FUNDING APPLICATION FOR YOUNG RESEARCH TEAMS - PN-II-RU-TE-2014-4

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CUPRINS

B. Project leader ...... 3

B1. Important scientific achievements of the project leader ...... 3

B2. Curriculum vitae ...... 5

B3. Defining elements of the remarkable scientific achievements of the project leader ...... 7 B3.1 The list of the most important scientific publications from 2004-2014 period ...... 7 B3.2. The autonomy and visibility of the scientific activity...... 9

C.Project description ...... 11

C1. Problems...... 11

C2. Objectives...... 13

C3. Impact...... 14

C4. Methodology...... 15

C5. Resources and budget...... 18

2 B. Project leader B1. Important scientific achievements of the project leader (maximum 2 pages) Please present the most important contributions of the project leader in his/her research field.

The main scientific achievement of the project leader (as main investigator or as coordinator of research grants) within the proposed topic (parasitic , vector-borne diseases, wild medicine):

 Obtaining the title of Diplomate of European College of Zoological Medicine, being the only Romanian Diplomate recognized by the European Board of Veterinary Specialisation.  Within the COST European Network “European Network for Neglected Vectors and Vector- Borne Infections (EURNEGVEC)” coordinated by the project leader, the first exhaustive identification keys for ticks from Europe have been produced and are under publication in the form of a monograph.  Description of four species new to science (nov. sp.): Isospora farahi nov. sp. (from the lizard Agama rueppelli in Kenya) (Mihalca et al. 2009), Ixodes ariadnae nov. sp. (from bats in Europe) (Hornok et al. 2014), samburi nov. sp. (from the antelope Madoqua guentheri in Kenya) (Durden et al. 2014), Stachiella roumanicus nov. sp. (from the European mink Mustela lutreola in ) (Mey et al. in prep).  Morphological redescription and genetic characterization of some rare species of ticks: Ixodes auriculaelongae (Central African Republic), Ixodes aulacodi (Ivory Coast) and Rhipicephalus camicasi (Kenya).  First report of Borrelia burgdorferi sensu lato (Lyme Disease agent) infection in endagered carnivore species: the Marbled polecat (Vormela peregusna) and European mink (Mustela lutreola) (Gherman et al. 2012b).  First highlight of the medical importance of Rhipicephalus rossicus tick in areas sympatric with R. sanguineus (Mihalca et al. 2014, Dumitrache et al. 2014, Sándor et al. 2014a).  First experimental proof for solving the transmission cycle of Borrelia turcica (Kalmár et al. 2014).  First report of Trichinella spiralis infection in carnivores of Mustelidae family in Europe (Oltean et al. 2014).  First report in Europe of Knemidocoptes intermedius (scaly leg mites in wild birds) (Dabert et al. 2011).  First report in Eastern Europe of Thelazia callipaeda (zoonotic vector-borne nematode with ocular localization in dogs) (Mihalca et al. submitted).

3  First exhaustive analysis and introduction of the concept of co-endangered tick species in literature (Mihalca et al. 2011).  First nationwide study regarding the diversity and distribution of ticks in Romania (Mihalca et al. 2012a, c).  First national distribution maps of two major tick-borne zoonoses: Lyme disease (caused by Borrelia burgdorferi sensu lato) (Kalmár et al. 2013) and granulocytic anaplasmosis (caused by Anaplasma phagocytophilum) (Matei et al. submitted), the first pan-national study regarding the epidemiology of canine filariases (Ionică et al. in press) and the first nationwide study on the distribution and epidemiology of the main vector-borne zoonoses in dogs (Mircean et al. 2012).  First report of an autochthonous case of canine leishmaniasis in Romania after 80 years of apparent absence (Mircean et al. 2014).  First report in Romania of certain parasites from domestic or wild : Cercopithifilaria bainae (agent of canine subcutaneous filariasis) (Ionică et al. 2014), Knemidocoptes jamaicensis (scaly leg mites in wild birds) (Dabert et al. 2013) etc.  First extensive survey on the role of foxes as reservoir hosts for major zoonoses (Șuteu et al. 2014b), for the role of wild birds (Sándor et al. 2014) and micromammals as reservoirs for major vector-borne pathogens (Mihalca et al. 2012b; Dumitrache et al. 2011, 2013).  First study from Romania on the diversity of ticks parasitic on humans (Briciu et al. 2011).  Development of a new innovative method for collecting ticks from the environment (Gherman et al. 2012a).  First approach to the concept of conservation medicine in Romania by reporting some potentially pathogenic infections in endangered species: Alaria alata parasitic polymyositis in European minks (Mustela lutreola) (Tăbăran et al. 2013), mesothelial metaplasia caused by the nematode Spiroxys contortus in European pond turtles (Emys orbicularis) (Miclăuș et al. 2013; Miclăuș et al. 2014), vector-borne zoonoses infection in Greek tortoises (Testudo graeca) (Paștiu et al. 2012), the ecology of vector-borne parasite in reptiles (Mihalca et al. 2008; Majláthová et al. 2008; Široký et al. 2006, 2009).

4 B2. Curriculum vitae (maximum 2 pages) This must contain at least the following categories of information: (a) information about the degrees and diplomas; (b) information about the professional experience and jobs. This will include, in particular, the professional positions where the project leader coordinated a team, a group or a research laboratory, if any.

