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THE 1ST CONFERENCE ON NEGLECTED VECTORS AND VECTOR-BORNE DISEASES (EURNEGVEC) WITH MANAGEMENT COMMITTEE AND WORKING GROUP MEETINGS OF THE COST ACTION TD1303

CLUJ-NAPOCA, APRIL 8-11 2014 WWW.EURNEGVEC.ORG

ABSTRACT BOOK

EUR NEG partners: VEC SECTION 1

Abstracts published in Parasites & Vectors 2014 7(Suppl 1) Chaligiannis et al. Parasites & Vectors 2014, 7(Suppl 1):O1 http://www.parasitesandvectors.com/content/7/S1/O1

ORALPRESENTATION Open Access Ticks feeding on ruminants and humans in Greece I Chaligiannis1,2,3*, A Papa3, S Sotiraki2 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Ticks are important vectors of disease and transmit an H. excavatum 8 (5.6%); H. dromedarii 67 (47.2%); extensive range of viral, bacterial and protozoan pathogens H. rufipes 4(2.8%);H. impeltatum 1 (0.7%); H. anatolicum to livestock in a wide variety of habitats. In recent years, 3(2.1%)andH. turanicum 3(2.1%). diseases such as babesiosis, ehrlichiosis and anaplasmosis Finally, 701 ticks were coming from humans and identi- have all shown evidence of increased prevalence and dis- fied as: R. sanguineus 562 (80.17%); R. bursa 23 (3.28%); tribution in various parts of Europe. However data con- R. turanicus 34 (4.85%); R. annulatus 5(0.71%);H. margin- cerning the prevalence of ticks and tick borne diseases atum 30 (4.28%); H. excavatum 2 (0.28%); H. rufipes 11 present in livestock and humans in Greece are limited. (1.57%); Dermacentor marginatus 2 (0.28%); Ixodes ricinus In order to fill this gap we performed the current study 6 (0.85%); I. gibosus 6 (0.85%) and Rhipicephalus nymphs to define the existence and prevalence of different tick spe- 20 (2.85%). cies found in farm animals and humans. As regards live- In conclusion, the majority of ticks found in both animal stock, we focused on ruminants (mainly sheep and goats species and humans examined belonged in the Rhipice- and, in a lesser extent, cattle) since they are the only ones phalus sanguineus group which is the main vector of spend time on pastures. A sufficient number of farms all Rickettsia conorii, while Hyalomma marginatum, the vec- over the country were visited during 2 tick seasons (from tors of CCHF virus, were also present. The above results March to October), taking different habitats and animal were more or less anticipated given the climatic conditions density around Greece into account. Ticks collected from of the area, fact that also explains the low prevalence of humans originated from infected individuals who visited Ixodes spp. hospitals.

In total, 2676 ticks were collected from 26 different Authors’ details prefectures (mainland and islands) all over Greece. 1State Veterinary Laboratory of Thrace, Ministry of Rural Development and From those, 1,883 were coming from sheep (1201) and Food, Komotini 691 00, Greece. 2Veterinary Research Institute of Thessaloniki, 3 goats (681) and identified as: Rhipicephalus sanguineus HAO-Demeter (former NAGREF), Thermi 57001, Thessaloniki, Greece. A’ Department of Microbiology, Medical School, Aristotle University of 1,216 (64.65%); R. bursa 495 (26.3%); R. camicasi 12 (0.6%); Thessaloniki, Thessaloniki, Greece. R. turanicus 70 (3.7%); Ixodes ricinus 1(0.05%);Dermacen- tor marginatus 47 (2.5%); Hyalomma marginatum 5 Published: 1 April 2014 (0.3%); H. excavatum 2 (0.1%), H. dromedarii 31 (1.6%) H. rufipes 2(0.1%);H. impeltatum 1(0.05%);andRhipicepha- lus nymph 1 (0.05%). More than half (54.3%) of the above doi:10.1186/1756-3305-7-S1-O1 Cite this article as: Chaligiannis et al.: Ticks feeding on ruminants and were found in an altitude of 0-300 meters, 37.1% in an humans in Greece. Parasites & Vectors 2014 7(Suppl 1):O1. altitude of 301-800m and 3.2% in an altitude of >800m. 142 ticks originated from cattle and were identified as: R. sanguineus 15 (10.5%); R. bursa 6 (4.2%); R. camicasi 5 (3.5%); R. turanicus 6(4.2%);H. marginatum 24 17%);

* Correspondence: [email protected] 1State Veterinary Laboratory of Thrace, Ministry of Rural Development and Food, Komotini 691 00, Greece Full list of author information is available at the end of the article © 2014 Chaligiannis et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Gewehr et al. Parasites & Vectors 2014, 7(Suppl 1):O2 http://www.parasitesandvectors.com/content/7/S1/O2

ORALPRESENTATION Open Access Risk assessment for West Nile Virus in Northern Greece (2010-2013)

S Gewehr*, S Kalaitzopoulou, L Slavi, S Mourelatos From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Since the large WN fever epidemic in Central Macedonia habitat monitoring are permanently used to optimize in 2010 caused by WNV Lineage 2, with a total of 262 vector control measures that are implemented in the cases (197 neuroinvasive, incidence rate 1:140), WNV region of Central Macedonia. cases are reported every year from different areas through- Funding: Ecodevelopment, Hellenic Center for Disease out Greece (2011: 100 cases/75 neuroinvasive, 2012: 161/ Control & Prevention, Integrated Surveillance and Control 109, 2013: 86/51). WN fever has become one of the most Programme for West Nile Virus and Malaria in Greece. important issues for the National Health Authorities in terms of vector-borne diseases. Published: 1 April 2014 From 2011 and onwards, for the surveillance of WNV in Central Macedonia, two major networks were established by Ecodevelopment in collaboration with the Hellenic doi:10.1186/1756-3305-7-S1-O2 CDC, the region of Central Macedonia and four specia- Cite this article as: Gewehr et al.: Risk assessment for West Nile Virus in lized laboratories: 1) A network of 60 CO2-traps for adult Northern Greece (2010-2013). Parasites & Vectors 2014 7(Suppl 1):O2. mosquitoes at fixed sites monitored biweekly for 4 months every year. Pools of 10-50 Culex spp. are forwarded weekly to the laboratories for the detection of WNV. 2) A network of sentinels (domestic pigeons and/or backyard chickens, 40 -50 hencoops or pigeon coops, 400-450 samples/year) for blood sampling in early summer and/or at the end of the hot season. In the plain of Thessaloniki, in early summer (June) the seroconversion in chickens reached 11,9% (28 positive/236 chickens) in 2011 versus 4,1% (8 positive/197 chickens) in 2013. The corresponding average weekly Minimum Infec- tion Rate (M.I.R.) for the period mid June-end August was Submit your next manuscript to BioMed Central 1.73 in 2011 and 0.39 in 2013 respectively. These data and take full advantage of: seem to support the hypothesis that it is possible to relate the level of WNV circulation (infected mosquitoes and • Convenient online submission animal sentinels) with the upcoming human WNV cases: • Thorough peer review 16 human neuroinvasive cases were recorded in 2011 • No space constraints or color ﬁgure charges versus 5 cases in 2013. • Immediate publication on acceptance The follow up of the epidemiological risk through these • Inclusion in PubMed, CAS, Scopus and Google Scholar two networks in combination with the weekly epidemiolo- • Research which is freely available for redistribution gical reports of the Hellenic CDC and the results of larval Submit your manuscript at * Correspondence: [email protected] www.biomedcentral.com/submit Ecodevelopment S.A., Thessaloniki, Greece © 2014 Gewehr et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. IgnjatovićĆupina et al. Parasites & Vectors 2014, 7(Suppl 1):O3 http://www.parasitesandvectors.com/content/7/S1/O3

ORALPRESENTATION Open Access Outbreaks of blackflies and related problems in Serbia: past and present situation A IgnjatovićĆupina1*, D Werner2, M Kúdela3, L Vujanović4, T Brúderová3, A Giannelli5, M Zgomba1, D Petrić1 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Due to the repeated outbreaks of blackflies, consequent eco- Present state of blackfly fauna composition indicates nomical losses and health problems, Serbia was considered the existing risk of outbreaks of some species in the as the most threatened European country in the past. Dur- future. ing the last century (up to `60s), Simulium colombaschense Acknowledgements: The study was supported by The caused enormous losses of livestock. Significant losses in Ministry of Education, Science and Technological Devel- poultry production caused by S. maculatum were also opment of the Republic of Serbia (projects TR31084 and reported in 1958, while S. erythrocephalum caused severe III43007). dermatological problems in humans in 1965 and 1970.

In the last fifteen years, repeated outbreaks of black- Authors’ details flies and reemerging of bite related problems in humans 1Faculty of Agriculture, University of Novi Sad, Novi Sad, Serbia. 2Leibniz were recorded in some parts of Serbia. Centre for Agricultural Landscape Research (ZALF), Müncheberg, Germany. 3 The research objective was to update the knowledge of Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia. 4Department for Dermatology and Venereology, Clinical Center of Vojvodina, blackfly pest species distribution in Serbia, with a special Novi Sad, Serbia. 5Department of Veterinary Medicine, University of Bari, attention to endangered regions in the present and the Valenzano (Bari), Italy. past. Samplings were conducted in the period 2003-2012. Published: 1 April 2014 Immature stages were collected from submerged substrates: in the Danube river and its tributaries, the Nera river and the Nišava river. Adults were sampled close to the breeding doi:10.1186/1756-3305-7-S1-O3 sites by application of CO2 baited traps or by light traps. Cite this article as: IgnjatovićĆupina et al.: Outbreaks of blackflies and In the lowlands 11 blackfly species were recorded. Two related problems in Serbia: past and present situation. Parasites & mammophilic species have been dominant: S. erythrocepha- Vectors 2014 7(Suppl 1):O3. lum in the Danube and S. ornatum (complex) in confluent streams. In 2010, S. erythrocephalum was recorded for the first time in the hilly area in southeastern Serbia. Periods of high adult population density of those two species coin- Submit your next manuscript to BioMed Central cided with the bite cases reports. and take full advantage of: In the Iron Gate region, 21 mainly mammophilic species were recorded in the Danube confluents exclusively. • Convenient online submission S colombaschense . , the main pest species in the past, was • Thorough peer review detected only in the Nera river, about 30 km upstream • No space constraints or color ﬁgure charges from the entrance of the Iron Gate. • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution

* Correspondence: [email protected] Submit your manuscript at 1Faculty of Agriculture, University of Novi Sad, Novi Sad, Serbia www.biomedcentral.com/submit Full list of author information is available at the end of the article © 2014 IgnjatovićĆupina et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Hildebrand et al. Parasites & Vectors 2014, 7(Suppl 1):O4 http://www.parasitesandvectors.com/content/7/S1/O4

ORALPRESENTATION Open Access Co-occurrence of Babesia microti, Bartonella spp., Borrelia burgdorferi s.l. and Anaplasma phagocytophilum in rodents from Lower Silesia, Poland

J Hildebrand, K Buńkowska-Gawlik, A Perec-Matysiak* From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

This study aims to establish the relative contribution of the occurrence of these pathogens in both blood and rodent populations from diverse habitats to the occurrence spleen was only detected in 13% of the rodents. of rodent-borne pathogens of public health significance. While examining the blood and spleen-derived DNA, Rodents (n = 492) represented by Apodemus agrarius, it was found that as many as 92.7% of A. agrarius har- A. flavicollis and Myodes glareolus, were captured in live bored at least one pathogen. Co-occurrence of 3-4 traps in four localities of south-western Poland (2009- pathogens was most common in this rodent species 2012). For the analysis of co-occurrence of pathogens, (32.1% infected). Babesia microti, Borrelia burgdorferi s.l., Bartonella spp. By examining the spleen and blood samples of rodents and Anaplasma phagocytophilum, both blood and spleen at the same time, we estimate that the prevalence of samples were obtained from selected rodent specimens. pathogens in these rodents is higher than the literature The choice of genetic markers and primers was based on indicates. This would implicate the examined rodent the literature data and our preliminary results. Conven- species as a significant reservoir of pathogens with zoo- tional PCR was used for the detection of DNA of exam- notic potential. Additionally, the role of A. agrarius, ined pathogens. Selected PCR positive products were now widespread in some regions of Europe, as a reser- purified and sequenced. BLAST searches were conducted voir host needs to be emphasized. A. agrarius can act as in order to elucidate any homologies with previously a bridge between woodland habitats and periurban deposited sequences in GenBank. environments frequented by humans. The DNA of pathogens was detected in 66.7% of the rodents tested. We observed that among infected rodents, Published: 1 April 2014 40.5% were infected with at least two pathogens, while only 4.7% with all four pathogens. All three of the rodent species were infected with each of the examined patho- doi:10.1186/1756-3305-7-S1-O4 gens. In examined rodent populations the prevalence of Cite this article as: Hildebrand et al.: Co-occurrence of Babesia microti, Bartonella spp., Borrelia burgdorferi s.l. and Anaplasma phagocytophilum in B. microti was 40.0%, Bartonella spp. 37.7%, B. burgdorferi rodents from Lower Silesia, Poland. Parasites & Vectors 2014 7(Suppl 1):O4. s.l. 28.2% and A. phagocytophilum 17.7%. In each of the tissue samples (blood or spleen), the pre- valences of Babesia microti and Bartonella spp. were recorded as comparable, on rather high levels. Interestingly,

* Correspondence: [email protected] Department of Parasitology, University of Wrocław; Przybyszewskiego Str. 63, 51-148 Wrocław, Poland © 2014 Hildebrand et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Polanska et al. Parasites & Vectors 2014, 7(Suppl 1):O5 http://www.parasitesandvectors.com/content/7/S1/O5

