# The future of research on tick-borne pathogens: shifting from the 'one pathogen-one disease' vision to the pathobiome paradigm

Muriel Taussat, Maria Kazimirova, Zdenek Hubalek, Sándor Hornok, Róbert Farkas, Sarah Bonnet, Gwenaël Vourc'h, Andrei D. Mihalca, Olivier Plantard, Cornelia Silaghi, et al.

► To cite this version:

Muriel Taussat, Maria Kazimirova, Zdenek Hubalek, Sándor Hornok, Róbert Farkas, et al. The future of research on tick-borne pathogens: shifting from the ‘one pathogen-one disease’ vision to the pathobiome paradigm. The impact of a decade (2004-2015) of research on vector-borne diseases, 2015. **hal-02798722**

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# EDEN & EDENext: the impact of a decade of research (2004-2015) on vector-borne diseases

BOOK · JANUARY 2015

DOI: 10.13140/RG.2.1.4382.6400

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<figure><img src="image_2.png" /></figure>

Ozge erisoz kasap

Hacettepe University

23 PUBLICATIONS

186 CITATIONS

SEE PROFILE
<figure><img src="image_3.png" /></figure>

EDEN

<figure><img src="image_4.png" /></figure>

EDENext

forest
abundance
culicoides trap
plasmodium tick-borne
genome
protein
bird

parasite rna blood
prevalence vector
natural
model vole phlebotomus
perniciosus
leishmania density focus

virus
infection
zoonosis
rodent
saliva
canine
phagocytophilum
questing serology

# The impact of a decade (2004-2015) of research on vector-borne diseases

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

horse presence
complex landscape
environment
deer borrelia
ecology
season
emergence
human
infection
population
glareolus
phagocytophilum
dna
mosquito
outbreak
control
fever
spatial
risk
wild temperature
incidence
phlebovirus
spread
diptera
mouse
dynamics
identification
molecular
lineage
season
pattern
sequence
africa
cycle
anopheles map
variation
animal
distribution
climate dog

<figure><img src="image_7.png" /></figure>

bluetongue
myodes sandfly antibody
vector-borne
change nymph tick-borne-encephalitis
reservoir
species pathogen
hantavirus
europe
infantum
detection
epidemiology
tick public-health
transmission
circulation

<figure><img src="image_8.png" /></figure>

SIXTH FRAMEWORK PROGRAMME

SEVENTH FRAMEWORK PROGRAMME
**Authors:**

Neil Alexander, Alberto Allepuz, Bulent Alten, Rene Bødker, Sarah Bonnet, Simon Carpenter, Catherine Cêtre-Sossah, Emilie Chirouze, Jérôme Depaquit, Kerstin Dressel, Els Ducheyne, Vit Dvorak, Ozge Erisoz Kasap, Yvonne Gall, Assane Gueye Fall, Robert Farkas, Jordi Figuerola, Claire Garros, Martin H. Groschup, Petr Halada, Guy Hendrickx, Heikki Henttonen, Kristyna Hlavackova, Sándor Hornok, Zdenek Hubalek, Nicole Iltis, Maria Kazimirova, Nils Kley, Marie-Christine Lambert, Renaud Lancelot, Andrei Daniel Mihalca, Miguel Miranda, Sebastian Napp, Matthias Niedrig, Olivier Plantard, Bethan V. Purse, Annapaola Rizzoli, David J. Rogers, Katja Schmidt, Kate R. Searle, Cornelia Silaghi, Tarja Sironen, Muriel Vayssier-Taussat, Petr Volf, Jan Votypka, Gwenaël Vourch, Steven White, William Wint.

**Co-Editors:** Renaud Lancelot, Sylvie Laurens and Andrew Lewer

With the collaboration of: Marieta Braks, Simon Carpenter, Guy Hendrickx and Heikki Henttonen

**Graphic design and layout:** Laurence Laffont

EDEN (GOCE-CT-2003-010284) is an Integrated Project (2004-2009) of the 6th Framework Programme (FP6) funded by the European Commission under the Research DG, Environment Directorate, priority 6.3: Global Change and Ecosystems EDENext (261504) is a research project (2011-2014) of the 7th Framework Programme (FP7) funded by the European Commission under the Health component.

This book was funded by EU grant FP7-261504 EDENext and is catalogued by the EDENext Steering Committee as EDENext420 (http://www.edenext.eu). The contents of this publication are the sole responsibility of the authors and don't necessarily reflect the views of the European Commission.

© CIRAD 2015 – ISBN: 978-2-87614-707-2
The impact
of a decade (2004-2015)
of research
on vector-borne diseases
Preface

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 book 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 2015. 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.

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 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 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.

In this book, we present the impacts of these two projects. We start with an interview with EDENext coordinator Renaud Lancelot (CIRAD, France) and examine the work of the vector and rodent groups, presented at the GERI conference (Genes, Ecosystems and Risk of Infection) in 2015, accompanied by updates of PUBLICise Health, EDENext's telegrams reporting on the project's research results with a particular relevance for Public Health. We also examine in greater detail the works of the data management, modelling and Public Health teams, interspersed with interviews with various leading figures who have been involved in the projects. In order to measure the academic impact of these projects, we also present a bibliometric study, encompassing more than 500 papers published in scientific journals over the past 10 years, and examples of other kinds of impacts of these projects.

We hope you find this book an interesting and stimulating example of what can be achieved when highly motivated teams are given the opportunity to work together for a long period of time. This opportunity would not, of course, have been possible without funding under the European Commission's Sixth and Seventh Framework programmes and we sincerely thank the Commission for the confidence shown in the EDEN and EDENext projects. We hope you find the book thought-provoking, too, with regards to the future. Ongoing environmental, social and economic changes mean that vector-borne diseases – and other diseases sensitive to these changes – will be high on the agenda for the foreseeable future.
# TABLE OF CONTENTS

<table><tr><td>Introduction: 10 years, 58 partners, 23 countries</td><td>7</td></tr><tr><td>Interview: Renaud Lancelot, EDENext coordinator (CIRAD, France)</td><td>8</td></tr></table>

## SCIENTIFIC ADVANCES FOR BETTER PUBLIC HEALTH

<table><tr><td>Many faces of rodent-borne infections in Europe</td><td>10</td></tr><tr><td>PUBLICise Health Public Health Telegram: Rodent and insectivore-borne diseases</td><td>17</td></tr><tr><td>Interview: Heikki Henttonen (LUKE, Finland)</td><td>34</td></tr><tr><td>The future of research on tick-borne pathogens: shifting from the 'one pathogen-one disease' vision to the pathobiome paradigm</td><td>35</td></tr><tr><td>PUBLICise Health Public Health Telegram: Tick-borne diseases</td><td>43</td></tr><tr><td>Molecular taxonomy of sand flies: which species are out there and how can we find out?</td><td>53</td></tr><tr><td>PUBLICise Health Public Health Telegram: Phlebotomine-borne diseases</td><td>60</td></tr><tr><td>Interview: Maria Antoniou (University of Crete, Greece)</td><td>73</td></tr><tr><td>Understanding the ecology of West Nile virus transmission in Europe</td><td>74</td></tr><tr><td>PUBLICise Health Public Health Telegram: Mosquito-borne diseases</td><td>79</td></tr><tr><td>Ecology and eco-epidemiology of Culicoides-borne diseases: what have we learnt and where do we go from here?</td><td>83</td></tr><tr><td>PUBLICise Health Public Health Telegram: Culicoides-borne diseases</td><td>90</td></tr></table>

## EXAMPLES OF EDEN AND EDENEXT IMPACTS

<table><tr><td>Four kinds of EDEN and EDENext impacts</td><td>94</td></tr><tr><td>Sharing scientific data: 1+1=3</td><td>101</td></tr><tr><td>Vector-borne disease models: R₀ and beyond</td><td>109</td></tr><tr><td>Interview: Guy Hendrickx (Avia-GIS, Belgium)</td><td>117</td></tr></table>

## PUBLIC HEALTH PERSPECTIVES

<table><tr><td>Public Health perspectives on vector-borne infections in Europe</td><td>120</td></tr><tr><td>Interview: Marieta Braks (RIVM, Netherlands)</td><td>129</td></tr><tr><td>Interview: Hervé Zeller (ECDC, Sweden)</td><td>131</td></tr></table>

<footnote n="1">¹ Endnote</footnote>

<footnote n="2">² Bio</footnote>
Introduction: 10 years,
58 partners, 23 countries

EDEN<sup>1</sup> (2004-2010) and EDENext<sup>2</sup> (2011-2015) were two large collaborative research projects funded by the European Commission (Directorate-General for Research and Innovation) in Framework Programmes 6 and 7. They were aimed at improving knowledge as well as surveillance and control tools regarding vector-borne infections in Europe and neighbouring territories. In EDEN, more emphasis was put on understanding the effect of environmental changes on the emergence of these infections, and characterising those ecosystems at high risk of emergence. In EDENext, the focus was more on understanding the biological processes, and preparing the transfer of research results to Public Health.

(a) Geographical reach

<figure><img src="image_9.png" /></figure>

(b) Organisation of the work

<figure><img src="image_10.png" /></figure>

**WP:** subdivision of EDENext work programme into work packages

**TBD, RBD, MBD, PhBD, CBD:** vector or wild host groups for (i) ticks, (ii) rodents, (iii) mosquitoes, (iv) Phlebotomine sand flies and (v) *Culicoides* biting midges, respectively.

Figure 1. The geographical reach of EDEN and EDENext (a) and work organisation (b). The coloured layer on the map represents biomes covered by the two projects. The organisational diagram refers to EDENext (the EDEN diagram was very similar).

As a whole, EDEN and EDENext involved 58 partners from 23 countries in Europe, the Middle East, and Africa (Figure 1a). To implement and manage the work programme negotiated with the Commission, a matrix-like structure was adopted (Figure 1b) with vector groups as the 'columns', and integrative teams as the 'rows'. This structure was adopted to facilitate the harmoni-sation of field and laboratory work, as well as the dissemination of methods and tools.

<footnote n="1">¹ Emerging diseases in a changing European environment</footnote>

<footnote n="2">² Biology and control of vector-borne infections in Europe</footnote>
## Interview: Renaud Lancelot

<figure><img src="image_11.png" /></figure>

Renaud Lancelot is a French veterinary epidemiologist with a PhD in population biology and ecology. He is the deputy director, and epidemiology team leader, of the INRA-CIRAD Emerging and Exotic Animal Disease Control research unit (Montpellier, France, see umr-cmaee.cirad.fr). He has 30 years of experience in the epidemiology of animal, human and zoonotic diseases, including 20 years of field work in French Guiana, Mauritania, Chad, Senegal and Madagascar. He has been coordinating the large collaborative European projects FP6 EDEN (Emerging diseases in a changing European environment) and FP7 EDENext (Biology and control of vector-borne infections) since 2006 (www.edenext.eu). His current research interests are in the epidemiology and modelling of peste des petits ruminants, Rift Valley fever, and tsetse flies. He is the author of more than 160 scientific publications, communications, books and statistical software.

### Why conduct so much research on vector-borne diseases?

**Renaud Lancelot (EDENext coordinator, CIRAD, France):** "The big question at the beginning of EDEN was to assess the possible role of environmental changes on the emergence of new diseases. We decided to work on vectors and vector-borne diseases because vectors, insects or ticks for example, are very sensitive to environmental conditions and therefore are supposed to be very sensitive to changes in these conditions. That is the basic reason we decided to work on vectors and vector-borne infections. The other reason was that at this time, there was a poor availability of scientists working on vectors and vector-borne infections. Moreover, they had few opportunities to work together. It was a neglected area in European research. EDEN allowed the gap to be filled and increased European capacity in this field."

### Can you tell us something about the impact of EDEN and EDENext?

**Renaud** "In such huge projects it is quite difficult to make a list of all the possible impacts. I would say that there are three main domains. The first one is the impact on the scientific audience, via papers in peer-reviewed journals. During EDEN and EDENext we have published more than 500 papers in scientific journals and their average impact factor is above three. It is a very good score indeed, and we have had several outstanding papers in journals like Nature. The second domain is expertise and the impact on Public Health. Because the project was huge, involving lots of different partners from most European countries, we had the possibility to construct a network of all these scientists. This network was efficient at doing research, but it was also quite visible at the European level. It was used by the European Commission to form an expertise network to assess the risk of disease introduction and spread. The first-generation network, called VBORNET, ran between 2008 and 2013. Recently it was turned into a veterinary and Public Health network called VECTORNET. It brings together most of the European teams working on vectors and vector-borne diseases. It is used to answer specific questions asked by ECDC (European Centre for Disease Prevention and Control) and EFSA (European Food Safety Authority). The third domain is capacity building and we were able to implement and support more than 90 PhD theses. This means a whole generation of young scientists has been raised by EDEN and EDENext. We can consider that the impact of the project will be very large over the next 20 to 30 years because all these young people have used the same methods, the same network and they know each other. We can be sure that when applications are to be made for projects they will contact each other and enter into the projects together."

### What about the future?

"In our quickly changing world, it is quite important that the Commission funds long-term research on issues related to environment and health. Vector-borne diseases are only a part of the problem. There are many other diseases which are not borne by vectors but are related to the environment and we know we will have many such disease emergences in the future. This makes it important for the scientific community to be ready to address the questions and, for the Commission, it is important to have large networks at hand, which can be reactive when there is a disease emergence and can be mobilised to find solutions to solve the problem when and where it occurs. I would also add that the geographical scope is much wider than Europe. In particular, we must help developing countries to control emerging diseases on their own, to protect local populations and limit the risk of spread across their borders."