Personal data  Date of birth: 20.01.1978 (36 years)  Place of birth: Cluj-Napoca  Marital status: married, 2 children.

Degrees and diplomas  2014: Diplomate of the European College of Zoological Medicine, Wildlife Population Health specialization (European Board of Veterinary Specialisation).  2013: Habilitation (PhD coordinator): University of Agricultural Sciences and Veterinary Medicine of Cluj-Napoca (USAMV CN).  2007: Doctorate (PhD) in Medical Sciences (Veterinary Medicine - Parasitology specialisation): USAMV CN.  2003: Master of Science (MSc) (Pathology and Laboratory Diagnosis): USAMV CN  2002: Doctor of Veterinary Medicine (DVM): USAMV CN.  1996: Baccalaureat Degree: “Avram Iancu” high school of Cluj-Napoca

Professional experience  2002-2003 PhD Student, USAMV CN.  2003-2006 pre-Assistant Lecturer, Parasitology and Parasitic Diseases, USAMV CN.  2006-2009 Assistant Lecturer, Parasitology and Parasitic Diseases, USAMV CN.  2009-2013 Lecturer, Parasitology and Parasitic Diseases, USAMV CN.  2013-2014 Associate Professor, Parasitology and Parasitic Diseases, USAMV CN.  2012-2014 Vice-rector for International Relations, USAMV CN.

Relevant prizes  In Hoc Signo Vinces (Best Young Scientist), National Research Council (CNCSIS) (2010)

5 International stages and mobility  10 teaching mobility stages under Erasmus: Bari, Teramo (Italy), Brno, Prague (Czech Republic), Lisbon (Portugal), Valencia (Spain).  Scholarships: CEEPUS (), Erasmus (Portugal), Nicolae Titulescu (Czech Republic).  Practical stages: Lisbon Oceanarium, Lisbon Zoo, Budapest Zoo.

Overview of research activity  Project leader of two European Grants and three international research service contracts.  Project leader in three national grants; principal investigator in one national grant.  Team member in four European projects; Invited expert in CALLISTO (FP7), VectorNet (ECDC), ESCMID (Veterinary Microbiology Steering Group).  Team member in 12 national grants.  Main organizer of 6 scientific expeditions in Africa (Kenya, Uganda, Ivory Coast, Central African Republic).

Overview of publications  52 papers published in peer-reviewed journals with impact factor (total IF 114.5) of which 33 as principal author.  12 ISI proceedings papers.  25 other scientific papers and 80 conference abstracts.  Editor and author of the monograph: [Lyme Disease in Romania] [in Romanian], author of 4 chapters in international monographs, author of 3 textbooks (of which one in English).  Member in the Editorial Board of two ISI journals: BMC Veterinary Research, North Western Journal of Zoology and several other international peer-reviewed journals.  Reviewer for 15 journals with impact factor.

Invited speaker  Academic lectures: University of Veterinary and Pharmaceutical Sciences Brno (Czech Republic), Uppsala University (Sweden), University of Teramo (Italy).  Conferences: Mexico City, Mexico: XIII International Congress of Parasitology; Chernivtsi, Ukraine: XIV Conference of the Ukrainian Scientific Society of Parasitologists; , Romania: The Sixth Annual Zoological Congress of “Grigore Antipa”; Cluj-Napoca, Romania: XI European Multicolloquium of Parasitology; Bucharest, Romania: The Second Annual Zoological Congress of “Grigore Antipa” Museum etc.

6 B3. Defining elements of the remarkable scientific achievements of the project leader(max 4 pages) B3.1 The list of the most important scientific publications from 2004-2014 period (articles, monographs, patents, books, chapters, critical editions, dictionaries or encyclopaedias).

During 2004-2014, the project leader has published 52 papers in journals with impact factor (with a cumulative IF of 114.5). The journals include: Emerging Infectious Diseases (IF=7.327), PloS One (IF=3.534), International Journal of Medical Microbiology (IF=3.420) or Parasites & Vectors (IF=3.251). Among the most representative as principal author we mention:

1. Mihalca AD, Kalmár Z, Dumitrache MO. 2014. Rhipicephalus rossicus, a neglected tick at the margin of Europe: a review of its distribution, ecology and medical importance. Medical and Veterinary Entomology, accepted, in press. 2. Kalmár Z, Cozma V, Sprong H, Jahfari S, D’Amico G, Mărcuțan DI, Ionică AM, Magdaş C, Modrý D, Mihalca AD. 2014. Transstadial transmission of Borrelia turcica in Hyalomma aegyptium ticks. PLoS One, accepted, in press. 3. Sándor AD, Mărcuţan DI, D’Amico G, Gherman CM, Dumitrache MO, Mihalca AD. 2014. Do the ticks of birds at an important migratory hotspot reflect the seasonal dynamics of Ixodes ricinus at the migration initiation site? A case study in the Danube Delta. PLoS One 9(2):e89378. 4. Hornok S, Kontschán J, Kováts D, Kovács R, Angyal D, Görföl T, Polacsek Z, Kalmár Z, Mihalca AD. 2014. Bat ticks revisited: Ixodes ariadnae sp. nov. and allopatric genotypes of I. vespertilionis in caves of Hungary. Parasites & Vectors 7:202. 5. Mircean V, Dumitrache MO, Mircean M, Bolfă P., Györke A, Mihalca AD. 2014. Autochthonous canine leishmaniasis in Romania: neglected or (re)emerging? Parasites & Vectors 7:135. 6. Dumitrache MO, Kiss B, Dantas-Torres F, Latrofa MS, D’Amico G, Sándor AD, Mihalca AD. 2014. Seasonal dynamics of Rhipicephalus rossicus attacking domestic dogs from the steppic region of southeastern Romania. Parasites & Vectors 7:97. 7. Sándor AD, Dumitrache MO, D’Amico G, Kiss BJ, Mihalca AD. 2014. Rhipicephalus rossicus and not R. sanguineus is the dominant tick species of dogs in the wetlands of the Danube Delta, Romania. Veterinary Parasitology 204(3-4):430-432. 8. Ionică AM, D’Amico G, Mitková B, Kalmár Z, Annoscia G, Otranto D, Modrý D, Mihalca AD. 2014. First report of Cercopithifilaria spp. in dogs from Eastern Europe with an overview of their geographic distribution in Europe. Parasitology Research 113:2761-2764.

