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Biology and control of vector-borne infections in Europe

Final Report Summary - EDENEXT (Biology and control of vector-borne infections in Europe)

Executive Summary:
British author L.P. Hartley famously wrote, “The past is a foreign country: they do things differently there.” And while a literary introduction to a report focused on science may seem incongruous, it offers a neat summary of the changes we have seen in European research on vector-borne diseases since the launch of the EDEN (Emerging diseases in a changing European environment) project in 2004 and the end of its follow-up project, EDENext (Biology and control of vector-borne infections in Europe), in 2014.
EDEN set out to identify and catalogue the European ecosystems and environmental conditions which can influence the spatial and temporal distribution and dynamics of human pathogenic agents. It developed generic methods, tools and skills such as predictive models early warning and monitoring tools that can be used by decision makers for risk assessment, decision support for intervention and Public Health policies. EDENext took up the baton with a focus on investigating the biological, ecological and epidemiological components of vector-borne disease introduction, emergence and spread, and the creation of new tools to control them. Vector groups focused on Culicoides midges, Phlebotomine sand flies, mosquitoes, rodents and ticks, while a fifth team examined rodents and insectivore-borne diseases. They were supported by modelling and data management teams, plus a team focused on ensuring the project had a real impact on improving Public Health.
These major projects, bringing together a total of more than 50 partners from across Europe, North and West Africa, have changed the way research on vector-borne diseases is conducted. The creation of international teams working on the same problems using the same methods, albeit in different locations, the routine sharing of data between institutes, the increased use of sophisticated models and the training of a new generation of scientists used to working with each other all mark a break with the past.

Project Context and Objectives:
Future environmental and socio-economic changes, mean that emerging vector-borne diseases will become an increasing challenge for human and veterinary public-health in Europe, and, indeed, in the World as a whole. In a retrospective analysis of emerging infectious diseases (EID), Jones et al (2008) have shown that while EID events were most often detected in Europe or North America, the major risks were in southern countries, where many demographic, environmental, social and economic factors favor the emergence of vector-borne diseases (VBD), and where there are limited health facilities to prevent, monitor or control their spread. Controlling VBD must therefore be considered as a global public good: European research into VBD should be seen as a means of protecting and improving public health and the welfare of its citizens, as well as being part of a coordinated international effort.
In the project “Biology and control of vector-borne infections in Europe” (EDENext) launched by the European Commission, we have implemented research to address two main goals: (i) to investigate the biological, ecological and epidemiological components of VBD introduction, emergence and spread, and - using the knowledge acquired, (ii) to propose new tools for controlling them.
Human behavior and risk perception are an important, if neglected, component of VBD emergence and spread. The impact of VBD on human and veterinary public health in Europe is beginning to emerge into the public awareness, as illustrated by recent events such as the chikungunya outbreak in Italy, or the spread of Crimean-Congo hemorrhagic fever in Europe. The unprecedented spread of the bluetongue epizootics in western and northern Europe between 2006 and 2009, with its huge consequences on animal health and the livestock industry, have raised similar concerns in farmers, veterinary services, other stakeholders, and as well as the general public. We also addressed these aspects of human and veterinary public health in our proposal.
All aspects of the project have benefited from, and amplify the strong scientific results, capacity building, and research networks established by the FP6 EDEN project on emerging VBD in a changing European environment.
Finally, the set of innovative research methods, tools and results delivered during the project was a substantial contribution toward a generic approach the monitoring and early warning of VBD, and reinforced the general regional framework needed for an effective integrated pest and disease management system.

EDEN and EDENext
The EDENext project built on the concepts, methods, tools and results of the FP6 EDEN project (Emerging diseases in a changing European environment). We adopted the same general approach of understanding and explaining the biological, ecological and epidemiological processes to develop a set of state-of-the-art methods and tools to improve prevention, surveillance and control of vector populations, and VBD. The EDEN project has been focusing on the effects of environmental changes on the emergence of VBD. Here, the goals were to explain and model the processes leading to the establishment and spread of vectors and/or VBD, and assess the possible control strategies to break the epidemiological cycles of vector-borne diseases.
The project structure followed that of EDEN, with a set of vertical disease-related activities linked by horizontal themes providing integrated technical input to all vertical groups, thereby minimizing duplication and ensuring a coordinated approach throughout the project. In EDENext, we have chosen to focus on vector groups rather than on disease groups: ticks, mosquitoes, sandflies, Culicoides and rodents.
Vector point of view
The “vectors” addressed in EDENext include not only arthropods but also rodents and insectivores which harbor a wide range of pathogens, some of them being infective to humans without the intervention of arthropod vectors (e.g. Hantaviruses, Bunyaviridae). We have therefore selected for study rodents and insectivores as well as the main arthropod vector groups of human and animal diseases in Europe: hard ticks (Acari, Ixodidae), mosquitoes (Diptera, Culicicae), sand flies (Diptera, Psychodidae), and biting midges (Diptera, Ceratopogonidae). Each constituted “vertical” group structuring EDENext research activities. With such a structure, the EDENext consortium was able to provide expertise and useful information regarding prevention of human or animal infection, control measures for vector populations, and implementation of vector surveillance networks, for any new emerging, VBD transmitted by vector / rodent / insectivore species belonging to these groups.
Model diseases
To focus the project objectives and produce specific results regarding VBD in Europe, we have selected a range of relevant diseases. The selection criteria used are (i) diseases with insufficient epidemiological knowledge or control measures to produce efficient intervention programs, and (ii) priority diseases for European public-health agencies. For the latter, we have used the results of the V-Borne expert consultation which was launched in 2008 by the European Centre for Disease Prevention and Control (ECDC, Stockholm): “Assessment of the magnitude and spread of vector-borne diseases in Europe”, ECDC public tender OJ/2207/04/13 – PROC/2007/003.
• For tick-borne diseases (TBD), we have chosen to focus on (i) Crimean-Congo hemorrhagic fever (CCHF) with an on-going epidemic in Turkey, and poorly documented spread in south-eastern Europe (Vorou 2009) and on (ii) newly emerging diseases, mainly borne by Ixodes ricinus, caused by different bacteria and parasites: Anaplasma spp., Babesia spp., Bartonella spp, and Rickettsia spp. (Vorou et al 2007). Little information is available about the occurrence, genetic variability and the possible future spread of these pathogens in the EU, and the range of possible reservoir hosts has yet to be established. In addition, the vector competence of I. ricinus for various strains of these pathogens is still unknown.
Consecutively, tick-borne encephalitis (TBE) and Lyme borreliosis (LB) were not the main research topics here because a lot has been done in EDEN, and scientific efforts are still devoted to them in other projects. However Borrelia spp. and TBE virus were included wherever further eco-epidemiological information was useful to fill knowledge gaps identified at the end of Project EDEN.
• Mosquito-borne diseases (MBD): though West Nile (WN) was addressed in EDEN, a lot of research is still needed for a better understanding of WN epidemiology and the risk of large-scale spread in Europe. The recent outbreaks in Italy (Rezza, 2009), and the emergence of WN virus lineage 2 in Hungary and Austria has raised public-health concerns (Erdélyi et al 2007, Weissenböck et al., 2009). In addition, the recent chikungunya outbreak in Italy has revealed the epidemic potential of Aedes albopictus (and other invasive mosquito species like Ae. japonicus) as a vector for Chikungunya virus, and other arboviruses like dengue (Chretien & Linthicum 2007, Sambri 2008).
• Sandfly-borne diseases (PBD): leishmaniasis and Phlebovirus infections. EDEN results have shown that sand fly distribution has changed in European countries (e.g. Dereure et al 2009; Martin-Sanchez et al 2009). Jointly, an increase of human and canine leishmaniasis infections, as well as a spread of human infections by Toscana Phlebovirus has been reported (Charrel et al, 2005). Moreover, following intensification of migration and travelling, environmental changes, and other aspects of globalization, there is an increased risk for the introduction and spread of infections by Leishmania species like L. tropica or L. donovani in Europe (Antoniou et al 2009) and in the newly emerging Leishmania hybrids (Ravel et al, 2006; Volf et al, 2007).
• Culicoides-borne diseases (CBD). A striking feature of veterinary epidemiology during the last decade was the accelerated occurrence of waves of CBD epizootics in the Mediterranean Basin and Europe, with huge economic consequences. Bluetongue (domestic and wild ruminants) has been the most important so far, but other CBD are lurking on Europe’s borders: epizootic hemorrhagic disease (ruminants), African horse sickness and horse encephalosis (equids)... For years, the northward spread of these diseases, from sub-Saharan Africa and the Middle East to Europe, has been associated with the extension of the distribution of their main Afro-asiatic vector, Culicoides imicola (Purse et al 2005). The emergence of several bluetongue virus (BTV) serotypes in Northern Europe as from 2006 has confirmed that endemic Culicoides species are able to transmit BTV efficiently, and that overwintering mechanisms are able to maintain the virus in animal hosts and/or infected vectors (Carpenter et al 2009).
• Rodent- and insectivore-borne diseases (rainbo): Hantavirus (Bunyaviridae) infections have a significant public health impact in Europe and globally. Apart from the high case fatality rate caused by some Hantaviruses, they cause considerable disease burden (hemorrhagic fever with renal syndrome: HFRS). Hantavirus infections are emerging infections: they are found in new areas, and the incidence has grown in several established endemic regions, (Heyman & Vaheri 2008). Recently, new groups of potential zoonotic agents borne by rodents and other small mammals have been found in Europe.
Rodents beyond rodent-borne diseases
Beyond their role in rainbo diseases, rodents and insectivores are also the hosts of many arthropod species. In EDENext, we assessed the joint diversity and abundance of small (rodents and insectivores) and larger (e.g. cervids or domestic ruminants) reservoir hosts, arthropod vectors, and pathogen agents, for a better understanding of their joint dynamics. We assessed the influence of major environmental factors such as land cover / land use, or masting in deciduous-forest regions (during mast years, vegetation produces a significant abundance of fruit). Assembling the different pieces of knowledge for the ecosystems of interest will provide a better basis for planning more efficient disease monitoring and early warning systems.
Emergence and spread of vectors and pathogens
Emergence and spread of vectors and pathogens are the sequence of events leading to epidemics of vector-borne diseases. Their understanding is crucial to develop science-based and data driven suite of preventative, control, or monitoring measures (intervention and control). However, the underlying mechanisms of each stage are different, and so were investigated by using different model diseases to investigate the different stages.
To understand and model the emergence and spread of vector-borne and rainbo diseases, the description and explanation of vector and host competence and capacity were the connecting thread of most research activities implemented in EDENext. The identification of biological mechanisms and ecological processes involved in competence and capacity are of particular importance to ensure a broad application of the results and a predictive power for the quantitative models that will be developed. For this purpose, the infection's natural cycle was thoroughly studied for each host-vector-pathogen system, including basic biology of vectors and diseases reservoirs. The spread of vectors, hosts (rodents and insectivores), and pathogens was studied, either using proxies like wind analysis and dispersion models (biting midges), or with field studies (population genetics of ticks and tick-borne pathogens, pathogens and ectoparasites borne by migrating birds, etc.). A coordinated research network was developed to replicate field studies with harmonized protocols in strategic places (disease-emergence or vector-invasion frontline, contrasted environmental or control conditions...). Laboratory experiments to study the life traits of vectors and pathogens, and vector / host competence were also coordinated at the project scale.
A common goal for all the disease systems under study was to develop predictive, quantitative models of vector-population dynamics, or disease transmission and spread. For this purpose, we benefited from the datasets, experience, and capacity gained after the EDEN project. An important step forward was to model vector and disease spread in a context of changing environment in space and time. Mathematical tools are now available and have already been applied for some VBD we want to address (Hartemink et al 2009: EDEN result). Moreover, methods and tools have been developed in EDEN to characterize climatic and environmental changes, including landscapes, at various resolution scales (e.g. Linard et al 2007, Scharlemann et al 2008). Beyond epidemiological studies to assess the role of these environmental features in disease incidence, progress has been made to integrate the environmental, landscape and (process-based) mathematical approaches to explain disease spread. This integration was developed further within EDENext.
Vector control intervention
Many methods are available to control vector populations and break epidemiological cycles. However, the massive use of insecticides is less and less acceptable from environmental and societal viewpoints. The effectiveness of other control methods depends on the biology and ecology of the target vectors, as well as the nature of the pathogen agents as they affect, for example, the availability of vaccine or efficient prophylactic / curative treatments. The wide distributions of wild or domestic reservoir hosts further complicate the identification and implementation of effective control methods. Most vector- or VBD-control programs therefore follow an integrated pest-control strategy, in which several concepts and methods are used together.
In EDENext, research was implemented to develop new vector-control research tools which allowed missing information on the efficacy of specific control methods to be assessed using laboratory or field experiments. Simulation models were used to assess control strategies (pest management or disease control, including vaccination, or host medical treatment) according to various scenarios (emerging disease, climate or environmental change, etc.) and control strategies which were defined and selected with animal / human health stakeholders. Depending on the specific cases, field or laboratory experiments were carried out to assess the efficacy of one or more components of the integrated control strategy. Then, we used the predictive models, to assess the efficacy, or other indicators such as the cost/benefit ratio, of these control strategies.
Public health
A thorough understanding of biological, ecological and epidemiological processes and the availability of predictive quantitative models provide important tools to implement efficient preventive, surveillance and control programs. However, we also need relevant and accurate data on risk perception in the public health agencies and in the exposed human population categories, as well as a clear understanding of the importance of these human population segments. Moreover, the human or veterinary public-health messages to be delivered must be adequately formatted in their content and style, to reach the targeted populations most at risk. These activities were implemented in the Public-health work package, in close collaboration with human / animal health stakeholders, as well as national and international public-health agencies. We focused these activities on two of the model diseases proposed for study as they are particularly important and could provide sufficient data to feed Public Health activities: CCHF in south-eastern Europe, and HFRS in Fennoscandia, and Belgium, north of France, Luxembourg, and Germany.