ORALPRESENTATION Open Access The role of Sergentomyia schwetzi in epidemiology of visceral leishmaniasis in Ethiopia N Polanska*, I Rohousova, P Volf From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Leishmaniasis is caused by a protozoan of the genus Our results suggest that sand flies of the genus Sergento- Leishmania and transmitted by the bites of phleboto- myia frequently bite domestic animals in Ethiopia. mine sand flies. During the blood feeding, sand fly However, further studies are needed to investigate the role females inject saliva into the host thus affecting Leish- of Sergentomyia in transmission cycle of veterinary impor- mania transmission; in a naive host saliva enhances tant pathogens, including Leishmania sp. parasite virulence, in preexposed host it acts as the pro- The study was supported by following grant projects: tective immunogenic agent by eliciting anti-saliva speci- Grant Agency of Charles University 675012/2012, Czech fic cellular and antibody immune response. Interestingly, Science Foundation 13-05292S and Bill and Melinda anti-saliva antibodies in bitten hosts can be used in epi- Gates foundation. demiological studies as the marker of exposure and the risk marker of Leishmania transmission. Published: 1 April 2014 Ethiopia is endemic for visceral leishmaniasis caused by Leishmania donovani and transmitted mainly by Phleboto- mus orientalis. However, the most abundant sand flies in doi:10.1186/1756-3305-7-S1-O5 the area belong to the genus Sergentomyia. Sergentomyia Cite this article as: Polanska et al.: The role of Sergentomyia schwetzi in females prefer to feed on reptiles, but several studies epidemiology of visceral leishmaniasis in Ethiopia. Parasites & Vectors reported mammals as the additional blood source. The 2014 7(Suppl 1):O5. main aim of this study was to determine, whether S. schwetzi frequently bite domestic animals and thus may play some role in the pathogen transmission. Sera of domestic animals collected in three leishmaniasis foci were tested for anti-S. schwetzi IgG antibodies by ELISA using S. schwetzi salivary gland homogenate as an antigen. Altogether we tested 603 serum samples from five species: cattle, sheep, goats, donkeys and dogs. Sera of ani- Submit your next manuscript to BioMed Central mals from nonendemic countries served as a negative con- and take full advantage of: trol and the results were statistically evaluated. Significant seropositivity for anti-S. schwetzi IgG was • Convenient online submission found in about one third of domestic animals tested. • Thorough peer review The highest seropositivity was found in sheep (115/181), • No space constraints or color ﬁgure charges cattle (25/108) and goats (26/144), followed by donkeys • Immediate publication on acceptance (2/24) and dogs (10/37). • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution

* Correspondence: [email protected] Submit your manuscript at Department of Parasitology, Faculty of Science, Charles University in Prague, www.biomedcentral.com/submit Prague, Czech Republic © 2014 Polanska et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Seidel et al. Parasites & Vectors 2014, 7(Suppl 1):O6 http://www.parasitesandvectors.com/content/7/S1/O6

ORALPRESENTATION Open Access Sustainable and multifunctional mosquito pest management: a pull opportunity and a push advice B Seidel1,2*, F Allerberger3, P Hufnagl3, A Indra3 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Since 2001, we have repeatedly detected pathogens during doi:10.1186/1756-3305-7-S1-O6 mosquito field studies in Austria (e. g. West-Nile-, Cite this article as: Seidel et al.: Sustainable and multifunctional mosquito pest management: a pull opportunity and a push advice. Parasites & Vectors Tahyna-, Usutu-virus and Plasmodium sp.). Recent sur- 2014 7(Suppl 1):O6. veillance discovered two invasive and two thermophilic mosquito species that were new for the Austrian fauna (Aedes japonicus, Ae. albopictus, Anopheles hyrcanus and Culiseta longiareolata). These species increase the possible spectrum of vector-borne communicable diseases in Aus- tria. But until today Flaviviridae and Bunyaviridae have only been detected in context with the endemic mosquito species. Little attention was given to control pests and to develop sustainable, useful and affordable projects for con- trolling the dynamic of these indigenous mosquito populations. We emphasize the need for integration of mosquito control aspects, which up to now were ignored within contemporary Austrian river construction projects and we ask for a Europe wide consideration of mosquito control within the EU-Waterframework Directive (WFD); in particular we recommend mosquito control elements within fish ladders which are prescribed for water storage structures all over Europe. We also report on a material which allows the pulling of mosquitoes in the dimension of landscape management. We present our experience and emphasize the great advantage of actions working in connection with constructive river engineering. Submit your next manuscript to BioMed Central and take full advantage of: Authors’ details 1Technical Office of Ecology and Landscape Assessment, Persenbeug, • Convenient online submission 2 Austria. Department of Theoretical Biology, University of Vienna, Vienna, • Thorough peer review Austria. 3Austrian Agency for Health and Food Safety, Vienna, Austria. • No space constraints or color ﬁgure charges Published: 1 April 2014 • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution

* Correspondence: [email protected] Submit your manuscript at 1Technical Office of Ecology and Landscape Assessment, Persenbeug, Austria www.biomedcentral.com/submit Full list of author information is available at the end of the article © 2014 Seidel et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tilibaşa and Dărăbuş Parasites & Vectors 2014, 7(Suppl 1):O7 http://www.parasitesandvectors.com/content/7/S1/O7

ORALPRESENTATION Open Access Preliminary studies on dynamics of Culicoides spp. In western Romania in conjunction with some environmental factors

EM Tilibaşa*,GDărăbuş From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Culicoides spp. play an important role as vectors for of Culicoides is influenced by abiotic factors such as bluetongue and their dynamics is markedly influenced maximum temperature, minimum temperature, average by the environmental changes. Based on these consid- temperature, wind speed and relative humidity. erations, a study was conducted during May-June 2013 Of all the abiotic factors monitored, the average tem- in the west area of Romania, which aimed capturing and perature, relative humidity and wind speed have a major identifying Culicoides spp. and tracking their population role in the variability of the total number of Culicoides. dynamics. We used two types of traps: (i) CDC Ondes- Dynamics of Culicoides population is positively corre- tepoort mobile light and (ii) unconventional type (hand- lated with minimum temperature (11.5-16 °C). made). Traps were placed in different areas and local- The results obtained in previous studies conducted in ities in the counties of Timiş,AradandCaraş-Severin. this part of Romania show that the maximum temperature They were followed over three consecutive nights in explained most variability in the total number of Culi- each location. In total 28 samples were taken. coides and wind speed at least. Comparing to them, the In addition, abiotic parameters monitoring was per- results obtained in this study render that during May-June formed (minimum temperature, maximum temperature, 2013 increased values of wind speed (1.8-2.5 m/s) and low relative humidity and wind speed), evaluating their relative humidity (60%) had a more negative influence on influence on Culicoides population dynamics. Data on insect population dynamics, as compared with the maxi- atmospheric temperature (maximum and minimum tem- mum (30 °C) and average (15-16 ° C) temperatures. perature), wind speed and relative humidity were taken at each trap using an anemometer and an ambient thermo- Published: 1 April 2014 hygrometer; for a more precise expression of data a conjunction with data taken from the website of the National Agency of Meteorology of the two months was made. doi:10.1186/1756-3305-7-S1-O7 A total of 4534 Diptera specimens were captured during Cite this article as: Tilibaşa and Dărăbuş: Preliminary studies on this research in Timiş, Arad and Caraş-Severin Counties, dynamics of Culicoides spp. In western Romania in conjunction with including three species having the role of potential vectors some environmental factors. Parasites & Vectors 2014 7(Suppl 1):O7. for bluetongue, namely Culicoides obsoletus 409 (9.02%), Culicoides pulicaris 239 (5.27%) and Culicoides nubeculo- sus 183 (4.03%). Based on the preliminary data obtained in Western Romania, it is considered that the population dynamics

* Correspondence: [email protected] Department of Parasitology and Parasitological Diseases, Faculty of Veterinary Medicine,Banat’s University of Agricultural Sciences and Veterinary Medicine “King Michael I” Timisoara, Romania © 2014 Tilibaşa and Dărăbuş; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zintl et al. Parasites & Vectors 2014, 7(Suppl 1):O8 http://www.parasitesandvectors.com/content/7/S1/O8

ORALPRESENTATION Open Access Changing epidemiology of the tick-borne bovine parasite, Babesia divergens A Zintl1*, G McGrath1,LO’Grady1, J Fanning2, K Downing3, D Roche4, M Casey2, JS Gray5 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

3 4 Bovine babesiosis is caused by the tick-borne blood Ireland. Irish Cattle Breeding Federation, Bandon, Co Cork, Ireland. Growth from Knowledge, GfK-Ireland, Market Research Company, Ireland. 5School of parasite, Babesia divergens. A survey of veterinary prac- Biology and Environmental Science (Emeritus Professor), University College titioners and farmers in Ireland in the 1980’s revealed Dublin, Ireland. an annual incidence of 1.7% associated with consider- Published: 1 April 2014 able economic losses. However, two subsequent surveys in the 1990’s indicated a decline in clinical babesiosis. Recent evidence from continental Europe suggests doi:10.1186/1756-3305-7-S1-O8 that, probably due to climate change, the distribution of Cite this article as: Zintl et al.: Changing epidemiology of the tick-borne the tick vector of B. divergens, Ixodes ricinus is extend- bovine parasite, Babesia divergens. Parasites & Vectors 2014 7(Suppl 1):O8. ing to more northerly regions and higher altitudes. In addition, milder winters are thought to increase the win- dow of tick activity. In order to determine whether any such changes have affected the incidence of bovine babesiosis in Ireland, a questionnaire survey of farmers and veterinarians was carried out and compared against data from previous surveys. Our results indicate that while the incidence of clinical disease has continued to decline, cases occurred at any time of year. In contrast to previous surveys, affected farms were the same size as unaffected ones and there was no correlation between disease risk and the presence of deer on the land. Disease severity and mortality rates were increased because many infections were advanced by the time they were detected and treated. While the precise reasons for the decline in the incidence of redwater are Submit your next manuscript to BioMed Central unknown, a reversal of the trend could be devastating, as and take full advantage of: vigilance among farmers and veterinarians is flagging and the national herd is losing its protective immunity to • Convenient online submission disease. • Thorough peer review • No space constraints or color ﬁgure charges Authors’ details • Immediate publication on acceptance 1 2 School of Veterinary Medicine, University College Dublin, Ireland. Central • Inclusion in PubMed, CAS, Scopus and Google Scholar Veterinary Laboratory, Department Agriculture, Food & the Marine, Dublin, • Research which is freely available for redistribution

* Correspondence: [email protected] Submit your manuscript at 1School of Veterinary Medicine, University College Dublin, Ireland www.biomedcentral.com/submit Full list of author information is available at the end of the article © 2014 Zintl et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bhide et al. Parasites & Vectors 2014, 7(Suppl 1):O9 http://www.parasitesandvectors.com/content/7/S1/O9

ORALPRESENTATION Open Access Vector borne pathogens and host complement system: a tangled tale M Bhide*, S Dolinska, E Bencurova From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Complement-mediated lysis of pathogens is the effective ovine CRPs and insufficiently protect bacteria against ovine immune mechanism developed in the mammalian hosts, complement attack, which correlates with intermediate nevertheless, pathogens have also evolved complement competence nature of sheep for both pathogens. None of evasion strategies to infect the hosts. Study of complement the Borrelia and Francisella strains, except B. coriaceae, evasion mechanisms in various host help to understand possessed factor H binding protein. This also correlates many aspects like, pathogenesis, reservoir competence, with the fact that only B. coriaceae effectively evade bovine association and host selectivity of vector-borne pathogens complement system and infect cattle. This corresponds etc., and thus forms unique benchmarking to prevent the with non-competence nature of cattle for both pathogens. infection within the ‘One Health’ concept. The most effec- In summary, study of tangled association between surface tive complement evasion mechanisms employed by patho- proteins of pathogens, CRPs, complement evasion in gens are: 1. binding of the host complement-regulatory/ various hosts and host-selectivity of vector born organisms inhibitory proteins (CRPs) on their surface and 2. expres- is crucial to understand basic molecular principles of sion of CRP mimicking molecules. A comparative study of host-pathogen relationship. Moreover, identification and complement-pathogen interaction was performed, that characterization of the molecules circumventing host com- included three different hosts (human as susceptible, cattle plement system is also important aspects to develop thera- as resistant and sheep as intermediate susceptible host) peutic or preventive approaches. and a repertoire of Borrelia (twelve species) and Franci- sella strains (three subspecies). Series of experiments like Acknowledgements affinity ligand binding experiments, pull-down assays and Work was supported by APVV-0036-10 and VEGA-1/0054/12. E.B and L.B. are mass spectrometry based protein identification revealed an funding for post-doctoral studies from ITMS 26220220185. array of surface proteins of Borrelia (37 proteins) and Published: 1 April 2014 Francisella (6 proteins) that bind human factor H or C4BP or vitronectin to block complement activation. We also found that some stains of Borrelia possess human CD46 Borrelia Francisella doi:10.1186/1756-3305-7-S1-O9 mimicking molecule, while some and Cite this article as: Bhide et al.: Vector borne pathogens and host may express human CD59-homologous protein. Results complement system: a tangled tale. Parasites & Vectors 2014 7(Suppl 1): shows that these vector-borne pathogens possess multiple O9. proteins that can bind or mimic various CRPs and block human complement. It is noteworthy that, two proteins of Francisella (LVS, Tul4 strains) bound vitronectin only, whereas, only two proteins of Borrelia (of strains SKT-2 and DN127) bound ovine C4BP or factor H. It shows that both pathogens possess very few proteins that can bind

* Correspondence: [email protected] Laboratory of biomedical microbiology and immunology, University of veterinary medicine and pharmacy, Komenskeho -73, Kosice, Slovakia © 2014 Bhide et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bichaud et al. Parasites & Vectors 2014, 7(Suppl 1):O10 http://www.parasitesandvectors.com/content/7/S1/O10