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SCIENTIFIC ADVANCES
FOR BETTER PUBLIC
HEALTH
Tarja Sironen¹, Heikki Henttonen²
and the EDENext rodent and
insectivore-borne disease group

¹ Department of Virology, University of Helsinki, Finland

² Natural Resources Institute, Finland

Many faces of rodent diseases

INTRODUCTION

During the EDEN and EDENext projects, the rodent-borne and subsequent rodent and insectivore-borne disease groups have, in collaboration, assessed the current diversity, distribution and prevalence of important rodent-borne pathogens in Europe. Among the rodent and insectivore-borne diseases, hantavirus (family Bunyaviridae) infections pose a significant threat to public health in Europe and elsewhere. Some hantaviruses have a high case-fatality rate and many others represent a considerable disease burden. Hantavirus infections are emerging in new areas and outbreaks have been reported in established endemic regions such as Germany and Sweden. Beyond their role in transmitting infections directly, rodents and insectivores also serve as amplifying hosts of many arthropod-borne microbes, in particular tick-borne pathogens, many of which are spreading in Europe due to environmental change. What are the key characteristics in the life cycle of a rodent-borne virus that determine its persistence and ability to cause epidemics in humans? How are different rodent-borne viruses maintained in their reservoir host population? What factors affect spillover to humans? How do changes in the environment (for example, due to climate change or anthropogenic activities) affect the prevalence and distribution of pathogens? Can we model the dynamics and predict the behaviour of rodent-borne infections? Answers to these questions are sought at the level of (see Figure 1): the (1) virus, (2) individual host, (3) reservoir host species, and (4) host populations in different geographical regions

THE VIRUS

Rodent-borne viruses have a world-wide distribution with significant disease burden caused by hantaviruses and arenaviruses in particular (Goijenbier 2013). Both of these viral families are considered to be highly host-specific and almost exclusively rodent-borne. These viruses typically exhibit asymptomatic life-long infections in rodent hosts but can induce severe disease if they spill over into the local human population. Recent findings have suggested new host taxa for both viral families, for example, novel arenaviruses have been found in snakes and hantaviruses are now known to occur in shrews, moles and bats. Whether these new hosts carry viruses pathogenic to humans is unknown, and in most cases we are still lacking the tools to address this question.

Hantaviruses cause hantavirus cardiopulmonary syndrome (HCPS) in the Americas and haemorrhagic fever with renal syndrome (HFRS) in Europe and Asia (Jonsson 2010). In the Americas, hantavirus infections are rare but severe, and the case-fatality rate of Andes and Sin Nombre hantavirus infections is 30-40%. In Europe and Russia, Puumala virus (PUUV) carried by bank voles (*Myodes glareolus*) is the major hantavirus pathogen, causing thousands of infections every year with an emerging pattern in Central Europe (Heyman et al. 2011). The mild HFRS due to PUUV is often called nephropathia epidemica (NE). In Europe, Dobrava (DOBV) and Seoul (SEOV) hantaviruses also infect humans. SEOV is a global pathogen due to the worldwide distribution and movement of its host, the brown rat (*Rattus norvegicus*). During the winter of 2012-13, human cases of severe HFRS caused by SEOV were reported in the UK and France. Some of these infections were associated with pet rats, and SEOV has also been detected in domestic rats in Sweden (Lundkvist et al. 2013). In contrast to the persistent infection of rodent reservoirs, human hantavirus infection is transient and self-limiting. Humans typically inhale infectious aerosols of animal excreta. While capillary leakage is a hallmark of hantaviral diseases and it appears to be caused by direct viral effects and immunopathological mechanisms, specific details of pathogenesis are poorly understood (Vaheri 2013).

Are rodent reservoir hosts considered to have a role in the spread of zoonotic viruses? A review of the literature shows that the majority of rodent reservoir hosts are thought to be responsible for spread of zoonotic viruses. During the last decade, however, a number of studies have shown that reservoir hosts may play a more important role in the spread of zoonotic viruses. This review aims to provide an overview of the current understanding of the role of reservoir hosts in the spread of zoonotic viruses.
## Euro dent-borne infections in Europe

Arenaviruses comprise a genus in the family Arenaviridae and have all been considered rodent-borne viruses with the exception of Tacaribe virus, which has been isolated from bats (Charrel et al. 2011). Similar to hantaviruses, arenaviruses are considered to be quite host-specific. Recent analyses have suggested that, on an evolutionary time-scale, arenaviruses have crossed the species barrier more frequently and easily than previously thought and that diversification might have been influenced by geographical proximity more than the relatedness of hosts (Irwin et al. 2012). Serological evidence for arenavirus infections is frequently reported for many different rodent species, but arenavirus genomes are rarely recovered. This might be due to low viral load in spillover infections or there might be a variety of arenaviruses circulating that are simply not detected with the current techniques. During EDEN and EDENext, lymphocytic choriomeningitis virus-like sequences have been detected at a low prevalence in bank voles (unpublished observations) and wood mice (*Apodemus sylvaticus*) (Ledesma et al. 2009).

Arenaviruses can be transmitted to humans primarily through aerosolized rodent excreta, but transmission via contaminated food or drink and through wounds or skin abrasions is possible (Moraz et al. 2011). Several arenaviruses are known human pathogens, with Lassa virus being responsible for most human infections. Of the known arenaviruses, lymphocytic choriomeningitis virus (LCMV) is found in Europe. Recently, we and others - to our great surprise - have detected arenaviruses in boid snakes where infections manifest as boid inclusion body disease (BIBD) (Hetzel et al. 2013). The prevalence of the arenaviruses that cause the disease in wild snake populations and the origins of BIBD remain unknown. It has been hypothesised that snakes caught the virus from rodents they had eaten, but the known lineages infecting these taxa are distantly related and inferences remain unclear.

Cowpox virus (CPXV) belongs to the genus Orthopoxvirus (OPV) and shares a cross-reacting immunoresponse with smallpox, monkeypox and vaccinia (VACV) viruses. As vaccinations against smallpox have been scaled back, the incidence of OPV infections seems to be rising. CPXV is transmitted to humans sporadically from its rodent reservoirs and a high CPXV seroprevalence has been observed in field voles (*Microtus agrestis*), bank voles and wood mice. In Finland, CPXV antibodies can be locally common in field voles, and the antibody prevalence shows a delayed density dependence in spring and direct density dependence in the autumn. In Europe, it seems that the bank vole has the highest seroprevalence although many other

## REFERENCES

Bennett SN, Gu SH, Kang HJ, Arai S, Yanagihara R (2014). Reconstructing the evolutionary origins and phylogeography of hantaviruses. *Trends Microbiol* 22, 473-482.

Charbonnel N, Pages M, Sironen T, Henttonen H, Vapalahti O, Mustonen J, Vaheri A (2014). Immunogenetic factors affecting susceptibility of humans and rodents to hantaviruses and the clinical course of hantaviral disease in humans. *Viruses* 6, 2214-2241.

Charrel RN, Coutard B, Baronti C, Canard B, Nougairede A, Frangeul A, Morin B, Jamal S, Schmidt CL and other authors (2011). Arenaviruses and hantaviruses: From epidemiology and genomics to antivirals. *Antiviral Res* 90, 102-114.

Forbes KM, Voutilainen L, Jääskeläinen AJ, Sironen T, Kinnunen PM, Stuart P, Vapalahti O, Henttonen H, Huitu O (2014). Serological survey of rodent-borne viruses in Finnish field voles. *Vector Borne Zoonotic Dis* 14, 278-283.

Goeijenbier M, Wagenaar J, Goris M, Martina B, Henttonen H, Vaheri A, Reusken C, Hartskeerl R, Osterhaus A, Van Gorp E (2013). Rodent-borne hemorrhagic fevers: Under-recognized, widely spread and preventable - epidemiology, diagnostics and treatment. *Crit Rev Microbiol* 39, 26-42.

Guivier E, Galan M, Henttonen H, Cosson JF, Charbonnel N (2014). Landscape features and helminth co-infection shape bank vole immunoheterogeneity, with consequences for Puumala virus epidemiology. *Heredity* (Edinb) 112, 274-281.

Hetzel U, Sironen T, Laurinmaki P, Liljeroos L, Patjas A, Henttonen H, Vaheri A, Artelt A, Kipar A and other authors (2013). Isolation, identification, and characterization of novel arenaviruses, the etiological agents of boid inclusion body disease. *J Virol* 87, 10918-10935.

Heyman P, Ceianu CS, Christova I, Tordo N, Beersma M, Joao Alves M, Lundkvist A, Hukic M, Papa A and other authors (2011). A five-year perspective on the situation of haemorrhagic fever with renal syndrome and status of the hantavirus reservoirs in Europe, 2005-2010. *Euro Surveill* 16, 19961.

Irwin NR, Bayerlova M, Missa O, Martinkova N (2012). Complex patterns of host switching in new world arenaviruses. *Mol Ecol* 21, 4137-4150.

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during the first month after seroconversion and continued throughout the study period with only a slight decline. The quantity of PUUV shed over time was constant. Our findings contradict the prevailing view of a decline in virus shedding after the acute phase, and of a short viraemia - assumptions widely made in epidemiological models of hantaviruses. Life-long shedding could enable hantaviruses to survive the population bottlenecks that are commonly observed in their rodent hosts, and promote virus dispersal in fragmented habitats where local host and/or virus populations face frequent extirpation. These findings suggest that the abundance of infective hosts, which is the most important driver of human hantavirus epidemics, may be underestimated in current simulation models. Whatever the underlying reasons for the discrepancy between experimental and natural infections, our results stress the importance of studying zoonotic infections in natural settings. Further investigation of hantavirus shedding by wild hosts in other virus-host pairs is urgently needed and the assumptions in transmission models need to be updated. Embracing these recommendations will lead to improved techniques to predict the threat posed by hantavirus to Public Health.

## RESERVOIR HOST SPECIES

Rodent species exhibit different capacities to act as reservoirs of hantaviruses. Hantavirus infections are believed to be asymptomatic and chronic in their rodent hosts. Interestingly, hantavirus has yet to be detected in several rodent species that appear to be suitable reservoirs. This may be due to a lack of exposure to the virus or inadequate sampling. Indeed, hantavirus infections in new reservoir hosts have recently been reported in poorly sampled regions, for example, Sangassou and other hantaviruses in African rodents. A variety of new hantaviruses has been detected in shrews, moles and bats (Yanagihara et al. 2014), and these have been established as important reservoirs for hantaviruses. Non-reservoir species may also be those that die soon after infection, hence limiting the probability of detecting positive individuals in wild populations, or those in which hantavirus is unable to enter and/or replicate within cells. This would also include spillover infections in which the virus infects a non-reservoir host, as evidenced by an antibody response, but the virus is not transmitted further. Concepts of primary and secondary species, reservoir and spillover hosts are ambiguous, and detecting antibodies or RNA should not be considered as evidence of a role in the circulation of hantavirus.

Hantaviruses have been widely believed to have co-evolved with their reservoir hosts (Plyusnin and Sironen 2014). During active surveillance, numerous spillover events were observed and multiple hosts for a given hantavirus, or multiple hantavirus species in a single host species, have been proposed. The common vole (*Microtus arvalis*) is considered to be the reservoir host of Tula hantavirus (TULV). Since the first reports, TULV has been found in a number of *Microtus* species including the field vole (*M. agrestis*), the sibling vole (*M. levis*) and water vole (*Arvicola amphibius*) (Schlegel et al. 2012). The field vole might also host the recently detected

Klempa B, Avsic-Zupanc T, Clement J, Dzagurova TK, Henttonen H, Heyman P, Jakab F, Kruger D H, Maes P and other authors (2013). Complex evolution and epidemiology of Dobrava-Belgrade hantavirus: Definition of genotypes and their characteristics. *Arch Virol* 158, 521-529.

Korpela K, Helle P, Henttonen H, Korpimäki E, Koskela E, Ovaskainen O, Pietäinen H, Sundell J, Valkama J, Huitu O (2014). Predator-vole interactions in northern Europe: the role of small mustelids revised. *Proc Biol Sci* 281, 10.1098/rspb.2014.2119.

Ledesma J, Fedele CG, Carro F, Lledo L, Sanchez-Seco MP, Tenorio A, Soriguer RC, Saz JV, Dominguez G and other authors (2009). Independent lineage of lymphocytic choriomeningitis virus in wood mice (*Apodemus sylvaticus*), Spain. *Emerg Infect Dis* 15, 1677-1680.

Ling J, Vaheri A, Hepojoki S, Levanov L, Jääskeläinen AJ, Henttonen H, Vapalahti O, Sironen T, Hepojoki J (2015). Serological survey of Seewis virus (SWSV) antibodies in patients suspected for hantavirus infection in Finland - A cross-reaction between Puumala virus antiserum with seewis virus N protein? *J Gen Virol* Mar 18.

Ling J, Sironen T, Voutilainen L, Hepojoki S, Niemimaa J, Isoviita VM, Vaheri A, Henttonen H, Vapalahti O (2014). Hantaviruses in Finnish soricomorphs: Evidence for two distinct hantaviruses carried by *Sorex araneus* suggesting ancient host-switch. *Infect Genet Evol* 27, 51-61.

Lundkvist Å, Verner-Carlsson J, Plyusnina A, Forslund L, Feinstein R, Plyusnin A (2013). Pet rat harbouring Seoul hantavirus in Sweden, June 2013. *Euro Surveill* 18, 20521.

Moraz ML and Kunz S (2011). Pathogenesis of arenavirus hemorrhagic fevers. *Expert Rev Anti Infect Ther* 9, 49-59.

Plyusnin A and Sironen T (2014). Evolution of hantaviruses: Co-speciation with reservoir hosts for more than 100 MYR. *Virus Res* 187, 22-26.

Pounder KC, Begon M, Sironen T, Henttonen H, Watts PC, Voutilainen L, Vapalahti O, Klempa B, Fooks AR, McElhinney LM (2013). Novel hantavirus in field vole, United Kingdom. *Emerg Infect Dis* 19, 673-675.

Schlegel M, Kindler E, Essbauer SS, Wolf R, Thiel J, Groschup MH, Heckel G, Oehme RM, Ulrich RG (2012). Tula virus infections in the Eurasian water vole in central Europe. *Vector Borne Zoonotic Dis* 12, 503-513.

Tersago K, Verhagen R, Vapalahti O, Heyman P, Ducoffre G, Leirs H (2011). Hantavirus outbreak in Western Europe: reservoir host infection dynamics related to human disease patterns. *Epidemiol Infect.* 139(3):381-90.
Tatenale hantavirus (Pounder et al. 2013). These findings emphasise the need for further investigation of the host-specificity of hantaviruses and the potential for host-switching that may be associated with changes in the virulence of these pathogens.

Over the past decade a wide variety of hantaviruses have been detected in shrews and moles throughout the world. Four sori-
comorph-borne hantaviruses circulate in Finland (Ling et al. 2014), including Boginia virus in the water shrew (*Neomys fodiens*)
and Asikkala virus in the pygmy shrew (*Sorex minutus*). Common shrews (*S. araneus*) were found to harbour two different
hantaviruses: Seewis virus (SWSV) and a new distinct and genetically divergent virus (Altai-like virus) which cluster with the
most basal hantaviruses known. This was the first evidence of the coexistence of two clearly distinct hantavirus species circu-
lating simultaneously in a single host species population. These results contradict earlier hypotheses concerning rodent-borne
hantaviruses, and suggest an ancient host-switching event from a yet unknown host to *S. araneus*. These four insectivore-borne
hantaviruses are reported from many European countries, with the addition of Nova virus in common moles (*Talpa europaea*).

