Particularly in relation to rainfall events, land use and soil type has a marked effect on mosquito abundance. These findings underscore the importance of local environmental parameters for studies focusing on predicting or mitigating disease risk.

First Detections of Culiseta longiareolata (Diptera: Culicidae) in Belgium and the Netherlands.

Culiseta (Allotheobaldia) longiareolata (Macquart) (Diptera: Culicidae) is an ornithophilic mosquito species that occurs in the southern Palaearctic Region from the Azores to Central Asia, the Ethiopian Region, India, and Pakistan. Although it has a widespread distribution range, the species was only recently reported in Western and Central Europe. Between 2017 and 2020, larvae, pupae, and adults of Cs. longiareolata (n = 161) were found at 13 distinct locations in Belgium (n = 4) and The Netherlands (n = 9). Collected mosquitoes were morphologically identified and the identification was then validated by COI DNA barcoding. These are the first records of the species in the above-mentioned countries. The present results suggest that Cs. longiareolata could be increasing its distribution range in temperate regions, indicating a warming climate. As the species might be a potential vector of bird pathogens (e.g., West Nile virus), its spread in Western Europe is noteworthy.

Imported Hyalomma ticks in the Netherlands 2018-2020.

BACKGROUND: Ticks of the genus Hyalomma, which are vectors for several tick-borne diseases, are occasionally found in areas outside their endemic range including northern parts of Europe. The objective of this study was to analyse adult Hyalomma ticks that were recently found in the Netherlands. METHODS: Hyalomma ticks were morphologically identified. Cluster analysis, based upon sequence data (cox1 barcoding) for molecular identification, and pathogen detection were performed. Additionally, a cross-sectional survey of horses was conducted to actively search for Hyalomma ticks in summer 2019. Analysis of temperature was done to assess the possibility of (i) introduced engorged nymphs moulting to adults and (ii) establishment of populations in the Netherlands. RESULTS: Seventeen adult Hyalomma ticks (one in 2018, eleven in 2019, five in 2020) were found by citizens and reported. Fifteen ticks were detected on horses and two on humans. Twelve were identified as H. marginatum, one as H. rufipes and four, of which only photographic images were available, as Hyalomma sp. No Crimean-Congo haemorrhagic fever virus or Babesia/Theileria parasites were detected. One adult tick tested positive for Rickettsia aeschlimannii. In the cross-sectional horse survey, no Hyalomma ticks were found. Analysis of temperatures showed that engorged nymphs arriving on migratory birds in spring were able to moult to adults in 2019 and 2020, and that cumulative daily temperatures in the Netherlands were lower than in areas with established H. marginatum populations. CONCLUSIONS: Our results show that Hyalomma ticks are regularly introduced in the Netherlands as nymphs. Under the Dutch weather conditions, these nymphs are able to develop to the adult stage, which can be sighted by vigilant citizens. Only one human pathogen, Rickettsia aeschlimannii, was found in one of the ticks. The risk of introduction of tick-borne diseases via Hyalomma ticks on migratory birds is considered to be low. Establishment of permanent Hyalomma populations is considered unlikely under the current Dutch climatic conditions.

Spatial risk analysis for the introduction and circulation of six arboviruses in the Netherlands.

BACKGROUND: Arboviruses are a growing public health concern in Europe, with both endemic and exotic arboviruses expected to spread further into novel areas in the next decades. Predicting where future outbreaks will occur is a major challenge, particularly for regions where these arboviruses are not endemic. Spatial modelling of ecological risk factors for arbovirus circulation can help identify areas of potential emergence. Moreover, combining hazard maps of different arboviruses may facilitate a cost-efficient, targeted multiplex-surveillance strategy in areas where virus transmission is most likely. Here, we developed predictive hazard maps for the introduction and/or establishment of six arboviruses that were previously prioritized for the Netherlands: West Nile virus, Japanese encephalitis virus, Rift Valley fever virus, tick-borne encephalitis virus, louping-ill virus and Crimean-Congo haemorrhagic fever virus. METHODS: Our spatial model included ecological risk factors that were identified as relevant for these arboviruses by an earlier systematic review, including abiotic conditions, vector abundance, and host availability. We used geographic information system (GIS)-based tools and geostatistical analyses to model spatially continuous datasets on these risk factors to identify regions in the Netherlands with suitable ecological conditions for arbovirus introduction and establishment. RESULTS: The resulting hazard maps show that there is spatial clustering of areas with either a relatively low or relatively high environmental suitability for arbovirus circulation. Moreover, there was some overlap in high-hazard areas for virus introduction and/or establishment, particularly in the southern part of the country. CONCLUSIONS: The similarities in environmental suitability for some of the arboviruses provide opportunities for targeted sampling of vectors and/or sentinel hosts in these potential hotspots of emergence, thereby increasing the efficient use of limited resources for surveillance.