7 9. Mihalca AD, Sándor AD. 2013. The role of rodents in the ecology of Ixodes ricinus and associated pathogens in Central and Eastern Europe. Frontiers in Cellular and Infection Microbiology 3:56. 10. Mihalca AD, Dumitrache MO, Sándor AD, Magdaş C, Oltean M, Györke A, Matei IA, Ionică A, D’Amico G, Cozma V, Gherman CM. 2012. Tick parasites of rodents in Romania: host preferences, community structure and geographical distribution. Parasites & Vectors 5:266. 11. Mihalca AD, Gherman CM, Magdaş C, Dumitrache MO, Györke A, Sándor AD, Domşa C, Oltean M, Mircean V, Mărcuţan DI, D’Amico G, Păduraru AO, Cozma V. 2012. Ixodes ricinus is the dominant questing tick in forest habitats from Romania: the results from a countrywide dragging campaign. Experimental and Applied Acarology 58:175-182. 12. Mihalca AD, Dumitrache MO, Magdaş C, Gherman CM, Domşa C, Mircean V, Ghira IV, Pocora V, Ionescu DT, Sikó Barabási S, Cozma V, Sándor AD. 2012. Synopsis of the hard-ticks (Acari: Ixodidae) of Romania with update on host associations and geographical distribution. Experimental and Applied Acarology 58:183-206. 13. Paştiu AI, Matei IA, Mihalca AD, D’Amico G, Dumitrache MO, Kalmár Z, Sándor AD, Gherman CM, Cozma V. 2012. Zoonotic pathogens associated with Hyalomma aegyptium in endangered tortoises: evidence for host-switching behaviour in ticks? Parasites & Vectors 5:301. 14. Gherman CM, Mihalca AD, Dumitrache MO, Györke A, Oroian I, Sandor M, Cozma V. 2012. CO2 flagging - an improved method for the collection of questing ticks. Parasites & Vectors 5:125. 15. Mihalca AD, Gherman CM, Cozma V. 2011. Coendangered hard-ticks: threatened or threatening? Parasites & Vectors 4:71.

Among the papers as co-author, we list:

1. Briciu VT, Meyer F, Sebah D, Ţăţulescu DF, Coroiu G, Lupșe M, Cârstina D, Mihalca AD, Hizo-Teufel C, Klier C, Huber I, Fingerle V. 2014. Real-time PCR based identification of Borrelia burgdorferi sensu lato species in ticks collected from humans in Romania. Ticks and Tick-Borne Diseases 5(5):575-581. 2. Durden LA, Mihalca AD, Sándor AD, Kanyari PWN. 2014. A new species of (Phthiraptera: Anoplura: Linognathidae) from Günther’s dikdik (Madoqua guentheri) in Kenya. Journal of Parasitology, accepted, in press. 3. Kalmár Z, Mihalca AD, Dumitrache MO, Gherman CM, Magdaș C, Mircean V, Oltean M, Domşa C, Matei IA, Mărcuţan DI, Sándor AD, D’Amico G, Paștiu A, Györke A, Gavrea R,

8 Marosi B, Ionică A, Burkhardt E, Toriay H, Cozma V. 2013. Geographical distribution and prevalence of Borrelia burgdorferi genospecies in questing Ixodes ricinus from Romania: a countrywide study. Ticks and Tick-borne Diseases 4:403-408. 4. Mircean V, Dumitrache MO, Györke A, Pantchev N, Jodies R, Mihalca AD, Cozma V. 2012. Seroprevalence and geographic distribution of Dirofilaria immitis and tick-borne infections (Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato and Ehrlichia canis) in dogs from Romania. Vector-Borne and Zoonotic Diseases 12:595-603.

B3.2. The autonomy and visibility of the scientific activity. Please indicate a number of maximum 5 factual arguments of the high degree of autonomy of the research activity developed by the project leader. The arguments can belong to one or more of the following categories, but not necessarily from all categories: (1) the articles where the project leader is a main author (2) research fellowships, academic grants where the project leader was the 1 principal investigator (PI) or project coordinator (3) total number of citations; (4) international prizes (5) oral presentations at international conferences (6) status of invited speaker to prestige 2 universities .

Factual reason 1: High publication output with a significant number of citations  The project leader is author for 52 papers in journals with impact factor, of which 33 as main author, a Hirsch index H=8, with a total number of 189 citations (according to Web of Science).