References
Antoniou et al (2009). Increased incidence of zoonotic visceral leishmaniasis on Crete, Greece. Emerg Infect Dis, 15: 1-3.
Carpenter et al (2009). Culicoides and the emergence of bluetongue virus in northern Europe. Trends Microbiol, 17: 172-178.
Charrel et al (2005). Emergence of Toscana virus in Europe. Emerg Infect Dis, 11: 1657-1663.
Chretien et al (2007). Chikungunya in Europe: what's next? Lancet, 370: 1805-1806.
Dereure et al (2009). The potential effects of global warming on changes in canine leishmaniasis in a focus outside the classical area of the disease in southern France. Vector Borne Zoonotic Dis, 9: 687-694.
Erdélyi et al (2007). Clinical and pathologic features of lineage 2 West Nile virus infections in birds of prey in Hungary. Vector Borne Zoonotic Dis, 7: 181-188.
Hartemink et al (2009). Mapping the basic reproduction number (R0) for vector-borne diseases: A case study on bluetongue virus. Epidemics, 1: 153-161.
Heyman P & Vaheri A (2008). Situation of Hantavirus infections and haemorrhagic fever with renal syndrome in European countries as of December 2006. Eurosurveillance, 13: 1-7.
Jones et al (2008). Global trends in emerging infectious diseases. Nature, 451: 990-993.
Linard et al (2007). Determinants of the geographic distribution of Puumala virus and Lyme borreliosis infections in Belgium. Int J Health Geogr, 6: 15.
Martin-Sánchez J et al (2009). Canine leishmaniasis in southeastern Spain. Emerg Infect Dis, 15: 795-798.
Purse et al (2005). Climate change and the recent emergence of bluetongue in Europe. Nature Rev Microbiol, 3: 171-181.
Ravel et al (2006). First report of genetic hybrids between two very divergent Leishmania species: Leishmania infantum and Leishmania major. Int J Parasitol, 36: 1383-1388.
Rezza, G. 2009. Chikungunya and West Nile virus outbreaks: what is happening in north-eastern Italy? Eur J Public Health, 19: 236-237.
Sambri et al (2008). The 2007 epidemic outbreak of Chikungunya virus infection in the Romagna region of Italy: a new perspective for the possible diffusion of tropical diseases in temperate areas? New Microbiol, 31: 303-304.
Scharlemann al (2008). Global data for ecology and epidemiology: a novel algorithm for temporal Fourier processing MODIS Data. PLoS ONE, 3: e1408.
Toledo et al (2009). Tick-borne zoonotic bacteria in ticks collected from Central Spain. Am J Trop Med Hyg, 81: 67-74.
Volf P et al (2007). Increased transmission potential of Leishmania major/Leishmania infantum hybrids. Int J Parasitol, 37: 589-593.
Vorou R (2009). Crimean-Congo hemorrhagic fever in south-eastern Europe. Int J Inf Dis, 13: 659-662.
Vorou et al (2007). Emerging zoonoses and vector-borne infections affecting humans in Europe. Epidemiol. Inf., 135: 1231-1247.
Weissenböck et al (2009). Zoonotic mosquito-borne flaviviruses: worldwide presence of agents with proven pathogenicity and potential candidates of future emerging diseases. Vet. Microbiol, 140: 271-280.

Project Results:
EDENext’s Public Health Telegrams (PUBLICise Health) were established to inform interested individuals and institutions about research results on vector-borne diseases with direct or indirect impact on Public Health issues. Edited and produced by EDENext’s Public Health team, each issue concentrates on the work of one of the project’s five specialist groups. Here we present versions of each of these telegrams which are otherwise available as stand-alone documents at http://www.edenext.eu/.
1. Tick-borne diseases
Tick-borne viruses - an overview
Ticks are recognized as vectors for several pathogens of Public Health relevance. Hubálek and Rudolf (2012) offer an extensive overview of the currently known European tick-borne viruses with a special focus on their taxonomy, host and tick vector range, pathogenicity for vertebrates including humans, and their significance for Public Health. Besides the known highly pathogenic Crimean-Congo haemorrhagic fever virus (CCHFV) and tick-borne encephalitis virus (TBEV), the authors also report other tick-borne virus pathogens such as looping-ill and African swine fever virus.
Hubálek Z and Rudolf I (2012). Tick-borne viruses in Europe. Parasitol. Res. 2012, 111: 9-36. Doi: 10.1007/s00436-012-2910-1.

Effect of deer density on tick infestation of rodents and TBE hazard, Part I and II
The occurrence and population dynamics of wild ungulates, in particular deer, represent some of the most important risk factors for the risk assessment of diseases transmitted by the pan-European tick species Ixodes ricinus. The spatial and temporal variation in the abundance of deer does not only affect the number of ticks in the environment, but also the rate of ticks infected with various pathogens. In the case of tick borne encephalitis, the probability of TBEV circulation is enhanced above a certain threshold deer density as shown in a joint study carried out in Italy and Slovakia and published within two EDENext publications (Cagnacci et al. 2012; Bolzoni et al. 2012). The effect of variable deer population density also has consequences on the pattern of tick infestation of the yellow-necked mouse (Apodemus flavicollis), one of the most important small mammal hosts supporting high TBEV circulation through co-feeding ticks in the natural habitat. By comparing sites in Italy and Slovakia with moderate and low deer population density to the presence and/or absence of TBEV occurrence in these areas, researchers have obtained quantitative estimates of the variation of the number of co-feeding ticks on rodents (Cagnacci et al. 2012) and included these estimates within a mathematical model (Bolzoni et al. 2012) that allows for the computation of the basic reproductive number of the infection and various host densities.
Cagnacci et al (2012). Effect of deer density on tick infestation of rodents and the hazard of tick-borne encephalitis. Part I: empirical assessment. International Journal for Parasitology 42(2012) 365-372.
Bolzoni et al (2012). Effect of deer density on tick infestation of rodents and the hazard of tick-borne encephalitis. Part II: population and infection models. International Journal for Parasitology 42(2012) 373-381

The impact of Crimean-Congo hemorrhagic fever (CCHF) on Public Health
CCHF is a hemorrhagic fever in humans, caused by infection with the CCHF virus (CCHFV). It is the only biosafety level 4 agent currently present in Europe. Every year, more than 1,000 human clinical cases and up to 50 fatalities are reported from more than 30 countries in Asia, the Middle East, south-eastern Europe and Africa. CCHF can be considered a major emerging disease threat to the European Union due to its high infectiousness, potential of human-to-human transmission and present lack of suitable prophylaxis and therapeutic interventions. Additionally, the ongoing range expansion of its tick vectors within Europe underlines the imminent threat this disease poses to Public Health in Europe. The EDENext study by Mertens et al. (2013) summarizes the current state of knowledge about CCHF and provides guidance for Public Health analysts and managers. Special emphasis is given to the importance of vector and virus distribution, the impact of climate and the different modes of virus transmission, as well as to the current limitations in diagnostics, treatment and vaccination. Based on this knowledge, recommendations for protection measures for individuals and the population at large, including case management options, and Public Health management and communication strategies are proposed. CCHFV is primarily transmitted by ticks of the genus Hyalomma, which can be found in many regions of southern and south-eastern Europe (see 4). Up to 20% of these ticks have been found positive for CCHFV in endemic areas of Turkey. Mammals such as hedgehogs, hares, sheep and cattle are infested by these ticks and thereby may become infected and amplify the virus, yet without suffering from any clinical disease. The virus circulates in a tick-vertebrate-tick cycle, but can also be transmitted horizontally and vertically within the tick population. Most human infections occur by tick bites and by crushing infected ticks, but infections are also possible through contact with blood and other body fluids of viraemic animals. Moreover, there are several reports about person-to-person transmissions following unprotected handling of infected blood samples from patients, by accidental needle stick injuries, and by accidents during surgery. Currently, there is no CCHFV vaccine available and the therapy is restricted to symptomatic treatment.
Mertens et al (2013). The impact of Crimean-Congo haemorrhagic fever virus on public health. Antiviral Res. 2013 May; 98(2):248-60. Doi: 10.1016/j.antiviral.2013.02.007. Epub 2013 Feb 28.

Discovery of CCHF tick vector in Hungary
CCHFV is transmitted primarily by tick species of the genus Hyalomma. These ticks are endemic to regions with (sub)tropical or Mediterranean climates, such as Albania, Turkey and Uzbekistan. Migratory birds may occasionally carry Hyalomma larvae and nymphs from their original habitat to European countries north of the Mediterranean basin. However, it was previously assumed that the cool climate in these areas of Europe would prevent the ticks from completing their life cycle successfully and therefore both prohibit the development of adult ticks possible of transmitting CCHF and the establishment of Hyalomma populations. In the EDENext study by Hornok and Horváth (2012) ticks were collected from cattle and wild ruminants in a region of southern Hungary, where, according to prediction models, Hyalomma ticks are most likely to occur and establish themselves in the future. Adult Hyalomma marginatum rufipes males were found on two occasions. This tick species was therefore detected for the first time in Hungary, marking the (so far) northern-most occurrence of this CCHF vector in Central Europe. A human case of CCHF as well as CCHF virus seropositive animals have already been reported in Hungary. The northern spread of Hyalomma ticks and thus potentially of the CCHF virus is of uttermost importance to European Public Health. To monitor and possibly prevent the further expansion of this zoonotic disease beyond its original range, further investigations of the current distribution of CCHF tick vectors in Europe have to be undertaken.
Hornok S and Horváth G (2012). First report of adult Hyalomma marginatum rufipes (vector of Crimean-Congo haemorrhagic fever virus) on cattle under a continental climate in Hungary. Parasit. Vectors 2012; 5:170. Doi: 10.1186/1756-3305-5-170
High TBEV infection rate in Austrian horses
To evaluate the status of West Nile virus (WNV) within the domestic horse population in Austria, the sera of 257 horses of the same breed, distributed among three federal states, were screened via a commercial ELISA for flaviviruses antibodies. ELISA-positive sera were further tested by virus-specific neutralisation assays for the three flaviviruses circulating in Austria: WNV, Usutu virus (USUV) and tick-borne encephalitis virus (TBEV). While no specific WNV antibodies could be determined, a comparatively high seropositivity rate of 40.4% for TBEV-specific antibodies was detected, with a significantly higher antibody prevalence in younger horses and in stallions. This result is remarkable as comparable studies in Austria and Germany show significantly lower TBEV prevalence in horses and other domestic animals. The age and gender aspect is equally surprising, and is contradictory to results from comparable studies. TBEV is a virus associated with tick-borne encephalitis, a viral infectious disease causing inflammations of the central nervous system in various vertebrate species, including humans, but very little is known about the disease in horses. Overall, the results of the study by Rushton et al. (2013) indicate the necessity of further investigations of TBE prevalence in equines and other domestic animals in Central Europe for the sake of evaluating the Public Health risk posed by TBEV.
Rushton J et al (2013). Tick-borne Encephalitis Virus in Horses, Austria, 2011. Emerg Infect Dis. 2013 April; 19(4): 635–637. Doi: 10.3201/eid1904.121450

Tick-borne diseases in Romania
The geopolitical position of Romania as a bridge connecting southern and central Europe has to be respected in regard to its implication for the Public Health system of the European Union, of which Romania has been a member since 2007. In this country, cases of Lyme disease and tick-borne encephalitis have been identified. However, little is known about the Public Health impact of these diseases, and none of the other tick-borne pathogens present in Europe have been reported as causes of infection in Romania. Therefore, the EDENext study by Paduraru et al. (2012) in which various zoonotic tick-borne pathogens (including Rickettsia monacensis, R. helvetica, Anaplasma phagocytophilum, Ehrlichia muris, Francisella tularensis, and Babesia sp. EU1) are described for the first time in Romania can be considered an important step towards future evaluations of the prevalence of these diseases in the country and the implications of these results for national and international Public Health surveillance.
Paduraru et al (2012). Zoonotic transmission of pathogens by Ixodes ricinus ticks, Romania. Emerg Infect Dis. 18(12):2089-90. Doi: 10.3201/eid1812.120711.

Driving forces for tick range expansion
The distribution of the tick Ixodes ricinus, which serves as a vector of a large variety of pathogens of medical and veterinary importance (among others, babesiosis, Lyme borreliosis and tularaemia), has increased significantly all over Europe over the past decades. I. ricinus specimens are now detected in more northern areas and habitats at higher altitudes than their prior range. Medlock et al. (2013) assume ongoing climatic change to be a driving factor of the tick range expansion among several other anthropogenic factors. Milder climatic conditions may allow one of the tick’s major host species, the European roe deer (Capreolus capreolus), whose abundance has increased in several regions as a consequence of current wildlife management, to spread to and inhabit previously inhospitable areas at higher altitude, thereby serving as a means of transport for the ticks. Furthermore, increasing temperatures reduce the length of snow cover, impacting positively the survival and spread of the ticks. Among other driving factors, land use changes due to agriculture and forestry management in various European countries over the past decades, and thereby suitable habitat expansion for I. ricinus, including urbanized areas, are discussed in regard to their effects on the tick range expansion. In conclusion, the authors note that multiple factors drive the range expansion of I. ricinus in Europe and that enhanced tick surveillance with harmonised approaches for international data comparison would allow better follow-up of tick population trends at the European Union level, thereby improving the prediction of health risks related to tick-borne diseases.