In Finland, PUUV (carried by *M. glareolus*) co-circulates with SWSV (carried by *S. araneus*). While PUUV causes 1,000 to 3,000 nephropathia epidemica cases annually, the pathogenicity of SWSV (or any insectivore-borne hantavirus) to man is unknown. To study the prevalence of SWSV antibodies in hantavirus fever-like patient sera, 486 samples (sent for PUUV diagnostics) were screened, but no evidence of SWSV infection was found among them (Ling et al. 2015). It is difficult to prove a negative result, but this data strongly suggests that insectivore-borne hantaviruses do not pose a threat to humans.

THE HOST POPULATION

Epidemiological patterns of human hantavirus infections are related to changes in the reservoir rodent population. Voles typically exhibit pronounced population cycles or more irregular outbreaks with high amplitude. Populations may also display prolonged but transient periods of weak cyclicity (Korpela et al. 2013), which may be reflected in the occurrence of zoonotic diseases. In temperate Europe, masting is an important driver of hantavirus epidemics (mostly due to PUUV) and warm summers are known to induce this effect (Tersago et al. 2011). In contrast, specialist predation is thought be the main factor affecting bank vole populations in northern Europe (Korpela et al. 2014).

During the EDEN and EDENext projects, one of our main aims was to understand the differences in the epidemiology of human hantavirus infections in the different climatic and biotic conditions of boreal and temperate Europe. Clear seasonal differences have been documented in the transmission dynamics of PUUV between the two biomes, and this is also reflected in the seasonality of human epidemics. Transmission of PUUV among bank voles in Finland is seasonal and peak seroprevalences are found in old overwintered voles in late spring-early summer and also in low-density years. Peak densities of positive voles are found in late autumn-early winter, one to two months prior to the peak in human infections. As a factor on which to gauge the imminent threat to Public Health, we recommend the abundance of positive rodents be used rather than rodent seroprevalence alone. Bank vole density, as such, is a reasonable proxy for infection risk.

The potential role played by landscape homogeneity and heterogeneity has been examined in rodent and virus dispersal and with respect to differences in the presence or absence of host threshold densities for PUUV. Landscape genetics have been used to analyse the dispersal of rodent hosts and viruses at different geographic scales, for example, indicating an efficient dispersal in non-fragmented boreal forests. Also, within geographic regions, higher genetic diversity is related to higher PUUV seroprevalence (Cosson et al. ms), reflecting the role of dispersal in homogenous landscapes. The quality of habitat in terms of resources, parasitism, predation and fragmentation may trigger trade-offs affecting the investment of individuals in alternative immunological pathways.

In nature, animals usually have multiple infections and the interactions among pathogens and their succession, including PUUV and CPXV, has been documented (Guivier et al. 2014). Landscape immunogenetics has been used to study immune gene variability among landscapes and its causes, and has provided critical information concerning parasite epidemiology. Several landscape features (for example, habitat quality, metapopulation dynamics) and helminth co-infection were identified and may participate in shaping the immunoheterogeneity of wild host populations (Guivier et al. 2014). In the future, the integration of such information in the analysis of PUUV epidemiology will improve our understanding of the impacts of co-infection on the evolution of PUUV tolerance in bank voles.
One of the main aims has been to develop predictive models of the spread or dispersal of a disease and a spatially explicit model for PUUV infection dynamics has been built. It was used to investigate PUUV infection patterns during vole population cycles, both in the vole (PUUV) and human populations. The model reproduced the natural dynamics observed in the field and showed that the temporal existence of maternal antibodies in the offspring is an important factor influencing the observed infection patterns and the consequent infection risk to humans. Also, it showed that the density dependent spatial behaviour of voles seems to play a less important role than one might expect (Rejniers et al. ms).

Small mammal communities vary geographically and the impact of community structure and diversity of small mammal species, often expressed via the so-called dilution effect, has been of interest. The presence of other species can affect intraspecific contact rates in the reservoir species and thereby viral transmission rates. This effect can be seasonal because breeding animals are typically more territorial and aggressive than nonbreeding individuals in autumn or winter. In boreal Finland, when we controlled for host density, a significant dilution effect was observed for breeding bank voles in early summer but not among non-breeding individuals in autumn. A similar effect was observed for the wood mouse (*Apodemus sylvaticus*) on PUUV transmission among bank voles in Belgium. These results suggest that the species composition of the small mammal community could indeed play a role in PUUV infection dynamics (Voutilainen et al. 2012).

## CONCLUSION

A vast amount of new data has been gathered during EDEN and EDENext and is now being used to understand the different factors that influence the dynamics of rodent-borne infections and host-pathogen relationships. The new knowledge is improving our ability to predict the range and population dynamics of hosts and the pathogens they spread in relation to environmental change. Understanding these processes and changes at the ecosystem scale will provide the basis for more efficient disease monitoring and the development of early warning systems.

## ACKNOWLEDGEMENT

We would like to thank all the researchers who worked in the EDEN rodent-borne and subsequent EDENext rodent and insectivore-borne disease groups. We thank the following institutions:

Institut National de la Recherche Agronomique (INRA), France

Institute of Zoology, Slovak Academy of Sciences, Slovakia

Natural Resources Institute Finland (ex METLA), Finland

University of Antwerp, Belgium

Uppsala University (ex Swedish Institute for Infectious Disease Control), Sweden

Vaheri A, Strandin T, Hepojoki J, Sironen T, Henttonen H, Makela S, Mustonen J (2013). Uncovering the mysteries of hantavirus infections. *Nat Rev Microbiol* 11, 539-550.

Voutilainen L, Sironen T, Tonteri E, Tuiskunen Back A, Razzauti M, Karlsson M, Wahlstrom M, Niemimaa J, Henttonen H, Lundkvist A (2015). Life-long shedding of Puumala hantavirus in wild bank voles (*Myodes glareolus*). *J Gen Virol* Feb 2015.

Voutilainen L, Savola S, Kallio ER, Laakkonen J, Vaheri A, Vapalahti O, Henttonen H (2012). Environmental change and disease dynamics: Effects of intensive forest management on Puumala hantavirus infection in boreal bank vole populations. *PLoS One* 7, e39452.
# DETERMINANT OF HUMAN EPIDEMICS OF RODENT-BORNE ZOONOSIS?

Hantavirus particles, © Jussi Hepojoki, Pasi Laurinmäki and Sarah Butcher.

<figure><img src="image_13.png" /></figure>

## VIRUS

> Survival of the virions outside the host

> Virulence

> Ability to cross species barriers

## INDIVIDUAL HOST OF INFECTION

Bank vole (Myodes glareolus), © Heikki Henttonen, LUKE.

<figure><img src="image_14.png" /></figure>

> Persistence or clearance

> Susceptibility to infection

> Susceptibility to severe forms of infections (versus resistance)

## RESERVOIR

Bank vole distribution (Myodes glareolus), © EDENext DMT.

<figure><img src="image_15.png" /></figure>

> Longevity of shedding-natural versus laboratory settings

> Reservoir host versus spillovers hosts

> Co-divergence with the reservoir host

## POPULATION

> Cyclic populations - predation in the boreal Europe and masting in temperate Europe

> Landscape structure

> Dilution effect

> Co-infections

> Changing environment
PUBLICise Health Public Health
Telegram: rodent-borne diseases

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 an updated version of the issue dedicated to the work of the team examining rodents and insectivore-borne diseases, first published in April 2013.

# 1. 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, no infections in either humans or animals have yet been reported in France. Remarkably, the genotype of CNM isolated from the rodents, trapped in proximity to residential areas in the French Ardennes, was identical to those found earlier to cause disease in humans and animals. CNM infections might have hitherto been unrecognised 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 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.

Vayssier-Taussat M, Maaoui N, Le Rhun D, Buffet J-P, Galan M, Guivier E, Charbonnel N, Cosson J-F (2012). *Candidatus* Neoehrlichia mikurensis in bank voles, France. *Emerg Inf Dis* 18: 2063-5. Doi: 10.3201/eid1812.120846.

<figure><img src="image_16.png" /></figure>

CNM was discovered in French voles.

© LUKE, Finland.
## 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 has 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 EDENext, the presence, distribution and genetic variability of SWSV was investigated in various Sorex species captured 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, 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 harboured strains of two very different hantaviruses: Seewis virus and a new distinct,

Ling J, Sironen T, Voutilainen L, Hepojoki S, Niemimaa J, Isoviita, V.-M., Vaheri, A., Henttonen, H. and Vapalahti O (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.
genetically distant (identity 57% at amino acid level) virus (Altai-like virus). This is the first ever evidence 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.

## 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 a pet rat in the UK, 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 haemorrhagic fever (HFRS) with renal syndrome than Puumala hantavirus, the agent for the most common form of HFRS in Europe. The potential for HFRS due to Seoul virus is not well understood in Europe, and diagnostic tools are not widely optimised for this hantavirus. Recognising the emerging character of Seoul hantavirus could have significance for Public Health in large areas of 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 in 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 three years, for antibodies against four rodent-borne, potentially

Schlegel M, Radosa L, Rosenfeld UM, Schmidt S, Triebenbacher C, Löhr P-W, Fuchs D, Heroldová M, Jánová E, Stanko M, Mošanský L, Fričová J, Pejčoch M, Suchomel J, Purchart L, Groschup MH, Krüger DH, Klempa B and Ulrich RG (2012). Broad geographical distribution and high genetic diversity of shrew-borne Seewis hantavirus in Central Europe. Virus Genes 2012 Aug 45: 48-55. DOI: 10.1007/s11262-012-0736-7. Epub 2012 Mar 31.

Goeijenbier M, Verner-Carlsson J, van Gorp ECM, Rockx B, Kooptmans MPG, Lundkvist Å, van der Giessen JWB and Reusken CBEM (2015). Seoul hantavirus in brown rats in the Netherlands: implications for physicians - Epidemiology, clinical aspects, treatment and diagnostics. *The Netherlands Journal of Medicine*, May 2015, Vol. 73, No. 4.

<figure><img src="image_17.png" /><figcaption>Field voles (*Microtus agrestis*). © E. Oksanen.</figcaption></figure>
zoonotic viruses or virus groups - hantaviruses, lymphocytic choriomeningitis virus (LCMV), Ljung-gan 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 south-east Finland. This is interesting because in 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 the autumn. Higher seroprevalence was found in spring than autumn. 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 - 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 1,587 tested rodents, 286 (18.0%) harboured OPV-specific antibodies. Seroprevalence was highest in bank voles (71.4%) and striped field mice (66.7%). Due to a masting event in the autumn of 2011 across Central Europe, the abundance of bank voles increased dramatically 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 the risk for Public Health.

## 2. 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

Forbes KM 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). Serological Survey of Orthopoxvirus Infection Among Rodents in Hungary. *Vector-borne Zoon. Dis.* 15:317-322. Doi: 10.1089/vbz.2014.1731.
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 EDENext, 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 a 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 characterising 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 1,000-3,000 nephropathia epidemica cases annually, 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 characterising 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

Schlegel M, Tegshduuren E, Yoshimatsu K, Petraityte R, Sasnauskas K, Hammerschmidt B, Friedrich R, Mertens M, Groschup MH, Arai S, Endo R, Shimizu K, Koma T, Yasuda S, Ishihara C, Ulrich RG, Arikawa J and Köllner B (2012). Novel serological tools for detection of Thottapalayam virus, a Soricomorpha-borne hantavirus. Arch Virol 2012 Nov 157: 2179-87. Doi: 10.1007/s00705-012-1405-9. Epub 2012 Jul 25.
fected 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-in-
fections in bank voles was analysed. Results sug-
gest that even though habitat alterations through
intensive forest management can potentially re-
duce the hantavirus load in the environment, PUUV
is commonly found in both old forests and young
plantations. In conclusion, despite intensive ma-
nagement measures in forests, the majority of the
Fennoscandian boreal forest landscape constitutes
a source for hantavirus infection of humans.

Voutilainen L, Savola S, Kal-
lio ER, Laakkonen J, Vaheri A,
Vapalahti O and Henttonen H
(2012). Environmental Change
and Disease Dynamics: Effects
of Intensive Forest Management
on Puumala Hantavirus Infection
in Boreal Bank Vole Populations.
PLoS One 2012; 7: e39452. Doi:
10.1371/journal.pone.0039452.
Epub 2012 Jun 20.

4. How long can wild rodents excrete
hantaviruses?

The knowledge of viral shedding patterns and vi-
raemia in the reservoir host species is a key
factor in assessing the human risk of zoonotic
viruses. The shedding of hantaviruses (Bunyaviri-
dae) 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 viraemia
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 the longest follow-up history for
the presence and relative quantity of PUUV RNA in
the excreta and blood from two months before up to
eight months after seroconversion. The proportion
of animals shedding PUUV RNA in saliva, urine and
faeces peaked during the first month after sero-
conversion, but continued throughout the study
period with only a slight decline. The quantity
of shed PUUV in reverse transcription quantita-
tive PCR (RT-qPCR) positive excreta was constant
over time. In blood, PUUV RNA was present for
up to seven months but both the probability of
viraemia and the virus load declined with time.
The findings contradict the current view of a de-
cline in virus shedding after the acute phase and
a short viraemic period in hantavirus infection
- an assumption widely adopted in current epide-
miological models. The authors suggest life-long
shedding as a means for 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 set-
tings. Go to the field!

Voutilainen et al. (2015). Life-
long shedding of Puumala
hantavirus in wild bank voles
(*Myodes glareolus*). *J. Gen.
Virology* (in press). Doi: 10.1099/
vir.0.000076

5. Analysing the effects of climate changes on rodent populations

Rodents play a key role in many ecosystems, for example 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 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 were 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 such as 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

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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 subni-vean (under-snow) predators. Using national long-term population data on voles, predators, birds of prey and climate, collected throughout Finland from 1986 to 2011, they analysed the spatio-thermal variation in the interactions between popula-tions 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 interac-tions during winter. The results strongly support the importance of small mustelids for the vole cycle. However, in the models weakening specia-list 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 summer 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 col-lapsing small mammal population cycles. Changing climate may affect this interaction between voles and specialist predators.