Factual reason 2: Coordinator and main investigator in European or national projects  The project leader is the coordinator of FP7/H2020 COST TD1303 “European Network for Neglected Vectors and Vector-Borne Infections (EURNEGVEC)” project that includes 289 researchers from 44 countries (cca. 617.000 EUR).  The project leader is coordinator of the Tender ECDC/2014/023 “Guidance, data collection and scientific advice on tick‐borne diseases” (174.000 EUR).  The Project leader coordinated several national grants (selection): (i) IDEI PNII-PCE 236/2011: ” Geospatial approach to the ecology, distribution and vectorial role of parasitic Arthropoda” (UEFISCDI, 327875 EUR); (ii) TD 196/2005 „The study of Dracunculus oesophageus parasitism in Natrix snakes from Histria area” (UEFISCDI, 2485 EUR).

1 for each grant will be given the following concise information: title, funding agency, total amount in euros, the ongoing period and web link; 2 only 500 top universities, according to ARWU 2011 Ranking, www.arwu.org

9 Factual reason 3: Invited expert in European Scientific Committees  The project leader is Management Committee member of COST Action TD1404 “Network for Evaluation of One Health (NEOH)”, COST Action TD1302 “European Network on Taeniosis/Cysticercosis” and COST FA1408 “A European Network for Foodborne Parasites (Euro-FBP)”.  The project leader is invited expert in FP7-KBBE-2011-5 “Companion Animals multisectoriaL interprofessionaL Interdisciplinary Strategic Think tank On zoonoses”.  The project leader is invited expert in OC/EFSA/AHAW/2013/02 “VectorNet: a European network for sharing data on the geographic distribution of vectors, transmitting human and animal disease agents”.

Factual reason 4: Invited speaker and oral presentations in international conferences  The project director was invited/keynote speaker at the following scientific meetings (selection): (i) XIII International Congress of Parasitology (Mexico City, Mexico, 2014); (ii) The Sixth Annual Zoological Congress of “Grigore Antipa” Museum (Bucharest, Romania, 2014); (iii) XIV Conference of the Ukrainian Scientific Society of Parasitologists (Chernivtsi, Ukraine, 2013); (iv) XI European Multicolloquium of Parasitology (Cluj- Napoca, Romania, 2012).  The project leader has delivered oral presentations at the following scientific meetings (selection): (i) 2nd Global Conference on Entomology (Kuching, Malaysia, 2013); (ii) 23rd International Conference of the World Association of Veterinary Parasitology (Buenos Aires, Argentina, 2011); (iii) 68th Midwest Fish & Wildlife Conference (Madison, WI, USA, 2007).

Factual reason 5: The project leader is PhD coordinator The project leader defended his habilitation thesis in July 2012 and obtained the Habilitation certificate in 2013, presently coordinating 5 PhD students and 2 post-doctoral fellows who are among the authors of the papers from the lists presented above. This status of PhD coordinator renders the project director an increased autonomy in research activities and in involving the PhD students in the project. Thus, the involvement of another PhD coordinator who is not part of the proposed team is no longer required.

10 C. Project description (maximum 10 pages) In this section the project leader will detail the scientific context, the goals and objectives, the approach to these objectives and human resources. C1. Problems. The scientific motivation will clearly delimit the approached problem within the framework of the state of the art. Will be revealed the following three aspects: (1) the importance of the problem from the scientific, technologic, socio-economic or cultural point of view; (2) the difficulty elements of the problem; (3) the limits of the current approaches in the context of the state of the art in the field. The modern society is characterized by an increasing trend of globalization in most of the fields of human activity. From the public health point of view, more aspects of this trend are relevant. The international trade (especially with live animals and animal products) and the increase of global travelling (business or tourist trips) have an important role. Secondary, the demographic growth, climate changes and the way how humans interact with the environment are overlapping with globalization. All these components have an important impact on the spreading of infectious diseases to humans and animals (Dantas-Torres et al. 2012). Vector-borne diseases have gained an increasing importance among all infectious diseases mainly because of their complex life cycles and diverse host spectrum. In tropical areas, mosquitos play the major vectorial role. However, in temperate areas, including Europe, ticks are the most important vectors from the public health point of view (Baneth, 2014). All the infectious agents transmitted by ticks to humans are zoonotic, with vertebrate animals being in general the natural reservoir hosts. For this reason, any epidemiological or ecological study on vector-borne diseases should be focused on vectors and hosts, especially on wild vertebrates (Baneth 2014). In Romania, 25 hard tick species are known to occur (Mihalca et al. 2012a). Several of them have a major medical importance, being vectors for zoonotic pathogens. The most important tick from this point of view is Ixodes ricinus, being also the species with the largest geographical distribution in Romania (Mihalca et al. 2012c). The zoonotic importance of Ixodes ricinus is high also because of the broad hosts range. In Romania, this tick was collected from more than 70 different species of vertebrate hosts, including humans (Mihalca et al. 2012a). The majority of hosts were micromammals and birds, with a crucial impact on the natural epidemiological cycles. Moreover, our previous studies have shown the important role of Ixodes ricinus in Romania, as the most common tick in rodents (Mihalca et al. 2012c), migratory birds (Sándor et al. 2014b) and humans (Briciu et al. 2011). Ixodes ricinus is considered to be a major vector for several bacterial, viral or parasitic agents with a high pathogenic potential risk for humans and animals. The most important tick-borne pathogens vectored by Ixodes ricinus are the TBE virus (Tick borne encephalitis), Borrelia burgdorferi sensu