Medlock et al (2013). Driving forces for changes in geographical distribution of Ixodes ricinus ticks in Europe. Parasit Vectors. 2013; 6: 1. Doi: 10.1186/1756-3305-6-1

Woodland fragmentation increases Lyme disease risk
Over the past decades, the increase in the number of reported cases of Lyme disease throughout Europe has led to growing Public Health concern regarding the pathogenicity of this disease. Lyme disease, is the most frequent vector-borne disease of humans in the Northern Hemisphere. The causative agent is the spirochaete Borrelia burgdorferi sl, which is transmitted in Europe mainly by the tick species Ixodes ricinus. Lyme disease is strongly connected to various environmental factors, particularly in forest and agricultural landscapes, where the activity ranges of ticks and their hosts (including humans) overlap. However, these factors are not homogeneously distributed among the different landscapes. An understanding of the underlying processes in the transmission system is crucially needed to explain the spatial distribution risk of Lyme disease.
Li et al. (2012) demonstrate a cellular automata model for Lyme disease. The study includes a heterogeneous landscape model with three interactive components (hosts, tick population and a disease transmission function). Scenarios of various landscape configurations are simulated and compared. Based on the simulation results, the authors conclude that Lyme disease risk correlates to the density, shape and aggregation level of woodland areas, among others due to the latter’s impact on the local host population and thus spread of the disease-carrying ticks. This correlation is useful for understanding and predicting the occurrence of tick-borne diseases in dynamic forest landscapes.

Li S et al (2012). Consequences of landscape fragmentation on Lyme disease risk: a cellular automata approach. PLoS One. 2012; 7(6):e39612. Doi: 10.1371/journal.pone.0039612. Epub 2012 Jun 25.

Detection of Borrelia miyamotoi in vectors and reservoir of Lyme disease spirochete in France.
In France, as elsewhere in Europe, the most prevalent TBD in humans is Lyme borreliosis, caused by different bacterial species belonging to the Borrelia burgdorferi sensu lato complex and transmitted by the widespread tick species, Ixodes ricinus. However, the diagnosis of Lyme disease is not always confirmed and unexplained syndromes occurring after tick bites have become an important issue. Recently, Borrelia miyamotoi belonging to the relapsing fever group and transmitted by the same Ixodes species has been involved in human disease in Russia, the USA and the Netherlands. In this study, researchers identified B. miyamotoi in ticks and bank voles in France and demonstrated that its genotype is identical to those already described in ticks from Western Europe, as well as to the one identified from a sick person in The Netherlands (Cosson et al. 2014). These results suggest that even though no human cases have been reported in France, surveillance has to be improved. Moreover, researchers show that ticks could simultaneously carry B. miyamotoi and Lyme disease spirochetes, increasing the problem of co-infection in humans.

Cosson et al (2014). Genetic characterization of the human relapsing fever spirochete Borrelia miyamotoi in vectors and animal reservoirs of Lyme disease spirochetes in France. Parasites & Vectors 2014, 7:233. Doi:10.1186/1756-3305-7-233.

Ixodes ricinus and its transmitted pathogens in urban and peri-urban areas in Europe: new hazards and relevance for Public Health
Tick-borne diseases represent major public and animal health issues worldwide. Ixodes ricinus, primarily associated with deciduous and mixed forests, is the principal vector of causative agents of viral, bacterial, and protozoan zoonotic diseases in Europe. Recently, abundant tick populations have been observed in European urban green areas, which are of Public Health relevance due to the exposure of humans and domesticated animals to potentially infected ticks. In urban habitats, small and medium-sized mammals, birds, companion animals (dogs and cats) and larger mammals (roe deer and wild boar) play a role in the maintenance of tick populations and as reservoirs of tick-borne pathogens. The presence of ticks infected with tick-borne encephalitis virus and high prevalence of ticks infected with Borrelia burgdorferi s.l. causing Lyme borreliosis, have been reported from urbanized areas in Europe. Emerging pathogens, including bacteria of the order Rickettsiales (Anaplasma phagocytophilum, Candidatus Neoehrlichia mikurensis, Rickettsia helvetica and R. monacensis), Borrelia miyamotoi, and protozoans (Babesia divergens, B. venatorum, and B. microti) have also been detected in urban tick populations. Understanding the ecology of ticks and their associations with hosts in a European urbanized environment is crucial to quantify the parameters necessary for risk pre-assessment and identification of Public Health strategies for control and prevention of tick-borne diseases (Rizzoli et al, 2014).

Rizzoli et al. (2014). Ixodes ricinus and Its Transmitted Pathogens in Urban and Peri-Urban Areas in Europe: New Hazards and Relevance for Public Health. Frontiers in Public Health. 2014;2:251. Doi:10.3389/fpubh.2014.00251.

Birds as potential reservoirs of tick-borne pathogens: first evidence of bacteraemia with Rickettsia helvetica.
Birds have long been known as carriers of ticks, but data from the literature are lacking on their role as a reservoir in the epidemiology of certain tick-borne pathogens. Therefore, the aim of a study carried out by Hornok et al. (2014) was to evaluate the presence of three emerging, zoonotic pathogens in blood samples and ticks of birds and to assess the impact of feeding location preference and migration distance of bird species on their tick infestation. Blood samples and ticks of birds were analysed with TaqMan real-time PCRs and conventional PCR followed by sequencing. During the spring and autumn bird migrations, 128 blood samples and 140 ticks (Ixodes ricinus, Haemaphysalis concinna and a Hyalomma specimen) were collected from birds belonging to 16 species. The prevalence of tick infestation and the presence of tick species were related to the feeding and migration habits of avian hosts. Birds were shown to be bacteraemic with Rickettsia helvetica and Anaplasma phagocytophilum, but not with Candidatus Neoehrlichia mikurensis. The prevalence of rickettsiae was high (51.4%) in ticks, suggesting that some of them may have acquired their infection from their avian host. Based on the present results birds are potential reservoirs of both I. Ricinus-transmitted zoonotic pathogens, R. helvetica and A. phagocytophilum, but their epidemiological role appears to be less important concerning the latter.

Hornok et al (2014). Birds as potential reservoirs of tick-borne pathogens: first evidence of bacteraemia with Rickettsia Helvetica. Parasites & Vectors 2014, 7:128. Doi:10.1186/1756-3305-7-128.

2. Rodent-borne diseases
Detection of novel/emerging pathogens in new areas
Candidatus Neoehrlichia mikurensis
The occurrence of the previously described novel human pathogen Candidatus Neoehrlichia mikurensis (CNM) was confirmed for the first time in trapped wild rodents in France, during the course of an EDENext study (Vayssier-Taussat et al., 2012). CNM is an intracellular bacterium that belongs to the Anaplasmataceae family. DNA sequences had been found before in ticks and rodents originating from other European countries and from Asia. While the first human infection was reported from Sweden in 2010, followed by five cases described for Germany, Switzerland and the Czech Republic and a canine infection reported from Germany, in France no infections in either humans or animals have yet been reported. Remarkably, the genotype of CNM isolated from the rodents, trapped in proximity to residential areas in the French Ardennes, was identical to those having been found earlier to cause disease in humans and animals. CNM infections might have hitherto been unrecognized or have been mistaken for other diseases displaying mild or unspecific symptoms (body aches, chills, sweats, fatigue and high fever) such as hantavirus-induced nephropathia epidemica, which is endemic in the study region (French Ardennes).
Because of the absence of serological tests and the gap in knowledge of medical authorities regarding this new pathogen, the relevance of CNM for Public Health is difficult to estimate. In order to provide fundamental data for a reliable assessment and evaluation of this Public Health risk, adequate diagnostic tools need to be developed and surveillance measures implemented.

Seewis virus and other soricomorph-borne hantaviruses
Until recently, only rodent-borne hantavirus species were known in Central Europe. The development of advanced molecular techniques led to the identification of numerous new soricomorph (insectivore) -borne hantavirus species in Asia, Africa, North America and Europe. In 2007, a Soricinae-borne hantavirus was detected in a common shrew (Sorex araneus) from Switzerland, the Seewis virus (SWSV). Further SWSV strains were later found in Hungary, Finland and Russia. Within the framework of EDENext, the presence, distribution and genetic variability of SWSV was investigated in various Sorex species captured at different trapping sites in Germany, the Czech Republic and Slovakia between 2004 and 2011. The results confirmed the presence of SWSV in Germany, as well as in the Czech Republic and Slovakia (Schlegel et al., 2012). Furthermore, the first comprehensive sequence analyses of SWSV strains from these countries were achieved indicating a broad geographical distribution and high genetic divergence of SWSV in Central Europe. A more detailed analysis of the molecular evolution of SWSV may shed more light on the complex evolution and host adaptation processes of hantaviruses in general. In light of these results, particularly in respect to the widespread distribution of SWSV in Europe, future investigations should aim at assessing the zoonotic potential and pathogenicity of SWSV to evaluate their potential threat for Public Health.
In Finland, Seewis virus was reported in 2009 from archival common shrew (Sorex araneus) samples collected in 1982. To elucidate the diversity of hantaviruses in soricomorphs in Finland, 180 individuals were screened by Ling et al. (2014), representing seven species captured from 2001 to 2012: hantavirus RNA was screened using RT-PCR, and hantaviral antigen using immunoblotting with polyclonal antibodies raised against truncated SWSV nucleocapsid protein. The overall hantavirus RNA prevalence was 14% (26/180), and antigen could be demonstrated in 9 of 20 SWSV RT-PCR positive common shrews. Genetic analyses revealed that four soricomorph-borne hantaviruses circulate in Finland, including Boginia virus (BOGV) in water shrew (Neomys fodiens) and Asikkala virus (ASIV) in pygmy shrew (Sorex minutus). Interestingly, on two study sites, common shrews harbored strains of two very different hantaviruses: Seewis virus and a new distinct, genetically distant (identity 57% at amino acid level) virus (Altai-like virus). This is the first evidence ever of coexistence of two clearly distinct hantavirus species circulating simultaneously in one host species population. The findings suggest an ancient host-switching event from a yet unknown host to S. araneus. In addition, phylogenetic analyses of partial S and M segment sequences showed that SWSV in Finland represents a unique genotype in Europe.

Ling et al (2014). Hantaviruses in Finnish soricomorphs: evidence for two distinct hantaviruses carried by Sorex araneus suggesting ancient host-switch. Infection, Genetics and Evolution 27:51-61.

Seoul hantavirus in rats
Until recently, very little was known of the globally distributed Seoul hantavirus in brown rats (Rattus norvegicus) in Europe. Following the observation of a human case that originated from pet rat shop in the UK, a screening in Sweden also revealed Seoul hantavirus in pet rats. The virus has also been found in Belgium, the Netherlands (Goeijenbier et al. 2015) and France. Seoul hantavirus causes a more severe form of hemorrhagic fever (HFRS) with renal syndrome than Puumala hantavirus, the agent for most common form of HFRS in Europe. Potential for HFRS due to Seoul virus is not well understood in Europe, and diagnostic tools are not widely optimized for this hantavirus. Recognizing the emerging character of Seoul hantavirus in Europe could have significance for Public Health in large areas in Europe. Special attention should be paid to safety with pet rats.

Cowpox virus in voles in Hungary and Finland
As a result of discontinuing vaccination against smallpox after the late 1970s, different orthopoxviruses (OPVs), such as cowpox virus (CPXV), have become a re-emerging healthcare threat among zoonotic pathogens. Two recent EDENext articles have analysed OPVs in Finland and Hungary.
In northern Europe, rodent populations display cyclic density fluctuations that can be correlated with the human incidence of zoonotic diseases they spread. During density peaks, field voles (Microtus agrestis) become one of the most abundant rodent species in northern Europe, yet little is known of the viruses they host. Forbes et al. (2014) screened 709 field voles in Finland, trapped from 14 sites over 3 years, for antibodies against four rodent-borne, potentially zoonotic viruses or virus groups—hantaviruses, lymphocytic choriomeningitis virus (LCMV), Ljungan virus (LV), and orthopoxviruses (OPV). Antibodies against all four viruses were detected. However, seroprevalence of hantaviruses, LV, and LCMV was low in field voles. On the other hand, OPV antibodies (most likely cowpox, CPWV) were more common but restricted geographically to southeastern Finland. This is interesting because in the sympatric bank voles cowpox virus occurs more widely. Within these positive field vole sites, antibody prevalence showed delayed density dependence in spring and direct density dependence in fall. Higher seroprevalence was found in spring than fall. These results substantially increase knowledge of CPXV infection dynamics.
In Hungary, data on OPV prevalence among its rodent host species have been absent. Rodents belonging to four species, i.e. striped field mouse (Apodemus agrarius), yellow-necked mouse (A. flavicollis), wood mouse (A. sylvaticus) and bank vole (Myodes glareolus), were live-trapped by Oldal et al. (2015) at 13 sampling plots in the Mecsek Mountains, Hungary, from March to September in 2011 and 2012. Among the 1587 tested rodents, 286 (18.0%) harbored OPV-specific antibodies. Seroprevalence was the highest for the bank vole (71.4%) and the striped field mouse (66.7%). Due to a masting event in the autumn of 2011 across Central Europe, the abundance of bank voles increased drastically in the 2012 season, raising the overall OPV seroprevalence. These are the first data on OPV occurrence and seroprevalence in rodents in Hungary. The common circulation of OPV in rodents in densely populated areas warrants further studies to elucidate the zoonotic potential of OPVs and risk for Public Health.