Korpela K, Delgado M, Henttonen H, Korpimäki E, Koskela E, Ovaskainen O, Pietiäinen H, Sundell J, Yoccoz NG and Huitu O (2012). Non-linear effects of climate on boreal rodent dynamics: warm winters do not negate high-amplitude cycles. *Global Change Biology* 2013 Mar 19: 697–710.

Korpela K, Helle P, Henttonen H, Korpimäki E, Koskela E, Ovaskainen O, Pietiäinen H, Sundell J, Valkama J and Huitu O (2014). Predator-vole interactions in boreal Europe: the role of small mustelids revised. *Proc. R. Soc. B* 281. 20142119.

# 6. Analyses of the evolution and molecular epidemiology of hantaviruses

## A fatal case of nephropathia epidemica (NE)

Despite the usually mild course of NE (case fatality rate is less than 0.1%), fatal cases have been reported, and 5% of patients need dialysis. In EDENext, the first complete PUUV gene sequence was directly recovered from a fatal case from Finland. In order to find out whether the fatal outcome of the disease was due to a particular genetic variant of PUUV, wild-type PUUV isolates originating from bank voles trapped in and near the house of the patient were searched for identical or closely related genetic variants (Plyusnina et al. 2012). Indeed, one completely sequenced rodent-originated PUUV genome was identical to the fatal human case PUUV genome.
Subsequent phylogenetic analysis confirmed that this virus was closely related to genetic variants identified earlier, approximately 60km north-west from where the infection occurred. These findings demonstrate that the fatal human NE case and local wild-type PUUV strains are genetically linked. The results of the sequence analyses also revealed that no mutations had been acquired in the genome of PUUV during the transmission of the virus to the patient resulting in a fatal outcome. In conclusion, the fatal outcome was not due to a unique or even rare genetic hantavirus variant causing the infection but may rather be a consequence of a possible defect in the anti-viral defence mechanisms in the patient's immune system. The patient showed a HLA (human leukocyte antigen)-B8-DR3 haplotype which is associated with a more severe outcome of NE, including a C4A null allele, i.e. a major antivirus defence system complement was impaired. Furthermore, the patient had been a heavy smoker, which has been shown to be a real risk factor in contracting NE. In regard to Public Health, identifying the risk factors that may lead to a fatal outcome of a hantavirus infection is of great importance in reducing the disease burden.

## Proposal of a new classification of Dobrava-Belgrade virus

More than 20 years ago, a human pathogenic hantavirus was isolated from a patient with clinically severe haemorrhagic fever with renal syndrome (HFRS) and, at the same time, from a yellow-necked mouse (*Apodemus flavicollis*) captured in Slovenia. After it had become evident that both isolates were identical, it was named Dobrava-Belgrade virus (DOBV). Subsequently, DOBV and DOBV-like viruses were found in various rodent species of the *Apodemus* genus in several European countries and Russia. Phylogenetic analyses of the isolates from *A. flavicollis* and *A. agrarius*, occurring largely sympatric in Slovenia and Slovakia, clearly showed that DOBV isolates cluster in distinct evolutionary lineages according to their reservoir hosts. This was confirmed when virus sequences from a third host, *A. ponticus*, were analysed. As a result of EDENext research, a novel intra-species classification of DOBV into four related genotypes, i.e. Dobrava, Kurkino, Saaremaa, and Sochi, has been proposed based on phylogenetic analyses of the S segment (Klempa et al. 2013). The four genotypes show characteristic

Plyusnina A, Razzauti M, Sironen T, Niemimaa J, Vapalahati O, Vaheri A, Henttonen H and Plyusnin A (2012). Analysis of complete Puumala hantavirus genome, Finland. *Emerg Infect Dis* 2012 Dec 18: 2070-2. DOI: 10.3201/eid1812.120747.

<figure><img src="image_18.png" /><figcaption>Yellow-necked mouse (*Apodemus flavicollis*).
© Gianfranco Scieghi.</figcaption></figure>
<figure><img src="image_19.png" /><figcaption>ollis), andeghi.</figcaption></figure>

differences in their phylogeny, host reservoirs, geographical distribution and in their pathogenicity in humans. According to the current stage of knowledge, the order of virulence in humans of the DOBV genotypes is assumed to be as follows: Dobrava > Sochi > Kurkino > Saaremaa. In terms of Public Health, it will be important to determine which genetic differences between the four virus genotypes are responsible for the different virulence, and whether differences in the susceptibilities of resident populations influence the severity of disease.

Klempa B, Avsic-Zupanc T, Clement J, Dzagurova TK, Henttonen H, Heyman P, Jakab F, Kruger DH, Lundkvist Å, Maes P, Nemirov K, Papa A, Tkachenko EA, Ulrich RG, Vapalahti O and Vaheri A (2012). Complex evolution and epidemiology of Dobrava-Belgrade hantavirus: implications for taxonomy. *Arch Vir. Doi* 10.1007/s00705-012-1514-5.

# 7. Reviews on hantavirus and other rodent-borne pathogens causing haemorrhagic fever virus infections

## Hantavirus infections in Europe and their impact on Public Health

A review by Vaheri et al. (2013) highlights the most important Public Health issues of hantavirus infections in Europe. 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 hospitalised 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.

Vaheri A, Henttonen H, Voutilainen L, Mustonen J and Vapalahti O (2012). Hantavirus infections in Europe and their impact on public health. *Rev Med Virol. 2013* Jan 23: 35-49.
Doi: 10.1002/rmv.1722. Epub 2012 Jul 3.
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

A review by Goeijenbier et al. (2013) 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, and New and Old World arenaviruses. 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 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 trials. 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.

Goeijenbier M, Wagenaar JFP, Goris MGA, Martina BEE, Henttonen H, Vaheri A, Reusken CBEM, Hartskeerl R, Osterhaus ADME and van Gorp ECM (2013). Rodent-borne haemorrhagic fevers: under-recognised, widely spread and preventable - epidemiology, diagnostics and treatment. *Crit Rev Microbiol* 2013 Feb; 39: 26-42.

## 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 case they can cause two diseases: 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 syndrome (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 characterised 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 A, Strandin T, Hepojoki J, Sironen T, Henttonen H, Mäkelä S and Mustonen J (2013). Uncovering the mysteries of hantavirus infections. *Nature Reviews in Microbiology* 11:539-550. DOI:10.1038/nrmicro3066.

## 8. 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 tumour 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 hanta-
viruses in reservoir hosts. Persistence was as-
sociated 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 analysed. These genes were selected based
on the literature of rodent or human/hantavirus
interactions. The comparison of genetic differen-
tiation estimated between bank vole populations
sampled over Europe, at neutral and candidate
genes, has allowed to evidence signatures of se-
lection for *Tnf*, *Mx2* and the *Drb Mhc* class II
genes. Altogether, these results corroborated the
hypothesis of an evolution of tolerance strate-
gies in rodents. In other words, the bank vole
has quite different immunogenetic responses to
Puumala virus in different parts of Europe, which
could partly explain the variable epidemiology of
Puumala virus in Europe.

Charbonnel N, Pagès M, Sironen
T, Henttonen H, Vapalahti O,
Mustonen J and Vaheri A (2014).
Immunogenetic Factors Affec-
ting Susceptibility of Humans
and Rodents to Hantaviruses
and the Clinical Course of Hanta-
viral Disease in Humans. *Viruses*
2014 (6) 2214-2241; 2014.
Doi:10.3390/v6052214.

9. From single infection studies to
understanding of multiple infections and
interactions among parasites/pathogens

Until quite recently most epidemiological stu-
dies on wild pathogens and reservoirs were done
using single pathogen models. During the past
decade it has been increasingly understood that
parasites/pathogens interact and these interac-
tions affect the immunology and susceptibility of
hosts. In this EDENext article, the authors des-
cribed spatial heterogeneity in immunity in bank
vole populations with respect to landscape fea-
tures and parasite coinfections, the focus being
on Puumala hantavirus and the bank vole's common
parasite, the nematode *Heligmosomum mixtum*. They
assessed the consequences of this heterogeneity
for the risk of Puumala hantavirus (PUUV) infec-
tion. The authors had earlier found a positive
association between infection with *H. mixtum* and
PUUV. They found that the tumor-necrosis factor
a (Tnf-a) was more strongly expressed in voles
infected with PUUV than in uninfected voles or in
voles co-infected with the nematode *H. mixtum* and
PUUV. Indeed, *H. mixtum* may limit the capacity of
the vole to develop pro-inflammatory responses.
This effect may increase the risk of PUUV infec-
tion and replication in host cells. Overall, the
results suggest that close interactions between
landscape features, co-infections and immune gene
expression may shape PUUV epidemiology.

Guivier E, Galan M, Henttonen
H, Cosson JF and Charbonnel N
(2014). Landscape features and
helminth co-infection shape
bank vole immunoheterogeneity,
with consequences for Puuma-
la virus epidemiology. *Heredity*
112: 274-281. Doi: 10.1038/
hdy.2013.103
## 10. 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 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 of this study 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 stay at their current values, the models predict that NE may intensify but should not spread (although southern Sweden, the Norwegian coast and The Netherlands should be kept under watch). Models indicate that large-scale modelling can lead to a very high predictive power. At a 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 CB, Olsson GE, Ahlm C and Vanwambeke SO (2012). Modelling zoonotic diseases in humans: comparison of methods for hantavirus in Sweden. *Int J Health Geogr* 2012 Sep 11: 39. doi: 10.1186/1476-072X-11-39.

Zeimes CB, Quoilin S, Henttonen H, Lyytikainen O, Vapalahti O, Reynes JM, Reusken C, Swart A, Vainio K, Hjertqvist M and Vanwambeke S (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
## Interview: Heikki Henttonen

<figure><img src="image_20.png" /></figure>

Heikki Henttonen, a professor of forest zoology at the Finnish Forest Research Institute (METLA), part of the Natural Resources Institute Finland (Luke), has more than 200 scientific publications to his name. A Fulbright Scholar in the 1980s. Heikki continues to have strong links with the United States of America and is an invited research associate at the University of Alaska Museum and the University of New Mexico's Museum of Southwestern Biology. Heikki is on the editorial board of the journals *Mammalia and Zoonotic and Vector-borne Diseases*. Heikki was a member of both the EDEN and EDENext steering committees (and was president of the latter) and coordinator for rodent-borne diseases.

**In your opinion, what have been the notable achievements of EDEN and EDENext?**

**Heikki Henttonen (Natural Resources Institute Finland):** "I think European collaboration is one important point because EDEN and EDENext have made possible so much European collaboration, which would not have happened without them, without that financing. Another point is working in this network of collaboration opens up the training of a young generation, who from the beginning have learned to work on a European or global-scale network and they know each other. 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, they are too competitive, they cannot collaborate as we do. In EDEN, at least in rodent-borne systems, everyone has understood the value of collaboration from the very beginning. In many cases, teams collected material and then one team would data analyse it, but all are co-authors, so that makes it efficient and high quality. Starting from the first contacts, maybe 13 years ago, it has been extremely interesting and it has really given a lot to me...It is one of the happier things to be old yourself but see the young people developing and then you see some of the really smart ones appearing. It's so nice to help these young students."

**What have you liked best?**

**Heikki:** "Maybe it is this European-scale collaboration. We have been able to look, say, at Puumala hantavirus and the host, bank vole, in Europe, but now we have been able to look at the disease pattern in different climatic biomes and we have been able to compare. Many people have said, 'one host, one virus, there is one model' but that is exactly what it is not! We need comparative models from different parts of Europe and this is what we have been trying to do in the EDEN and EDENext rodent programmes."

<figure><img src="image_21.png" /></figure>

**Outstanding research moments?**

**Heikki:** "We have had many interesting findings, we have found a lot of new things. One of the most interesting things was the life-long shedding of Puumala hantavirus by voles, because we had no idea about how long it was shedded. We knew it was a chronic infection in bank vole and some lab experiments suggested it was two months and then it was over. We followed individual animals for almost a year and we had blood samples, then urine, faeces, saliva samples, every month and we could establish that it is life-long shedding, there wasn't even a decline in shedding. It is also interesting that it wasn't continuous through one route. You could have virus in the saliva, then a break, then again in the saliva or it could be in the urine, every time they were shedding through one route at least."
# The future of research on tick-borne pathogens:

## shifting from the 'one pathogen-one disease' vision to the pathobiome paradigm

Muriel Vayssier-Taussat<fnref n="1" />, Maria Kazimirova<fnref n="2" />, Zdenek Hubalek<fnref n="3" />, Sándor Hornok<fnref n="4" />, Robert Farkas<fnref n="4" />, Sarah Bonnet<fnref n="1" />, Gwenaël Yourch<fnref n="5" />, Andrei Daniel Mihalca<fnref n="6" />, Olivier Plantard<fnref n="6" />, Cornelia Silaghi<fnref n="8" />, Annapaola Rizzoli<fnref n="9" />

## INTRODUCTION

Since the discovery of the Lyme disease agent in 1982 by Willy Burgdorfer (see Burgdorfer 1984), the importance of *Ixodes* ticks as vectors of zoonotic pathogens has continued to rise. The fact that *Borrelia* species responsible for Lyme borreliosis (LB) have been discovered so recently may not be because they did not exist before but because, at that time, no accurate tools were available to discover them. Indeed, the genus *Borrelia* has been recently found in the Tyrolean Iceman, demonstrating that it was already associated with man 5,300 years ago (Keller et al. 2012).

Although the most frequent tick-borne disease reported in European citizens is LB, with more than 65,000 new cases each year (Rizzoli et al. 2011), patients bitten by ticks can also be exposed to many other micro-organisms. These include viruses, bacteria, and parasites, some of which (for example, *Borrelia miyamotoi*) were identified in ticks decades prior to their association with human disease, whereas others were discovered only very recently and their Public Health importance remains so far unknown (for example, *Rickettsia helvetica*, *Candidatus* Neoehrlichia mikurensis, *Babesia venatorum* (formerly referred to as sp. EU1)).