11 lato (the Lyme diseases agent), Anaplasma phagocytophilum (the agent of granulocytic anaplasmosis), Rickettsia helvetica, R. monacensis (agents of spotted fevers) and the zoonotic protozoa Babesia spp. (B. divergens, B. microti and B. venatorum). During the last years, new bacterial species were isolated in Europe. Among these, with high pathogenic and zoonotic risk are Borrelia miyamotoi and the enigmatic ”Candidatus Neoehrlichia mikurensis” (Rizzoli et al. 2014). In the last years, our team conducted an extensive, nation-wide epidemiologic screening based on modern molecular techniques, with high sensitivity and specificity (PCR, Real-Time PCR, RFLP, RLB and sequencing). As a result, a countrywide mapping was produced for two major tick-borne diseases: Lyme borreliosis and granulocytic anaplasmosis (Kalmár et al. 2013; Matei et al. submitted). Human granulocytic anaplasmosis has been reported in USA in 1994 (Chen et al. 1994) and in Europe in 1997 (Petrovec et al. 1997). Since then, the diseases had an emerging trend in regions with temperate climate, including Europe. The clinical signs in humans are diverse, varying from subfebrile to lethal evolution. The common symptoms are fever, headaches and nausea (Stuen et al. 2013). Only few data are available concerning the epidemiology of A. phagocytophilum in Romania. The first molecular identification was in 2012, when it was found in I. ricinus collected from roe deer and goats (Păduraru et al., 2012). Subsequently, several other reports on its presence or prevalence were published in dogs (Hamel et al., 2012; Mircean et al., 2012) and wild boars (Kiss et al., 2014) as well as in ticks collected from livestock (Ioniţă et al., 2013), hedgehogs (Dumitrache et al., 2013), tortoises (Paştiu et al., 2013) and birds (Mărcuţan et al., 2014). Recent approaches are focused on molecular identification of the different A. phagocytophilum genotypes, the genetic variability being correlated with still unclear factors (Stuen et al. 2013). Some genotypes were isolated from humans and animals with asymptomatic infections while others were isolated from patients with severe symptomatology. One of the hypothesis, based on molecular studies from USA, suggests the presence of different A. phagocytophilum ecotypes, with each genotype circulating between hosts which share similar ecological niches (Stuen et al. 2013). In Europe, this approach is at the beginning, and in Romania is completely overlooked. Moreover, Romania could play an important role for elucidating the ecology of the A. phagocytophilum genotypes since it is the only country from Europe with five different ecoregions (Cogălniceanu and Cogălniceanu 2010). This high ecological variety is correlated with a rich biodiversity, especially in terrestrial vertebrate species and associated with high probability with more complex ecocycles of vector-borne diseases. Anaplasma platys is transmitted by the tick Rhipicephalus sanguineus s.l. and it causes cyclic thrombocytopenia in dogs. The infection with A. platys was reported also in humans (Maggi et al. 2013). In Romania, the infection with this bacterium was recently reported in dogs (Andersson et al.

12 2013). Despite the increasing medical importance and the possible zoonotic potential, there are no large scale epidemiological studies on A. platys infection in Eastern Europe. The third pathogen of Anaplasmataceae family to be studied is “Candidatus Neoehrlichia mikurensis”. Its ecology is poorly known and the only suspected vector until now is Ixodes ricinus. During the last 10 years, the bacterium was reported in ticks from few European countries, being involved in the pathology of human febrile syndromes (Rizzoli et al. 2014). Another emerging zoonotic pathogen transmitted by ticks is Borrelia miyamotoi. This bacterium was isolated for first time in 1995 from Ixodes spp. ticks and its implication in human pathology was demonstrated only in 2011 in Russia and 2013 in Netherlands (Rizzoli et al. 2014). In Europe, the vector of B. miyamotoi is I. ricinus but the epidemiological studies are scarce. These bacteria were isolated only in Netherlands and France (Rizzoli et al. 2014). None of these two zoonotic diseases were studied in the South-Eastern Europe (including Romania). However, the vector for both of them, Ixodes ricinus is the most abundant tick in the region (Mihalca et al. 2012c), being also the most common species found on humans (Briciu et al. 2011) and on important reservoir hosts (Mihalca et al. 2012b, Dumitrache et al. 2013). Borrelia turcica is phylogenetically related to the Lyme diseases (B. burgdorferi s.l.) and relapsing fever spirochetes (Borrelia spp.). It is a relatively new organism, being described only recently from Hyalomma aegyptium ticks (Güner et al. 2004). The vectorial importance of this tick for B. turcica was very recently demonstrated by our team (Kalmár et al. in press). Moreover, since H. aegyptium is feeding also on humans, additional studies on the ecology of this pathogen are required. Currently the vertebrate hosts for B. turcica are not known, but since the immature stages of the only known vector are feeding mainly on small mammals (Mihalca et al. 2012), their investigation can confirm the hypothesis of natural foci. Given the zoonotic potential of all these tick-borne pathogens, understanding their natural cycle is essential for evaluating the risk of infection in humans and animals.