Forbes, K.M. et al. 2014. Serological Survey of Rodent-Borne Viruses in Finnish Field Voles. - Vector-borne Zoon Dis. 14:278- 283. DOI: 10.1089/vbz.2013.1526
Oldal, M. et al. 2015. Serologic Survey of Orthopoxvirus Infection Among Rodents in Hungary. – Vector-borne Zoon. Dis 15:317-322. DOI: 10.1089/vbz.2014.1731

Development of novel diagnostic tools for recently detected Soricomorpha-borne hantaviruses
Despite numerous recent reports on shrew- and mole-associated hantaviruses from America, Europe, Asia and Africa, since the identification of the first non-rodent borne hantavirus - Thottapalayam virus (TPMV) in 1971 - little is so far known about the antigenic and immunological characteristics of TPMV and the newly isolated Soricomorpha-borne hantaviruses. The differences in antigenicity compared to that of rodent-borne hantaviruses, including the prototype Hantaan virus (HTNV), has not yet been studied, and the understanding of the pathogenicity and the course of infection, as well as the humoral immune response in shrews is poor. Until now, limits in diagnosis, particularly due to the lack of commercially available species-specific secondary antibodies, have hampered progress in this matter.
Within the EDENext project, TPMV-specific monoclonal antibodies and anti-shrew immunoglobulin G (IgG) antibodies were generated (Schlegel et al., 2012). Using these antibodies, serological tools for the detection of hantavirus-specific antibodies and hantavirus antigens in shrews were subsequently developed. The novel monoclonal antibodies have proved to represent useful tools for the detection of TPMV, and antigenically related hantaviruses, in cell culture whereby the shrew anti-TPMV-antisera could serve as positive control. The generation of mouse anti-shrew-IgG allowed the detection of hantavirus-specific antibodies in transudates and sera derived from immunized and experimentally infected shrews. Hence, the established serological tools are assumed to be helpful in discovering novel insectivore-associated hantaviruses and in characterizing the humoral immune response and antigen expression in hantavirus-infected insectivores. Moreover, they may be used for diagnosis and surveillance purposes in order to estimate the potential threat which TPMV and antigenically related hantaviruses pose to Public Health.

Human antibodies to shrew-borne hantaviruses?
Puumala virus carried by the bank vole (Myodes glareolus) co-circulates with Seewis virus (SWSV, carried by the common shrew (Sorex araneus) in Finland. While PUUV causes annually 1 000 - 3 000 nephropathia epidemica cases, the pathogenicity of SWSV to man is unknown. To study the prevalence of Seewis antibodies among hantavirus fever-like patients sera, Ling et al. (2015) used recombinant SWSV nucleocapsid (N) protein as the antigen in enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay (IFA), and immunoblotting (IB). While characterizing the recombinant SWSV N protein, they observed that a polyclonal rabbit antisera against PUUV N cross-reacts with SWSV N, and vice versa. They initially screened in SWSV N IgG ELISA 486 (450 PUUV-seronegative and 36 PUUV seropositive) samples sent to Helsinki University Central Hospital Laboratory for PUUV serodiagnosis during 2002 and 2007. In total 4.2% (19/450) of PUUV-seronegative were reactive in SWSV N IgG ELISA, and none of 58 PUUV seronegative samples were reactive in SWSV N IgM ELISA. None of these reactions could be confirmed by IFA or IB. Furthermore, out of 36 PUUV-seropositive serum samples, three showed reactivity in SWSV N IgG and 10 in SWSV N IgM ELISA. One of the PUUV-seropositive samples was IgG positive in IFA and four in IB. Finally, we used a competitive IgG ELISA to confirm that the observed reactivity toward SWSV N protein is actually a cross-reaction and not a true SWSV response. In conclusion, no evidence of SWSV infection was found among a panel of 486 patient serum samples. However, we could demonstrate a cross-reaction anti-PUUV serum with shrew-borne hantavirus N protein.

Ling et al (2015). Serological survey on patients with febrile illness: Puumala virus antiserum cross-reacts with Seewis virus N protein. J. Gen. Virol., 96: 1664-1675.

Analysing the effects of environmental change on rodent populations
Anthropogenic disturbance of ecosystems may, inter alia, result in a changed distribution and abundance of vector species harbouring zoonotic pathogens. The bank vole, Myodes glareolus, is the rodent reservoir of Puumala virus (PUUV), the pathogen that causes nephropathia epidemica (NE), a mild form of haemorrhagic fever with renal syndrome (HFRS), the most common disease caused by hantavirus infections in Europe. Its incidence is highest in the boreal zone in Northern and North-eastern Europe. Here, human NE epidemic peaks correlate with high bank vole abundance, reflecting the seasonal and cyclic multiannual dynamics of the host. Apart from factors causing fluctuations of rodent populations as such, an important reason for this epidemiological pattern regarding human NE cases has been hypothesized to result from differences in landscape structures between temperate and boreal Europe. In boreal Finland, forests, the preferred habitat of bank voles, cover more than 77% of land, whereas in temperate Belgium it is only 23%. In consequence, the NE incidence rate is more than ten times higher in Finland than in Belgium.
An EDENext study investigated the connection between forest age and the abundance of PUUV-infected bank voles, and what influence the intensive management of boreal forests has on the dynamics of PUUV in bank voles (Voutilainen et al., 2012). Furthermore, the correlation between the age of forests, bank vole abundance and the abundance of non-host species with the likelihood of PUUV-infections in bank voles was analysed. Results suggest that even though habitat alterations through intensive forest management can potentially reduce the hantavirus load in the environment, PUUV is commonly found in both old forests and young plantations. In conclusion, despite intensive management measures in forests, the majority of the Fennoscandian boreal forest landscape constitutes a source for hantavirus infection of humans.

How long wild rodents can excrete hantaviruses?
The knowledge of viral shedding patterns and viremia in the reservoir host species is a key factor in assessing the human risk of zoonotic viruses. The shedding of hantaviruses (Bunyaviridae) by their host rodents has widely been studied experimentally, but rarely in natural settings. Voutilainen et al. (2015) studied the dynamics of Puumala hantavirus (PUUV) shedding and viremia in naturally infected wild bank voles (Myodes glareolus) in the field. In a monthly capture–mark–recapture study they collected shedding data from more than a hundred voles. They analysed those 18 bank voles with longest follow-up history for the presence and relative quantity of PUUV RNA in the excreta and blood from 2 months before up to 8 months after seroconversion. The proportion of animals shedding PUUV RNA in saliva, urine and faeces peaked during the first month after seroconversion, but continued throughout the study period with only a slight decline. The quantity of shed PUUV in reverse transcription quantitative PCR (RT-qPCR) positive excreta was constant over time. In blood, PUUV RNA was present for up to 7 months but both the probability of viremia and the virus load declined with time. The findings contradict the current view of a decline in virus shedding after the acute phase and a short viremic period in hantavirus infection – an assumption widely adopted into current epidemiological models. The authors suggest the life-long shedding as a means of hantaviruses to survive over host population bottlenecks, and to disperse in fragmented habitats where local host and/or virus populations face temporary extinctions. The results indicate that the kinetics of pathogens in wild hosts may differ considerably from those observed in optimal conditions in laboratory settings.

Voutilainen et al. 2015. Life-long shedding of Puumala hantavirus in wild bank voles (Myodes glareolus). J. Gen. Virology, 96: 1238-1247 DOI 10.1099/vir.0.000076

Analysing the effects of climate changes on rodent populations
Rodents play a key role in many ecosystems, e.g. as prey animals for a variety of predators and by affecting the structure of plant communities through their grazing. They also affect human societies, not only by causing economic losses in forestry and agriculture, but also because they pose a serious concern for Public Health as reservoir hosts for a number of zoonotic diseases. The hypothesis that warmer winters cause the disappearance of multiannual vole cycles was tested in the course of an EDENext study (Korpela et al., 2013). A reliable analysis of population dynamics and of effects of climate thereon requires extensive spatial and temporal data. Therefore, all data available on vole abundances and climate collected at 33 locations throughout Finland during the period 1970 to 2011 was combined. The results of this study allow the assumption that vole population dynamics exhibit geographic and temporal variations that are associated with a variation in climate. Reduced cyclicity should be observed when winter temperatures are mild. The spatiotemporal variation in dynamics correlated with seasonal temperatures: cyclicity was weakened by increasing temperatures in the north, but strengthened in the south. Further, the study results do not support the hypothesis that warm winters are uniformly leading to irregular or more stable dynamics in boreal vole populations. Long and cold winters were not a prerequisite for high amplitude multiannual cycles, nor were warm winters with a reduced duration of snow cover associated with reduced winter growth rates.
Small mammalian specialist predators, i.e. small mustelids like least weasels and stoats, have commonly been attributed as the important cause of pronounced cyclicity of voles in the boreal zone. Korpela et al. (2014) analysed the impact of climate on the long-term trends in cyclicity in Finland. In several parts of Europe reports of collapsing rodent cycles have attributed the changes to warmer winters, which weaken the interaction between voles and their specialist subnivean (under-snow) predators. Using national long-term population data on voles, predators, birds of prey and climate, collected throughout Finland during 1986–2011, they analysed the spatio-temporal variation in the interactions between populations of voles and specialist, generalist and avian predators, and investigated by simulations the roles of the different predators in the vole cycle. They tested the hypothesis that vole cyclicity is dependent on predator–prey interactions during winter. The results strongly support the importance of the small mustelids for the vole cycle. However, in the models weakening specialist predation during winters, or an increase in generalist predation, was not associated with the loss of cyclicity. Strengthening of delayed density dependence coincided with strengthening small mustelid influence on the summer population growth rates of voles. In conclusion, a strong impact of small mustelids during summers appears highly influential to vole population dynamics, though this importance between winter and summer may vary geographically, and deteriorating winter conditions are not a viable explanation for collapsing small mammal population cycles. Changing climate may affect this interaction between voles and specialist predators.

Korpela et al ( 2014). Predator-vole interactions in boreal Europe: the role of small mustelids revised. Proc. R. Soc. B 281. 20142119.

Reviews on hantavirus and other rodent-borne pathogens causing haemorrhagic fever virus infections
Hantavirus infections in Europe and their impact on public health
This review highlights the most important Public Health issues of hantavirus infections in Europe (Vaheri et al., 2013). There are several hantavirus species in Europe causing haemorrhagic fever with renal syndrome (HFRS). The most common is Puumala virus that causes a mild form of HFRS called nephropathia epidemica (NE). Dobrava-Belgrade virus (DOBV) consists of four genotypes that differ in pathogenicity: Dobrava causes severe symptoms and a high case fatality rate (up to 12%), Sochi causes HFRS of medium severity, Kurkino causes a rather mild disease and Saaremaa a possibly mild or no disease. Seoul virus, carried by rats, causes HFRS with medium severity but is rare. In addition to renal changes, cardiac, pulmonary, ocular and hormonal disorders are quite common during the acute stage of a PUUV infection, the major cause of HFRS in Europe. Although PUUV-associated HFRS has a low (0.1-0.4 %) case fatality rate, complications and long-term consequences regularly occur. About 5% of hospitalized patients require dialysis and some need prolonged intensive care treatment. In this context, prospective studies are needed to define the role of hantavirus infections in the development of chronic kidney diseases, hyper-tension, hormonal disorders and other possible chronic diseases. Research is also needed to understand the mechanism of shock and vascular leakage in order to properly treat the severe forms of HFRS.
Despite the availability of diagnostic tools in many endemic areas in Europe, there are still several countries where HFRS has not yet been recognised by the medical community and may therefore be worryingly underdiagnosed. Field investigations are therefore needed to evaluate the occurrence and distribution of hantaviruses within rodent populations in addition to risk based sero-surveys of patients to detect new endemic areas. In Europe thousands of HFRS cases are diagnosed annually, and the numbers are increasing. As no vaccine or specific antiviral therapy is in general use in Europe, the development of vaccines containing components of a vole-derived virus (PUUV) and of a mouse-derived virus (DOBV) must be recommended.