ORALPRESENTATION Open Access Virus discovery in sandflies, from field studies to phylogenetic tree building: classic methods versus novel methods, what do we need to address public health impact? L Bichaud1, C Alkan1, G Piorkowski1, E Zhioua2, I Bitam3, A Izri1, V Moin Vaziri4, B Alten5, Y Ozbel6, OE Kasap5, X De Lamballerie1, RN Charrel1* From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Phlebotomine sandflies are vectors of a large variety of follows: France (n = 3,087), Tunisia (n = 8,206), Algeria arboviruses, mostly in the genus Phlebovirus. Many were (n = 1,283), Turkey (n = 12,000), Iran (n = 5,089). We iso- discovered during the last decade, but were only partially lated 9 virus strains in France (2 Toscana, 7 Massilia), sequenced because of extremely high genetic diversity. 10 strains in Tunisia (2 Toscana, 5 Punique, 3 new viruses Next-Generation sequencing (NGS) is a key tool to achieve still unnamed), 1 strain in Algeria (1 Toscana), 2 strains in rapid sequencing of these viruses. Here we present the Turkey (new phlebovirus, provisionally named Damyeri), results obtained with a custom-made automated platform 1straininIran(newphlebovirus, provisionally named for high throughput processing (HTP) of field-trapped Dashli). Analysis using complete gene sequences allowed sandflies. to produce full protein phylograms for isolated virus The aim is to present an integrated approach to discover strains. Seroprevalence studies were performed to define new/existing viruses vectored by sandflies, to characterize whether these viruses infect humans and non human ver- these viruses by complete genome sequencing, and to tebrates in regions where sandflies were trapped or not. study their potential public health impact for humans and Seroprevalence results will be presented for France and non human vertebrates. Tunisia. Sandflies were collected in France, Tunisia, Algeria, A total of 23 strains were isolated, 6 of which corre- Turkey and Iran. They were processed on a custom- spond to new sandfly-borne phleboviruses that have been made automated HTP platform. RT-PCR assays targeting completely sequenced through NGS. Screening field- conserved regions within the genus Phlebovirus were per- trapped sandflies is suitable to inventory recognized and formed both directly on sandfly extracted RNA and on new viruses. Combining cell culture for isolation and NGS supernatant of Vero cells, and further sequenced. for sequencing is cost-effective and adapted to process Sandflies and cell culture that were PCR-positive, as well high quantities of insects. Serological tools are a rapid and as supernatant of flasks with cytopathic effect were easy method to address potential public health impact sequenced using a de novo protocol onto a Ion Torrent which must be confirmed by molecular diagnostic derived PGM sequencer. from complete sequence data obtained through NGS. A total of 29,664 sandflies were collected and processed onto the platform. Origin and number of specimens are as Authors’ details 1Aix Marseille Universite, IRD French Institute of Research for Development, * Correspondence: [email protected] EHESP French School of Public Health, EPV UMR_D 190 “Emergence des 1Aix Marseille Universite, IRD French Institute of Research for Development, Pathologies Virales”, 13385, Marseille, France. 2Institut Pasteur, Tunis, Tunisia. EHESP French School of Public Health, EPV UMR_D 190 “Emergence des 3Institut Pasteur, Algiers, Algeria. 4Shahid Beheshti University of Medical Pathologies Virales”, 13385, Marseille, France Sciences, Tehran, Iran. 5Hacettepe University, Ankara, Turkey. 6Ege University, Full list of author information is available at the end of the article Izmir, Turkey. © 2014 Bichaud et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bichaud et al. Parasites & Vectors 2014, 7(Suppl 1):O10 Page 2 of 2 http://www.parasitesandvectors.com/content/7/S1/O10

Published: 1 April 2014

doi:10.1186/1756-3305-7-S1-O10 Cite this article as: Bichaud et al.: Virus discovery in sandflies, from field studies to phylogenetic tree building: classic methods versus novel methods, what do we need to address public health impact? Parasites & Vectors 2014 7(Suppl 1):O10.

Submit your next manuscript to BioMed Central and take full advantage of:

• Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution

Submit your manuscript at www.biomedcentral.com/submit Mathis et al. Parasites & Vectors 2014, 7(Suppl 1):O11 http://www.parasitesandvectors.com/content/7/S1/O11

ORALPRESENTATION Open Access Rapid protein profiling for arthropod vector identification A Mathis1*, P Müller2, V Pflüger3, F Schaffner1 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Identification of insect vectors is primarily carried out programs of container-inhabiting mosquitoes in Europe, using morphological features, which can be a time-con- i.e. species usually breeding in tree holes or rock pools in suming and difficult task. PCR-based approaches have southern Europe (Ae. berlandi, Ae. echinus, Ae. gilcolladoi, been developed for the identification of a number of vec- Ae. mariae, Ae. pulcritarsis, Ae. zammitii,andOrthopodo- tor species. We have recently established matrix-assisted myia pulcripalpis).Takentogether,proteinprofilingby laser desorption/ionization time of flight mass spectrome- MALDI-TOF MS is a quick and inexpensive tool to accu- try (MALDI-TOF MS), which has come of age for the rately identify adult and immature stages of insect vectors high throughput identification of microorganisms, to iden- collected in the field, and the technique has the potential to tify imagos and larvae of biting midges (Culicoides spp.), become the method of choice for a centralized, robust and and the technique has proven its suitability to accurately high throughput screening of insect vector populations in identify field-collected biting midges on a large scale. We connection with surveillance programs. The simple sample have extended our insect vector reference database to also preparation can be done in peripheral laboratories and the include culicid mosquito and phlebotomine species. Cur- slides be sent to the measuring laboratory. The future appli- rently, biomarker mass sets are determined for the imagos cation of the method will include the accomplishment of 15 phlebotomines (9 Phlebotomus spp., 3 Lutzomyia of the measurement with a mass spectrometry device spp., 3 Sergentomyia spp.), as well as for both imagos and anywhere and the identification via our online platform. larvae of 38 Central European culicid species, including all established and presumptive Aedine invasive species such Authors’ details as the Asian tiger mosquito Aedes albopictus or the Asian 1National Centre for Vector Entomology, Institute of Parasitology, University bush mosquito Ae. japonicus. In addition, biomarker mass of Zürich, Zürich, Switzerland. 2Swiss TPH, Basel, Switzerland. 3Mabritec SA, sets of eggs were determined for indigenous and invasive Riehen, Switzerland. container-inhabiting Aedine mosquitoes (Ae. aegypti, Ae. Published: 1 April 2014 albopictus, Ae. atropalpus, Ae. cretinus, Ae. geniculatus, Ae. japonicus, Ae. koreicus, Ae. phoeniciae, Ae. triseriatus) allowing to identify eggs collected in oviposition traps in doi:10.1186/1756-3305-7-S1-O11 the framework of surveillance programs. Eggs can be iden- Cite this article as: Mathis et al.: Rapid protein profiling for arthropod tified by MALDI-TOF MS either singly or in pools vector identification. Parasites & Vectors 2014 7(Suppl 1):O11. (demonstrated for pools of 10 eggs, but possibly also for larger ones) for those species of which at least 3 eggs are present in the sample. We are continuously expanding our database, e.g. to also include the remaining mosquito species whose eggs can be encountered in surveillance

* Correspondence: [email protected] 1National Centre for Vector Entomology, Institute of Parasitology, University of Zürich, Zürich, Switzerland Full list of author information is available at the end of the article © 2014 Mathis et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Michelet et al. Parasites & Vectors 2014, 7(Suppl 1):O12 http://www.parasitesandvectors.com/content/7/S1/O12

ORALPRESENTATION Open Access Multiple detection of pathogens in ticks: development of a high throughput real time PCR chip used as a new epidemiologic investigative tool L Michelet1, S Delannoy2, E Devillers1, G Umhang3, A Aspan4, M Juremalm5, J Chirico5, FJ Van der Wal6, H Sprong7 , TP Boye Pihl8, K Klitgaard8, R Bødker8, P Fach2, S Moutailler1* From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Worldwide, ticks transmit more pathogens than other has been tested on a dilution range of reference DNAs of arthropods. Around 60 bacteria, 30 parasites and 100 the targeted pathogens on a Lightcycler 480. Specificity viruses have been registered as tick-borne pathogens; a then has been tested on a Biomark™dynamic array. The third of these pathogens are responsible for zoonoses. chip was secondly evaluated on field samples correspond- Usually, detection of tick-borne pathogens depends on ing to 47 pools of 25 nymphs collected in two sites in the tick species collected: assays are performed for a France, the Netherlands and Denmark (corresponding to restricted number of pathogens that are known to be 7050 nymphs in total). We succesfully detected and deter- transmitted by a particular tick species collected at a mined the prevalence of Anaplasma phagocytophilum, particular site. To better understand the epidemiology Neoehrlichia mikurensis, Rickettsia helvetica, Bartonella of tick-borne pathogens, it will be important to detect henselae, five different genospecies of Borrelia burgdorferi for each sample (one tick or one pool of ticks) most of s.l., the recently identified pathogen Borrelia miyamotoi, the diseases they potentially transmit, regardless of the and two parasite species Babesia divergens and Babesia tick species. The aim is therefore to develop a new epi- venatorum. This fast and low-cost tool allows comprehen- demiologic investigative tool which could detect high sive testing of tick-borne pathogens and can be custo- number of tick-borne pathogens by real time PCR. mized to fit regional demands or to accommodate new or We developed a chip (BioMark™ dynamic arrays, emerging pathogens. The tool represents a major improve- Fluidigm Corporation) targeting pathogens of worldwide ment for surveillance and future epidemiological studies. distribution transmitted by ticks. The designed epidemiologic arrays may detect 48 pathogens in 48 samples corre- Authors’ details 1USC BIPAR, Animal Health Laboratory, ANSES, Maisons-Alfort, France. sponding to 2304 qPCR reactions on the same time. 2IdentyPath Platform, Food Safety Laboratory, ANSES, Maisons-Alfort, France. Specific primers and probe have been designed for each 3Nancy Laboratory for Rabies and Wildlife, Wildlife EcoEPIdemiology & pathogen and their specificity have been tested in silico Surveillance Unit, ANSES, Malzéville, France. 4Department of Bacteriology, National Veterinary Institute (SVA), Uppsala, Sweden. 5Department of with Blast. To begin, we targeted: (i) 37 pathogens whose Virology, Immunobiology and Parasitology, National Veterinary Institute Francisella tularensis, Coxiella burnetii, Neoehrlichia (SVA), Uppsala, Sweden. 6Department of Infection Biology, Central Veterinary mikurensis, 5 species of Anaplasma, 3 species of Ehrlichia, Institute, Wageningen UR, Lelystad, the Netherlands. 7Laboratory for Zoonoses and Environmental Microbiology, National Institute for Public 8speciesofBorrelia, 2 species of Bartonella, 4 species of Health and Environment (RIVM), Bilthoven, the Netherlands. 8National Rickettsia, 10 species of Babesia and 2 species of Theileria, Veterinary Institute, DTU, Copenhagen, Denmark. (ii) 5 species of ticks whose 3 species of Ixodes and 2 species of Dermacentor. Sensitivity of primers and probe Published: 1 April 2014

doi:10.1186/1756-3305-7-S1-O12 Cite this article as: et al * Correspondence: [email protected] Michelet .: Multiple detection of pathogens in ticks: development of a high throughput real time PCR chip used as a new 1USC BIPAR, Animal Health Laboratory, ANSES, Maisons-Alfort, France epidemiologic investigative tool. Parasites & Vectors 2014 7(Suppl 1):O12. Full list of author information is available at the end of the article © 2014 Michelet et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Qablan et al. Parasites & Vectors 2014, 7(Suppl 1):O13 http://www.parasitesandvectors.com/content/7/S1/O13

ORALPRESENTATION Open Access Next generation sequencing as a novel tool for diagnostics of apicomplexan pathogen in ticks and mammalian hosts MA Qablan1,2*, F Boyer3, C Miquel3,GD’Amico4, AD Mihalca4, F Pompanon3, D Modrý1,2 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Authors’ details Among apicomplexan parasites, ticks are known vector of 1 Babesia Department of Pathology and Parasitology, University of Veterinary and several species belongs to three protozoan genera ( , Pharmaceutical Sciences, Brno, Czech Republic. 2CEITEC-Central European Theileria and Hepatozoon). During their life cycle, tick- Institute of Technology, University of Veterinary and Pharmaceutical Sciences, 3 transmitted apicomplexan parasites alternate between asex- 61242 Brno, Czech Republic. Laboratoire d’Ecologie Alpine, Université Joseph Fourier, Grenoble, France. 4Department of Parasitology and Parasitic ual (in vertebrate host) and sexual (in ticks) developmental Diseases, University of Agricultural Sciences and Veterinary Medicine, 400372 stages. The major constraint for the proper diagnostics of Cluj-Napoca, Romania. those pathogens is the high possibility of mix infection, Published: 1 April 2014 both in ticks and vertebrate hosts, with several species or genotypes. The aim of this study was to apply the Next Generation Sequencing (NGS) as a method of choice for doi:10.1186/1756-3305-7-S1-O13 simultaneous determination of the full spectrum of api- Cite this article as: Qablan et al.: Next generation sequencing as a novel tool for diagnostics of apicomplexan pathogen in ticks and mammalian complexan pathogens in ticks and the mammalian hosts. hosts. Parasites & Vectors 2014 7(Suppl 1):O13. Therefore, A pair of universal primers were designed to flank a 167 bp barcode region of the 18s rRNA gene of all Babesia, Theileria and Hepatozoon species. The new protocol was evaluated on DNA samples isolated from 195 dogs and 144 ticks (Rhipicephalus armatus and R. pulchellus) collected from Northern Kenya. In total 301 sample (89%) were positive for apicomplexan infections; ranging from single to multiple infection with one species or several species and/or genotypes in a single sample. The most abundant apicomplexan pathogens were Hepatozoon Babesia Theileria followed by and , respectively. Further, Submit your next manuscript to BioMed Central the result shows that the barcode region entails enough and take full advantage of: variability that allows identifying the pathogens up to the subspecies and genotypes level. The exact methodological • Convenient online submission and results detailed will be presented later. This work was • Thorough peer review supported by the project OP VK CZ.1.07/2.3.00/30.0014. • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution * Correspondence: [email protected] 1 Department of Pathology and Parasitology, University of Veterinary and Submit your manuscript at Pharmaceutical Sciences, Brno, Czech Republic www.biomedcentral.com/submit Full list of author information is available at the end of the article © 2014 Qablan et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Razzauti et al. Parasites & Vectors 2014, 7(Suppl 1):O14 http://www.parasitesandvectors.com/content/7/S1/O14