Ticks represent an important and increasing threat for Public Health in Europe for three main reasons. Firstly, ticks are hæmatophagous during their three life stages and ingest a huge volume of blood (compared to other vectors) on many species, which increases the likelihood of acquiring and therefore transmitting pathogens. Indeed, ticks are hosts to a great variety of micro-organisms. Secondly, spending most of their life off-host in the environment, ticks are very sensitive to any changes which impact the environment. Furthermore, socio-economic changes may lead people to be more frequently present in tick habitats, increasing their exposure to tick-borne pathogens which in turn may lead (or have already led) to an important burden of tick-borne diseases. Thirdly, because of the large variety of pathogens that ticks are able to harbour and transmit, clinical and laboratory diagnosis of tick-borne diseases (TBD) is challenging.

Recently the use of next-generation sequencing (NGS) technology revealed that ticks harbour, in addition to pathogens, many other micro-organisms which co-exist and might interact with pathogens. Our vision of tick-borne pathogens therefore needs

I think Next have given us a great example of how scale doesn't matter. They aren't about having to make a first decision. They're about smart, efficient, effective.

I want to be clear that I agree with that.

It's not that I disagree with it. I agree with it. I just want to make sure that we're clear on what we're talking about.

<footnote n="1">¹INRA, UMR BIPAR, INRA, ANSES, ENVA Maisons-Alfort, France</footnote>

<footnote n="2">²Institute of Zoology, Slovak Academy of Sciences, Bratislava, Slovakia</footnote>

<footnote n="3">³Institute of Vertebrate Biology, Academy of Sciences of the Czech Republic, Brno, Czech Republic</footnote>

<footnote n="4">⁴Department of Parasitology and Zoology, Faculty of Veterinary Science, Szent István University, Budapest, Hungary</footnote>

<footnote n="5">⁵INRA, UR 346 Épidémiologie Animale, Saint Genès Champanelle, France</footnote>

<footnote n="6">⁶University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Department of Parasitology and Parasitic Diseases, Cluj-Napoca, Romania</footnote>

<footnote n="7">⁷INRA, UMR 1300 BioEpAR, Nantes, France</footnote>

<footnote n="8">⁸National Centre for Vector Entomology, Institute of Parasitology, Vetsuisse-Faculty, University of Zurich, Zürich, Switzerland</footnote>

<footnote n="9">⁹Fondazione Edmund Mach, Research and Innovation Centre, San Michele all'Adige, Trento, Italy</footnote>
to evolve to a more integrated view which considers the 'pathobiome', representing the pathogenic agent integrated within its
biotic environments including other pathogens, commensals or symbionts. Shifting the paradigm from pathogens to pathobiome
has many consequences in terms of research. The most demanding challenges of the coming years will be:

1. To investigate whether all new micro-organisms revealed by NGS cause human and/or animal disease following tick bites, or whether they are non-pathogenic tick-endogenous micro-organisms.

2. To identify whether interactions exist between pathogens or between pathogens and other tick-endogenous micro-organisms, as well as the consequences in terms of pathogen transmission, persistence, virulence and evolution.

3. Based on the new knowledge, the development of adequate strategies to better diagnose and combat tick-borne diseases.

In this review, we analyse how this new vision will revolutionise our understanding of tick-borne diseases, whose number has grown considerably since the discovery of the Lyme disease agent, and we discuss the implications in terms of research plans for the future in order to efficiently prevent and control the threat posed by ticks in Europe.

STATE OF THE ART OF THE 'OLD VISION' OF TICK-BORNE PATHOGENS: AN EVER-INCREASING LIST

In Europe, the most prevalent tick-borne disease in humans is LB, caused by a group of bacteria belonging to the *Borrelia burgdorferi* sensu lato group with at least five different species infecting humans in Europe (Rizzoli et al. 2011). Recently, *B. miyamotoi*, belonging to the relapsing fever group, has been detected in patients in USA, Japan, Russia and The Netherlands (Platonov et al. 2011, Chowdri et al. 2013, Krause et al. 2013, Hovious et al. 2013) and is transmitted by the tick species involved in LB. Ticks, especially *Ixodes ricinus*, can also be infected with other pathogens that might be transmitted to humans (Heyman et al. 2010). Amongst them, *Anaplasma phagocytophilum* is responsible for granulocytic anaplasmosis, *Candidatus Neoerhichia mikurensis* may cause severe febrile illness in immunocompromised patients (Welinder-Olsson et al. 2010, von Loewenich et al. 2010), and Rickettsiae of the spotted fever group are known (*R. monacensis*, *R. conorii*) or suspected (*R. helvetica*) to cause rickettsioses (Parola and Raoult 2001). Another bacterial pathogens, *Francisella tularensis*, causes tularaemia. The Q fever agent *Coxiella burnetii* has also been detected in *I. ricinus*, but the role of this tick species in the epidemiology of these diseases is probably not significant (Duron et al. 2014, Angelakis and Raoult 2010).

Protozoans of the genus *Babesia*, mainly *B. divergens*, cause babesiosis in immunocompromised humans, and *B. venatorum* pathogenicity to humans has been confirmed (Hildebrandt and Hunfield 2014). *B. microti*, a well-known human tick-borne pathogen in the USA, has also been identified in ticks with, until now, one single human case in Europe (Hildebrandt et al. 2007). *Ixodes* species also transmit arboviruses, tick-borne encephalitis virus being the most important in terms of Public Health in Europe (Charrel et al. 2004). An increasing number of new species, strains or genetic variants of other micro-organisms are being detected in ticks, resulting in an ever-increasing list of (potential) pathogens capable of infecting livestock, companion animals and humans. However, we need to take into account that a significant portion of these 'new' species/genotypes are not truly emerging, but are only newly detected. This increasing biodiversity of pathogens is not generating answers but rather complex questions regarding the ecological cycles of pathogens, their interspecific interactions, cross-influence on infection mechanisms and differential diagnosis and synergic clinical importance.

Identification of micro-organisms in ticks has been largely dominated by the use of conventional molecular approaches mostly based on the use of specific primers combined with (real-time) PCR, and less frequently by culture-dependent methods. However, pathogen detection in an arthropod is not sufficient to validate its vector competence.

Following new or unexpected pathogen detections in ticks, it is therefore essential to conduct vector competence studies, and to evaluate the risk of exposure for both humans and animals. These types of studies require living ticks raised under controlled conditions, though because of their complex biological cycle and feeding biology, maintenance of tick colonies and their infection with micro-organisms is not easy. However, several methods have been successfully developed and used to infect hard ticks with pathogens, for example, feeding ticks on infected animals, injecting pathogens through the cuticle, by using thin capillary tubes and feeding ticks on infected blood through artificial or animal-derived membranes (Liu and Bonnet 2014). These me-
thods have been successfully employed to validate vector competence for a number of tick-borne pathogens, including Lyme spirochetes (Burgdorfer 1984), *A. phagocytophilum* (Massung et al. 2003), *Babesia* sp. EU1 (or *B. venatorum*) (Bonnet et al. 2009) and *Bartonella* sp. (Cotte et al. 2008, Reis et al. 2011). However, for some established tick-borne pathogens such as *Ca. N. mikurensis* (for which no cultivable strain has so far been obtained) and *R. helvetica*, the vector competence of ticks has not yet been proven. They became de facto tick-borne pathogens under more or less strong 'epidemiological evidence'.

## The problem posed by the detection/diagnosis of TBDs

When a clinical history of tick bites is mentioned, Lyme disease is the primary diagnosis, but in some cases the borreliosis diagnosis remains unconvinced by conventional serological tests (Perronne 2014). People bitten by ticks can also be infected by tick-borne encephalitis virus (TBEV), causing severe encephalitis, which is well diagnosed by serological tests. No specific treatment is currently available but TBE can be successfully prevented by active immunisation (Kaiser 2012).

As already mentioned, among vectors ticks are capable of transmitting the largest variety of pathogens, and pathogens other than Lyme or TBE agents might be involved in TBDs. Interestingly, the majority of those pathogens have been discovered during the past 20 years. The symptoms induced by those pathogens are often mild (high fever, fatigue, body aches, chills etc.) and can be confused with symptoms caused by infection with other agents. This is probably the reason why medical practitioners have a poor knowledge of them and they are almost never detected in humans while being frequently identified in ticks and/or reservoir animals. A striking example is *B. miyamotoi*. This *Borrelia* species was first isolated from Japanese *Ixodes* ticks in 1995 and was considered a non-pathogenic endogenous tick bacteria until the first human cases of *B. miyamotoi* infection were reported in Russia in 2011 (Platonov et al. 2011). Since then, human infections have been described in the USA and most recently in The Netherlands (Chowdri et al. 2013, Krause et al. 2013, Hovius et al. 2013, Gugliotta et al. 2013). In European countries such as France, Estonia, Poland and Switzerland, circulation of *B. miyamotoi* in *I. ricinus* and in wild animals has been confirmed (Vayssier-Taussat et al. 2013), with the French genotype identical to an isolate from a Dutch patient (Cosson et al. in press). No human cases of *B. miyamotoi* infections have been reported in these countries, but the absence of serological or molecular tests for *B. miyamotoi* combined with the lack of knowledge on these bacteria among medical practitioners makes diagnosis particularly difficult. Thus it is likely that the absence of human infections is rather due to missed diagnoses than to an actual absence of infection.

Patients bitten by ticks can also be co-infected by several pathogens. For example, Horowitz et al. (2013) described co-infection rates ranging from 2 to 5% for *Borrelia* species and *A. phagocytophilum* in patients with erythema migrans who were diagnosed with Lyme disease. Co-infections between *B. afzelii* and *R. monacensis* were also identified in skin biopsies of erythema migrans patients in The Netherlands (Tijssse-Klasen et al. 2013). However, co-infections are rarely diagnosed in routine practice, raising the problem of

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co-infection in humans as a relevant, albeit poorly studied issue, with strong implications for Public Health.

As a result, after a tick bite, if people are infected by pathogens other than Lyme disease spirochetes or TBEV, the cause of their disease is rarely identified. In recent years unexplained syndromes occurring after tick bites have become an increasingly important issue leading to considerable discord between scientists, patients and infectious disease institutes.

## THE TECHNOLOGY-DRIVEN REVOLUTION OF TICK-BORNE PATHOGENS' VISION: FROM PATHOGEN TO PATHOBIOME

Until now, most studies aiming to identify tick-borne pathogens in ticks have been able to assess simultaneously only a limited number (Halos et al. 2010, Reis et al. 2011). This is partly due to technological limitations. Indeed, complete screens of micro-organisms in natural host populations were out of reach using standard laboratory procedures. Within the past few years, the rapid development of Next-Generation Sequencing (NGS) methods has revolutionised epidemiology and the diagnosis of infectious diseases, facilitating the complete screening of pathogens in their hosts, the discovery of new pathogens or the detection of unexpected ones. NGS has recently been successfully used to identify the bacterial communities associated with *I. Ricinus* (Carpi et al. 2011, Nakao et al. 2013, Hawlena et al. 2013, Williams-Newkirk et al. 2014) based on the amplification and sequencing of hyper-variable regions of the 16SrRNA encoding genes (metagenomic profile), illustrating a highly diverse microbial community (108 genera representing all bacterial phyla). As expected, these approaches have made it possible to find without a priori the best known tick-borne pathogens such as the *Borrelia*, *Anaplasma*, *Coxiella*, *Francisella* and *Rickettsia* genus. Among these genera, mostly known as pathogenic for vertebrates, some species are considered as endosymbionts (for example, the *Rickettsia*-endosymbiont of *I. Scapularis*) (Gillespie et al. 2012) and the distinction between pathogens and endosymbionts is often tricky. For instance, some authors consider *Rickettsia* species as endosymbionts that are transmitted vertically in arthropods, and only secondarily as pathogens of vertebrates (Perlman et al. 2006).

For the *Coxiella* genus, the species *C. burnetii* is mostly considered as a vertebrate pathogen while numerous other *Coxiella* species have been found to be associated with ticks. Phylogenetic analyses combined with experimental approaches suggested that all of them might also be considered as endosymbionts of ticks (Duron et al. 2014, Almeida et al. 2012). Thus the pathogenic nature of *C. burnetii* could be an exception within the genus (Machado-Ferreira et al. 2011). Besides the well-known vertebrate pathogenic species *F. tulariensis* (occasionally found in ticks), *Francisella*-like-endosymbionts associated with *Dermacentor* spp. have been described but their pathogenic nature remains to be investigated (Kreizinger et al. 2013). The *Wolbachia* and *Arsenophonus* genera are also bacteria associated with arthropods (mostly insects) and affect the reproduction and/or immunity of their hosts (Rudolf et al. 2009, Werren et al. 2008). They have also been found associated with ticks (Ahantarig et al. 2013). However, a recent experimental approach has revealed that at least in *I. ricinus*, the finding of *Wolbachia* is due to parasitism by a parasitoid wasp (*Ixodaphagus hookeri*) (Plantard et al. 2012). The role of *Arsenophonus* as tick endosymbionts has still to be demonstrated. Finally, the *I. ricinus* endosymbiont *Midichloria mitochondrii* constitutes a particular example. This bacterium was initially observed within tick cells, especially in ovarian cells of *I. ricinus* (Lewis 1979). The use of molecular probes specific for this alphaproteobacteria have made it possible to detect them in almost 100% of *I. ricinus* females derived from natural populations (Sassera et al. 2006), but also in other tick species (Epis et al. 2008). Recently *M. mitochondrii* is increasingly considered as a potential vertebrate pathogen (Bazzocchi et al. 2013).

The use of NGS technology will undoubtedly shed new light on the intriguing bacterial communities associated with ticks (Hawlena et al. 2013). The clear-cut frontiers between the so-called 'vertebrate pathogens', 'arthropod pathogens' or 'arthropod symbionts' may thus vanish into a more subtle and complex vision of bacterial-vector-vertebrate communities. Additional investigations, including experimental approaches, should be conducted to define their role in ticks. A better knowledge of these bacteria could even constitute useful resources for developing anti-vectorial control methods.