Epidemiology, diagnostics and treatment of rodent-borne haemorrhagic fever virus infections
This review article gives a practical overview of the epidemiology, diagnosis and treatment of the most important rodent-borne haemorrhagic fever pathogens that are transmitted directly from rodents to humans: Leptospira, hantaviruses, New and Old World arenaviruses (Goeijenbier et al., 2013). These belong to an important group of zoonotic pathogens causing severe disease all over the world. Having become a serious challenge for clinicians and laboratory researchers they are of particular importance for Public Health due to increases in international travel and adventure tourism in regions where the pathogens occur. Clinical symptoms, transmission routes and other epidemiological features of the above mentioned pathogens are often similar and they often have an emerging pattern. High case fatality rates are characteristic for a fulminant manifestation of the diseases. The rather non-specific clinical picture of rodent-borne haemorrhagic fevers, particularly in the early phase, may lead to misdiagnosis. Besides, treatment strategies are usually based on the success in treatment of individual patients or on case reports rather than on randomised controlled trails. Consequently, the compilation of treatment protocols is more anecdotal than evidence based. Moreover, vaccines are often not available or are not approved by the authorities. Because of the rather unspecific clinical symptoms and similar laboratory parameters, along with the partial overlapping geographical distribution, the accurate diagnosis of rodent-borne haemorrhagic fevers depends mainly on the availability of sensitive and specific tests and high-level clinical practice. Preventive Public Health measures include rodent control, the development of vaccines and education of the public directed at avoiding contact with rodents or rodent excreta. For applying these measures, a fundamental understanding of the pathogens and their specific rodent hosts is necessary. Compilation of the present data on rodent-borne haemorrhagic fever pathogens will provide valuable information for clinicians.

Uncovering the mysteries of hantavirus infections – a review of hantavirus disease pathology and immunology
Hantaviruses are negative-sense single-stranded RNA viruses that infect many species of rodents, shrews, moles and bats. Infection in these reservoir hosts is almost asymptomatic, but some hantaviruses can be transmitted via aerosols of rodent excreta to humans, in which they can cause two dis¬eases: haemorrhagic fever with renal syndrome (HFRS), which is primarily caused by Hantaan virus (HTNV) and related viruses in Asia, Puumala virus (PUUV) and Dobrava virus (DOBV) in Europe, and Seoul virus (SEOV) worldwide; or hantavirus cardiopulmonary syn¬drome (HCPS), which is caused by Sin Nombre virus (SNV) and related viruses in North America, and Andes virus (ANDV) and related viruses in Latin America. Vaheri et al. (2013) discuss the basic molecular properties and cell biology of hantaviruses and offer an overview of virus-induced pathology, in particular vascular leakage and immunopathology. These diseases are characterized by increased capillary permeability (causing vascular leakage) and thrombocytopenia. These pathologies are thought to be caused by viral infection of endothelial cells, which does not disrupt the endothelium but nonetheless leads to dramatic changes in both the barrier function of the endothelium as a whole and the function of infected endothelial cells. It has also been suggested that cytotoxic CD8+ T cells (CTLs) trigger capillary leakage and that cytokines contribute to the increased capillary permeability. The terminal soluble complement complex can also increase vascular permeability, and complement activation is linked to severity of hantavirus infection.
Hantavirus infections and the diseases that they cause are a growing public health problem, and there is currently no therapy or vaccine in global use. Moreover, by the time the symptoms of HFRS or HCPS appear, viraemia has already started fading, so the administration of antiviral drugs might not be beneficial. Thus, a better understanding of the viral infection and of the involvement of factors such as vascular leakage, cytokine storm and CTL proliferation in disease pathology will be essential to the development of new therapies. Detailed knowledge about the exact contribution of these different host response factors to hantavirus pathology is currently limited, and it is unclear whether one factor acts as the primary cause of pathogenesis and others are secondary causes. It is possible that insights into the pathogenesis of hantavirus infections will also be gained by studying the pathological mechanisms which lead to vascular leakage in infections with other viruses causing haemorrhagic (flaviviruses, filoviruses and arenaviruses).

Vaheri et al (2013). Uncovering the mysteries of hantavirus infections. – Nature Reviews in Microbiology 11:539-550. doi:10.1038/nrmicro3066.

The importance of immunogenetics for hantavirus infection in rodents and humans
It is often not well understood that both in humans and animals there is great genetic and immunogenetic variation in disease susceptibility. Charbonnel et al. (2014) reviewed the associations of immunity-related genes with susceptibility of humans and rodents to hantaviruses, and with severity of diseases in humans. Several class I and class II human leucocyte antigens (HLA) haplotypes are linked with severe or benign human hantavirus infections, and these haplotypes varied among localities and hantaviruses. The polymorphism of other immunity-related genes including the C4A gene and a high-producing genotype of tumor necrosis factor (TNF) gene associated with severe PUUV infection. Additional genes that may contribute to disease or to PUUV infection severity include non-carriage of the interleukin-1 receptor antagonist (IL-1RA) allele 2 and IL-1β (-511) allele 2, polymorphisms of plasminogen activator inhibitor (PAI-1) and platelet GP1a.
In addition, immunogenetic studies have been conducted to identify mechanisms that could be linked with the persistence/clearance of hantaviruses in reservoir hosts. Persistence was associated during experimental infections with an upregulation of anti-inflammatory responses. Using natural rodent population samples, polymorphisms and/or expression levels of several genes have been analyzed. These genes were selected based on the literature of rodent or human/hantavirus interactions. The comparison of genetic differentiation estimated between bank vole populations sampled over Europe, at neutral and candidate genes, has allowed to evidence signatures of selection for Tnf, Mx2 and the Drb Mhc class II genes. Altogether, these results corroborated the hypothesis of an evolution of tolerance strategies in rodents. In other words, the bank vole has quite different immunogenetic responses to PUUV in different parts of Europe, which could partly explain the variable epidemiology of PUVV.

Charbonnel et al (2014). Immunogenetic Factors Affecting Susceptibility of Humans and Rodents to Hantaviruses and the Clinical Course of Hantaviral Disease in Humans. – Viruses 2014 (6) 2214-2241; doi:10.3390/v6052214.

Modelling using the example of Puumala hantavirus
Even though the data are similar in nature, modelling zoonotic diseases in humans differs from modelling the distribution of individual species because the distribution of pathogens with zoonotic potential may depend on the presence of more than one species. An EDENext study used data from Sweden on Puumala hantavirus (PUUV) infections in humans, reported between 1991 and 1998, as a case study in order to compare existing approaches when modelling spatial distributions recorded as points. In Sweden, PUUV, the pathogen causing of nephropathia epidemica (NE), is the only hantavirus and is hosted by bank voles (Myodes glareolus). The majority of NE cases (90%) are notified from the four northernmost counties.
Previous studies have shown that the prevalence of NE is linked to host abundance and human activities with an increased risk of exposure to rodents, such as forestry, farming, wood cutting, construction work, camping, cleaning and/or redecorating buildings where rodents live. Virus prevalence and transmission depend on the local environmental, anthropogenic, genetic, behavioural and/or physiological factors. This study focused on three different groups; environmental factors influencing the distribution of NE in relation to bank vole habitat, ex vivo virus survival, human presence and exposure. Logistic binomial regression and boosted regression trees were used to model ‘presence’ and ‘absence’ data. Occurrence and potential sites where the disease may occur were modelled using cross-validated logistic regression. Finally, the ecological niche model Maxent, based on ‘presence’-only data, was used. The results showed that zoonotic diseases can be modelled with diverse methods (Zeimes et al., 2012). The final purpose of the model, if explicative or predictive, and the availability of data, as well as the specificity of the system at hand, determines the choice.
Zeimes et al. (2015) also investigated the European spatial distribution of NE with a rich set of environmental variables. The influence of variables at the landscape and regional level was studied through multilevel logistic regression, and further information on their effects across the different European ecoregions was obtained by comparing an overall niche model (boosted regression trees) with regressions by ecoregion. The presence of NE is likely in populated regions with well-connected forests, more intense vegetation activity, low soil water content, mild summers, and cold winters. In these regions, landscapes with a higher proportion of built-up areas in forest ecotones and lower minimum temperature in winter are expected to be more at risk. Climate and forest connectivity have a stronger effect at the regional level. If variables are staying at their current values, the models predict that NE may know intensification but should not spread (although southern Sweden, the Norwegian coast, and the Netherlands should be kept under watch). Models indicate that large-scale modeling can lead to a very high predictive power. At large scale, the effect of one variable on disease may follow three response scenarios: the effect may be the same across the entire study area, the effect can change according to the variable value, and the effect can change depending on local specificities. Each of these scenarios impacts large-scale modelling differently.

Zeimes et al. 2015. Landscape and regional environmental analysis of the spatial distribution of hantavirus human cases in Europe. – Front. Public Health, March 2015, 3, 54:1-10. doi:10.3389/fpubh.2015.00054

3. Mosquito-borne diseases
New adhesive traps to monitor urban mosquitoes and assess efficacy of control strategies
An Italian research team has assessed the potential of three adhesive traps for passive monitoring of urban mosquito adult abundance and seasonal dynamics, and for assessing the efficacy of commonly used control measures. They note that urban mosquitoes in temperate regions may have a high nuisance value and are associated with the risk of arbovirus transmission. In highly infested urban areas in Italy, for example, this leads to calendar-based larvicide treatments of street catch basins - the main non-removable urban breeding site - and/or insecticide ground spraying. The planning of these interventions, as well as the evaluation of their effectiveness, rarely benefits from adequate monitoring of mosquito abundance and dynamics. Therefore the team designed two novel adhesive traps to monitor Aedes albopictus and Culex pipiens adults visiting and/or emerging from catch basins in order to evaluate the efficacy of insecticide-based control strategies. The mosquito emerging trap (MET) was used to assess the efficacy of larvicide treatments. The catch basin trap (CBT) was used together with the sticky trap (ST, commonly used to collect ovipositing / resting females) to monitor adult abundance on the campus of the University of Rome 'Sapienza', where catch basins had been treated with insect growth regulators (IGR) bi-monthly and low-volume insecticide spraying was carried out before sunset, and in a nearby control area. Their results from MET showed that, although all monitored diflubenzuron-treated catch basins were repeatedly visited by Ae. albopictus and Cx. pipiens, adult emergence was inhibited in most basins. Results obtained by ST and CBT showed a significant lower adult abundance in the treated area than in the untreated one after the second adulticide spraying, which was carried out during the major phase of Ae. albopictus population expansion in Rome. They conclude that all three adhesive traps have potential for passive monitoring of urban mosquito adult abundance and seasonal dynamics, and in assessing the efficacy of control measures.

Caputo et al (2015). New adhesive traps to monitor urban mosquitoes with a case study to assess the efficacy of insecticide control strategies in temperate areas. Parasites & Vectors 2015, 8:134. Doi:10.1186/s13071-015-0734-4 (EDENext291)

West Nile virus in Europe: knowledge gaps and research priorities
A joint initiative from European experts on West Nile virus (WNV) has seen them analyze 118 scientific papers published between 2004 and 2014 to provide the state of the art on knowledge on WNV. Their work highlights the existing knowledge and research gaps that need to be addressed as a high priority in Europe and neighboring countries. Noting the continual spread of WNV both in Europe and other regions, they point out that the high genetic diversity of the virus, with remarkable phenotypic variation, and its endemic circulation in several countries, requires an intensification of the integrated and multidisciplinary research efforts built under the 7th Framework Program. It is important, they say, to better clarify several aspects of WNV circulation in Europe, including its ecology, genomic diversity, pathogenicity, transmissibility, diagnosis and control options, under different environmental and socio-economic scenarios. They add that identifying WNV endemic areas, as well as infection-free areas, is becoming necessary for the development of human vaccines and therapeutics, and for the application of blood and organ safety regulations.

Rizzoli et al (2015). The challenge of West Nile virus in Europe: knowledge gaps and research priorities. Euro Surveill. 2015;20(20):pii=21135

The effects of climate change on mosquito abundance in Mediterranean wetlands
Noting that the impact of climate change on vector-borne diseases is highly controversial, an EDENext team has tackled one of the principal points of debate, investigating whether or not climate influences mosquito abundance, a key factor in disease transmission. Researchers analyzed 10 years of data (2003–2012) from bi-weekly surveys to assess inter-annual and seasonal relationships between the abundance of seven mosquito species known to be vectors of various pathogens, and several climatic variables in two wetlands in south-west Spain. They report that within-season abundance patterns were related to climatic variables (i.e. temperature, rainfall, tide heights, relative humidity and photoperiod) that varied according to the mosquito species in question. Rainfall during winter months was positively related to the annual abundance of Culex pipiens and Ochlerotatus detritus. Annual maximum temperatures were non-linearly related to annual Cx. pipiens abundance, while annual mean temperatures were positively related to annual Ochlerotatus caspius abundance. The team then modelled shifts in mosquito abundance using temperature and rainfall climate change scenarios for the period 2011–2100. They note that while Oc. caspius, an important anthropophilic species, may increase in abundance, no changes are expected for Cx. pipiens or the salt-marsh mosquito Oc. detritus. Their results highlight that the effects of climate are species-specific, place-specific and non-linear and that linear approaches will therefore overestimate the effect of climate change on mosquito abundances at high temperatures. Climate warming does not necessarily lead to an increase in mosquito abundance in natural Mediterranean wetlands, they say, and will affect, above all, species such as Oc. caspius whose numbers are not closely linked to rainfall but are influenced by local tidal patterns and temperatures. They conclude that the final impact of changes in vector abundance on disease frequency will depend on the direct and indirect effects of climate and other parameters related to pathogen amplification and spillover on humans and other vertebrates.