ORALPRESENTATION Open Access Zoonotic bacterial survey assessed by next-generation sequencing M Razzauti1*, M Galan1, M Bernard2, J Cheval3, S Maman4, N Charbonnel1, M Vayssier-Taussat5,MEloit3,6,7, J-F Cosson1 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Rodents represent one of the major sources of patho- DNA/RNA of the following bacteria genera were gens;mostofthemarevectored by ticks. Tick-borne detected by both approaches, RNAseq and DNA diseases are very diverse and cause a wide range of dis- 16S-metabarcoding: Bartonella, Leptospira, Borrelia, eases in livestock and human populations. Rodents, car- Rickettsia, Treponema, Neisseria, Spiroplasma, Klebsiella, rying ticks, are distributed across a vast range of natural Listeria and Shigella. Some unexpected genera were habitats and they often live in close contact with detected; such as Orientia,uptonowonlyfoundinAsia humans and their domestic animals, exposing them to or Helicobacter, generally thought to be restricted to zoonoses circulating in natural ecosystems. In this study, animal guts. Several bacterial pathogens explored by we analyse the potential of Next-Generation Sequencing RNAseq passed undetected by 16S-metabarcoding: (NGS) technologies as a tool for large-scale survey of Anaplasma, (Neo)Ehrlichia, Wolbachia, Brucella, Coxiella, bacterial zoonotic pathogens carried by rodents. We Campylobacter, Mycoplasma, Salmonella, Yersinia,and combined two NGS approaches in order to establish a Francisella. Furthermore, 16S meta-barcoding allowed to list of zoonotic bacteria and to identify their distribution specify prevalence of bacteria within our sample, and in individuals of rodents in natural populations. revealed high level of coinfection in wild rodents. Briefly, RNA/DNA were extracted from the spleen of 192 Our data demonstrate that NGS allows having a rather rodents collected in Northeast France. RNA from all sam- complete screening of pathogenic bacteria present in ani- ples was pooled and submitted to high throughput RNA mal reservoirs without any apriorion their presence, sequencing (RNAseq). Succeeding de novo assembly, bac- while having a price compatible with cohort studies. NGS terial contigs were assigned to the closest already-known approaches are becoming the new routine approaches in taxa, revealing a list of zoonotic bacteria for the whole sam- large-scale epidemiological studies. ple. Parallel, DNA samples were submitted to meta-barcoding approach: each sample was amplified by PCR using Authors’ details universal primers tagged at the V4 region of the 16S rRNA. 1INRA, UMR CBGP (INRA ⁄ IRD ⁄ Cirad ⁄ Montpellier SupAgro), France. 2INRA, The amplified templates were multiplexed and submitted to GABI, Sigenae, Domaine de Vilvert, 78352 Jouy-en-Josas, France. 3PathoQuest 4 454-pyrosequencing. The resulting dataset was demulti- SAS, Paris, France. INRA, LGC, Sigenae, Chemin de Borde rouge 31326 Castanet Tolosan, France. 5INRA, UMR Bipar, Enva, Anses, USC INRA, Maisons- plexed using a home-made pipeline that assigns each read Alfort, France. 6Ecole Nationale Vétérinaire d’Alfort, UMR 1161 Virologie ENVA, to a sample using the tagged primers, following these were INRA, ANSES, Maisons-Alfort, France. 7Institut Pasteur, Laboratory of Pathogen processed using Mothur pipeline to construct OTUs and Discovery, Paris, France. classify them using the RDP database. These methods Published: 1 April 2014 allowed listing bacteria detected in each rodent and, so derive the prevalence, coinfections and bacteria interactions.

doi:10.1186/1756-3305-7-S1-O14 Cite this article as: Razzauti et al.: Zoonotic bacterial survey assessed by next-generation sequencing. Parasites & Vectors 2014 7(Suppl 1):O14.

* Correspondence: [email protected] 1INRA, UMR CBGP (INRA ⁄ IRD ⁄ Cirad ⁄ Montpellier SupAgro), France Full list of author information is available at the end of the article © 2014 Razzauti et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Moutailler et al. Parasites & Vectors 2014, 7(Suppl 1):O15 http://www.parasitesandvectors.com/content/7/S1/O15

ORALPRESENTATION Open Access To be or not to be co-infected

S Moutailler1, L Michelet1, J Chotte1, F Féménia1, E Le Naour1, M Cote1, ML Poulle2, E Vaumourin1,3, P Gasqui3, G Vourc’h3, JF Cosson4, D Raoult5, M Vayssier-Taussat1* From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

4 Ticks can transmit a large spectrum of pathogens including INRA, Saint-Genès Champanelle, France. CBGP, Campus International de Baillarguet, Montpellier, France. 5URMITE, Université de Médecine Aix- bacteria, viruses and parasites with a significant number of Marseille, Marseille, France. these pathogens being agents of emerging infectious diseases. In Europe, the most prevalent tick-borne disease Published: 1 April 2014 is Lyme Borreliosis, caused by the bacteria Borrelia burgdorferi s.l. In most cases, Lyme Borreliosis is well diag- doi:10.1186/1756-3305-7-S1-O15 nosed. However, beside these typical cases, patients bitten Cite this article as: Moutailler et al.: To be or not to be co-infected. by ticks can be infected by many other pathogens (bacteria: Parasites & Vectors 2014 7(Suppl 1):O15. Anaplasma spp., Bartonella spp., Rickettsia spp.; parasites: Babesia spp., Theileria spp.; and arboviruses: TBEV) that aremoredifficulttodiagnose. Moreover, co-infections between several of these pathogens might also occur. Clinical surveys show that patients coinfected by several tick-borne pathogens present more severe symptoms and a longer duration of illness than those infected by a single pathogen. The overall objective of our study was to evaluate tick-borne pathogen coinfection in ticks and the consequence of those coinfections for human health. Using the high throughput real-time PCR chip, we detected the DNA of the 37 major tick-borne pathogens in a cohort of questing adult of Ixodes ricinus ticks collected in Ardennes (France). We identified that 60% of ticks were infected by at least one pathogen and half of the infected ticks were coinfected. We then evaluated the risk for simultaneous infection of those pathogens to humans by detecting the DNA of the most prevalent tick-borne Submit your next manuscript to BioMed Central pathogens in the blood of patients bitten by ticks. Our and take full advantage of: data illustrated the importance of coinfection, and highlighted the necessity to evaluate coinfection in the context • Convenient online submission of tick-borne diseases. • Thorough peer review • No space constraints or color ﬁgure charges Authors’ details • Immediate publication on acceptance 1 2 UMR Bipar, Enva, Anses, USC INRA, Maisons-Alfort, France. URCA, SFR Cap • Inclusion in PubMed, CAS, Scopus and Google Scholar Santé-PROTAL EA3800 et CERFE, France. 3UR 346 Epidémiologie Animale, • Research which is freely available for redistribution

* Correspondence: [email protected] Submit your manuscript at 1UMR Bipar, Enva, Anses, USC INRA, Maisons-Alfort, France www.biomedcentral.com/submit Full list of author information is available at the end of the article © 2014 Moutailler et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Alho et al. Parasites & Vectors 2014, 7(Suppl 1):O16 http://www.parasitesandvectors.com/content/7/S1/O16

ORALPRESENTATION Open Access Transmission risk of Dirofilariosis in Portugal AM Alho1*, T Nunes1, L Rinaldi2, J Meireles1, S Belo3, P Deplazes4, L Madeira de Carvalho1 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Dirofilariosis is an important and potential fatal vector- number of potential transmission days (179.8) across the borne canine disease, endemic in Portugal as well as in selected stations. During the last decade, 130 DDUs were other Mediterranean countries, particularly due to its geo- inclusively registered from June to November in Porto and graphic and climatic conditions. Despite improved diag- Azores Island, from April to November in Lisbon and nostic methods and effective preventives, infections of Faro, and an uncommonly extended period from April to Dirofilaria spp. continue to increase. Forty-one species of January in Madeira Island. An average Dirofilaria seasonal mosquitoes have been detected in mainland Portugal risk period ranged from a minimum of 5 months/year in although only Culex theileri was found naturally infected Porto, 5.6 months/year in Azores, 6.4 months/year in with Dirofilaria larvae. Therefore, a study was addressed Lisboa, 6.9 months/year in Faro and 8 months/year in to assess Dirofilaria transmission risk in Portugal based on Madeira. air temperatures, estimating the potential days with These results are in accordance with the existing preva- temperature values compatible with the transmission of lence data and reinforce the value of geospatial tools, map- Dirofilaria larvae between mosquito and reservoirs. A ping the risk and helping to monitor and forecast future degree-days model based on Dirofilaria Development epidemiological trends, ensuring a continued surveillance Units (DDUs) was used, considering minimum and maxi- and a “One Medicine-One Health” integrated approach. mum daily temperatures registered in five Portuguese meteorological stations, obtained in the platform wunder- Funding ground.com. Preconditions for the model were: a thresh- PhD research grant SFRH/BD/85427/2012; Project PTDC/ old temperature of 14°C below which Dirofilaria SAU-SAP/113523/2009 supported by Fundação para a development will not proceed in mosquitoes; 130 cumula- Ciência e a Tecnologia (FCT), Portugal. tive DDUs for larvae to reach infectivity; and a maximum life expectancy of 30 days for mosquito vectors. Authors’ details DDU was evaluated in three areas of Portugal mainland 1Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de - Porto (North, 41°9’0"N/8°37’0"W), Lisbon (Centre, 38° Medicina Veterinária, Universidade de Lisboa, Portugal. 2Unit of Parasitology 43’0"N/9°8’0"W) and Faro (South, 37°1’0"N/7°56’0"W), as and Parasitic Diseases, Department of Veterinary Medicine and Animal Productions, University of Naples Federico II, Naples, Italy. 3Unidade de well as in two different Portuguese islands in the Atlantic Parasitologia Médica, Instituto de Higiene e Medicina Tropical, Universidade Ocean - São Miguel, Azores (37°44’0"N/25°40’0"W) and Nova de Lisboa, Portugal. 4Institute of Parasitology, Vetsuisse Faculty, Madeira (32°38’ 0"N/16°54’0"W), over the period from University of Zurich, Switzerland. 2003 to 2013. The results show that the highest number of Published: 1 April 2014 potential days with suitable conditions for Dirofilaria transmission was registered in Madeira with an average of 209.9 days/year, followed by Faro 175.2 days/year, Lisbon doi:10.1186/1756-3305-7-S1-O16 163.5 days/year, Azores 140 days/year and Porto 117.2 Cite this article as: Alho et al.: Transmission risk of Dirofilariosis in days/year. The year 2006 was the one with a maximum Portugal. Parasites & Vectors 2014 7(Suppl 1):O16.

* Correspondence: [email protected] 1Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, Portugal Full list of author information is available at the end of the article © 2014 Alho et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Duscher et al. Parasites & Vectors 2014, 7(Suppl 1):O17 http://www.parasitesandvectors.com/content/7/S1/O17

ORALPRESENTATION Open Access Roe deer used as indicator species for a country wide survey for the occurrence of Tick Borne Encephalitis in Austria

GG Duscher1*, R Baumgartner2, G Walder2 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Although 85% of the Austrian citizens are vaccinated Authors’ details 1Department of Pathobiology, Institute of Parasitology, Vienna, Austria. against Tick Borne Encephalitis (TBE), still ~100 clinical 2Dr. Gernot Walder GmbH, Außervillgraten, Austria. cases are reported each year. Until now, risk maps are created by using human cases only, albeit this source is Published: 1 April 2014 biased due to the high level of vaccinated persons, uncertainties about the location of infection and undo- cumented cases because of inapparent infections. doi:10.1186/1756-3305-7-S1-O17 Cite this article as: Duscher et al.: Roe deer used as indicator species for To overcome this problem, several solutions are a country wide survey for the occurrence of Tick Borne Encephalitis in proposed: Investigations of ticks by PCR seems to be rea- Austria. Parasites & Vectors 2014 7(Suppl 1):O17. sonable, when looking on small areas. Another possibility to gain knowledge about TBE occurrence is to investigate the sera of wildlife animals for antibodies against TBE. Due to the fact that roe deer can be found all around the whole Austrian landscape, but its habitat is restricted to a relatively small area, we chose this species as indicators for the monitoring of antibodies against TBE. Together with the hunting organisations blood samples from whole Austria was collected starting in September 2013 and screened for the occurrence of antibodies against TBE using an indirect immunofluorescence assay test. A total of 928 serum samples from roe deer were collected so far. The analysis of ~100 samples showed Submit your next manuscript to BioMed Central positivity to TBE in 2 roe deer. The shooting/finding and take full advantage of: location of each roe deer was georeferenced and a geographical information system was constructed. The • Convenient online submission findings of this survey on roe deer will be used to update • Thorough peer review the TBE risk map in Austria. • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution

* Correspondence: [email protected] Submit your manuscript at 1Department of Pathobiology, Institute of Parasitology, Vienna, Austria www.biomedcentral.com/submit Full list of author information is available at the end of the article © 2014 Duscher et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Estrada-Peña Parasites & Vectors 2014, 7(Suppl 1):O18 http://www.parasitesandvectors.com/content/7/S1/O18

ORALPRESENTATION Open Access Methodological caveats in the environmental modelling and projections of climate niche for ticks, with examples for Ixodes ricinus (ixodidae) A Estrada-Peña From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

There is a growing interest in inferring the associations of like projected climate scenarios is also pointed in health-threatening arthropods to capture the climate the results. niche to which they associate, projecting such inference on a territory. This is intended to predict the range of Published: 1 April 2014 distribution of the tick and to understand their responses to climate scenarios, using the so-called correlative models. However, some methodological gaps might prevent doi:10.1186/1756-3305-7-S1-O18 to obtain an adequate background against which test Cite this article as: Estrada-Peña: Methodological caveats in the hypotheses. We explore, describe and illustrate these pro- environmental modelling and projections of climate niche for ticks, cedural inaccuracies with examples focused on the tick with examples for Ixodes ricinus (ixodidae). Parasites & Vectors 2014 7(Suppl 1):O18. Ixodes ricinus, and how these may affect the modelling outcomes. We aim to provide a background of rules against which develop reliable models for these parasites. The use of partial sets of occurrences of the tick might produce unreliable associations with climate because the algorithms cannot capture the complete niche to which the tick is associated. Reliability measures of the model cannot detect these inaccuracies, and undesirable estima- tions of the niche will prevail in the chain of further calculations. The use of inadequate environmental variables (covariates) may lead to inflation of the results of the model through two statistical processes, called autocorrelation and colinearity. The high colinearity Submit your next manuscript to BioMed Central existing in climate products derived from interpolation of and take full advantage of: climate recording stations is demonstrated, and it is explicitly advised the training of climate models with • Convenient online submission satellite-derived information of climate, of which coli- • Thorough peer review nearity of the time series has been removed through a • No space constraints or color ﬁgure charges harmonic regression. The high uncertainty if inference • Immediate publication on acceptance on the climate niche is applied into different time slices, • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution

Submit your manuscript at Correspondence: [email protected] www.biomedcentral.com/submit Faculty of Veterinary Medicine. Zaragoza, Spain © 2014 Estrada-Peña; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ivović et al. Parasites & Vectors 2014, 7(Suppl 1):O19 http://www.parasitesandvectors.com/content/7/S1/O19

ORALPRESENTATION Open Access Illegal waste sites as potential micro foci of Mediterranean Leishmaniasis V Ivović1,2*, K Kalan1, S Zupan1,2, VE Buzan1,2 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Apart from being against the law, illegal waste dumping and distribution of such occurrences so that we may better also poses a threat to human health and to the environ- classify the role of rodents as potential reservoirs of ment. Solid and decomposing waste is an ideal breeding leishmaniasis in the Mediterranean basin. ground for a number of rodents, insects, and other vermin that pose a health risk through the spread of infectious dis- Authors’ details eases. The main objective of this study was to survey dis- 1Science and Research Centre, University of Primorska, Koper - Capodistria, ease vectors and rodents for the presence of Leishmania Slovenia. 2Faculty of Mathematics, Natural Sciences and Information sp. from illegal waste sites along the Istrian Peninsula in Technologies University of Primorska, Koper - Capodistria, Slovenia. Slovenia and Croatia. Published: 1 April 2014 The entomological and rodent survey was carried out between April 2011 and May 2013, at 12 locations, considering only illegal waste sites which consist of at least 2 m3 doi:10.1186/1756-3305-7-S1-O19 of garbage. Cite this article as: Ivović et al.: Illegal waste sites as potential micro foci A total of 119 specimens of Phlebotomine sandflies were of Mediterranean Leishmaniasis. Parasites & Vectors 2014 7(Suppl 1):O19. collected. Five species were identified as follow: Sergento- myia minuta (48.7%), Phlebotomus perniciosus (30.3%), P. papatasi (13.4%), P. neglectus (5%) and P. mascitii (2.6%). Additionally, 173 small rodents were trapped at the same sites including following species: Rattus rattus (3.5%), Mus musculus (44%), Apodemus agrarius (27%), A. flavicollis (15%) and A. sylvaticus (10.5%). A geospatial analysis software ArcView was used to map the distribution of both vectors and rodents. Sandflies and rodents were screened using a molecular probe to amplify an approximately 120 bp fragment of the Submit your next manuscript to BioMed Central kinetoplast DNA (kDNA) minicircle for the detection of and take full advantage of: Leishmania spp. parasites. While not recorded in the tested sandflies, L. infantum DNA was detected in the • Convenient online submission spleen of one juvenile black rat (R. rattus). • Thorough peer review Despite few published records on Leishmania spp. infec- • No space constraints or color ﬁgure charges tion in black rats, the addition of our record highlights the • Immediate publication on acceptance importance for further investigation into the frequency • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution * Correspondence: [email protected] 1 Science and Research Centre, University of Primorska, Koper - Capodistria, Submit your manuscript at Slovenia www.biomedcentral.com/submit Full list of author information is available at the end of the article © 2014 Ivović et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Rinaldi et al. Parasites & Vectors 2014, 7(Suppl 1):O20 http://www.parasitesandvectors.com/content/7/S1/O20

ORALPRESENTATION Open Access Mapping and modeling Dirofilaria infections in Europe L Rinaldi1,4*, V Musella2, G Marzatico1, M Mortarino3, Genchi Claudio3, G Cringoli1,4 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Climate change and increasing temperatures are a global Authors’ details 1Unit of Parasitology and Parasitic Diseases, Department of Veterinary phenomenon that can influence the dynamics of a number Medicine and Animal Productions, University of Naples Federico II, Naples, of hematophagous arthropods, vectors of pathogens with Italy. 2Department of Health Sciences, University Magna Graecia of 3 importance in human and veterinary medicine. In fact, cli- Catanzaro, Italy. Department of Veterinary Science and Public Health, University of Milan, Italy. 4CIRPAR, Italy. matic changes, together with an increase in the movement of dogs across Europe, have caused an increase in the geo- Published: 1 April 2014 graphical range of Dirofilaria immitis and D. repens infections. A Geographic Information System based on thermal regimen was constructed to identify areas potentially suita- doi:10.1186/1756-3305-7-S1-O20 ble for Dirofilaria transmission in Europe. These models Cite this article as: Rinaldi et al.: Mapping and modeling Dirofilaria infections in Europe. Parasites & Vectors 2014 7(Suppl 1):O20. are based on evidence that: i) there is a threshold of 14 °C below which Dirofilaria development will not proceed in mosquitoes; ii) there is a requirement of 130 growing degree-days for larvae to reach infectivity, and; iii) there is a maximum life expectancy of 30 days for a mosquito vector. The output of these models predicted that the summer temperatures (with peaks in July and August) are sufficient to facilitate extrinsic incubation of Dirofilaria even at high latitudes. Recently, an additional model was constructed to verify the influence of temperature in the course of three decades (1980-1989, 1990-1999 and 2000-2012) on the risk of infection by Dirofilaria in Italy. The results showed an expected increasing trend of temperatures, an increase of the Dirofilaria generation numbers into the mosquitoes and a significant extension Submit your next manuscript to BioMed Central of the infection risk from 5-6 months (1980-1989) to and take full advantage of: 6.5 months (1990-1999), up to more than 7 months (2000-20012). These findings show that geospatial tools • Convenient online submission are very useful for mapping, monitoring, forecasting • Thorough peer review and surveillance of both heartworm and subcutaneous • No space constraints or color ﬁgure charges dirofilariasis. • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar * Correspondence: [email protected] • Research which is freely available for redistribution 1Unit of Parasitology and Parasitic Diseases, Department of Veterinary Medicine and Animal Productions, University of Naples Federico II, Naples, Submit your manuscript at Italy www.biomedcentral.com/submit Full list of author information is available at the end of the article © 2014 Rinaldi et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kasap Erisoz et al. Parasites & Vectors 2014, 7(Suppl 1):O21 http://www.parasitesandvectors.com/content/7/S1/O21

ORALPRESENTATION Open Access Phlebotomine sand flies on the crossroads of Anatolia: transmitted diseases and vectors

O Kasap Erisoz1, A Belen2, C Alkan2, F Gunay1, V Dvorak3, K Ergunay4, S Aydın5, J Votypka3, A-L Banuls6, R Charrel2, A Özkul7, Y Özbel8, P Volf3, B Alten1* From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

The Western Palearctic (WP) is composed of Europe, Studies were supported by EU-FP7 Edenext project, Middle East and North Africa. In this territory, the HU-Scientific Research Foundation and Turkish Scientific Mediterranean Sea, and the land under the influence of and Technical Research Council. the Mediterranean Sea is the most important geographical character for both migration and dispersion of Authors’ details organisms; especially for invertebrates including sand 1Faculty of Science, Hacettepe University, Department of Biology, Ecology flies. Anatolia (Asia-Minor) takes place on the cross- Section, Beytepe-Ankara-Turkey. 2Unite des Virus Emergents, Marseille roads of this area and these events. Universite, UMR190 “Emergence des Pathologies Virales, Faculte de Medecine, Marseille, France. 3Parasitology Department, Charles University, The phlebotomine sand flies (Diptera: Psychodidae, Faculty of Science, Prague, Czech Republic. 4Department of Medical Phlebotominae) are vectors of several infectious pathogens Microbiology, Hacettepe University, Faculty of Medicine, Virology Unit, 5 causing leishmaniases and arbovirus infections due to Sihhiye-Ankara-Turkey. Department of Communication Sciences, Hacettepe University, Faculty of Communication, Beytepe-Ankara-Turkey. 6IRD (IRD 224- phleboviruses. Several of these diseases have wide geogra- CNRS 5290-UM1-UM2), MIVEGEC, Montpellier, France. 7Department of phical distributions in the WP, and give rise to occasional Virology, Ankara University, Faculty of Veterinary Medicine, Diskapi-Ankara- 8 epidemic outbreaks. In numerous countries, increasing Turkey. Parasitology Department, Ege University, Faculty of Medicine, Bornova, Izmir, Turkey. risk factors are making sand fly-borne diseases a major public and veterinary health problem. Many studies on Published: 1 April 2014 phylogenetic relationship among sand fly taxa, their distribution, population structure and diseases of phlebotomine species have been already published, but there are still doi:10.1186/1756-3305-7-S1-O21 many gaps waiting to be filled up in, especially, Anatolia. Cite this article as: Kasap Erisoz et al.: Phlebotomine sand flies on the crossroads of Anatolia: transmitted diseases and vectors. Parasites & In this point, scientists have to discuss some deficiencies Vectors 2014 7(Suppl 1):O21. under cover of geography, history and phylogenetic studies to understand the mechanisms of distribution of both sand fly species and their pathogens in Anatolia. Submit your next manuscript to BioMed Central In this presentation, updates in distribution of sand fly and take full advantage of: species with state of art maps of EU-VBORNET project, possible new species, leishmaniasis and phleboviruses epi- • Convenient online submission demiology will be discussed with an emphasis on several • Thorough peer review studies performed by our group between 2000 and present • No space constraints or color ﬁgure charges in Anatolia. • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution * Correspondence: [email protected] 1 Faculty of Science, Hacettepe University, Department of Biology, Ecology Submit your manuscript at Section, Beytepe-Ankara-Turkey www.biomedcentral.com/submit Full list of author information is available at the end of the article © 2014 Kasap Erisoz et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Gallusová et al. Parasites & Vectors 2014, 7(Suppl 1):O22 http://www.parasitesandvectors.com/content/7/S1/O22

ORALPRESENTATION Open Access A molecular survey on host specificity of feline and canine Hepatozoon in model site of northern Kenya

M Gallusová1,5*, G Baneth2, MA Qablan1,5, AD Mihalca4, D Modrý1,3,5 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Species of the genus Hepatozoon are apicomplexan para- were confirmed as Hepatozoon felis, 26 samples was sites transmitted by variety of hematophagous arthropods proven to be Hepatozoon sp.andasinglesamplewas to a wide range of intermediate hosts. Hepatozoon infec- determined as Hepatozoon canis with an identity of 96%. tions are broadly distributed around the world and the Regarding dogs, 121 out of 258 (47%) were positive and presence of this parasite is confirmed both in cats and we gained 107 sequences of which 105 showed similarity dogs. Until now, two species from canines (H. canis, H. to Hepatozoon canis and the remaining two proved to americanum) and a single species from felines (H. felis) be Hepatozoon sp. Despite a close contact of hosts, have been reported. While the canine Hepatozoon spp. are obviouslycatsanddogsdonotsharethesameparasite, transmitted by ticks, the vector for H. felis remains which demonstrates a great majority of clearly identified unknown. The aim of presented study was to determine Hepatozoon canis/Hepatozoon felis. Mentioned facts could the prevalence and the diversity of Hepatozoon in popula- be explained either by strict host specificity or by presence tion of domestic cats and dogs living in close contact in of different vector or by both statements. rural communities of Samburu pastoralists in northern

Kenya and to evaluate the existence of possible cross- Authors’ details transmissions between both hosts. Between years 2007- 1Department of Pathological Morphology and Parasitology, University of 2012 in total, 135 and 258 blood samples from cats and Veterinary and Pharmaceutical Sciences Brno, Czech Republic. 2School of 3 dogs from the area of Mt. Kulal (5 localities) was collected, Veterinary Medicine, Hebrew University of Jerusalem, Israel. Institute of Parasitology, Biology Centre, and Faculty of Sciences, University of South respectively. The DNA from blood (preserved in ethanol) Bohemia, České Budějovice, Czech Republic. 4Department of Parasitology was extracted using phenol-chlorophorm method and fol- and Parasitic Diseases, University of Agricultural Sciences and Veterinary 5 lowed by conventional PCR screening. First round of PCR Medicine, Cluj-Napoca, Romania. CEITEC - Central European Institute of Technology (CZ.1.05/1.1.00/02.0068) from European Regional Development was done by using Piroplasmid-F and Piroplasmid-R Fund, Brno, Czech Republic. primers, amplifying 18S rRNA gene of Hepatozoon spp. (400 bp). A second PCR assay was performed in samples Published: 1 April 2014 positive by Piroplasmid primers to amplify a larger fragment (1400 bp) of the 18S rRNA gene. All PCR amplicons comming from positive samples were sequenced and doi:10.1186/1756-3305-7-S1-O22 Cite this article as: Gallusová et al.: A molecular survey on host determined according to the BLAST match. In cats, this specificity of feline and canine Hepatozoon in model site revealed 110 out of 135 (81.5%) positive samples, of which of northern Kenya. Parasites & Vectors 2014 7(Suppl 1):O22. we obtained 104 sequences with an identity of 98% -100% to an existing GenBank accession. In total, 77 sequences

* Correspondence: [email protected] 1Department of Pathological Morphology and Parasitology, University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic Full list of author information is available at the end of the article © 2014 Gallusová et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Mitková et al. Parasites & Vectors 2014, 7(Suppl 1):O23 http://www.parasitesandvectors.com/content/7/S1/O23

ORALPRESENTATION Open Access Questing for the identity of Hepatozoon in foxes B Mitková1*, MA Qablan1, AD Mihalca2, D Modrý1,3 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Apicomplexan parasites of genus Hepatozoon invade the Authors’ details 1Department of Pathological Morphology and Parasitology, University of blood cells of many mammalian species, being transmitted Veterinary and Pharmaceutical Sciences Brno, Czech Republic. 2Department by range of arthropods. In domestic carnivores, three spe- of Parasitology and Parasitic Diseases, Faculty of Veterinary Medicine, cies of Hepatozoon were described to date: H. canis and University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Romania. 3Institute of Parasitology, Biology Centre, and Faculty of Sciences, H. americanum in dogs and H. felis in cats. The classifica- University of South Bohemia, České Budějovice (Budweis), Czech Republic. tion of Hepatozoon in wild carnivores is still not complete due to lack of field and experimental data as well as phylo- Published: 1 April 2014 genetic studies. The aim of this study is to carry out a survey on the prevalence and diversity of Hepatozoon sp. in red foxes Vulpes vulpes. Samples of tissues were doi:10.1186/1756-3305-7-S1-O23 Cite this article as: Mitková et al.: Questing for the identity of collected from dead foxes in 11 counties of Romania; 91 Hepatozoon in foxes. Parasites & Vectors 2014 7(Suppl 1):O23. samples of liver tissue were examined in total. DNA extraction was performed with commercial kit according to the manufacture’sprotocol.Hepatozoon sp. DNA was detected by PCR using primers amplifying 400-600 bp long part of 18S rRNA gene. These primers are commonly used for diagnostic purposes in dogs and in some studies also for detection of Hepatozoon sp. in wild carnivores. PCR products were sequenced to validate positive results of reaction. DNA of parasite was confirmed in 55% of examined samples. Recent findings classified Hepatozoon sp. in foxes and other wild canids in Europe as H. canis. However, Rhipicephalus sanguineus, the only known vector of H. canis in Europe, is absent in most of our sampling sites. Moreover, this tick is typical for the dogs but rare or even absent in foxes. In order to clarify the identity of the parasite, the next step of our study is to focus on amplification of longer or full segment of 18s rRNA gene Submit your next manuscript to BioMed Central to allow more accurate phylogenetic analyses and compar- and take full advantage of: ison with H. canis sequences from dogs and Hepatozoon • Convenient online submission isolates from other carnivores. • Thorough peer review This study was supported by IGA UVPS Brno, project • No space constraints or color ﬁgure charges 115/2013/FVL. • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution * Correspondence: [email protected] 1 Department of Pathological Morphology and Parasitology, University of Submit your manuscript at Veterinary and Pharmaceutical Sciences Brno, Czech Republic www.biomedcentral.com/submit Full list of author information is available at the end of the article © 2014 Mitková et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Stuen et al. Parasites & Vectors 2014, 7(Suppl 1):O24 http://www.parasitesandvectors.com/content/7/S1/O24