C2. Objectives. The approach of the project at the level of principle will be presented, underlining the following two aspects: (1) the concrete objectives of the project; (2) the elements of originality and innovation that the implementation of the objectives bring to the field, related to the state of the art in the field and to the previous projects developed by the project leader. Objectives 1. Evaluating the territorial distribution, host spectrum and genetic diversity of zoonotic Anaplasmataceae in Romania: 1.1. Assessing the genetic diversity of Anaplasma phagocytophilum and the territorial distribution and host spectrum of the identified genotypes

13 1.2. Evaluating the prevalence, distribution and host spectrum of Anaplasma platys 1.3. Epidemiology of ”Candidatus Neoehrlichia mikurensis” in its vectors and possible reservoir hosts 2. To elucidate key eco-epidemiologic features of two neglected species of genus Borrelia: 2.1. Determining the prevalence and host spectrum of Borrelia miyamotoi in Romania 2.2. Identification of the vertebrate species which act as reservoir hosts for Borrelia turcica 3. To produce spatial models and predictive maps for the distribution and epidemiological risk of the identified pathogens: 3.1. Spatial modelling for all species 3.2. Predictive maps for Anaplasma platys. Elements of originality The proposed study is a continuation of the research on the molecular epidemiology and ecology of tick-borne pathogens, initiated in 2010 on Borrelia burgdorferi s.l. (PCCE 7/2010) and in 2011 on the spatial distribution and modelling of arthropod vectors (PCE 236/2011). Under the frame of these two research grants, a large number of samples have been collected. They have been examined so far only for the pathogens targeted in the respective grants. However, the stored DNA could be further investigated for the detection and genotyping of other important zoonotic and emerging tick-borne pathogens, some of which were unknown in Europe at the time of samples collection. The proposal brings also other original elements, as it is the first which attempts to elucidate the ecology and epidemiology of A. platys, ”Candidatus Neoehrlichia mikurensis” and Borrelia miyamotoi and the first to evaluate the genetic diversity of A. phagocytophilum in Romania. Another original element is the attempt to elucidate the natural cycle of B. turcica, so far unknown at its vertebrate host level.

C3. Impact. Will be discussed the aspects related to the expected impact of the project in a broader frame of the scientific field, focusing on the following aspects: (1) the potential of significantly influence the scientific field by new concepts or approaches and, if it’s the case, by opening new themes or research directions; (2) the discussion of the potential impact of the project in the scientific, social, economic or cultural environment and/or of the applicable directions which will be explored in the project (when this is relevant, in the context of the proposed research direction). The current proposal aims research topics with major impact on the public and environmental health, under the umbrella of One Health. Understanding the epidemiology of new, emerging zoonotic pathogens, with unknown ecology even at European level, is particularly concerning the risk awareness and differential diagnosis with other infections. The clinical importance of such eco- epidemiological research is highlighted by the possibility of multiple (co)infection following a tick

14 bite and the necessity of a complex diagnostic and therapeutic approach of such patients. The impact from the fundamental research perspective results from the first approach to elucidate the ecology of certain pathogens and the translation of results on a wider geographic scale. We estimate to publish 7 papers in impacted journals, which will enhance the international scientific visibility of the team members, of the institution and of Romania.

C4. Methodology. The research methodology will be detailed, mentioning, as much as possible, some milestones. In the elaboration of this section the following aspects will be revealed: (1) choosing the investigation methods and tools, by relating to the newest approaches in the field and also the way these will be integrated; (2) a work plan, well scheduled, which will describe the way of organizing and planning the project, related to the proposed objectives; (3) describing the potential risks and the approaches for mitigation these risks. The methodology for implementing the project consists in field activities (sample collection), laboratory methods (morphological identification of ticks and hosts, molecular biology and genetics) and computer modelling. For each specific objective of the proposal, we are proposing the following activities (Gantt chart) as detailed below.

Objective 1.1  Activity 1.1.a: MLVA (Multiple-Locus Variable-number tandem repeat Analysis) analysis (Dugat et al. 2014) of DNA probes extracted from unengorged ticks known as PCR-positive for the AnkA gene of A. phagocytophilum (Matei et al. submitted).  Activity 1.1.b: DNA extraction from tissues (heart, spleen) collected from wild vertebrates (the samples are already collected within previous grants).  Activity 1.1.c: Conventional PCR examination of the tissue samples (targeting the AnkA gene of A. phagocytophilum) followed by MLVA for genotyping.

15  Activity 1.1.d: Conventional PCR examination of the blood samples (samples already collected - Mircean et al. 2012) targeting the AnkA gene of A. phagocytophilum followed by MLVA for genotyping.  Activity 1.1.e: Statistical and phylogenetic analysis of the results, followed by ecocycle possibility evaluation.  Activity 1.1.f: Publication of one research paper in an impacted journal on the ecocycles of A. phagocytophilum in Romania. Objective 1.2  Activity 1.2.a: Morphologic identification of ticks collected from dogs from Palearctic and Afrotropical regions (samples already collected).  Activity 1.2.b: Extraction of the DNA from the identified ticks.  Activity 1.2.c: Nested PCR examination of ticks (targeting the 16S rRNA gene) followed by purification and sequencing (Martin et al. 2005).  Activity 1.2.d: Extraction of DNA from the blood collected from dogs and cats in the Palearctic and Afrotropical regions (samples already collected).  Activity 1.2.e: PCR examination of the blood samples (targeting the 16S rRNA gene) followed by purification and sequencing (Martin et al. 2005).  Activity 1.2.f: Editing, alignment and și phylogenetic analysis of the relevant DNA sequences.  Activity 1.2.g: Publication of two papers in impacted journals a on the epidemiology of A. platys infection in vectors and reservoir hosts. Objective 1.3  Activity 1.3.a: Nested PCR and Real-Time PCR on tissue samples from wild vertebrates and unengorged ticks (Derdáková et al 2014) (samples already collected).  Activity 1.3.b: Sequencing of the positive samples (groEL and 16S rRNA genes).  Activity 1.3.c: Phylogenetic analysis of the sequences and statistical analysis of the epidemiological results.  Activity 1.3.d: Publication of one research paper in an impacted journal on the presence of ”Candidatus Neoehrlichia mikurensis” in Romania. Objective 2.1  Activity 2.1.a: Quantitative Real-Time PCR (Cosson et al 2014) on tick and tissues samples (samples already collected).  Activity 2.1.b: Sequencing the positive samples (16S rRNA, glpQ and p66 genes).  Activity 2.1.c: Phylogenetic analysis of the sequences and statistical analysis of the epidemiological results.