Roiz et al (2014). Climatic effects on mosquito abundance in Mediterranean wetlands. Parasites & Vectors 2014, 7:333. Doi:10.1186/1756-3305-7-333

The effect of landscape on mosquito populations in Mediterranean wetlands
An EDENext study by Roiz et al. has shown that landscape significantly affects mosquito distribution and abundance, and as a result may alter disease risk. The team analysed the relationship between environmental variables estimated by remote sensing and spatial distribution (presence, abundance and diversity) of seven mosquito species which are vectors of West Nile and other pathogens (Usutu, avian malaria and dirofilariasis) in the Doñana Natural Park, Spain. They sampled 972,346 female mosquitoes, the most abundant species being Culex theileri, Ochlerotatus caspius, Culex modestus, Culex perexiguus, Culex pipiens, Anopheles atroparvus and Ochlerotatus detritus. Their results suggest that: (1) hydroperiod, inundation surface and NDVI are strongly related to the spatial distribution of mosquitoes; (2) the spatial scales used to measure these variables affects the quantification of these relationships, the larger scale being more informative; (3) these relationships are species-specific; (4) hydroperiod is negatively related to mosquito presence and richness; (5) Culex abundance is positively related to hydroperiod; (6) NDVI is positively related to mosquito diversity, presence and abundance, except in the case of the two salt marsh species (Oc. caspius and Oc. detritus); and (7) inundation surfaces positively condition the abundance and richness of most species except the salt marsh mosquitoes. They conclude that while environmental conditions affect the distribution and abundance of mosquitoes, other factors such as human modification of landscapes may give rise to significant changes in mosquito populations and consequently disease risk.

Roiz et al (2015). Landscape Effects on the Presence, Abundance and Diversity of Mosquitoes in Mediterranean Wetlands. PLoS ONE 10(6): e0128112. Doi: 10.1371/journal.pone.0128112

4. Phlebotomine-borne diseases
The role of phlebotomine sand flies in spreading diseases of Public Health concern
The EDENext review by Maroli et al. (2013) describes the role of phlebotomine sand flies as vectors in spreading different diseases, i.e. leishmaniasis, sand fly fever, summer meningitis, vesicular stomatitis, Chandipura virus encephalitis and Bartonella (Carrión’s disease), and outlines their relevance for Public Health. The emergence of new diseases and pathogens or their re-emergence in areas previously considered to be free can be associated with ecological factors and changes in land use, such as deforestation, urbanisation or irrigation measures, that result in changes in the vector populations, and with human factors, for example an increase in global travel and trade that favours the spread of vector species to previously unaffected areas. Climate changes, for their part, may allow not only the northward expansion of vector species but also virus persistence during winter.
Among sand fly-borne diseases, leishmaniasis is the most widespread infection worldwide. Like other tropical infectious diseases, such as Chagas’ disease and sleeping sickness, it is generally regarded as a neglected disease, because of the lack of effective control and treatment measures. With few exceptions, phlebotomine sand flies (Diptera, Psychodidae, subfamily Phlebotominae) are the only haematophagous insects that transmit leishmaniasis. Among the more than 800 estimated phlebotomine sand flies species, around 100 species have been recorded as potential vectors of human leishmaniasis. The review provides a list of these species by endemic country, the Leishmania species agent transmitted (if known) and the clinical forms of leishmaniasis in humans. In addition, other human diseases and their growing impact on human health are outlined.
Phlebotomine sandflies are involved in the transmission of several viral agents, among which the most important are grouped into the Phlebovirus genus (family Bunyaviridae), which includes the sandfly fever Sicilian and Toscana viruses, and the Vesiculovirus genus (family Rhabdoviridae), which includes vesicular stomatitis, and the Chandipura and Isfahan viruses. Vesiculovirus, which is well-known to affect livestock animals in southeastern USA and Latin America, is increasingly reported to cause infection in humans in India. In addition Carrión’s disease caused by Bartonella bacilliformis, a motile, aerobic and Gram-negative bacterium, formerly restricted to elevated altitudes of Peru, Ecuador and Colombia, is spreading to non-endemic areas of the Amazon basin.
Finally, Maroli and co-authors discuss the spread of leishmaniasis as well as the other mentioned diseases and the lack of suitable treatment due to the rather poor awareness of national Public Health authorities, especially in poor countries. Though advances in phlebotomine research with regard to vector control have been made, in particular studies on the social and environmental variables relevant for Leishmania ecology and transmission, the development of geographical-spatial and analytical models are needed.

Maroli et al (2013). Phlebotomine sandflies and the spreading of leishmaniases and other diseases of public health concern. Med Vet Entomol. 27(2):123-47. doi: 10.1111/j.1365-2915.2012.01034.x.

Leishmaniasis in Greece
Leishmaniasis is a protozoan disease transmitted by phlebotomine sand flies in a zoonotic or an anthroponotic cycle, depending on the parasite species and the geographical location. Though leishmaniasis is regarded as a tropical or subtropical disease, it has become endemic in other geographical regions such as the Mediterranean Basin and has recently been reported to have spread to parts of Central Europe, for example Leishmania infantum in northern Italy, Hungary and southern Germany. Therefore, Leishmania constitutes a Public Health problem in Europe, not only in endemic areas but also in geographic regions that are at risk of introduction.
The first case of visceral leishmaniasis in the Mediterranean region was reported from the Greek island of Spetses in 1835. Though the frequency of mosquito and sand fly transmitted diseases decreased due to the use of DDT against malaria in the years 1946-1952, the disease has re-emerged and spread across Greece in the past 35 years with an increasing number of human and dog cases per year. The aim of the EDENext review by Ntais et al. (2013) was to investigate the current situation in Greece of leishmaniases: the geographical distribution of human and canine leishmaniases, Leishmania species as well as sand fly vector species and the distribution of the diseases in relation to epidemiological and environmental factors using geospatial tools.
Leishmania seropositive dogs were found in 43 of 54 prefectures with an average of 22 % indicating that the disease is widespread across Greece. Favorable factors were found to be altitude, presence of water bodies, land use, wind speed, mean land surface temperature, mean relative humidity and mean annual rainfall. Dogs kept outside (strays, guard or hunting dogs) are at higher risk of getting infected and, in turn, to infect sand flies. L. infantum zymodeme (Z) MON-1 found predominantly in the Mediterranean Basin was present in all parts of the country, while the rare ZMON-98 was less frequent and the dermatotropic L. tropica was found only in Crete. Thirteen different sand fly species are known to occur in Greece and are widely distributed across the country, 10 of which belong to the genus Phlebotomus and three to the genus Sergentomyia.
The importance of infected dogs lies in their role in transmitting the parasite L. infantum to sand fly vectors, which in turn can infect humans. Controlling the disease in the dog population is the best way to reduce the risk of infection in the human population. Changing human habits and living conditions, the movement and travel of humans and dogs, and changes in climatic and environmental conditions and in animal husbandry practices that increase vector abundance enhance leishmaniasis cases in humans and dogs and the geographic spread.
In another EDENext publication Ntais et al. (2014) reported the introduction of Leishmania tropica ZMON-58 to the island of Crete and the infection of a local dog. L. tropica ZMON-58 was isolated from a young Afghan refugee with cutaneous lesions who came to Crete a few months earlier. In the same area the same zymodeme was isolated from a local dog with symptoms of visceral leishmaniasis. Since L. tropica ZMON-58 has so far only been described in six human cases in Afghanistan, this is the first record of L. tropica in a dog and presents another example of the introduction of a vector-borne pathogen to an unaffected area that supplies a suitable vector population allowing new transmission cycles. In a closed ecosystem such as Crete, hosting nine different Phlebotomus species, there always is the risk of exchanging genetic material between L. infantum and L. tropica and the risk of zymodeme variants resulting in new hybrids with a potentially changed epidemiology, pathogenicity and drug resistance. Globalisation favours changes in the epidemiology of many pathogens and the introduction to new areas. Pathogens and vectors become established in new foci if they encounter the right conditions and hosts for spread. Therefore, monitoring vectors and reservoirs in areas at risk for introduction and providing information to Public Health authorities is of great importance to prevent the spread and establishment of a novel disease in new, so far unaffected areas.
Sifaki-Pistolla et al. (2014) evaluated the use of a veterinary questionnaire as a cost-effective tool for locating, recording and predicting the spread of canine leishmaniasis in an area, comparing the collected questionnaire data with the results of two epidemiological surveys accomplished in Greece (Ntais et al. 2013) and Cyprus. In order to assess the risk of human Leishmania infections in an area, surveillance studies in the local dog population are necessary. As epidemiological studies of dogs are costly and time-consuming, the aim of the study was to evaluate the questionnaire, consisting of 14 brief multiple-choice questions, in order to collect information on the disease which made it possible to reveal spatially-explicit indicators of canine leishmaniasis prevalence for use in predictive modelling. In addition, the questionnaire offers a two-way exchange of information; it provides information not only to scientists and specialists but also to veterinarians as they become aware of what is considered important by the specialists. Although the questionnaire data cannot provide a quantitative measure of canine leishmaniasis in an area, it demonstrates the dynamic of the disease. Overall, the veterinary questionnaire can be used as an early warning system and a screening tool to assess the risk of leishmaniasis in a country or region and therefore promoting Public Health.

Ntais et al (2013). Leishmaniases in Greece. Am J Trop Med Hyg. 89 (5): 906-15. Doi: 10.4269/ajtmh.13-0070.
Ntais et al (2014). Will the introduction of Leishmania tropica MON-58, in the island of Crete, lead to the settlement and spread of this rare zymodeme? Acta Trop. 132:125-30.
Sifaki-Pistola et al (2014). The Use of Spatial Analysis to Estimate the Prevalence of Canine Leishmaniasis in Greece and Cyprus to Predict Its Future Variation and Relate It to Human Disease. Am J Trop Med Hyg. 91(2): 336-341.

Risk factors for cutaneous leishmaniasis in Turkey
Cutaneous leishmaniasis (in the form of a cutaneous lesion at the infected sand fly bite site), is the most common form of human leishmaniasis, with millions of human cases reported from southern Europe, Asia, Africa, South and Central America every year. Other mammal species, such as dogs and rodents, play a role as reservoirs or hosts for Leishmania parasites and contribute to the spread of the disease. The EDENext study by Votýpka et al. (2012) evaluated risk factors for non-typical cutaneous leishmaniasis caused by L. infantum in the Cukurova region in South Anatolia, Turkey. By using structured questionnaires in interviews with local people, epidemiological and clinical characteristics, application of personal protection measures and knowledge about Leishmania parasites were analysed. Ownership of a dog, raising cattle and sleeping without a bed-net were associated with a significantly increased risk of Leishmania infection in statistical-univariate analyses, with dog ownership having the greatest correlation with leishmaniasis. The authors recommend further research on dogs to clarify the transmission cycle and the role of dogs in the epidemiology of cutaneous leishmaniasis in this area.
A medium-scale field trial with insecticide-treated bed-nets was conducted in south Anatolia to measure the protective efficacy of Olyset® Plus, a new long-lasting factory-treated insecticidal net (LLIN) incorporated with 2% permethrin and 1% of the synergist piperonyl butoxide (PBO), against cutaneous leishmaniasis (CL) in a new hyper-endemic focus caused by a Leishmania infantum / L. donovani hybrid parasite transmitted by proven vector Phlebotomus tobbi, between May 2013 and May 2014. The study area comprised eight villages; two of them were selected as an intervention village with Olyset® Plus net (Kizillar) and a control village without net application (Malihidirli). Six villages with surrounding allopatric barriers were utilized as a buffer zone cluster between intervention and control villages. Monthly entomological surveys were performed in the intervention and control villages and Damyeri, representing the other six villages, to collect adults of Phlebotomus tobbi. Results showed a significant reduction in cutaneous leishmaniasis incidence in the intervention village from 4.78% to 0.37%. The protective efficacy rate of LLIN was 92.2%. In contrast, incidence rates increased in the control village from 3.7% to 4.7%. We also evaluated residual insecticide levels of used nets after six and 12 months of usage. It was determined that the nets had retained full insecticidal strength. These results highlight the value of real-world data on bed net effectiveness and longevity to guide decisions regarding sand fly control strategies (Gunay et al. 2014).

Votýpka et al (2012). Risk factors for cutaneous leishmaniasis in Cukurova region, Turkey. Trans R Soc Trop Med Hyg. 2012 Mar; 106(3):186-90. Doi: 10.1016/j.trstmh.2011.12.004.
Gunay et al (2014). Evaluation of the efficacy of Olyset® Plus in a village-based cohort study in the Cukurova Plain, Turkey, in an area of hyperendemic cutaneous leishmaniasis. Journal of Vector Ecology, 39: 395–405. Doi: 10.1111/jvec.12115

Updates from phlebotomine sandfly-borne disease research in Portugal
Zoonotic leishmaniasis caused by L. infantum is endemic in Portugal. Within the scope of EDENext, surveys concerning sand fly, canine and feline diseases have been conducted in the internationally renowned tourist hot spot of the Algarve region in southern Portugal (Maia et al. 2013, 2014). L. infantum prevalence ranged from 10 % in cats and 16 % in dogs. The simultaneous presence of dogs, cats and Phlebotomus perniciosus infected with L. infantum in the Algarve region shows that the region continues to be an endemic area of this parasitic zoonosis.
In addition, DNA of Leishmania major, which is associated with cutaneous leishmaniasis, was detected in a Sergentomyia minuta specimen collected in the same geographic region (Campino et al. 2013). It is the first documented case of Leishmania major in a sand fly of the Sergentomyia genus in Europe. Further analysis of blood meals of sand flies from Algarve region using the cyt-b sequence showed that P. perniciosus fed on a wide range of domestic animals while human and lizard DNA was detected in engorged S. minuta. Detection of Leishmania infections by PCR using ITS-1 as the target sequence revealed in two specimens of Sergentomyia minuta and one Phlebotomus perniciosus (Maia et al. 2015).
Overall, the results reinforce the need for systematic investigations of the spatial distribution of phlebotomine populations and, at the same time, of pathogens in both invertebrate and vertebrate hosts in order to improve the understanding of the transmission, distribution and spread of Leishmania species.