ORALPRESENTATION Open Access Anaplasma phagocytophilum - pathogen with a zoonotic potential S Stuen1*, EG Granquist2, C Silaghi3 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Anaplasma phagocytophilum has for decades been known suspected to be competent reservoir hosts for human var- to cause the disease tick-borne fever (TBF) in domestic iants. However, the natural transmission cycles of various ruminants in Ixodes ricinus-infested areas in northern A. phagocytophilum strains, the involvement of their Europe. Later studies have shown that the infection is respective hosts and vectors, and in particular their zoono- widespread on the continent. A. phagocytophilum is able tic potential, have to be unravelled. Updated information to persist between seasons of tick activity in several concerning the zoonotic potential of A. phagocytophilum mammalian species and movement of hosts and infected will be presented. ticks on migrating animals or birds may spread the bacterium. A. phagocytophilum encompasses multiple Authors’ details geneticstrains,abroadhostrangeandhaspathogenic 1Department of Production Animal Clinical Sciences, Norwegian University of potential in several other mammalian species, including Life Sciences Sandnes, Norway. 2Department of Production Animal Clinical 3 humans. Identification and stratification into phyloge- Sciences, Norwegian University of Life Sciences, Oslo, Norway. Department of Veterinärwissenschaftliches,Comparative Tropical Medicine and netic subfamilies has been based on cell culturing, Parasitology, Ludwig-Maximillians-Universität München, Munich, Germany. experimental infections, PCR and sequencing techniques. The clinical symptoms vary from subclinical to Published: 1 April 2014 fatal conditions, and considerable underreporting of clinical incidents is suspected in both human and veterinary medicine. In human, the most common clinical doi:10.1186/1756-3305-7-S1-O24 Cite this article as: Stuen et al.: Anaplasma phagocytophilum - pathogen manifestations are flu-like symptoms two to three weeks with a zoonotic potential. Parasites & Vectors 2014 7(Suppl 1):O24. after tick attachment. In Europe, human granulocytic anaplasmosis (HGA) was first described in 1997. Since then only few cases of HGA have been reported. The reason for the suspected underreporting may be lack of awareness among physicians, unspecific clinical signs, lack of available adequate hosts and non-virulent strains Submit your next manuscript to BioMed Central of A. phagocytophilum involved. Phylogenetic studies and take full advantage of: indicate that stains isolated from humans, dogs and horses from Europe belongs to the same clonal complex, • Convenient online submission while similar studies on isolates from wild and domestic • Thorough peer review ruminants indicate that they are unlikely to harbour • No space constraints or color ﬁgure charges variants of A. phagocytophilum that are infectious to • Immediate publication on acceptance humans. In contrast, wild boar and hedgehogs are • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution * Correspondence: [email protected] 1 Department of Production Animal Clinical Sciences, Norwegian University of Submit your manuscript at Life Sciences Sandnes, Norway www.biomedcentral.com/submit Full list of author information is available at the end of the article © 2014 Stuen et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tomanović et al. Parasites & Vectors 2014, 7(Suppl 1):O25 http://www.parasitesandvectors.com/content/7/S1/O25

ORALPRESENTATION Open Access Strain diversity of Borrelia burgdorferi sensu lato in Serbia

S Tomanović1*,SĆakić1, Ž Radulović1, D Mihaljica1, R Sukara1, M Milutinović1,ERužić-Sabljić2 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

The geographic distribution of Borrelia burgdorferi sensu specimens were determined as: B. lusitaniae, B. afzelii, lato species in Europe shows very dynamic spatial and B. burgdorferi s.s., B. garinii and B. valaisiana.Forall temporal variations. Since different Borrelia species usually strains identified as B. lusitaniae, B. garinii, B. afzelii and correlate with different clinical manifestation of Lyme bor- B. valaisiana according to the MseI-RFLP method, results reliosis, knowledge of the geographic distribution of the of RT-PCR were in absolute agreement. However, pathogen is very important for understanding the ecology Tm values for all strains identified as B. burgdorferi s.s. and epidemiology of the disease. Our previous studies, according to RFLP patterns were in the range for B. lusita- based on direct molecular methods, revealed high diversity niae strains (64.17 to 64.58 °C). Thus, it was impossible to of species of the B. burgdorferi s.l. complex and unex- distinguish B. burgdorferi s.s. and B. lusitaniae strains by pected high prevalence of B. lusitaniae in Ixodes ricinus this method. Sequencing analysis for strains identified as ticks from Serbia. The aim of the present study was B. lusitaniae, B. garinii, B. afzelii and B. valaisiana was in further isolation and typization of viable B. burgdorferi s.l. agreement with MseI-RFLP and RT-PCR results. In cases strains from vectors in Serbia as a basis for studies of of uncoordinated MseI-RFLP and RT-PCR results, sequen- biological, genetic and ecological variations. cing analysis confirmed unclear strains as B. lusitaniae. A total of 248 adult I. ricinus ticks collected from 24 The results of this study showed B. lusitaniae to be the spe- localities were processed for cultivation. Prior to cultiva- cies with highest prevalence in I. ricinus ticks from Serbia. tion, all ticks were disinfected, then triturated in BSK H medium. The culture tubes were incubated at 33°C for Authors’ details 3 months. Cultures were periodically examined by dark 1Laboratory for Medical Entomology, Department for Parasitology, Center of field microscopy. For confirmation of isolated strains, Excellence for Toxoplasmosis and Medical Entomology, Institute for Medical 2 “seminested” PCR for the flaB gene was performed. Research, University of Belgrade, Belgrade. Institute of Microbiology and Immunology, Medical Faculty, University of Ljubljana, Ljubljana, Slovenia. Determination of B. burgdorferi s.l. species was carried out by the RFLP technique, using restriction enzymes Published: 1 April 2014 MseIandDraI on the previously amplified 5-23S rDNA intergenic spacer (Postic et al., 1994). For Borrelia identification by RT-PCR targeting, the hbb gene protocol of doi:10.1186/1756-3305-7-S1-O25 Portnoï et al. (2006) was followed. Sequencing of Cite this article as: Tomanović et al.: Strain diversity of Borrelia burgdorferi sensu lato in Serbia. Parasites & Vectors 2014 7(Suppl 1):O25. the 5-23S rDNA intergenic spacer and flaB gene was performed for phylogenetic analysis. Thirty-four spirochete cultures were isolated and subjected to further genotyping analyses. According to the RFLP patterns of the 5S-23S rDNA intergenic spacer,

1Laboratory for Medical Entomology, Department for Parasitology, Center of Excellence for Toxoplasmosis and Medical Entomology, Institute for Medical Research, University of Belgrade, Belgrade Full list of author information is available at the end of the article © 2014 Tomanović et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Alho et al. Parasites & Vectors 2014, 7(Suppl 1):O26 http://www.parasitesandvectors.com/content/7/S1/O26

ORALPRESENTATION Open Access Preliminary results on the seroprevalence of Angiostrongylus vasorum and co-infection with Dirofilaria immitis in shelter dogs from Portugal AM Alho1*, M Schnyder2, J Meireles1, S Belo3, P Deplazes2, L Madeira de Carvalho1 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Angiostrongylosis and dirofilariosis have recently revived only antigen-positive and 2.35% (8/341, CI: 1.1-4.6%) only the attention of the scientific community due to their antibody-positive. Positive dogs were from the three sur- emergence in several geographical areas. Angiostrongylus veyed areas. Concerning D. immitis, a total of 7.92% of the vasorum is responsible for verminous pneumonia and also animals were positive (27/341,CI:5.5-11.3%),alsofrom for neurological, cardiovascular and coagulation disorders the three areas. Out of the 341 sera analysed, one dog was while Dirofilaria immitis is responsible for right side heart positive for D. immitis circulating antigen and A. vasorum failure. Portugal is an endemic country for D. immitis. antibody-positive only. However, prevalence data concerning A. vasorum is These results show a slightly higher seroprevalence of scarce. Considering that documented cross-reactions angiostrongylosis in Portugal in comparison with seropre- between these parasites may lead to incorrect or misfit valence found in UK, Italy, Germany and Poland, probably diagnoses, we aimed to assess the prevalence and potential explained by the fact that this study was performed with co-infections of both canine heartworms. shelter dogs, usually not under prophylaxis and therefore An epidemiological survey was conducted, involving at a higher risk of infection. 341 sera collected from shelter dogs housed in three To the authors’ knowledge, this is the first study per- coastal districts of Portugal: Coimbra, Santarém and formed in Portugal to assess A. vasorum seroprevalence in Setúbal. Sera were tested for circulating A. vasorum anti- dogs and D. immitis co-infection. We expect that the gens by a sandwich-ELISA using mono and polyclonal obtained data will enhance the awareness of veterinary antibodies (sensitivity 95.7%, specificity 94.0%, Schnyder practitioners concerning these diseases and reinforce the et al., 2011) and for specific antibodies against purified A. importance of a more targeted preventive therapy for vasorum adult stage antigen (sensitivity 85.7%, specificity heartworms in companion animals in Portugal. 98.8%, Schucan et al. 2012). In order to detect the presence of D. immitis circulating antigens, a commercial kit Funding ® WITNESS Dirofilaria (Synbiotics Corp., Europe, sensi- PhD research grant SFRH/BD/85427/2012; Project PTDC/ tivity 100% and specificity 98.3%) based on rapid immu- SAU-SAP/113523/2009 supported by Fundação para a nomigration technology was used. Statistical analysis was Ciência e a Tecnologia (FCT), Portugal. performed with SPSS program and estimated prevalence was obtained using Agresti-Coull method. Authors’ details Regarding A. vasorum, a total of 1.17% (4/341, 95% Con- 1Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de fidence Intervals, CI: 0.3-3.1%) of the animals were positive Medicina Veterinária, Universidade de Lisboa, Portugal. 2Institute of 3 in both ELISAs, while 1.76% (6/341, CI: 0.7-3.9%) were Parasitology, Vetsuisse Faculty, University of Zurich, Switzerland. Unidade de Parasitologia Médica, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Portugal. * Correspondence: [email protected] 1Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, Portugal Full list of author information is available at the end of the article © 2014 Alho et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Alho et al. Parasites & Vectors 2014, 7(Suppl 1):O26 Page 2 of 2 http://www.parasitesandvectors.com/content/7/S1/O26

Published: 1 April 2014

doi:10.1186/1756-3305-7-S1-O26 Cite this article as: Alho et al.: Preliminary results on the seroprevalence of Angiostrongylus vasorum and co-infection with Dirofilaria immitis in shelter dogs from Portugal. Parasites & Vectors 2014 7(Suppl 1):O26.

Submit your next manuscript to BioMed Central and take full advantage of:

Submit your manuscript at www.biomedcentral.com/submit Baneth et al. Parasites & Vectors 2014, 7(Suppl 1):O27 http://www.parasitesandvectors.com/content/7/S1/O27

ORALPRESENTATION Open Access Leishmania tropica infection in wild and domestic canines

G Baneth1*, A Nasereddin2,3, Z Abdeen2, CL Jaffe3 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Leishmania tropica is a causative agent of cutaneous leish- nose was recently found to have mucocutaneous leishma- maniasis in the Middle East, North Africa and some parts niasis caused by L. tropica. of southeastern Europe. It has also been described as a In conclusion, domestic and wild canine infection with cause of human visceral leishmaniasis. Although cuta- L. tropica may be more prevalent in areas of endemic neous leishmaniasis caused by L. tropica is usually consid- human L. tropica cutaneous leishmaniasis than currently ered an anthroponotic infection transmitted between recognized, and canines should be evaluated as possible people directly by phlebotomine sand flies without the additional reservoirs for human infection. involvement of an animal reservoir, in Israel, Jordan and the Palestinian Authority it is a zoonosis with the rock Funding hyrax (Procavia capensis) as a main reservoir host. Golden This study was supported by supported by the U.S. jackals (Canis aureus) and red foxes (Vulpes vulpes) have Agency for International. also been found to be infected with L. tropica in Israel and Development (USAID) Middle East Regional Coopera- are assumed to have a role in spreading the infection to tion Program (MERC) project TA-MOU-08-M27-072 and distant locations, but clinical signs of infection in these by the Deutsche Forschungsgemeinschaft (DFG) as part of wild canids have not been detected. a German-Israeli-Palestinian cooperative project SCHO In the domestic dog, L. tropica infection has been 448/8-1. reported in only a few cases from Morocco and Iran where infection was mostly described as involving the Authors’ details visceral organs. While some surveys describe the detec- 1School of Veterinary Medicine, Hebrew University, P.O. Box 12, Rehovot tion of parasite infection from dog organs by culture or 76100, Israel. 2Al-Quds Nutrition and Health Research Institute, Al-Quds 3 PCR without much detail on the manifestations of dis- University, Abu Deis, Palestinian Authority. Department of Microbiology and Molecular Genetics, IMRIC, Hebrew University-Hadassah Medical School, ease, reports from Morocco described two dogs infected Jerusalem, Israel. with L. tropica with clinical manifestations similar to those found in canine L. infantum infection including Published: 1 April 2014 generalized lymphadenomegaly, onychogryphosis, alope- cia, keratoconjunctivitis, and also glomerulonephritis in one case. A report from northwestern Iran also doi:10.1186/1756-3305-7-S1-O27 L. tropica Cite this article as: Baneth et al.: Leishmania tropica infection in wild described in a dog with cutaneous and visceral and domestic canines. Parasites & Vectors 2014 7(Suppl 1):O27. involvement comparable to canine L. infantum infection. A young dog from a focus of L. tropica human cutaneous leishmaniasis near Jerusalem, with a large proliferative red mucocutaneous lesion between the upper lip and the