Besides the known micro-organisms (belonging either to pathogens, endosymbionts or both), NGS has also revealed that the majority of RNA/DNA sequences carried by ticks belonged to still unknown micro-organisms. For example, 80% of the viral nucleic sequences obtained from tick extracts may belong to as-yet unidentified micro-organisms (Vayssier-Taussat et al. unpublished data). Among these new viral sequences, we identified genera transmissible to humans and/or animals via arthropods, including Bunyaviridae (Nairovirus and Phlebovirus), Rhabdoviridae (Vesiculovirus) and Reoviridae (Coltivirus) (Vayssier-Taussat

and in the field of virology. By comparing micro-organism

and molecular phylogenetic analysis of the 16S rRNA gene, we have investigated the phylogenetic position of the 16S rRNA gene of *Wolbachia* and *Arsenophonus* and compared it with the phylogenetic position of the 16S rRNA gene of *I. ricinus*. In addition, we have compared the 16S rRNA gene of *Wolbachia* and *Arsenophonus* with the 16S rRNA gene of other bacteria, such as *M. mitochondrii* and *M. pneumoniae*.
et al. unpublished data). A similar study has been undertaken by Lipkin et al. in the USA to characterise the virome of different tick species. The presence of Powassan virus, a well-known human pathogenic tick-borne virus, and eight novel viruses belonging to Nairovirus, Phlebovirus and Mononegavirus genera were identified (Tokarz et al. 2014).

By having a picture of the entire tick microbial community, we can identify that pathogens are intimately associated with the vast community of micro-organisms (including other pathogens) that may influence tick biology, but also pathogen persistence, transmission and virulence, thereby justifying the need for a shift from pathogens to pathobiome.

## HOW CAN WE REVEAL MICRO-ORGANISM INTERACTIONS IN THIS VERY COMPLEX ECOSYSTEM?

Micro-organism interactions have mostly been considered as a one-to-one interaction, where the infection by one pathogen influences the acquisition of and/or dynamics of infection by a second pathogen. However, interactions between a set of pathogens are conceivable where different pathogens interact within a network or through 'cascade consequence' (Rigaud et al. 2010, Bordes and Morand 2011). In experimental studies, we can investigate how the presence of one pathogen may interfere with the infection by another. However, this is not possible in a pathobiome perspective where many pathogens and other micro-organisms are present, including poorly known ones.

In such cases, we can conduct population studies and try to seek whether the probability of finding these pathogens together is not random. The first step is therefore to look at the co-occurrence of micro-organisms, even though this can also result from confounding factors which create statistical associations between pathogens without true biological interactions. In population studies, longitudinal or time series data are useful for identifying pathogen associations, identifying whether the presence of one pathogen modifies subsequent infection by another (Telfer et al. 2010). However, such studies are resource-intensive. One-off cross-sectional studies are widely used to screen for the presence of several pathogens because they consume less time and money than longitudinal studies. This is especially the case when emerging or poorly known pathogens or host species are studied. Numerous approaches are available to detect pathogen associations in such contexts. For instance, multivariate analyses (principal component analysis, correspondence analysis, discriminant analysis, co-inertia analysis) (Gauch 1982) help evaluate which pathogens tend to group together. The drawback is that there is usually no statistical test associated with these analyses (but see, for example, permutation methods, Tollenaere et al. 2008, Salvador et al. 2011).

Recently, we developed a new association screening approach to detect overall and detailed multi-pathogen associations (Vaumourin et al. 2014). This method was shown to be very powerful, but still its use for micro-

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organisms >10 requires more than 1,000 samples. Strong methodological developments have been made in network analyses (Bascompte 2007) over the past few years in many fields (for example, metabolic pathways in medicine (Ravasz et al. 2002, Qin et al. 2012), peer-to-peer networks in computer science (Fox 2001) and scientific collaboration in social science (Newman 2004)). They also offer an attractive representation of multiple pathogen relationships. They provide indices of association such as connectedness (Yodzis 1980), nestedness (Bascompte et al. 2003) and betweenness (Freeman 1977). However, to date, statistical tests regarding network parameters have rarely been used, though developments in this field are promising.

## SURVEILLANCE AND DIAGNOSIS FOR TBD

Considering the large number of potential tick-borne pathogens that can be involved in diseases, either alone or in association, there is a need for the urgent development of adapted methods that take into account this variety but also the biology of tick-borne pathogens. For instance, many tick-borne pathogens colonise blood (infecting blood cells or not) of vertebrate hosts. Thus it makes sense to detect the presence of their DNA in the blood of infected human patients or animals. However, blood infection does not occur for all tick-borne pathogens. For example, Lyme spirochete species do not stably infect the blood of humans and therefore detecting their DNA in the blood of a patient bitten by ticks is tricky and requires either bacterial DNA detection in more specific samples (such as skin biopsies) or serological tests, even though their specificity and sensitivity are not always optimal. Molecular identification of tick-borne pathogens has mostly been based on the use of specific primers combined with real-time PCR, which can only detect a selected and limited number of species. To overcome these problems, new tools allowing high-throughput monitoring of tick-borne pathogens have to be urgently set up.

Based on data on circulation and the presence of tick-borne pathogens in ticks in different geographical regions in Europe obtained by NGS, we developed a tool using a so-called microfluidigm system allowing multiple parallel real-time PCRs for TBD surveillance that might be adapted to diagnosis (Michelet et al. 2014). This new tool has the unique ability to simultaneously analyse multiple pathogens (up to 48 different species of pathogens) in the same samples. It presents a major advantage and can be easily adapted to new situations as it is entirely possible to remove primers/probes sets in order to modify the panel of targeted pathogens. If developed by private companies, these kinds of tools will represent an important improvement in the diagnosis of TBD.

## VACCINATION

Considering the number of pathogens which could be transmitted by the same tick species, establishing tick vaccines could be a smart and environmentally friendly alternative to protect human and animal populations against tick-borne diseases through the control of vector infestations and reducing pathogen infection and transmission. For this purpose, research on molecular interactions between ticks and pathogens as well as the identification of suitable targets for vaccine development are major challenges for the implementation of new TBD control strategies (Liu and Bonnet 2014). Among these, target molecules playing key roles in tick infestation and vector capacity are particularly promising (de la Fuente and Merino 2013). To date, the only commercially available anti-tick vaccine is based on the *R. microplus* midgut protein BM86, interfering with tick feeding and subsequent egg production (de la Fuente et al. 2007). However, thanks to the combination of the techniques of tick infection in the laboratory and new methods of molecular investigation, promising candidates have been recently identified. These include tick proteins derived from *I. ricinus* (Decrem et al. 2008, Liu et al. 2014), *I. scapularis* (Dai et al. 2009), *Rhipicephalus microplus* (Merino et al. 2013, Labuda et al. 2006), as well as candidates common to several hard tick species (de la Fuente et al. 2006). Improving our understanding of molecular interactions between ticks and tick-borne pathogens is a pre-requisite for the conception of future generations of vaccines and for vector and disease control.
## ACKNOWLEDGEMENT

The authors thank the community of enthusiastic scientists exploring new insights in ticks and tick-borne pathogens in Europe, especially the ones participating in the EDENext project and the COST action Eurnegvec. We thank the following institutions:

Fondazione Edmund Mach, Italy
ID-VET, France

Institut of Vertebrate Biology, Academy of Sciences of the Czech Republic, Czech Republic

Institute of Zoology, Slovak Academy of Sciences, Slovakia

Kafkas Universitesi, Turkey

Ludwig-Maximilians Universität München, Germany

Research Center Borstel, Germany

Szent Istvan University, Faculty of Veterinary Science, Hungary

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PUBLICise Health Public Health
Telegram: Tick-borne diseases

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 an updated version of the issue dedicated to the work of the team examining tick-borne diseases, first published in December 2013.

1. Tick-borne viruses - an overview

Ticks are recognised as vectors for several
pathogens of Public Health relevance. Hubálek
and Rudolf (2012) offer an extensive over-
view of the currently known European tick-
borne viruses with a special focus on their
taxonomy, host and tick vector range, pathoge-
nicity for vertebrates including humans, and
their significance for Public Health. Besides
the known highly pathogenic Crimean-Congo hae-
morrhagic fever virus (CCHFV) and tick-borne
encephalitis virus (TBEV), the authors also
report other tick-borne virus pathogens such
as louping-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.

2. 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 (different *Cer-
vidae* species, for example roe deer *Capreolus
capreolus*), represent some of the most impor-
tant risk factors for the risk assessment of
diseases transmitted by the pan-European tick
species *Ixodes ricinus*. The spatial and tem-
poral variation in the abundance of deer does
not only affect the number of ticks in the
environment, but also the rate of ticks in-
fected with various pathogens. In the case of
tick borne encephalitis (TBE), 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 from the Fondazione Edmund Mach and the Slovak Academy of Sciences 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 F, Bolzoni L, Rosà R, Carpi G, Hauffe HC, Valent M, Tagliapietre V, Kazimirova M, Koci J, Stanko M, Lukan M, Henttonen H and Rizzoli A (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 L, Rosà R, Cagnacci F and Rizzoli A (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

### 3. THE IMPACT OF CRIMEAN-CONGO HAEMORRHAGIC FEVER (CCHF) ON PUBLIC HEALTH

Crimean-Congo haemorrhagic fever (CCHF) is a haemorrhagic fever in humans, caused by infection with the Crimean-Congo haemorrhagic fever 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) summarises 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 vac-
cination. Based on this knowledge, recommenda-
tions for protection measures for individuals
and the population at large, including case
management options, and Public Health mana-
gement and communication strategies are pro-
posed. CCHFV is primarily transmitted by ticks
of the genus *Hyalomma*, which can be found in
many regions of southern and south-eastern Eu-
rope. 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 horizontal-
ly 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-per-
son transmissions following unprotected hand-
ling 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 M, Schmidt K, Ozkul A and Groschup MH (2013). The impact of Crimean-Congo haemorrhagic fever virus on public health. *Antiviral Res.* 2013 May; 98(2):248-60. Doi: 10.1016/j.an-tiviral.2013.02.007. Epub 2013 Feb 28.

4. DISCOVERY OF CCHF TICK VECTOR
IN HUNGARY

Crimean-Congo haemorrhagic fever (CCHF) is a
zoonotic viral disease transmitted primarily by
tick species of the genus *Hyalomma*. These ticks
are endemic to regions with (sub)tropical or
Mediterranean climates, such as Albania, Tur-
key and Uzbekistan. Migratory birds may occa-
sionally 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.

<figure><img src="image_22.png" /><figcaption>Hyalomma rufipes from cattle.
© Sándor Hornok, Szent István University,
Hungary</figcaption></figure>

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

## 5. 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
<figure><img src="image_23.png" /></figure>

attle,
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ngary

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with tick-borne encephalitis (TBE), 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 investiga-
tions 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, Lecollinet S, Hubálek Z, Svobodová P, Lussy H and Nowotny N (2013). Tick-borne Encephalitis Virus in Horses, Austria, 2011. *Emerg Infect Dis.* 2013 April; 19(4): 635–637. Doi: 10.3201/eid1904.121450

6. 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 implica-
tion for the Public Health system of the Euro-
pean Union, of which Romania has been a member
since 2007. In this country, cases of Lyme
disease and TBE have been identified. However,
little is known about the Public Health im-
pact of these diseases, and none of the other
tick-borne pathogens present in Europe have
been reported as causes of infection in Roma-
nia. Therefore, the EDENext study by Paduraru
et al. (2012) in which various zoonotic tick-
borne pathogens (including *Rickettsia monacen-
sis*, *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 preva-
lence of these diseases in the country and the
implications of these results for national and
international (European Union) Public Health
surveillance.

Paduraru OA, Buffet JP, Cote M, Bonnet S, Moutailler S, Paduraru V, Femenia F, Eloit M, Savuta G and Vayssier-Taussat M. Zoonotic transmission of pathogens by *Ixodes ricinus* ticks, Romania. *Emerg Infect Dis.* 2012 Dec; 18(12):2089-90. Doi: 10.3201/eid1812.120711.

7. 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 im-
portance (among others, TBE, 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 J, Hansford K, Bormane A, Derdakov M, Estrada-Peña A, George JC, Golovljova I, Jaenson T, Jensen JK, Jensen P, Kazimirova M, Oteo J, Papa A, Pfister K, Plantard O, Randolph S, Rizzoli A, Santos-Silva M, Sprong H, Vial L, Hendrickx G, Zeller H and Bortel W (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

## 8. 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, or Lyme borreliosis, is the most frequent vector-borne disease of humans in the Northern Hemisphere. The causative agent is the spirochaete *Borrelia burgdorferi* sensu lato, 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 (in-
cluding humans) overlap. However, these fac-
tors are not homogeneously distributed among
the different landscapes. An understanding of
the underlying processes in the transmission
system is crucially needed to explain the spa-
tial distribution risk of Lyme disease.

In this EDENext study, Li et al. (2012) de-
monstrate a cellular automata model for Lyme
disease. The study includes a heterogeneous
landscape model with three interactive com-
ponents (hosts, tick population and a disease
transmission function). Scenarios of various
landscape configurations are simulated and com-
pared. Based on the simulation results, the
authors conclude that Lyme disease risk corre-
lates to the density, shape and aggregation le-
vel 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, Hartemink N, Speybroeck N and Vanwambeke SO (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.

9. 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 belon-
ging to the *Borrelia burgdorferi* sensu lato
complex and transmitted by the widespread tick
species, *Ixodes ricinus*. However, the diagno-
sis of Lyme disease is not always confirmed
and unexplained syndromes occurring after tick
bites have become an important issue. Recent-
ly, *Borrelia miyamotoi* belonging to the relap-
sing fever group and transmitted by the same
*Ixodes* species has been involved in human di-
sease in Russia, the USA and the Netherlands.
In this study, researchers identified B. *miya-
motoi* 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 J-F, Michelet L, Chotte J, Le Naour E, Cote M, Devillers E, Poulle M-L, Huet D, Galan M, Geller J, Moutailler S and Vays-sier-Taussat M (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.

## 10. 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 pro-tozoan 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 TBEV 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 pro-tozoans (*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 A, Silaghi C, Obiegala A, 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.
## 11. RICKETTSIAE IN QUESTING IXODES RICINUS TICKS IN THE CZECH REPUBLIC.

In a study implemented by Venclikova et al. (2014), a total of 1,473 *Ixodes ricinus* ticks (1294 nymphs, 99 males, and 80 females) were collected and screened for the presence of human pathogenic rickettsiae (*Rickettsia helvetica*, *R. monacensis*, *Candidatus* Neoehrlichia mikurensis, and *Anaplasma phagocytophilum*) in natural and urban ecosystems using molecular techniques. The minimum infection rate (MIR) for *Rickettsia* spp. was 2.9% in an urban park and 3.4% in a natural forest ecosystem; for *Candidatus* Neoehrlichia mikurensis, MIRs of 0.4% in the city park and 4.4% in natural habitat were observed, while for *A. phagocytophilum* the MIR was 9.4% and 1.9%, respectively. The study provides the first data on the occurrence of human pathogenic rickettsiae in questing *I. ricinus* ticks in the Czech Republic.