Campino et al (2013). The first detection of Leishmania major in naturally infected Sergentomyia minuta in Portugal. Mem Inst Oswaldo Cruz. 2013 Jun; 108(4):481-7. Doi: 10.1590/S0074-0276108042013014.
Maia et al (2013). Leishmania infection and host-blood feeding preferences of phlebotomine sand flies and canine leishmaniasis in an endemic European area, the Algarve Region in Portugal. Mem Inst Oswaldo Cruz. 108(4):481-7. Doi: 10.1590/S0074-0276108042013014.
Maia et al (2014). Bacterial and protozoal agents of feline vector-borne diseases in domestic and stray cats from southern Portugal. Parasit Vectors. 7(1):115. Doi: 10.1186/1756-3305-7-115.
Maia et al (2015). Molecular detection of Leishmania DNA and identification of blood meals in wild caught phlebotomine sand flies (Diptera: Psychodidae) from southern Portugal. Parasites & Vectors 8:173. Doi: 10.1186/s13071-015-0787-4.

A retrospective analysis of human leishmaniasis epidemics in Italy
The EDENext publication by Gramiccia et al. (2013) gives a retrospective analysis of the multi-annual human leishmaniasis epidemics by L. infantum that occurred between 1989 and 2009 in Italy. Starting from 1989, Italy has experienced an increase of visceral leishmaniasis cases that peaked between 2000 and 2004 with more than 200 cases per year and declined thereafter. The study was conducted to identify possible determinants that explain the recent trend of the disease in the country. The visceral leishmaniasis epidemic in Italy seems to be a complex phenomenon with several components: i) an outbreak involving infants and immune-competent adults in the Campania region that declined, presumably naturally; ii) a second outbreak affecting HIV-infected individuals throughout the country, that declined due to antiviral treatment; iii) a generalised increase of cases due to disease spreading within traditionally endemic areas as well as the appearance of a few autochthonous cases in previously non-endemic territories, starting from the early 1990s.
While the appearance of autochthonous visceral leishmaniasis cases in northern parts of Italy could be explained by the de novo colonization with phlebotomine vectors along with importing Leishmania-infected dogs from the endemic south and the generalized increase of cases in endemic areas due to changes in vector density, the causes for both the onset and the natural decline of the outbreak in the Campania region will remain unexplained. The immunity on a population level acquired during the epidemic may be one reason, but is hard to verify. No aggressive control measures were applied that could justify the general drop in incidence. To date there is no clear evidence for a direct association of canine leishmaniasis prevalence and incidence of human disease in a given territory. Although dogs are efficient sentinel hosts for L. infantum transmission, the prevalence rate of canine infections does not appear a useful parameter for explaining determinants of human visceral leishmaniasis trends in endemic areas as observed in the Campania region. While the presence of competent vectors in a given territory can be predictive for the occurrence of human visceral leishmaniasis together with the occurrence of canine leishmaniasis cases, the current entomological data is still insufficient for the analysis of epidemic visceral leishmaniasis trends.

Gramiccia et al (2013). The burden of visceral leishmaniasis in Italy from 1982 to 2012: a retrospective analysis of the multi-annual epidemic that occurred from 1989 to 2009. Euro Surveill. 18(29):20535.

Spanish leporids (hares and rabbits) as potential sylvatic Leishmania reservoirs
In Spain, dogs are considered to be the main reservoir of zoonotic leishmaniasis caused by L. infantum. In rural, periurban and suburban areas of the Madrid region, Leishmania is endemic. However, since 2010 the number of human cases of visceral and cutaneous leishmaniasis in four municipalities to the south-west of Madrid has drastically increased, presumably linked to the creation of a park adjacent to the urban area. These outbreaks have driven the investigation of local wild animals in the search for potential reservoirs for L. infantum. The low prevalence of leishmaniasis in dogs and the high population densities of hares and rabbits in the newly constructed periurban park have led to the assumption that leporids could sustain a large sand fly population in the area, suggesting the existence of a sylvatic transmission cycle linked to the urban periphery. In order to find the sources of infection, several xenodiagnosis studies focusing on wild leporids and Leishmania phlebotomine vectors were initiated:
Molina et al. (2012) delivered the first evidence by xenodiagnosis that Spanish hares (Lepus granatensis), a species endemic to the Iberian Peninsula, are used as hosts by P. perniciosus, and that L. infantum is successfully transmitted from hares to P. perniciosus sand flies. The study discussed the role of hares as potential sylvatic reservoirs for L. infantum. In conclusion, hares should be taken into account in future epidemiological studies when endemic sites of visceral leishmaniasis are investigated.
In line with these findings, Martín-Martín et al. (2014) observed that wild rabbits and hares captured in this area show high anti-sand fly saliva antibody levels, thus indicating they are frequently bitten by P. perniciosus.
A study conducted by Jiménez et al. (2014) in the same area demonstrated that rabbits can also contribute to the spread of leishmaniasis. Wild rabbits (Oryctolagus cuniculus) were found to serve as hosts for P. perniciosus and therefore are a source of L. infantum. Results of blood meal preference analyses of P. perniciosus, caught in the same area, also revealed that the majority of sand flies fed on hares and/or rabbits.
Jimenez et al. (2013) conducted a preliminary entomological survey in 2011, at the end of the seasonal transmission period when highest rates of infections in sand flies could be expected and before control measures were implemented, to determine the putative species involved. The data connect P. perniciosus as L. infantum vector to hares and humans as hosts and support the existence of an unusual sylvatic cycle as an alternative to the classical domestic one with dogs as the main reservoir in this area. Most noticeably, the percentage of L. infantum positive Phlebotomus sand flies was comparably high with approximately 60%. In addition, blood feeding preferences, analyzed by cytochrome b analysis, revealed that hares were preferred as hosts, with 60% of sand flies positive for hare blood, followed by humans with 30% and cats with 10%, whereas no dog blood was identified. This can explain the high transmission frequency of leishmaniasis in the green park where high population densities of hares are present (see above). Since blood feeding behavior plays a significant role in the transmission and maintenance of vector-borne pathogens in natural systems, analyses of blood feeding preferences will therefore help to improve our understanding of the role of a particular host or reservoir in urban foci, leading to more effective control strategies.
Using an ex vivo model of infection in murine bone marrow-derived cells, the virulence of the L. infantum isolates from Phlebotomus perniciosus captured in this area was evaluated, showing that L. infantum isolates captured from this endemic area exhibited high virulence in terms of infection index, cytokine production and enzymatic activity involved in the pathogenesis of visceral eishmaniosis (Domínguez-Bernal et al. 2014).
In summary, this exceptional scenario is likely to be a cause of the urbanization of leishmaniasis due to the coexistence of peri-urban and sylvatic transmission cycles involving different, domestic and sylvatic, reservoir species for sand fly vectors. These findings show the complexity of the eco-epidemiological factors that drive Leishmania transmission and, therefore, have important implications for Public Health in terms of taking effective measures to control the situation as soon as possible.

Jiménez et al (2013). Detection of Leishmania infantum and identification of blood meals in Phlebotomus perniciosus from a focus of human leishmaniasis in Madrid, Spain. Parasitol Res. 112(7):2453-9. Doi: 10.1007/s00436-013-3406-3.
Jiménez et al (2014). Could wild rabbits (Oryctolagus cuniculus) be reservoirs for Leishmania infantum in the focus of Madrid, Spain? Vet Parasitol. 2014. Doi: 10.1016/j.vetpar.2014.03.027.
Martín-Martín et al (2014). High levels of anti-Phlebotomus perniciosus saliva antibodies in different vertebrate hosts from the re-emerging leishmaniosis focus in Madrid, Spain. Vet. Parasitol. Doi: 10.1016/j.vetpar.2014.02.045.
Molina et al (2012). The hare (Lepus granatensis) as potential sylvatic reservoir of Leishmania infantum in Spain. Vet Parasitol. 2012; 190:268-271.
Domínguez-Bernal et al (2014). Characterisation of the ex vivo virulence of Leishmania infantum isolates from Phlebotomus perniciosus from an outbreak of human leishmaniosis in Madrid, Spain. Parasites & Vectors, 7:499. Doi: 10.1186/s13071-014-0499-1.

Isolation of (novel) sand fly-borne viruses
In countries around the Mediterranean basin, phlebotomine sand flies are involved in the transmission of several arthropod-borne viruses that belong to the genus Phlebovirus within the Bunyaviridae family, i.e. Toscana virus (TOSV; species Sandfly fever Naples virus), Arbia virus (species Salehebad virus) and Sandfly fever Sicilian virus. In southern European countries Toscana virus constitutes a threat for Public Health as it is one of the major viral pathogens causing central nervous system infections in humans. Findings of new sand fly-borne phleboviruses from Mediterranean countries indicated that the viral diversity in the genus Phlebovirus is higher than initially expected. In Tunisia, the recent isolation of Punique virus (PUNV; species Sandfly fever Naples virus) raised the question of its role in human infections and as a potential pathogen. The EDENext study by Sakhria et al. (2013) demonstrated the co-circulation of two related sand fly-borne phleboviruses, TOSV and PUNV, both belonging to the same species, Sandfly fever Naples virus. Serum samples from humans living in endemic areas for visceral leishmaniasis in northern Tunisia were tested by micro-neutralisation assay for neutralizing antibodies which allowed these closely related viruses to be differentiated. With seroprevalence rates of 41% for TOSV and 9% for PUNV found in the samples it was demonstrated that PUNV is capable of infecting humans but at a low rate, and that TOSV seems to be responsible for the majority of human infections by sand fly-borne phleboviruses in northern Tunisia. Therefore it should always be considered by physicians for patients with meningitis or unexplained fever.
TOSV was initially discovered in central Italy but it was not until 15 years later that its role as causative agent in human neuro-invasive disease was noticed. TOSV circulates in several European countries, for example Italy, Portugal, Spain, France, Croatia and Turkey, and is now recognized as the leading cause of aseptic meningitis during the warm season. Bichaud et al. (2014) reported the first detection of TOSV from P. perniciosus in Corsica demonstrating the presence of the virus on the island. TOSV poses a risk to the Public Health of locals and tourists and therefore always needs to be considered by physicians when patients present with aseptic meningitis and febrile illness during the warm season.
The EDENext publication by Remoli et al. (2014) described the isolation of a novel Phlebovirus, named Fermo virus, placed in the species Sandfly fever Naples virus. The importance of this finding lies in the virus’ ability to fill an ecological niche and to co-infect the same vector which in consequence may lead to the emergence of virus reassortants (with mixed gene segments) exhibiting different degrees of pathogenicity. Further studies need to focus on the distribution of Fermo virus and on its ability to cause infection and disease in humans.
A new Phlebovirus, Adana virus, was isolated from a pool of Phlebotomus spp. in the province of Adana, in the Mediterranean region of Turkey. Genetic analysis based on complete coding of genomic sequences indicated it belongs to the Salehabad virus species of the genus Phlebovirus (Bunyaviridae). Adana virus is the third virus of this species for which the complete sequence has been determined. To understand the epidemiology of Adana virus, a seroprevalence study using microneutralization assay was performed to detect the presence of specific antibodies in human and domestic animal sera collected in Adana as well as Mersin province, located 147 km west of Adana. The results demonstrate that the virus is present in both provinces. The significance of this Adana virus as a possible public health problem in exposed human populations deserves further studies (Alkan et al. 2015).

Bichaud et al (2014). First detection of Toscana virus in Corsica, France. Clin Microbiol Infect. 2014 Feb; 20(2):0101-4. Doi: 10.1111/1469-0691.12347.
Remoli et al (2014). Viral isolates of a novel putative phlebovirus in the Marche Region of Italy. Am J Trop Med Hyg. 2014 Apr; 90(4):760-3. Doi: 10.4269/ajtmh.13-0457.
Sakhria et al (2013). Co-circulation of Toscana virus and Punique virus in northern Tunisia: a microneutralisation-based seroprevalence study. PLoS Negl Trop Dis. 7(9):e2429. Doi: 10.1371/journal.pntd.0002429.
Alkan et al (2015). Isolation, genetic characterization, and seroprevalence of Adana virus, a novel phlebovirus belonging to the Salehabad virus complex, in Turkey. J Virol 89:4080 –4091. Doi:10.1128/JVI.03027-14.

Geographic spread of sand fly-borne phleboviruses
Sand fly-borne phleboviruses are widely distributed in the Mediterranean basin, North Africa, the Indian subcontinent, the Middle East and central Asia. Except for Toscana virus, the leading cause of aseptic meningitis in endemic regions, phleboviruses are inadequately considered by physicians and are frequently underestimated. However, climate changes, environmental conditions and anthropogenic factors, such as increased trade and travel of humans and animals, have a considerable impact on the distribution of vectors. This, together with the virus’ propensity for mutations, reassortment and recombinations of its three-segmented genome increases the risk for virus introduction and makes it probable that phleboviruses will extend their geographic range. Most studies document the distribution of sand fly-borne phleboviruses in Western Europe, while data for Eastern Europe, the Middle East and Africa are very limited. The EDENext review by Alkan et al. (2013) summarizes the geographic spread of sand fly-borne phleboviruses with a focus on understudied regions and discusses possible countermeasures and the need to conduct studies aimed at developing new antiviral drugs and vaccines. In terms of Public Health, their potential as emerging pathogens should raise our awareness and highlight the need to understand not only the complex nature of sand fly-borne viruses and but also the biological significance of possible interactions between Leishmania parasites and phleboviruses.