* Correspondence: [email protected] 1School of Veterinary Medicine, Hebrew University, P.O. Box 12, Rehovot 76100, Israel Full list of author information is available at the end of the article © 2014 Baneth et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Beck et al. Parasites & Vectors 2014, 7(Suppl 1):O28 http://www.parasitesandvectors.com/content/7/S1/O28

ORALPRESENTATION Open Access Identification of ‘Candidatus Neoehrlichia mikurensis’ and Anaplasma species in wildlife from Croatia

Relja Beck1*, Vlatka ČubrićČurik2,Račić Ivana1, Šprem Nikica2, Vujnović Anja1 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Vector borne diseases are classical emerging infectious dis- A. phagocytophilum, while muflons were infected with eases in human and animal populations. Ticks represent A. centrale, A. phagocytophilum, A. bovis and N. mikuren- perfect vectors for number of bacteria, parasites and sis. Martens and jackals were free from pathogens while viruses. The genera Anaplasma, Ehrlichia and recently sequencing failed in wolf samples. Foxes were infected specified cluster ‘Candidatus Neoehrlichia’ comprise all with both strains of A. phagocytophilum and interestingly bacteria from family Anaplasmataceae, transmitted by in single animal A. bovis was confirmed. Two isolates ixodid ticks to mammalian hosts causing infection in Ehrlichia sp. from roe deer and chamois were identical to humans and animals. ‘Candidatus Neoehrlichia mikurensis’ Ehrlichia sp. from Sika deer from Japan. Data obtained is one of emrgening vector borne zoonosis recently recog- from this study clearly present presence of two zoonotic nized as human pathogen. First case of human infection pathogens: A. phagocytophilum, causative agent of human was reported from Sweden in 2010 and up to now six granulocytic anaplasmosis (HGA) and newly recognized human infections were described in Czech Republic, Ger- pathogen N. mikurensis in various species. These results many and Switzerland. In 2011 first case of septicaemia in also imply importance of wildlife as potential reservoir for two dogs was reported indicating ability of Neoehrlichia to ticks and potential transmission to humans. infect various mammalian species, while different rodent species may act as reservoir host. The aim of this study Authors’ details was to identify species from family Anaplasmataceae from 1Croatian Veterinary Institute, Savska cesta 143,HR-10 000 Zagreb, Croatia. different species of wild animals (851). The presence of 2Faculty of Agriculture, Svetošimunska 25, 10 000 Zagreb, Croatia. selected pathogens from wild cervids, wild boars, small Published: 1 April 2014 rodents, muflons, chamois, martens, bears, badgers, wolves, jackals and foxes were determined by performing PCR on spleen samples and subsequent sequencing of doi:10.1186/1756-3305-7-S1-O28 fragment of 16S rRNA gene. Sequence analysis revealed Cite this article as: Beck et al.: Identification of ‘Candidatus Neoehrlichia presence of N. mikurensis, two strains of Anaplasma pha- mikurensis’ and Anaplasma species in wildlife from Croatia. Parasites & gocytophilum, Anaplasma centrale, A. bovis and Ehrlichia Vectors 2014 7(Suppl 1):O28. sp. Small rodents were infected mainly with N. mikurensis together with A. phagocytophilum. Neoehrlichia mikurensis was single species detected in wild boars, bears and badgers, which represent first finding in these mammalian species. In chamois all species have been found except A. bovis. Wild cervids were harbouring both strains of

* Correspondence: [email protected] 1Croatian Veterinary Institute, Savska cesta 143,HR-10 000 Zagreb, Croatia Full list of author information is available at the end of the article © 2014 Beck et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Napoli et al. Parasites & Vectors 2014, 7(Suppl 1):O29 http://www.parasitesandvectors.com/content/7/S1/O29

ORALPRESENTATION Open Access New insights into the biology and ecology of Acanthocheilonema reconditum (spirurida: onchocercidae) E Napoli1, G Gaglio1, L Falsone1, S Giannetto1, F Dantas-Torres2,3, D Otranto2, E Brianti1* From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Among filarioids infesting dogs, Acanthocheilonema recon- Authors’ details 1Dipartimento di Scienze Veterinarie, University of Messina, Messina, Italy. ditum has a global distribution and epidemiological data 2Dipartimento di Medicina Veterinaria, University of Bari, Bari, Italy. indicate that it is the most prevalent or even the sole filar- 3Department of Immunology, Aggeu Magalãhes Research Institute, Oswaldo ioid species-infecting dogs in some regions of the Mediter- Cruz Foundation, Recife, Brazil. ranean basin. For instance, in southern regions of Italy the Published: 1 April 2014 prevalence is as high as 13.3%, and an annual incidence of 5.9% was estimated in naturally exposed dogs. In spite of its wide distribution and its suspected zoonotic potential, doi:10.1186/1756-3305-7-S1-O29 scant information is available on the biology of this filar- Cite this article as: Napoli et al.: New insights into the biology and ioid. Nonetheless, recent studies have enhanced current ecology of Acanthocheilonema reconditum (spirurida: onchocercidae). Parasites & Vectors 2014 7(Suppl 1):O29. scientific knowledge on the biology and ecology of this nematode in naturally infected dogs. Recent data indicate the absence of any defined periodicity of blood circulating microfilariae with peaks recorded either diurnal or noctur- nal. With regard to the life cycle, fleas were confirmed to be vectors of A. reconditum,whereastheroleofixodid ticks (i.e., Rhipicephalus sanguineus sensu lato) as vectors of this filarioid species has been definitively rejected. The full development of microfilariae to infective forms occurs in the experimental infected cat flea Ctenocephalides felis felis in about 15 days. In addition, localization and size of developing larvae inside infected flea suggest that this arthropod might act as an intermediate host throughout Submit your next manuscript to BioMed Central the ingestion of infected fleas rather than the inoculation and take full advantage of: during the blood meal on dogs. If confirmed, this route of A. reconditum transmission is unique, differing from that • Convenient online submission of other filarioids affecting dogs such as Dirofilaria immi- • Thorough peer review tis and Dirofilaria repens, which are actively transmitted • No space constraints or color ﬁgure charges through the bites of mosquito vectors. • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution

* Correspondence: [email protected] Submit your manuscript at 1Dipartimento di Scienze Veterinarie, University of Messina, Messina, Italy www.biomedcentral.com/submit Full list of author information is available at the end of the article © 2014 Napoli et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Capelli et al. Parasites & Vectors 2014, 7(Suppl 1):O30 http://www.parasitesandvectors.com/content/7/S1/O30

ORALPRESENTATION Open Access How entomological studies can help the control of mosquito-borne diseases: a five-years experience in north-eastern Italy G Capelli1*, F Montarsi1, S Ravagnan1, S Cazzin1, M Mazzucato1, P Mulatti1, F Russo2, S Marangon1 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

North-eastern Italy is particularly suitable for mosquito population density and temperature in the 15 days prior to survival, due to its climate, landscape and abundance of sampling. Precipitation, number of rainy days and humidity wild/domestic animals. After the emergence of West Nile had limited importance. Linear models detected significant Virus (WNV) in 2008, entomological studies were imple- relationships between WNV in humans and mosquitoes. mented. Here we describe how entomological data were Spatial analysis detected clusters of WNV occurrences for managed to optimize surveillance and control of mos- all the hosts, identifying an area where to focus surveillance quito-borne pathogens. and promptly detect WNV re-activation. CDC-CO2 traps were used (May-October, 2009-2013). In long term studies the mosquito species composition, In one site, captures with CDC and gravid traps were seasonality, distribution, abundance and pathogen rate of organized every 2hrs for 24hrs. In other three sites infections were assessed. The blood-meal analyses indi- pre- and post-disinfestation captures were done. The mos- cated possible bird targets for surveillance. The control quitoes were screened by RT-PCR for Flaviviridae and a of the efficacy of disinfestations highlighted the need for sub-sample for Bunyaviridae. Host preference of Culex harmonic guidelines. Modelling indicated the need to pipiens was assessed by PCR blood meal analysis. incorporate density dependence in combination with key Mapping, modelling and spatial analyses were done using environmental factors for robust prediction of Cx.pipiens entomological data to identify correlations with climate, population expansion, helping to identify when and landscape, animal and human infections. where an increase in vector population size and WNV More than 700,000 mosquitoes were collected, with Cx. transmission risk should be expected. The results pipiens themostabundant(80%)andtheonlyvectorof stressed also the necessity to improve the density and the WNV and USUV. Tahyna virus was isolated once in frequency of mosquito captures. Ochlerotatus caspius. Cx.pipiens fed preferentially on birds (76%), mainly blackbird, sparrow, magpie and collared Funding dove, but not on humans. Diel activity showed that Cx. Veneto and Friuli Venezia Giulia regions, Italian Ministry pipiens changed its host searching activity according to of Health. the season and defined the favorite period for oviposition. The success of disinfestation measures in reducing Cx. pipiens Authors’ details density varied according to the methods used. The 1Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro (Padua), Italy. contribution of density-dependence in regulating vector 2Direzione Prevenzione, Veneto Region, Venice, Italy. population growth resulted greater than any environmen- Published: 1 April 2014 tal factor on its own. Overall the most significant predictors of Cx.pipiens dynamics included length of daylight, doi:10.1186/1756-3305-7-S1-O30 * Correspondence: [email protected] Cite this article as: Capelli et al.: How entomological studies can help 1Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro (Padua), Italy the control of mosquito-borne diseases: a five-years experience in Full list of author information is available at the end of the article north-eastern Italy. Parasites & Vectors 2014 7(Suppl 1):O30. © 2014 Capelli et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Duh et al. Parasites & Vectors 2014, 7(Suppl 1):O31 http://www.parasitesandvectors.com/content/7/S1/O31

ORALPRESENTATION Open Access Increased seroprevalence of lymphocytic choriomeningitis virus infection in mice sampled in illegal waste sites D Duh1*, E Varljen-Buzan1,2, S Hasic2, R Charrel3 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Lymphocytic choriomeningitis virus (LCMV), the prototy- Amplicons are in process of sequencing to obtain the pic member of the Arenaviridae family, was isolated from nucleotide sequence and confirm the presence of LCMV. a fatal case of aseptic meningitis during the St. Louis ence- Since the seroprevalence studies in Europe revealed up phalitis epidemic in 1933. Although it was among the first to 17% prevalence of LCMV infection in mice and voles isolated human pathogenic viruses, LCMV remained less sampled in natural host habitat, we concluded that illegal attractive through years due to its lack of clinical impor- waste sites accelerate the spread of rodent-borne patho- tance even though it is an important teratogen causing a genic LCMV. severe and permanent brain injury in newborns. Recently, this old neglected virus found a new niche in immunosup- Authors’ details pressed solid organs recipients. The natural host and 1Science and Research Centre, University of Primorska, Koper - Capodistria, reservoir of LCMV is a common house mouse, Mus mus- Slovenia. 2Faculty of Mathematics, Natural Sciences and Information 3 culus. Zoonotic transmission of LCMV to humans occurs Technologies University of Primorska, Koper - Capodistria, Slovenia. Faculty of Medicine, Aix-Marseille University, Marseille, France. worldwide by direct route with mice secretions. LCMV has been also isolated from several arthropods including Published: 1 April 2014 fleas, Culicoides flies, Aedes mosquitoes and ticks, but it is believed that arthropods play a minor role in LCMV transmission. However, in habitats where mice and arthropod doi:10.1186/1756-3305-7-S1-O31 vectors are abundant, a vector transmission of LCMV Cite this article as: Duh et al.: Increased seroprevalence of lymphocytic choriomeningitis virus infection in mice sampled in illegal waste sites. could be important. The illegal waste sites present optimal Parasites & Vectors 2014 7(Suppl 1):O31. conditions for spread of rodents, common house mice and rats in particular. Therefore, we tested 83 samples of Mus or Apodemus species sampled on illegal waste sites in Slovenia and Croatia. LCMV infection was determined Submit your next manuscript to BioMed Central with IFA and RT-PCR techniques. IFA slides and positive and take full advantage of: controls were kindly provided by Remi Charrel. The presence of IgG against LCMV was detected in 47% and 16% • Convenient online submission sera samples of Mus musculus and Apodemus species, • Thorough peer review respectively. Total RNA was extracted from spleen using • No space constraints or color ﬁgure charges RTP Pathogen Kit (Invitek-Stratec) and tested with nested • Immediate publication on acceptance RT-PCR specific for amplification of LCMV genome. • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution * Correspondence: [email protected] 1 Science and Research Centre, University of Primorska, Koper - Capodistria, Submit your manuscript at Slovenia www.biomedcentral.com/submit Full list of author information is available at the end of the article © 2014 Duh et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Hornok et al. Parasites & Vectors 2014, 7(Suppl 1):O32 http://www.parasitesandvectors.com/content/7/S1/O32

ORALPRESENTATION Open Access Epidemiological investigations of bovine besnoitiosis in Hungary S Hornok1*, W Basso2, A Fedák3, F Baska4, L Dencső5, T Abonyi5, B Dénes5 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