Detection of Exotic Mosquito Species (Diptera: Culicidae) at International Airports in Europe.

In Europe, the air-borne accidental introduction of exotic mosquito species (EMS) has been demonstrated using mosquito surveillance schemes at Schiphol International Airport (Amsterdam, The Netherlands). Based upon these findings and given the increasing volume of air transport movements per year, the establishment of EMS after introduction via aircraft is being considered a potential risk. Here we present the airport surveillance results performed by the Centre for Monitoring of Vectors of the Netherlands, by the Monitoring of Exotic Mosquitoes (MEMO) project in Belgium, and by the Public Health England project on invasive mosquito surveillance. The findings of our study demonstrate the aircraft mediated transport of EMS into Europe from a wide range of possible areas in the world. Results show accidental introductions of Aedes aegypti and Ae. albopictus, as well as exotic Anopheles and Mansonia specimens. The findings of Ae. albopictus at Schiphol airport are the first evidence of accidental introduction of the species using this pathway in Europe. Furthermore, our results stress the importance of the use of molecular tools to validate the morphology-based species identifications. We recommend monitoring of EMS at airports with special attention to locations with a high movement of cargo and passengers.

Practical management plan for invasive mosquito species in Europe: I. Asian tiger mosquito (Aedes albopictus).

Aedes albopictus, also known as the "Asian Tiger Mosquito", is an invasive mosquito species to Europe causing high concern in public health due to its severe nuisance and its vectorial capacity for pathogens such as dengue, chikungunya, yellow fever and Zika. Consequently, the responsible authorities implement management activities to reduce its population density, possibly to below noxious and epidemiological thresholds. In urban areas, these aims are difficult to achieve because of the species' ability to develop in a wide range of artificial breeding sites, mainly private properties. This document (Management Plan) has been structured to serve as a comprehensive practical and technical guide for stakeholders in organizing the vector control activities in the best possible way. The current plan includes coordinated actions such as standardized control measures and quality control activities, monitoring protocols, activities for stakeholders and local communities, and an emergency vector control plan to reduce the risk of an epidemic.

Risk-Based and Adaptive Invasive Mosquito Surveillance at Lucky Bamboo and Used Tire Importers in the Netherlands.

The detection of Aedes albopictus in Lucky bamboo (Dracaena sanderiana) greenhouses and Ae. atropalpus at used tire importers illustrates that the Netherlands is exposed to the risk of introductions of invasive mosquito species (IMS). In this study we implemented a risk-based and adaptive surveillance (2010-16) in order to detect introductions and prevent potential proliferation of IMS at these locations. Results at Lucky bamboo greenhouses show that interceptions of Ae. albopictus occurred every year, with 2010 and 2012 being the years with most locations found positive for this species (n = 6), and 2015 the year with the highest percentage of positive samples (4.1%). Furthermore, our results demonstrate that Ae. japonicus can also be associated with the import of Lucky bamboo. At used tire companies, IMS were found at 12 locations. Invasive mosquito species identified were Ae. albopictus, Ae. atropalpus, Ae. aegypti, and Ae. japonicus, of which Ae. albopictus has been found every year since 2010. The proportion of samples containing IMS was significantly higher before application of a covenant between the used tire importers and the Dutch government in 2013 (12.96%) than in the successive 3 years (2014 [6.93%], 2015 [4.24%], 2016 [5.09%], 1-sided binomial test, P < 0.01). It is concluded that risk-based and adaptive surveillance is an effective methodology for detection of IMS, and that application of governmental management measures in combination with mosquito control has stabilized the situation.

Detection of Aedes flavopictus (Yamada, 1921), Netherlands, June 2019.

In June 2019, a single specimen collected at a used tyre company was identified as Aedes flavopictus (Yamada, 1921), a sibling species of Ae. albopictus. Ae. flavopictus has not been recorded outside Japan and South Korea. Although it has only shown dengue virus vector competence under laboratory conditions, its detection demonstrates the value of active surveillance at risk locations and molecular tools for timely intervention against exotic mosquitoes with potential future public health impact.