16  Activity 2.1.d: Publication of one research paper in an impacted journal on the genetic diversity and eco-epidemiology of Borrelia miyamotoi in Romania. Objective 2.2  Activity 2.2.a: Obtaining the research permits and ethical approvals (collection of samples from wild animals) according to national and institutional regulations.  Activity 2.2.b: Trapping micromammals using Sherman traps and collection of tissues samples (skin biopsy, liver, spleen, heart) and ticks.  Activity 2.2.c: Catching tortoises (followed by release) and collection of ticks and blood samples.  Activity 2.2.d: Extraction of the DNA from the collected samples (ticks, tissues, blood).  Activity 2.2.e: Conventional PCR (targeting the genes rrf-rrl, flaB, glpQ and gyrB) (Kalmár et al. 2014), followed by sequencing.  Activity 2.2.f: Statistical analysis of the results using ecological variables.  Activity 2.2.g: Publication of one research paper in an impacted journal on the potential reservoir hosts for Borrelia turcica. Objective 3.1  Activity 3.1.a: Compiling the georeferenced databases using literature data and original results.  Activity 3.1.b: Creating the spatial models using maximal entropy (Maxent). Objective 3.2  Activity 3.2.a: Creating the predictive spatial models using the RCP (Representative Concentration Pathways) scenarios.  Activity 3.2b: Publication of one research paper in an impacted journal on the modelling of the pathogen distribution.

Potential risks and milestones The main risks consist in a too low prevalence which could decrease the value of the predictive and distribution models. In the same situation of a low prevalence, in the case of B. turcica, the infection of certain host species could be overlooked due to statistical probability. Another possible risk is the limited access to tissues from endangered vertebrate species, which could be limited to road kills. In order to dynamically evaluate the methodological limitations and the associated risks, we will follow some milestones: (1) DNA depository with monthly assessment of the associated database, number of samples and their origin. (2) Intermediate analysis of the preliminary molecular results and cross-checking these by repeating certain tests for suspicious results. (3) Presenting

17 intermediate results in international meetings to relevant experts followed by feed-back and the validation by the scientific community.

C5. Resources and budget. The existing, relevant infrastructure will be described, as well as the equipment which is to be acquired within the project. Please take into account particularly the following aspects: (1) the estimation of time allocated to the project, by each member of the project team, in person months/member, according to the work plan presented in C4; (2) explain why the project team and the research infrastructure available is appropriate for the project objectives in the allocated time; (3) the justification of the need of acquiring of new great value equipment (over 12.500 Euro, VAT included), by the relation to the project’s objectives.

Estimation for salaries Salary/ % of full Month Salary/month* Total*** Name Role h/day h (lei) time units* * (lei) (lei) MIHALCA Project leader 60 4 50% 12 5100 122400 Andrei Daniel DUMITRACHE Postdoctoral 36 4 50% 12 3060 73440 Mirabela Oana researcher D’AMICO PhD student 13 8 100% 24 2210 53040 Gianluca MATEI Ioana PhD student 13 8 100% 24 2210 53040 Adriana TOTAL 12580 301920 *Full-time equivallent; **with all taxes included; ***calculated for 24 months: 8 months in 2015, 12 months in 2016 and 4 months in 2017.

Project team justification. All team members are nominated and carry out research activities in the field of vector-borne diseases routinely. The competences and the achievements of the project leader are detailed in Section B of the proposal. The Gantt chart in Section C4 shows that each team member has a constant involvement in the project implementation and is active during all the 24 months.

Project leader (Andrei Daniel MIHALCA, age 36) is involved in activities 1.1.e, 1.1.f, 1.2.f, 1.2.g, 1.3.c, 1.3.d, 2.1.c, 2.1.d, 2.2.a, 2.2.b, 2.2.c, 2.2.f, 2.2.g, 3.1.a, 3.2.a, 3.2.a, 3.2.b (mainly designing the experimental protocols, interpretation of results, verification and validation of the working methods, interpretation of the results, writing the manuscripts, coordinating and administrative activities). Postdoctoral researcher (Mirabela Oana DUMITRACHE, age 31) has defended her PhD thesis in 2012 in the field of tick-borne diseases, being involved as a researcher in other research projects in the same domain (i.e. PCCE 7/2010). Currently, the researcher is employed on a postdoctoral