Venclikova K, Rudolf I, Mendel J, Betasova L and Hubalek Z (2014). Rickettsiae in questing *Ixodes ricinus* ticks in the Czech Republic. *Ticks and Tick-borne Diseases*, Volume 5, Issue 2, March 2014. Doi: 10.1016/j.ttb-dis.2013.09.008.

## 12. 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 tick-borne 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, at least in Central Europe.

Hornok S, Kováts D, Csörg T, Meli ML, Gönczi E, Hadnagy Z, Takács N, Farkas R and Hofmann-Lehmann R (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.

## 13. ISOLATION AND PROPAGATION OF A SPI-ROPLASMA SPECIES FROM SLOVAKIAN IXODES RICINUS TICKS IN IXODES SPP. CELL LINES

*Ixodes* spp. ticks may occasionally harbour spiroplasmas - helical mycoplasmas in the class Mollicutes; a previous study in Slovakia reported an overall prevalence of *Spiroplasma ixodetis* of 3% in *I. ricinus*. In the present study, extracts of unfed adult *I. ricinus* collected from vegetation in south-western Slovakia were added to a panel of cell lines derived from *I. ricinus* and *I. scapularis* embryos (Bell-Sakyi et al. 2015). The cultures were monitored at intervals over the subsequent 16-18 months. *Spiroplasma*-like microorganisms were detected in cultures of both tick species after 2-3 months and subcultured onto fresh, uninfected cells of the appropriate cell line up to seven times. Molecular analysis using PCR assays targeting fragments of the 16S rRNA, ITS and rpoB genes confirmed the identity of the microorganisms as a *Spiroplasma* species, with between 98.9% and 99.5% similarity to *S. ixodetis*. The sequences of the Spiroplasmas isolated from three different pools of ticks collected on two different occasions were identical for all three genes tested.

Bell-Sakyi L, Palomar AM and Kazimirova M (2015). Isolation and propagation of a *Spiroplasma* sp. from Slovakian *Ixodes ricinus* ticks in *Ixodes* spp. cell lines. *Ticks and Tick-borne Diseases*, Available online 11 May 2015, ISSN 1877-959X. Doi: 10.1016/j.ttbdis.2015.05.002.
Molecular taxonomy
of sand flies:

which species are out there
and how can we find out?

INTRODUCTION:
A TAXONOMIC PUZZLE

Phlebotomine sand flies (Diptera: Psychodidae, Phlebotominae) are the only proven vectors of leishmaniasis, a group of important but neglected human and animal diseases. These diseases are endemic in 88 countries, putting an estimated 350 million people at risk and afflicting 10 million people (World Health Organization 2010). In addition, sand flies also transmit other human and animal pathogens such as bacteria (Bartonella) and viruses (Bunyaviridae, Reoviridae and Rhabdoviridae families), further increasing their medical and veterinary significance. While the occurrence of bartonellosis is restricted to certain regions in the New World, current studies reveal an unexpectedly high diversity of sand fly-borne viruses and show that the transmission of different arboviruses occurs more often than previously thought across a wide range of the sand fly distribution area.

Vit Dvorak<fnref n="1" />, Ozge Erisoz Kasap<fnref n="2" />,
Bulent Alten<fnref n="2" />, Jérôme Depaquit<fnref n="3" />,
Kristyna Hlavackova<fnref n="1" />, Jan Votypka<fnref n="1" />,
Petr Halada<fnref n="4" /> and Petr Volf<fnref n="1" />

Despite this obvious and indisputable importance of sand flies in human and veterinary medicine, inadequate and inconsistent attention has been paid to their taxonomy and morphological identification. Several taxonomic reviews and keys for the identification of sand fly species have been serving as 'gold standard' references for several decades; however, these fundamental resources have become gradually outdated and nowadays do not reflect the current views on particular groups of sand fly species. Moreover, with a detailed morphological assessment of some vector species' populations, the actual discriminative power of some morphological characters that these keys rely on, and their ability to conclusively identify species, has been subject to critical debate. Nevertheless, proper identification of sand fly species is necessary not only for the purposes of faunistic entomological surveys. Its importance is considerably greater in epidemiological studies conducted in endemic areas of leishmaniasis, where the presence of morphologically similar species with different vectorial capacities and roles in the transmission of the pathogen can obscure vector-parasite relationships and their consequences for adequate control measures.

The conventional approach to the identification of sand fly species is based on morphological features and therefore requires the dissection of freshly collected or properly stored specimens and the mounting of their head and abdomen which bear the decisive characters (genitalia, cibarium and pharyngeal armature). Both the preparation of the slides and the identification are laborious and time-consuming, and demand a certain degree of proficiency and expertise. Moreover, dried or incomplete specimens are often impossible to identify. Determination is further obscured when specimens bear intermediate morphological characters. All these aspects of morphological analysis contribute to the conclusion that traditional identification of sand flies should be supported by complementary molecular methods.

<footnote n="1">¹ Department of Parasitology, Charles University,
Prague, Czech Republic</footnote>

<footnote n="2">²Department of Biology, Hacettepe University,
Ankara, Turkey</footnote>

<footnote n="3">³ Transmission vectorielle et épidémi-
surveillance de maladies parasitaires
(VECPAR), Université de Reims Champagne-
Ardenne, Reims, France</footnote>

<footnote n="4">⁴ Laboratory of Molecular Structure Characteriza-
tion, Institute of Microbiology, The Czech
Academy of Sciences, Prague, Czech Republic.</footnote>
accidental findings of immature sand fly life stages, there is no comprehensive morphological approach available and DNA barcoding would be much appreciated. Recently, the question of whether DNA barcoding is a suitable methodological approach for the discovery of new species has been much debated (Moritz and Cicero 2004). While the delimitation of a new species exclusively on the grounds of a divergence of mtDNA sequence is not considered reasonable for numerous reasons (Sites and Marschall 2003), divergent or discordant mtDNA sequences can stimulate taxonomic reassessment based on nuclear genes and morphology, in other words 'reverse taxonomy'. Thus DNA barcoding proves beneficial to research on groups like sand flies where complexities such as cryptic species are often encountered.

We investigated barcode sequences of specimens belonging to the subgenus *Transphlebotomus* originating from localities in Turkey and Crete. This subgenus comprises only three so far described species (*Phlebotomus mascittii*, *P. economidesi*, *P. canaaniticus*) which have a markedly restricted distribution in the Mediterranean basin. While *P. mascittii* is recorded from a broad area ranging from the western Mediterranean (Spain, Italy and France) and constitutes the northern limit of sand fly distribution in Europe (Belgium, Germany and Austria), the other two species are more restricted. *P. canaaniticus* is present in eastern Mediterranean countries (Syria, Lebanon, Israel and Palestine) and *P. economidesi* has been recorded only from its type locality in Cyprus. Species of this subgenus are never abundant in sand fly catches throughout their range of distribution, which is often attributed to their presumably cavernous habitats and low biting rates on humans. Therefore, their possible incrimination in transmission of leishmaniasis has never been studied, although it has been speculated that *P. mascittii* may be a vector in several small canine leishmaniasis foci in southern Germany, an assumption based on the fact that *P. mascittii* was the only species that was found in this region (Naucke et al. 2008).

Characterisation of COI barcode sequences of all three known *Transphlebotomus* species and their comparison with sequences from our investigated specimens from Crete and Turkey revealed important genetic differences which were corroborated by analyses of several other mitochondrial and nuclear genetic markers. They clearly demonstrated that specimens from several localities on the southern coast of Crete, as well as southern Anatolia in Turkey, in fact represent two putative new species. Their status was further confirmed by detailed morphological analysis of traditional features (pharynx, antennae and genitalia), which revealed minor, yet species-distinctive differences, that had not been noticed in the past (Erisoz Kasap et al. 2015). The description of two new sand fly species from a Mediterranean region, the first for several decades, represents a prime example of how molecular techniques, namely DNA barcoding, can help to reveal overlooked biodiversity and provide direction for taxonomists' investigations.

The key factor that affects (and often limits) the applicability and resolution power of DNA barcoding is the availability of a robust database of barcodes encompassing all major (and possibly also most of the minor and potentially present) sand fly species in a given region. Unfortunately, publicly available barcodes in the BOLD database and other resources, such as GenBank, represent only a fraction of species and do not constitute a collection that would be relevant for any particular endemic area of leishmaniasis transmission (Mediterranean basin, East Africa and the Indian subcontinent etc.). A research project dedicated to creating such a database for an epidemiologically important region for leishmaniasis transmission would create a strong tool enabling the routine and conclusive identification of sand fly species surveyed in that area.

## MOLECULAR MARKERS: HANDY TOOLS FOR SPECIES IDENTIFICATION

Besides DNA barcoding, molecular taxonomy methods can offer practical solutions to resolve particular problems in species identification within some difficult species complexes or closely related species without the necessity of analysing sequences of the studied specimens. Several methods of DNA fingerprinting can help to design tools for such tasks.

To characterise species-specific markers for their easy and rapid identification, we studied *Phlebotomus neglectus* and *P. syriacus*, closely related species which both belong to the *P. major* s.l. species complex. Established by Lewis (1982), this complex comprises up to six species or sub-species of similar morphology and a wide distribution area ranging from the Mediterranean basin to Russia, China and the Indian subcontinent. Until now there is no exact delineation of their actual distribution but the general concept assumes that only *Phlebotomus neglectus* and *P. syriacus* are present in the Mediterranean region, contributing to the transmission of *Leishmania infantum*. *P. neglectus* is a
<figure><img src="image_24.png" /><figcaption>Type locality of newly described species Ph. killicki in Crete, Greece.</figcaption></figure>

proven vector in several countries while *P. syriacus* is a probable vector based on circumstantial evidence (distribution, biting habits and taxonomic position). Nevertheless, it is very difficult to distinguish between these two species based on their morphology. The decisive characters in female genitalia are very difficult to assess in mounted specimens, while the length characters of male terminalia probably exhibit a continuum rather than the two distinctive and species-specific ranges that are given by traditional keys. Thus, these two closely related yet genetically distinct vector species are hot candidates for the development of molecular markers which would make it possible to conclusively identify the species.

When considering methods of DNA fingerprinting, one option is to resort to Random Amplified Polymorphic DNA (RAPD) markers. Comparing reproducible RAPD patterns of *P. neglectus* and *P. syriacus* acquired by a number of random decamer primers, we identified several candidate differential bands, sequenced them and designed specific primers for their amplification. We also exploited Restriction Fragment Length Polymorphism (RFLP) markers. By preparing an alignment of several gene sequences (namely cytB and EF alpha which are routinely used in phylogenetic studies) and predicting restriction sites within these sequences, we identified two restriction enzymes that find a single restriction site in the sequence of one species but lack any site in the sequence of the

second species. Therefore, after application of a restriction enzyme, a decisive pattern distinguishes the two species

By combining these two approaches, we have developed a panel of molecular markers that conclusively distinguish these two species. The main advantages of these markers are that they are rapid and cost-effective as they do not require sequencing analysis but only routine PCR amplification and, in the case of RFLP-based markers, quick enzyme restriction. However, the key element in developing such molecular markers is to test them on a large collection of different populations within each species. The general applicability of markers based on specimens stemming from a limited number of species populations can be severely hampered by intraspecific variability among different populations. Moreover, it is also important to apply the markers on other sand fly species that are present in the region to avoid possible cross-positivity. We successfully tested our markers on a large number of specimens from different populations (*P. neglectus*: Italy, Croatia, Hungary, Montenegro and Albania; *P. syriacus*: Turkey, Syria, Lebanon and Israel) and also several species of the subgenus *Larroussius* as well as other subgenera present in the Mediterranean region. We therefore believe they are applicable on the wide range of distribution of our desired species. While there are numerous examples of such species pairs or complexes in the Mediterranean region in the subgenus *Larroussius*, as well as other subgenera, such as hoc molecular markers could prove to be valuable tools for routine species differentiation.

## MALDI-TOF PROTEIN PROFILING: A NOVEL METHOD FOR SPECIES IDENTIFICATION

Protein profiling by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry has been established as a routine technique for the identification and successful discrimination of clinically important micro-organisms. Compared to classical phenotyping and molecular methods, this approach is simple, accurate and inexpensive. It requires minimal sample preparation and is especially useful for routine assessment of many field samples. All these qualities make this approach particularly attractive for applications on medically important insects of complicated taxonomy, including sand flies.

The procedure typically starts with acetonitrile/acid-based extraction of peptides and small proteins followed by mixing the resulting sample with a suitable aromatic acid serving as a MALDI matrix. After co-crystallization, the crystals are irradiated, desorbed

and the resulting mass spectrum can be used to distinguish species. Over the years, organophosphite and organobisphenol A have been identified as significantotoxins. In addition, the use of specific restriction enzymes has been shown to be effective in separating and identifying multiple alleles of a single gene. For example, the use of the P. aeruginosa aminopeptidase N (APN) enzyme has been shown to be effective in separating and identifying multiple alleles of the APN gene in P. aeruginosa isolates.
and ionized by a short laser pulse to generate peptide/protein ions in the gas phase, which are then measured by mass spectrometer. The obtained mass spectrum represents a characteristic and unique protein fingerprint which can either be compared with a reference spectra database for rapid and effective species identification, or can be evaluated using statistical methods for species discrimination and typing

Over the past decade, the use of MALDI-TOF MS protein profiling for species classification and identification has been expanded to many eukaryotic organisms including medically important haematophagous arthropods. Very recently, it was successfully applied on biting midges, tsetse flies, mosquitoes, fleas and ticks. We have demonstrated, for the first time, the applicability of MALDI-TOF MS to identify and distinguish Phlebotomine sand fly species, of both sexes, under different conditions of storage and homogenisation and to test its discriminatory power regarding subgenera, species and populations (Dvorak et al. 2014). The discriminatory power of the MS-based approach was initially tested on five Mediterranean species of four different subgenera, all proven vectors of important *Leishmania* parasites, namely *Phlebotomus papatasi* (specific vector of *L. major*), *P. sergenti* and *P. arabicus* (specific vectors of *L. tropica*), *P. perniciosus* and *P. tobbi* (vectors of *L. infantum*).