Alkan et al (2013). Sand fly-borne phleboviruses of Eurasia and Africa: epidemiology, genetic diversity, geographic range, control measures. Antiviral Res. 100(1):54-74. Doi: 10.1016/j.antiviral.2013.07.005.

5. Culicoides-borne diseases
The impact of biting midges on human Public Health in Europe
Carpenter et al. (2013) considers the impact of biting midges of the genus Culicoides on human health in Europe. Special focus is placed on their potential for transmitting human pathogenic arboviruses. To date, the only arboviruses identified as being primarily transmitted by Culicoides between humans are Oropouche virus (OROV) and the Iquitos virus, although the latter has not yet been confirmed. These two members of the family Orthobunyaviridae cause major epidemics of febrile illness in human populations of South and Central America and the Caribbean. The authors describe factors promoting sustained outbreaks of OROV in Brazil from an entomological perspective and assess areas of the epidemiology of this arbovirus that are currently poorly understood, but may influence the risk of incursion into Europe. Additionally, evidence implicating Culicoides in the transmission of other zoonotic infections is examined and placed in context with the presence of other vector groups in Europe.
Carpenter et al (2013). Culicoides biting midges, arboviruses and public health in Europe. Antiviral Res.:102-13. Doi: 10.1016/j.antiviral.2013.07.020.
Leishmania vector competence of biting midges
Leishmaniases are diseases caused by various species of the trypanosomatid protozoa genus Leishmania. They can cause various symptoms, ranging from a cutaneous form that leads to cutaneous ulcera at the bite site to a highly pathogenic visceral form. Millions of human cases are reported from Asia, Africa, South and Central America as well southern Europe every year. Female biting sand flies of the genus Phlebotomus in the Old World or Lutzomyia in the New World are currently the only known vectors of Leishmania sp. Recent observations of Australian midges transmitting Leishmania may indicate that biting midges might also play a role as potential vectors. To investigate this theory, Seblova et al. (2012) experimentally infected the colonized biting midge species Culicoides nubeculosus with two human pathogenic Leishmania species (L. infantum and L. major). An early stage of Leishmania development was demonstrated in the midge mid-gut until two days after feeding, but a subsequent loss of parasites occurred indicating that C. nubeculosus is unlikely to act as a competent vector.

Seblova et al (2012). Development of Leishmania parasites in Culicoides nubeculosus (Diptera: Ceratopogonidae) and implications for screening vector competence. J Med Entomol. 49(5):967-70.

Culicoides biology and ecology in Senegal
The first systematic survey of Culicoides has been implemented in Senegal using a wide range of monitoring techniques. In addition to providing detailed phylogenetic analyses (Bakhoum et al. 2013) both spatial and temporal surveys of Culicoides on livestock were successfully completed in a region where African horse sickness virus is of high epidemiological importance (Fall et al. 2015a; Fall et al. 2015b; Fall et al. 2015c; Fall 2015d; Diarra et al. 2014; Diarra et al. 2015). These studies are of Public Health interest in providing baseline data regarding Culicoides populations and their overlap with human populations. In central Africa, certain biting midge species may act as a huge nuisance for human populations and are able to transmit human filarial nematodes. It is highly likely that future studies will examine the impact of Culicoides in transmission of human pathogenic arboviruses within Africa following the implementation of virus discovery via next-generation sequencing and these studies provide a strong baseline for these approaches.

Bakhoum et al (2013). First Record of Culicoides oxystoma Kieffer and Diversity of Species within the Schultzei Group of Culicoides Latreille (Diptera: Ceratopogonidae) Biting Midges in Senegal. PLoS ONE 8(12): e84316. Doi: 10.1371/journal.pone.0084316.
Fall et al (2015). Culicoides (Diptera: Ceratopogonidae) midges, the vectors of African horse sickness virus – a host/vector contact study in the Niayes area of Senegal. Parasites & Vectors, 8:39. Doi: 10.1186/s13071-014-0624-1.
Fall et al (2015). Circadian activity of Culicoides oxystoma (Diptera: Ceratopogonidae), potential vector of bluetongue and African horse sickness viruses in the Niayes area, Senegal. Parasitology Research, 1-8.
Fall et al (2015). Host preferences and circadian rhythm of Culicoides (Diptera: Ceratopogonidae), vectors of African horse sickness and bluetongue viruses in Senegal. Acta Tropica, 2015, 149: 239-245.
Fall, M (2015). Ecologie et lutte contre les Culicoides vecteurs de la peste équine et de la fièvre catarrhale ovine au Sénégal (PhD thesis). Université Cheikh Anta Diop de Dakar, école doctorale Sciences de la Vie, de la Santé et de l'Environnement, Faculté des Sciences et Techniques.
Diarra et al (2014). Seasonal dynamics of Culicoides (Diptera: Ceratopogonidae) biting midges, potential vectors of African horse sickness and bluetongue viruses in the Niayes area of Senegal. Parasites & Vectors, 7:147. Doi: 10.1186/1756-3305-7-147.
Diarra et al (2015). Modelling the abundances of two major Culicoides (Diptera: Ceratopogonidae) species in the Niayes area of Senegal. PLoS ONE, 10 (6): e0131021.

Potential Impact:

1. Academic impact
For research projects, important impact indicators are the number and quality of articles published in international, peer-reviewed journals. With this respect, EDENext has been quite successful, with > 450 submitted papers and PhD dissertations as of August 2015, 329 of which being already released. Moreover, the quality of these papers fully complies with international standards. Indeed, 285 papers were published in international journal. Their median impact factor is 3.3 (vs. 2.6 in EDEN), with an interquartile range of [2.5; 3.5] ([2.3; 3.6] in EDEN), and outliers up to 42.4 (23.3 in EDEN) [3].
While the two most popular journals (Parasites & Vectors, and Vector-Borne and Zoonotic Diseases, totaling more than 16% of published papers) are specialized in the domain of vectors and vector-borne infections, this is not the case for many of the others. Indeed, journals from the PLoS family, as well as Eurosurveillance, or Emerging Infectious Disease are more general in their scope, with a strong orientation toward public health for the two latter. This is an indication of the special effort made by scientists involved in EDEN and EDENext to have a positive impact on public health.
With this respect, the most important findings with an actual or potential effect on public health are outlined in the former section of this document. The transmission of vector-borne infections in animal and human populations was addressed in many papers. Indeed, most vector-borne infections are zoonotic, i.e. common to animals and humans. The most cited pathogens belong to this category: Hantaviruses, tick-borne encephalitis virus (TBEV), West Nile virus and some Leishmania species (e.g. L. infantum). Moreover, though many publications focused on European ecosystems, Africa is well represented. For instance, Senegal is involved in 18 papers.

2. Networking and expertise
Improved networking is another major impact of EDEN and EDENext. We have several evidences of this positive impact, at different levels. A social network analysis of authors and their affiliation (institutions) linked in EDENext papers shows characteristic features of a scale-free network which is known to provide an optimal pattern for knowledge transfer. Therefore, institutions at the heart of the network are in a very good position to transfer knowledge and skills to their partners in the network.
Moreover, all network indices (number of institutions, number of links, degrees, closeness, betweenness...) improved between EDEN and EDENext: networking was more intense after 10 years of projects, with an obvious snowball effect aggregating much more research and public-health institutions than those which actually received funds from the Commission in the frame of EDEN and EDENext. For instance, 344 institutions were involved in the EDENext network of institutions while only 58 of them were funded in EDEN and EDENext, as a whole.
Beyond research, this network of scientists and institutions was formalized and utilized for expertizes at the European level. A “European network for arthropod vector surveillance for human public health” (VBORNET) was first set up and funded by the European Centre for Disease Prevention and Control (ECDC, Stockholm) to gather 400 medical entomologists and public-health specialists of vector-borne infections. It was coordinated by AVIA-GIS (Belgium) during its existence between 2009 and 2014. The network was used to collect, validate and map data regarding the present distribution of main vector species of public-health interest. Thus, maps were produced for 5 invasive mosquito species, 7 endemic mosquito species, 6 tick species, and 10 Phlebotomine sandfly species. They are available on ECDC website.
VBORNET has now being superseded by the “European network for sharing data on the geographic distribution of arthropod vectors, transmitting human and animal disease agents” (VECTORNET) – still coordinated by AVIA-GIS. VECTORNET has a wider scope than VBORNET, encompassing medical and veterinary public health and economics in the same domain. It is co-funded by ECDC and the European Food Safety Authority (EFSA) over a four-year period (2014-2018). Its geographical scope is wider, now covering the whole Mediterranean Basin. It also includes vectors of veterinary importance, such as Culicoides biting midges.
Products of these networks are now routinely used by ECDC, EFSA, and national public-health or veterinary services to write review papers, to feed risk analyses and more generally to provide information for a better control of vector-borne epidemics and epizootics, with a clear “One Health” perspective.

3. Capacity building
Increased European collaboration is one most important impact of EDEN and EDENext: it would not have happened without them, without that financing. Also, working in this network of collaboration opens up the training of a young generation of scientists, who from the beginning have learned to work on a European or global-scale network, and they know each other.
Indeed, before EDEN and EDENext, there was a huge gap in Europe of people working in the field. Especially because of the pressure on publications, there was more and more emphasis on lab work and having results as quickly as possible, publish in the biggest possible journals. It was all about genetics and bioinformatics. So, for a young student looking for a career, it was not very appealing to go the field and conducting two years of sampling and coming up with some graphs and linking this to a few factors was not very appealing.
Given this framework, the fact that EDEN/EDENext was such a large network, covering all of Europe, suddenly made the topic sexy in the sense that it became interesting for PhD students, especially because they were not working alone. As a matter of fact, it very quickly became obvious that a PhD network should be useful, and in the first year meeting it got organized (EDEN project, Rovaniemi, Jan 2006). PhD students got their specific time where they could discuss between themselves their results and the idea of having a PhD group became very concrete. Also, there was a PhD prize for the best presentation from a PhD student. It was a significant prize so for the PhD students it was very attractive. Progressively, this PhD meeting (held during the annual general meeting) became more like a summer school. In EDENext it was very much embedded in the project, the PhD group was even more like a group than in EDEN. As a whole, 93 PhD dissertations were written within the two projects (45 in EDEN, 48 in EDENext). It means that over the past 10 years, EDEN and EDENext have put a stamp of quality on a whole generation of PhD students. For any kind of research in the future they have a network ready. When they make applications, they can combine themselves in a reasonable way. This kind of European-scale collaboration would not be possible in the USA, for example.

4. Data sharing
In EDENext, everyone has understood the value of collaboration from the very beginning. In many cases, teams collected material and then one team would analyze it, but all are co-authors, so that makes it efficient and high quality.
Indeed, EDEN and EDENext both promoted scientific collaboration at a multi-disciplinary and cross-country level. The framework was ambitious, bringing together a wide variety of scientists from different disciplines and across Europe. What was even more ambitious was to convince them to share the data and to have a commercial company host this platform.
Sharing was not only via personal interaction and capacity building, but also on a more practical level: sharing of scientific data on a common platform now amounting to more than 2,000 datasets. At the onset of EDEN the idea of data sharing seemed frightening to some, idealistic to others, while at the end of EDENext it is accepted as normal.
Seeing other partners open up their historic databases collected for various scientific purposes, ranging from molecular research to spatial modelling, inspired others to do the same thing. Trust was built within the network creating an atmosphere where people were comfortable to discuss, share and publish together. Just looking at the number of papers published (450 submitted EDENext papers up to the end of August 2015, and 269 from EDEN) and the geographical range covered by field activities, it is undeniable that actively sharing information means a higher output can be achieved.
Having Euro-AEGIS, a European Economic Interest Grouping composed of two private companies, Avia-GIS (Zoersel, Belgium) and ERGO (Oxford, UK), hosting the platform seemed to benefit all too, although this may have seemed odd in the beginning. Still as it turned out, the scientists could focus on research and not worry about where which data was stored and if it would remain up-to -date as these technical tasks were taken care of. In addition, Euro-AEGIS was not a competitor in terms of initiating publications and provided training in GIS and spatial modelling to help improve the content of publications. Finally, using this database as a method to ensure that the data, collected with a lot of effort in the field or in the lab, remain available to all partners involved beyond the end of this or any other multinational and multidisciplinary project, and does not get lost in oblivion, which may even be the most important reason to build and maintain it.

List of Websites:
Public website address and contact details
Master website: http://www.edenext.eu/
Data portal: http://www.edenextdata.com/

EDENext coordination team

- Renaud Lancelot [renaud.lancelot@cirad.fr] Coordinator
- Olivier Pierre [olivier.pierre@cirad.fr] Financial and Administrative Officer
- Sylvie Laurens [sylvie.laurens@cirad.fr] Administrative Officer

CIRAD-Département Systèmes Biologiques
EDENext Secretariat
TA A-15/B Campus de Baillarguet
34398 Montpellier Cedex 5
France

Phone: 33 4 67 59 37 37
Fax: 33 4 67 59 37 98