Risk factors associated with sustained circulation of six zoonotic arboviruses: a systematic review for selection of surveillance sites in non-endemic areas.

Arboviruses represent a significant burden to public health and local economies due to their ability to cause unpredictable and widespread epidemics. To maximize early detection of arbovirus emergence in non-endemic areas, surveillance efforts should target areas where circulation is most likely. However, identifying such hotspots of potential emergence is a major challenge. The ecological conditions leading to arbovirus outbreaks are shaped by complex interactions between the virus, its vertebrate hosts, arthropod vector, and abiotic environment that are often poorly understood. Here, we systematically review the ecological risk factors associated with the circulation of six arboviruses that are of considerable concern to northwestern Europe. These include three mosquito-borne viruses (Japanese encephalitis virus, West Nile virus, Rift Valley fever virus) and three tick-borne viruses (Crimean-Congo hemorrhagic fever virus, tick-borne encephalitis virus, and louping-ill virus). We consider both intrinsic (e.g. vector and reservoir host competence) and extrinsic (e.g. temperature, precipitation, host densities, land use) risk factors, identify current knowledge gaps, and discuss future directions. Our systematic review provides baseline information for the identification of regions and habitats that have suitable ecological conditions for endemic circulation, and therefore may be used to target early warning surveillance programs aimed at detecting multi-virus and/or arbovirus emergence.

Detection of pathogens in Dermacentor reticulatus in northwestern Europe: evaluation of a high-throughput array.

BACKGROUND: The geographic distribution of Dermacentor reticulatus is expanding in Europe. Surveillance of this tick species and its pathogens is desirable, as it transmits pathogens of public and veterinary importance. A high-throughput real-time PCR-based array was used to screen 1.741 D. reticulatus ticks from Belgium, Germany, The Netherlands, and Great Britain for the presence of 28 tick-borne bacteria and twelve protozoan parasites. The presence of pathogen DNA was confirmed by conventional PCR followed by sequencing. RESULTS: The array detected the presence of DNA from Borrelia spp. (7%), B. afzelii (0.1%), B. garinii (0.1%), B. spielmanii (0.1%), B. miyamotoi (0.2%), Anaplasma marginale (0.1%), A. phagocytophilum (0.1%), Ehrlichia canis (2%), Rickettsia helvetica (0.2%), spotted fever group Rickettsia (9.6%), Francisella tularensis or Francisella-like endosymbionts (95%), Coxiella burnettii (0.1%), Babesia divergens (0.2%), B. canis (0.9%) B. vogeli (5.6%), and Theileria equi (0.1%). Only the presence of B. canis and spotted fever group Rickettsia could be confirmed by conventional PCR and sequencing. The spotted fever Rickettsia-positive samples were all identified as R. raoultii. CONCLUSIONS: We successfully detected and determined the prevalence of B. canis and R. raoultii in D. reticulatus. An high-throughput array that allows fast and comprehensive testing of tick-borne pathogens is advantageous for surveillance and future epidemiological studies. The importance of thorough validation of real-time PCR-based assays and careful interpretation is evident.

Pathogen communities of songbird-derived ticks in Europe's low countries.

BACKGROUND: Birds play a major role in the maintenance of enzootic cycles of pathogens transmitted by ticks. Due to their mobility, they affect the spatial distribution and abundance of both ticks and pathogens. In the present study, we aim to identify members of a pathogen community [Borrelia burgdorferi (s.l.), B. miyamotoi, 'Ca. Neoehrlichia mikurensis', Anaplasma phagocytophilum and Rickettsia helvetica] in songbird-derived ticks from 11 locations in the Netherlands and Belgium (2012-2014). RESULTS: Overall, 375 infested songbird individuals were captured, belonging to 35 species. Thrushes (Turdus iliacus, T. merula and T. philomelos) were trapped most often and had the highest mean infestation intensity for both Ixodes ricinus and I. frontalis. Of the 671 bird-derived ticks, 51% contained DNA of at least one pathogenic agent and 13% showed co-infections with two or more pathogens. Borrelia burgdorferi (s.l.) DNA was found in 34% of the ticks of which majority belong to so-called avian Borrelia species (distribution in Borrelia-infected ticks: 47% B. garinii, 34% B. valaisiana, 3% B. turdi), but also the mammal-associated B. afzelii (16%) was detected. The occurrence of B. miyamotoi was low (1%). Prevalence of R. helvetica in ticks was high (22%), while A. phagocytophilum and 'Ca. N. mikurensis' prevalences were 5% and 4%, respectively. The occurrence of B. burgdorferi (s.l.) was positively correlated with the occurrence of 'Ca. N. mikurensis', reflecting variation in susceptibility among birds and/or suggesting transmission facilitation due to interactions between pathogens. CONCLUSIONS: Our findings highlight the contribution of European songbirds to co-infections in tick individuals and consequently to the exposure of humans to multiple pathogens during a tick bite. Although poorly studied, exposure to and possibly also infection with multiple tick-borne pathogens in humans seems to be the rule rather than the exception.