Specimens of all five sand fly species analysed produced informative, reproducible and species-specific protein spectra that enabled conclusive species identification. The method also distinguished between two *P. sergenti* colonies originating from different geographical localities. Protein profiles within a species were similar for specimens of both sexes. We thoroughly tested the conditions of specimen storage and sample preparation in order to establish a standardised protocol that would be generally applicable and reflect the situations encountered in sand fly field surveys. We had in mind the utilisation of a single specimen for several approaches (i.e. MALDI-TOF protein profiling, DNA-based methods and traditional morphological analysis).

After the first successful application of the method on sand flies, we focused on processing more species from the Mediterranean basin and elsewhere, and our protein profile collection currently comprises 20 species of several genera and subgenera. We also tested the sensitivity of the method for distinguishing between different and geographically distant or close populations of the same species by analysing populations of *P. neglectus* from Croatia, Hungary and Romania and *P. perniciosus* from several catch sites in Spain. Preliminary results indicate that protein profiling is able to differentiate between distant populations while closer populations, with presumable gene flow, appear to produce almost identical protein spectra. Finally, we addressed the possibility of cross-analysing data produced by a machine made by one manufacturer (Bruker Daltonics) using software from another major manufacturer (Shimatzu) as it would much enhance the applicability and spread of the method. It was demonstrated that this is indeed possible (Mathis et al. 2015).

REFERENCES

Antoniou M, Gramiccia M, Molina R, Dvorak V, Volf P (2013). The role of indigenous phlebotomine sandflies and mammals in the spreading of leishmaniasis agents in the Mediterranean region. *Euro Surveillance* 18(30):pii=20540.

Dvorak V, Halada P, Hlaváčkova K, Dokianakis E, Antoniou M, Volf P (2014). Identification of phlebotomine sand flies (Diptera: Psychodidae) by matrix-assisted laser desorption/ionization time of flight mass spectrometry. *Parasites & Vectors* 7:21.

Erisoz Kasap O, Dvorak V, Depaquit J, Alten B, Votypka J, Volf P (2015). Phylogeography of the subgenus Transphlebotomus Artemiev with description of two new species, Phlebotomus anatolicus n. sp. and Phlebotomus killicki n. sp. *Infection, Genetics and Evolution*, in press.

Giorgobiani E, Lawyer PG, Babuadze D, Golidze N, Jochim RC (2012). Incrimination of *Phlebotomus kandelakii* and *Phlebotomus balcanicus* as vectors of *Leishmania infantum* in Tbilisi, Georgia. *PLoS Neglected Tropical Diseases* 6(4): e1609.

Hebert P, Cywinska A, Ball S, deWaard J (2003). Biological identification through DNA barcodes. *Proceedings of the Royal Society* 270: 313-321.

Lewis D (1982). A taxonomic review of the subgenus Phlebotomus (Diptera: Psychodidae). *Bulletin of the British Museum, Entomology series* 42: 1-95.

Killick-Kendrick R (1991). Phlebotomine vectors of the leishmaniasis: a review. *Medical and Veterinary Entomology* 4: 124.

Maroli M, Feliciangeli MD, Bichaud L, Charrel R, Gradoni L (2013). Phlebotomine sandflies and the spreading of leishmaniases and other diseases of public health concern. *Medical and Veterinary Entomology* 27(2):123-147.

Mathis A, Depaquit J, Dvorak V, Tuten H, Halada P, Zapata S, Lehrter V, Hlavackova K, Prudhomme J, Volf P, Sereno D, Kaufmann C, Pflüger V, Schaffner F (2015). Identification of phlebotomine sand flies using one MALDI-TOF MS reference database and two mass spectrometer systems. *Parasites and Vectors* 8:266.

Medlock J, Hansford K, Van Bortel W, Zeller H, Alten B (2014). A summary of the evidence for the change in European distribution of phlebotomine sand flies (Diptera: Psychodidae) of public health importance. *Journal of Vector Ecology* 39 (1): 72-77.

Moritz C, Cicero C (2004). DNA barcoding: promises and pitfalls. *PLoS Biology* 2: 1529-1531.
<figure><img src="image_25.png" /></figure>

MALDI-TOF MS protein profiles of five different species of the genus *Phlebotomus* showing species-unique peaks for conclusive species identification. The peaks in the mass spectra represent peptides or small proteins obtained from the sand fly bodies using acidic extraction.
Dvorak et al. *Parasites & Vectors* 2014 7:21 doi:10.1186/1756-3305-7-21

Species identification of sand flies using MALDI-TOF MS appears to be a feasible approach and will soon become a novel promising tool to improve biological and epidemiological studies on these medically important insects, being a rapid, simple, reproducible and cost-effective alternative, even to DNA-based methods. The key goal of ongoing research in this field is the construction of a robust reference database of species-specific protein spectra that would be publicly available and comprise an adequate amount of protein spectra to encompass intraspecific variability together with species biodiversity.

## CONCLUSION

Leishmaniasis are important yet neglected human and animal diseases that deserve adequate attention from both research and Public Health bodies. It has been clearly demonstrated that while they constitute a serious health problem in many regions of the world, they cannot be underestimated even in well-developed regions such as Europe. They are dynamic diseases and the circumstances of transmission are continually changing in relation to environmental, demographic and human behavioural factors. Recent studies show that the epidemiology of leishmaniasis in the Mediterranean basin is changing, old historical foci re-emerge and new foci are established. Introduction of new strains from other regions is a possibility that should be seriously considered (Antoniou et al. 2013).

Phlebotomine sand flies are the only proven vectors of *Leishmania* parasites and as such they should be under the spotlight of consistent and intensive research. Many sand fly species contribute to the transmission of leishmaniasis in natural foci, others
show the ability to transmit the causative agents, at least under laboratory conditions, and therefore represent possible vectors when the parasites are introduced into their natural environment (Volf and Myšková 2007). For the purposes of epidemiological studies, as well as appropriate control measures, it appears crucial to thoroughly study sand fly biology and understand their taxonomy as well as developing methods for accurate, conclusive and rapid species identification. Advances in understanding sand fly taxonomy as well as some practical tools for species identification have been possible due to the recent deployment of new molecular techniques. It must be emphasised that these novel methods do not replace but rather support and complement the traditional morphological approach as there is always a need for a baseline reference and 'gold standard'. The new methods for species identification described earlier must be based on broad and sufficient representations of sand fly species and populations from different areas.

Large collaborative projects such as EDEN and EDENext, which bring together researchers from many endemic countries, constitute the ideal platform for data collection, exchange of expertise and research coordination. We believe that the many scientific achievements of the phlebotomine-borne disease group within EDENext have contributed to the introduction and promotion of novel techniques and established a network of institutions which will continue to collaborate in the future.

Moreno, J and Alvar, J (2002). Canine leishmaniasis: epidemiological risk and the experimental model. *Trends in Parasitology* 18: 399–405.

Naucke TJ, Menn B, Massberg D, Lorentz S (2008). Sandflies and leishmaniasis in Germany. *Parasitology Research* 103 Suppl1: S65-68.

Ready P (2013). Biology of Phlebotomine sand flies as vectors of disease agents. *Annual Reviews in Entomology* 58:227–250.

Sites J, Marschall J (2003). Delimiting species: A Renaissance issue in systematic biology. *Trends in Ecology and Evolution* 18: 462-470.

Vogler A and Monaghan M (2006). Recent advances in DNA taxonomy. *Journal of Zoological Systematics and Evolutionary Research* 45: 1-10.

Volf P and Myskova J (2007). Sand flies and *Leishmania*: specific versus permissive vectors. *Trends in Parasitology* 23:91-92.

World Health Organization: *Control of the Leishmaniasis. WHO technical report series 949*. Geneva: WHO; 2010.

## ACKNOWLEDGEMENT

We would like to thank all the researchers who worked in the EDEN and EDENext Phlebotomine-borne diseases teams. We thank the following institutions:

Aix-Marseille Université, France

Charles University in Prague, Czech Republic

Ege University Medical School, Turkey

Hacettepe University. Faculty of Science, Turkey

Institut de Recherche pour le Développement, France

Instituto de Higiene e Medicina Tropical. Universidade Nova de Lisboa, Portugal

Instituto de Salud Carlos III, Spain

Istituto Superiore di Sanità, Italy

National Center for Disease Control and Public Health, Georgia

University of Crete, Greece
PUBLICise Health Public Health
Telegram: Phlebotomine-borne
diseases

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 an updated version of the issue dedicated to the work of the team examining Phlebotomine-borne diseases (Diptera, Psychodidae, subfamily Phlebotominae), first published in September 2014.

1. The role of phlebotomine sand flies in spreading diseases which are 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, phle-
botomine sand flies 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 (Bunyaviridae), which includes the sandfly fever Sicilian and Toscana viruses, and the *Vesiculovirus* genus (Rhabdoviridae), which includes vesicular stomatitis, and the Chandi-pura and Isfahan viruses. *Vesiculovirus*, which is well-known to affect livestock animals in southeastern USA and Latin America, is increas-singly 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 restric-ted 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 phlebo-tomine 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 analy-tical models are needed.

Maroli M, Feliciangeli MD, Bichaud L, Charrel RN and Gra-doni L (2013). Phlebotomine sandflies and the spreading of leishmaniasis and other di-seases of public health concern. *MedVetEntomol.2013Jun;27(2):* 123-47. Doi: 10.1111/j.1365-2915.2012.01034.x. Epub 2012 Aug.

## 2. 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 Medi-
terranean Basin and has recently been reported
to have spread to parts of Central Europe, for
example *Leishmania infantum* in northern Ita-
ly, 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 in-
troduction.

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 trans-
mitted 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 dis-
tribution 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 environmen-
tal factors using geospatial tools.

*Leishmania* seropositive dogs were found in 43 of 54 prefectures with an average of 22% indicating that the disease is widely spread across Greece. Favourable 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. Chan-
ging 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

Ntais P, Sifaki-Pistola D, Christodoulou V, Messaritakis I, Pratlong F, Poupalos G and Antoniou M (2013). Leishmaniasis in Greece. *Am J Trop Med Hyg*. 2013 Nov;89 (5): 906-15. Doi: 10.4269/ajtmh.13-0070. Epub 2013 Sep 23.

Ntais P, Christodoulou V, Tsirigotakis N, Dokianakis E, Dedet JP, Pratlong F and Antoniou M (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*. 2014 Apr; 132:125-30. Doi: 10.1016/j.actatropica.2014.01.003. Epub 2014 Jan 24
of dogs are costly and time-consuming, the aim
of the study was to evaluate the question-
naire, 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 leish-
maniasis prevalence for use in predictive mo-
delling. In addition, the questionnaire offers
a two-way exchange of information; it provides
information not only to scientists and specia-
lists 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 ca-
nine leishmaniasis in an area, it demonstrates
the dynamic of the disease. Overall, the ve-
terinary questionnaire can be used as an early
warning system and a screening tool to assess
the risk of leishmaniasis in a country or re-
gion and therefore promoting Public Health.

Sifaki-Pistola D, Ntais P, Christodoulou V, Mazeris A and Antoniou M (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*. 2014 Jun 23. pii: 13-0459.

3. Risk factors for cutaneous leishmaniasis
in Turkey

Cutaneous leishmaniasis (in the form of a cu-
taneous lesion at the infected sand fly bite
site), is the most common form of human leish-
maniasis, with millions of human cases reported
from southern Europe, Asia, Africa, South and
Central America every year. Other mammal spe-
cies, 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) eva-
luated 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 cli-
nical characteristics, application of perso-
nal protection measures and knowledge about
Leishmania parasites were analysed. Ownership
of a dog, raising cattle and sleeping wit-
hout a bed-net were associated with a signifi-
cantly 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 3.67% to 4.69%. 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).

### 4. 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.

Votýpka J, Kasap OE, Volf P, Kodym P and Alten B (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. Epub 2012 Jan 26.

Gunay F, Karakus M, Oguz G, Dogan M, Karakaya Y, Ergan G, Kaynas S, Kasap OE, Ozbel Y and Alten B (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

Campino L, Cortes S, Dionisio L, Neto L, Afonso MO and Maia C (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.
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 *L. 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.

**5. 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.

Maia C, Dionísio L, Afonso MO, Neto L, Cristóvão JM and Campino L (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.* 2013 Jun; 108(4):481-7. Doi: 10.1590/S0074-0276108042013014.

Maia C, Ramos C, Coimbra M, Bastos F, Martins A, Pinto P, Nunes M, Vieira ML, Cardoso L and Campino L (2014). Bacterial and protozoal agents of feline vector-borne diseases in domestic and stray cats from southern Portugal. *Parasit Vectors.* 2014 Mar 24; 7(1):115. Doi: 10.1186/1756-3305-7-115.

Maia C, Parreira R, Cristóvão JM, Freitas FB, Afonso MO and Leña Campino L (2015). Molecular detection of *Leishmania* DNA and identification of blood meals in wild caught phlebotomine sand flies (Diptera: Psychodidae) from southern Portugal. *Parasites & Vectors* 2015, 8:173. Doi: 10.1186/s13071-015-0787-4.
While the appearance of autochthonous visceral leishmaniasis cases in northern parts of Italy could be explained by the de novo colonisation with phlebotomine vectors along with importing *Leishmania*-infected dogs from the endemic south and the generalised 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 M, Scalone A, Di Mucchio T, Orsini S, Fiorentino E and Gradoni L (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.* 2013 Jul 18; 18(29):20535.

## 6. 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 peri- phery. In order to find the sources of infec- tion, 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 success- fully 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) could show that wild rabbits and hares captured in this area show high anti-sand fly saliva antibody levels which indicates that 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 leishmania- sis. 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. perni- ciosus*, caught in the same area, also revealed that the majority of sand flies fed on hares and/or rabbits.

In the framework of entomological studies ta- king place in this area, Jimenez et al. (2013) conducted a preliminary entomological survey in 2011, at the end of the seasonal transmis- sion 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 *Phebotomus* sand flies was comparably high with approxi- mately 60%. In addition, blood feeding pre- ferences, analysed by cytochrome b analysis,

Jiménez M, González E, Iriso A, Marco E, Alegret A, Fúster F and Molina R (2013). Detection of *Leishmania infantum* and identification of blood meals in *Phlebotomus perniciosus* from a focus of human leishmania- sis in Madrid, Spain. *Parasitol Res*. 2013; 112(7):2453-9. Doi: 10.1007/s00436-013-3406-3. Epub 2013 Mar 28.

Jiménez M, González E, Martín- Martín I, Hernández S and Molina R (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.