18 position, with the project leader as postdoctoral tutor. She has a remarkable and rich publishing activity, with a total of 17 publications in impacted journals (total impact factor 44.087), of which 7 as the principal author. Role in the project: Activities 1.1.d, 1.1.f, 1.2.a, 1.2.d, 1.2 a, 1.2.g, 2.1.b, 2.1.c, 2.1.d, 2.2.d, 2.2.g, 3.1 .a, 3.2.a, 3.2.a, 3.2.b (mainly DNA extraction, interpretation of the results, statistical analysis, manuscript writing). PhD student 1 (Gianluca D'AMICO, age 34) was enrolled as PhD student in 2012, having the project leader as PhD supervisor. His PhD research topic is the ecology and the vectorial role of ticks from genus Rhipicephalus. The PhD student has already published 13 articles in impacted peer-reviewed journal including one as first author. In addition, the PhD student participated with 16 abstracts in international conferences, being invited speaker at the 13th International Congress of Parasitology, Mexico City, 2014. Role in the project: Activities 1.1.d, 1.1., 1.1.f , 1.2.a, 1.2.b, 1.2.c, 1.2.g, 1.3.b, 1.3.c, 1.3.d, 2.2.a, 2.2.b, 2.2.c, 2.2.g, 3.1.a, 3.2 .a, 3.2.a, 3.2.b (mainly tick identification, DNA extraction, sample collection, compilation of georeferenced databases). PhD student 2 (Ioana Adriana Matei, age 26) was enrolled as PhD student, with the project leader as her PhD supervisor. Her PhD research topic is the ecoepidemiology of genus Anaplasma in Romania. The PhD student already has published 5 articles in impacted journals and other 7 abstracts in international conferences proceedings, among which one as first author with oral communication at the VIII International Conference on Ticks and Tick-borne Pathogens, South Africa, 2014. Role in the project: Activities 1.1.a, 1.1.b, 1.1.c, 1.1.d, 1.1., 1.1.f, 1.2.b, 1.2.c, 1.2.d, 1.2 a, 1.2.f, 1.2. g, 1.3.a, 1.3.b, 1.3.c, 1.3.d, 2.1.a, 2.1.b, 2.1.c, 2.1.d, 2.2.d, 2.2a, 2.2.g, 3.2.b (mainly performing the methods of molecular biology: PCR, DNA purification, Real Time PCR, MLVA, sequencing, phylogenetic analysis). Travel expenses argumentation. We plan field trips for collecting the samples (Dobrogea) and one practical training stage abroad for validating the MLVA method as well as travel to disseminate the results (oral presentations at conferences). Inventory costs argumentation. Since the infrastructure already exists and most samples were collected from the field during previous research projects, logistics costs represent, after staff expenses, the most important part of the budget. Our main purpose is to acquire consumables for molecular biology: DNA extraction kits, reagents for PCR, primers, purification kits, gel extraction kits, pipette tips, reaction tubes, electrophoretic migration solutions. Available research infrastructure. The laboratory facilities where the project leader and the proposed team are based, has the entire infrastructure required to carry out the proposed activities. Thus, we do not intend to purchase any equipment from the budget of this grant. The existing equipment is high-end, being purchased in the last 5 years from the budget of other research projects conducted in the Department of Parasitology and Parasitic Diseases and consist in:

19 Molecular diagnosis laboratory (DNA extractions, PCR, Real-Time PCR, RLB, RFLP): Thermal Cycler (simple and dual) PCR systems, Real-Time PCR System, Reverse Transcriptase PCR system, DNA/RNA hybridization, horizontal and vertical electrophoresis, GelDoc Molecular Imager, sterile and microbiological laminar flow hoods, Tissue Lyser homogenizer, ultrasonicator, vortexes, micro-centrifugation systems, centrifuge with cooling system, incubator with incorporated agitator, thermostat, electronic scales, pH meters, UV-VIS spectrophotometer for measuring the concentration of nucleic acids, Millipore ultra-pure water appliance, water distillation system, ice chips making machine, laboratory glassware washing machine, autoclave, ultra-freezers. Immunology diagnosis laboratory: ELISA system, sterile laminar flow hoods, immunofluorescence microscope, high precision scale, natural convection drying stove, thermostat, vortex. Medical entomology laboratory: in vitro maintenance chambers for vectors, microscopes, dark field microscope, professional digital micro-imaging systems, entomological dissection systems, necropsy kit for wild animals. Cell culture laboratory: sterile incubators, CO2 incubators, reversed microscope, sterile laminar flow hoods. Laboratory animals facilities: standard cages for mice, rats, rabbits, standard shelves, disinfection equipment, pressure pump, nebulizer. Mobile field laboratory for sample collection: 4x4 cars, GPS systems, portable cooling systems, generators, inverters, vector traps, traps for vertebrates, ornithological telescope, ornithological binoculars, satellite phone. Others: IT equipment, software licenses, laboratory glassware washing machine, refrigerators, freezers, laboratory glassware.

Budget Breakdown (lei) Budget chapter 2015 2016 2017 Total (expenses) (lei) (lei) (lei) (lei) Salaries 100640 150960 50320 301920 Inventory 66500 92000 15000 173500 Mobility 9500 12000 1500 23000 Overhead 17664 25496 6682 49842 Total 194304 280456 73502 548262

Budget Breakdown (euro) Budget chapter (expenses) Total (euro)* Salaries 68618 Inventory 39432 Mobility 5227 Overhead 11328 Total 124605 *1 Euro = 4.4 Lei

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