Environmental surveillance during an outbreak of tularaemia in hares, the Netherlands, 2015.

Tularaemia, a disease caused by the bacterium Francisella tularensis, is a re-emerging zoonosis in the Netherlands. After sporadic human and hare cases occurred in the period 2011 to 2014, a cluster of F. tularensis-infected hares was recognised in a region in the north of the Netherlands from February to May 2015. No human cases were identified, including after active case finding. Presence of F. tularensis was investigated in potential reservoirs and transmission routes, including common voles, arthropod vectors and surface waters. F. tularensis was not detected in common voles, mosquito larvae or adults, tabanids or ticks. However, the bacterium was detected in water and sediment samples collected in a limited geographical area where infected hares had also been found. These results demonstrate that water monitoring could provide valuable information regarding F. tularensis spread and persistence, and should be used in addition to disease surveillance in wildlife.

Winter Activity and Aboveground Hybridization Between the Two Biotypes of the West Nile Virus Vector Culex pipiens.

Culex (Cx.) pipiens mosquitoes are important vectors of West Nile virus (WNV). In Europe, the species Cx. pipiens consists of two biotypes, pipiens and molestus, which are morphologically identical, but differ in behavior. Typical behavior of the molestus biotype is the ability to remain active during winter, whereas the pipiens biotype enters diapause. The current paradigm is that the two biotypes occur sympatrically in southern Europe, but occur in isolated above- and belowground populations in northern Europe. In northern Europe, hybridization between biotypes is considered to be low because of the barrier that exists between typical habitats. Data on the occurrence of the biotypes and hybrids in northern Europe, however, are scarce, because identification to the level of biotype is often not performed. Our objective was to clarify the distribution of the Cx. pipiens biotypes and to determine hybridization rates in The Netherlands. Cx. pipiens mosquitoes were collected using three different approaches. First, traps were deployed randomly throughout The Netherlands during the summers of 2011 and 2012 (active surveillance). Second, using a web-based reporting platform and media campaign, Dutch citizens were asked to send dead mosquitoes to our laboratory during the winter and summer of 2014 (passive surveillance). Third, larvae and adults were collected during the summer of 2014 from aboveground locations in Amsterdam to identify molestus larval habitats. Real-time PCR was used for identification to the level of biotype. We found that biotype molestus and hybrids were feeding indoors during winter and summer in The Netherlands and that hybridization rates ranged between 6% and 15%. Larval habitats of biotype molestus were found to occur aboveground. The high percentage of hybridization has implications for assessing the risk of WNV transmission, because hybrids are thought to have ideal characteristics for bridging WNV between birds and humans.

Vector-borne disease intelligence: strategies to deal with disease burden and threats.

Owing to the complex nature of vector-borne diseases (VBDs), whereby monitoring of human case patients does not suffice, public health authorities experience challenges in surveillance and control of VBDs. Knowledge on the presence and distribution of vectors and the pathogens that they transmit is vital to the risk assessment process to permit effective early warning, surveillance, and control of VBDs. Upon accepting this reality, public health authorities face an ever-increasing range of possible surveillance targets and an associated prioritization process. Here, we propose a comprehensive approach that integrates three surveillance strategies: population-based surveillance, disease-based surveillance, and context-based surveillance for EU member states to tailor the best surveillance strategy for control of VBDs in their geographic region. By classifying the surveillance structure into five different contexts, we hope to provide guidance in optimizing surveillance efforts. Contextual surveillance strategies for VBDs entail combining organization and data collection approaches that result in disease intelligence rather than a preset static structure.