It usually gains proper attention, when blood-sucking belonged to the herd, is low. Based on ELISA results vener- arthropod species in Europe show northward spreading, in eal transmission (from imported, infected bulls to local, part due to climate change and global warming. Accord- uninfected cows) appears to be either rare or unlikely. The ingly, it is well known, that the geographical regions where seroprevalence of besnoitiosis decreased significantly pathogens transmitted by these as biological vectors are among calves born to the group of imported mother cows. endemic, also may extend towards the north. However, it The risk of infection seems to be high, when calves stay is mostly less emphasized, that for vector-borne pathogens with their mother during suckling (for 6-7 months), and if present in Western Europe the risk of eastward emergence animals are kept in the same stable (although physically is also possible, if they have competent vectors in the cen- separated) during the main fly season. Confirmation of the tral and/or eastern part of our continent. Besnoitia besnoiti ELISA results is done with immunoblot and IFAT. All ser- (Apicomplexa: Sarcocystidae) is a cyst-forming coccidian opositive cattle are now kept at a distance of several kilo- parasite that may cause severe lesions (with usually high metres from other groups of animals, prior to culling. seroprevalence, but low morbidity and mortality) in cattle Molecular and sero-epidemiological evaluation of the as intermediate hosts. Wild ruminants are also susceptible. situation continues with the aim of preventing the spread As a unique example among cystogenic coccidia, the main of the disease and regaining the epidemiological status of transmission route of B. besnoiti appears to be mechanical Hungaryasexemptofbovinebesnoitiosis. by blood-sucking dipterans (tabanid and stable flies), although it is also possible iatrogenically (with hypodermic Authors’ details needles) and most likely with close contact between ani- 1Department of Parasitology and Zoology, Faculty of Vet. Science, SZIU, mals. Bovine besnoitiosis has been endemic to South-Wes- Budapest, Hungary. 2Institute of Parasitology, Vetsuisse Faculty, University of 3 4 tern Europe for more than a century, but a significant Zürich, Switzerland. Veterinary Authority, Miskolc, Hungary. Department of Pathology, Faculty of Vet. Science, SZIU, Budapest, Hungary. 5NÉBIH, geographical expansion was observed during the last dec- Veterinary Diagnostic Directorate, Budapest, Hungary. ade to other parts of the formerly endemic countries and to countries neighboring France. Published: 1 April 2014 During the autumn of 2013 bovine besnoitiosis was diagnosed in a beef cattle herd in Hungary (for the first time in Central-Eastern Europe), following the import of Aubrac doi:10.1186/1756-3305-7-S1-O32 Cite this article as: Hornok et al.: Epidemiological investigations of heifers and bulls from France in the previous two years. bovine besnoitiosis in Hungary. Parasites & Vectors 2014 7(Suppl 1):O32. The preliminary serological herd screening with ELISA shows that even after the 2nd year of the presence of imported animals (with high prevalence of B. besnoiti infection), seropositivity among local animals, which originally

* Correspondence: [email protected] 1Department of Parasitology and Zoology, Faculty of Vet. Science, SZIU, Budapest, Hungary Full list of author information is available at the end of the article © 2014 Hornok et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ilie et al. Parasites & Vectors 2014, 7(Suppl 1):O33 http://www.parasitesandvectors.com/content/7/S1/O33

ORALPRESENTATION Open Access Occurrence of Hepatozoon spp. in red foxes (Vulpes vulpes) in Romania

MS Ilie1, M Imre1*, K Imre1, I Hotea1, S Morariu1, D Sorescu1, S Andrei1, C Mariş2,GDărăbuş1 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Hepatozoon is an apicomplexan parasite of carnivores Funding transmitted by the tick Rhipicephalus sanguineus trough This study was financially supported by CNCS Grant ingestion. Infections in red foxes (Vulpes vulpes)have TE_277_116/2010. been recorded in several parts of the world, including

European countries, with variable prevalence values. Authors’ details The study aimed to investigate the occurrence of 1Department of Parasitology and Parasitic Diseases, Faculty of Veterinary Hepatozoon spp. in red foxes originating from two coun- Medicine, BUASVM Timişoara, Romania. 2Department of Forestry, Faculty of ties (Arad and Timiş) of Western Romania. Horticulture and Forestry, BUASVM Timişoara, Romania. Fresh/coagulated blood samples from 28 red foxes, Published: 1 April 2014 killed in sanitary hunting during the routine rabies monitoring, were molecularly analyzed by a conventional PCR using the primers Hep F and Hep R for the presence of doi:10.1186/1756-3305-7-S1-O33 Hepatozoon spp. Cite this article as: Ilie et al.: Occurrence of Hepatozoon spp. in red Overall, 11/28 (39.2%) investigated samples contained foxes (Vulpes vulpes) in Romania. Parasites & Vectors 2014 7(Suppl 1):O33. theDNAoftheparasite.Mostofthepositivesamples were from Arad County (8/17), while in Timiş County only 3 out of 11 foxes tested were positive. Occurrence of Hepatozoon canis was reported in Romania in dogs but investigations in wildlife were not performed. The relatively high prevalence value of the infection recorded in the current survey can be related to the common occurrence of the brown dog tick (Rhipicephalus sanguineus) in the area, documented by previous surveys. The high prevalence of the parasite among red foxes Submit your next manuscript to BioMed Central confirms the presence of sylvatic cycle, highlights the and take full advantage of: role of wildlife as reservoirs and potential vectors for the infection in the screened region and also the emerging • Convenient online submission of this pathogen agent in the country. • Thorough peer review This is the first report of Hepatozoon spp. infection in • No space constraints or color ﬁgure charges red foxes from Romania. The findings support the global • Immediate publication on acceptance changing in vector borne diseases expansion. • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution * Correspondence: [email protected] 1 Department of Parasitology and Parasitic Diseases, Faculty of Veterinary Submit your manuscript at Medicine, BUASVM Timişoara, Romania www.biomedcentral.com/submit Full list of author information is available at the end of the article © 2014 Ilie et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Montarsi et al. Parasites & Vectors 2014, 7(Suppl 1):O34 http://www.parasitesandvectors.com/content/7/S1/O34

ORALPRESENTATION Open Access Laboratory evidence on vector competence of the invasive mosquito Aedes koreicus [Hulecoeteomyia koreica] for Dirofilaria immitis

F Montarsi1*, S Ciocchetta1, S Ravagnan1, G Simonato2, F Mutinelli1, S Camuffo1, A Frangipane di Regalbono2, G Capelli1 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Aedes (Finlaya) koreicus is an exotic invasive mosquito and L3 was 14.67, 8.56 and 3.15, respectively. Second stage detected for the first time in Italy in 2011. Little infor- larvae were observed only once (8 specimens on 13 dpi). mation on its vector competence for Dirofilaria immitis First stage larvae were first observed on 3 dpi whereas L3 are available. The area where this species is now estab- on 8 dpi. The latter were found in salivary glands and lished (Veneto Region, north-eastern Italy) is endemic proboscis starting on 16 till 28 dpi. for dirofilariosis. In this study, Ae. koreicus specimens Aedes koreicus seems to be a suitable intermediate host were experimentally infected with D. immitis to evaluate for D. immitis. Despite the low initial number of microfi- the development of filarial larval stages in different body lariae, the infective L3 stage was observed in all body dis- districts. tricts including the proboscis. An high mosquito Aedes koreicus were reared under laboratory standard mortality occurred during the first days, nevertheless one condition (temperature: 25 ± 1°C; relative humidity: 65 ± third of them survived and became infective. This results 5%; light-dark: 16-8 h). A test group (T) (n = 54 mosqui- show that Ae. koreicus maybeinvolvedinthenatural toes) and a control group (C) (n = 29 mosquitoes) were fed cycle of D. immitis, increasing the risk of exposure for by an artificial feeding system (Hemotek™)usingunin- dogs and humans. fected (in C) and naturally infected (in T) dog blood (3000 microfilariae/ml). Mosquitoes naturally dead and speci- Funding mens killed at 1, 13, 16, 22 and 28 days post infection (dpi) This work was sponsored by the Veneto Region and IZSVe. were dissected; head, thorax and abdomen were examined separately. Five specimens (3 from T and 2 from C) were Authors’ details selected for histology. In addition, molecular confirmation 1Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro (Padua), Italy. by real time PCR for Dirofilariae were performed. Each 2Department of Animal Medicine, Production and Health, University of larval stage was documented by pictures and videos. Padua, Legnaro (PD), Italy. The experiment lasted 28 days. A total of 46 mosqui- Published: 1 April 2014 toes fed in T (85%) and 24 mosquitoes in C (83%) groups. In T, 11 mosquitoes were killed and 32 were recovered dead. The mosquito mortalityrateinTwas52%during doi:10.1186/1756-3305-7-S1-O34 the first nine days, significantly higher compared to C Cite this article as: Montarsi et al.: Laboratory evidence on vector (8%) (p < 0.01). In total, 31 mosquitoes (67%) were competence of the invasive mosquito Aedes koreicus [Hulecoeteomyia koreica]forDirofilaria immitis. Parasites & Vectors 2014 7(Suppl 1):O34. infected. The average of microfilariae, L1 (sausage stage)

* Correspondence: [email protected] 1Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro (Padua), Italy Full list of author information is available at the end of the article © 2014 Montarsi et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Roelandt et al. Parasites & Vectors 2014, 7(Suppl 1):O35 http://www.parasitesandvectors.com/content/7/S1/O35

ORALPRESENTATION Open Access Autochthonous tick-borne encephalitis virus (TBEV) - seropositive cattle in Belgium: a risk-based targeted serological survey S Roelandt1*, V Suin2, F Riocreux3, S Lamoral2, S Van der Heyden4, Y Van der Stede1,5, B Lambrecht6, B Cay7, B Brochier2, S Roels4, S Van Gucht2 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Tick-borne encephalitis virus (TBEV) is the most impor- accuracy of the RFFIT-SNT was confirmed in a mouse tant arthropod-borne virus in Europe. The Western sub- inoculation test and by West Nile virus and Rabies virus type of this pathogenic neurotropic flavivirus is carried by serology. There was a positive correlation between the neu- Ixodes ricinus. Tick-borne encephalitis has become a con- tralizing antibody titer, determined by SN, and the median siderable public health risk in several European countries, survival time in mice inoculated intranasally with a mix of with currently 3000 hospitalized cases per year. The risk virus and serum. Lesions consistent of viral encephalitis of TBEV-introduction into Belgium remains high and the were demonstrated in histopathology. presence of infected wildlife in Belgium is suspected. The overall bovine TBEV-seroprevalence in the targeted Domestic animals such as dogs or cattle can serve as area was estimated between 2.6 and 4.3% and freedom excellent sentinels for TBEV-surveillance in order to could no longer be substantiated. Bovines with borderline install an early warning surveillance component for this results were often located close to confirmed seropositive emerging zoonotic disease of public health importance. animals. The geographical locations roughly coincided In a targeted, risk-based and cross-sectional sampling with the known Belgian hot spots for Lyme disease. design, serological screening was performed on Belgian cat- This risk-based cross-sectional serological survey in tle (n = 650), selected from the 2010 Belgian national cattle cattle, obtained through “one health” cooperation con- surveillance serum bank. The three most Eastern provinces firms the presence of infected foci in Belgium for the first of Belgium, which are geographically situated closest to time. Given the relevance of TBEV for the food chain known and/or recently emerging TBEV-endemic, were tar- through consumption of unpasteurized milk and cheese geted. These areas are also currently known as endemic for and through its considerable public health burden in Lyme disease (Borrelia burgdorferi), another disease trans- other European countries, further surveillance in cattle, mitted by the same tick. other sentinels, ticks and humans at risk is recommended Bovine sera were tested at the TBEV Belgian National to further determine the location and size of endemic foci Reference Centre at the WIV-ISP, by gold standard TBEV and the risk for public health. seroneutralisation test, based on the rapid fluorescent focus inhibition test (RFFIT) protocol. Using a conservative >1/ Authors’ details 15 cut-off titer for SN test, 17 bovines were seropositive 1Unit for Coordination of Veterinary Diagnosis, Epidemiology and Risk and six had borderline results (1/10 < titer < 1/15). The Assessment (CVD-ERA), Operational Directorate of Interactions and Surveillance, Veterinary and Agrochemical Research Centre (VAR-CODA- CERVA), Brussels, Belgium. 2National Reference Centre of TBEV and Rabies, Viral diseases, Communicable and Infectious Diseases, Scientific Institute of * Correspondence: [email protected] 3 1Unit for Coordination of Veterinary Diagnosis, Epidemiology and Risk Public Health (WIV-ISP), Brussels, Belgium. Unit for Data Management and Assessment (CVD-ERA), Operational Directorate of Interactions and Analysis (DMA), Operational Directorate of Interactions and Surveillance, Veterinary and Agrochemical Research Centre (VAR-CODA-CERVA), Brussels, Surveillance, Veterinary and Agrochemical Research Centre (VAR-CODA- 4 CERVA), Brussels, Belgium Belgium. Unit of Pathology and Prionology, Operational Directorate of Full list of author information is available at the end of the article Interactions and surveillance, Veterinary and Agrochemical Research Centre © 2014 Roelandt et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Roelandt et al. Parasites & Vectors 2014, 7(Suppl 1):O35 Page 2 of 2 http://www.parasitesandvectors.com/content/7/S1/O35

(CODA-CERVA), Brussels, Belgium. 5Department of Immunology, Faculty of Veterinary Medicine, University of Ghent, Merelbeke, Belgium. 6Unit of Avian Virology and Immunology, Operational Directorate of Virology, Veterinary and Agrochemical Research Centre (CODA-CERVA), Brussels, Belgium. 7Unit of Enzootic and (Re-)emerging Diseases, Operational Directorate of Virology, Veterinary and Agrochemical Research Centre (CODA-CERVA), Brussels, Belgium.

Published: 1 April 2014

doi:10.1186/1756-3305-7-S1-O35 Cite this article as: Roelandt et al.: Autochthonous tick-borne encephalitis virus (TBEV) - seropositive cattle in Belgium: a risk-based targeted serological survey. Parasites & Vectors 2014 7(Suppl 1):O35.

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Submit your manuscript at www.biomedcentral.com/submit Martello et al. Parasites & Vectors 2014, 7(Suppl 1):O36 http://www.parasitesandvectors.com/content/7/S1/O36

ORALPRESENTATION Open Access Use of small rodents for the surveillance of agents and vectors of tick-borne zoonoses in the northern Apennines, Italy E Martello1, A Mannelli1, C Ragagli2, M Selmi3, C Ambrogi2, E Grego1, LA Ceballos1, MC Stella1, L Tomassone1* From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Vector borne zoonoses are emerging threats in Europe. through co-feeding. While D. marginatus had been Data collection from animals may be useful to evaluate found at the same location in studies carried out in 1994, their occurrence and intensity of transmission, and to I.ricinus was very rare or absent. Data collection on small detect their introduction into previously free geographic rodents thus highlighted the recent range expansion of areas. Indeed, vertebrates serve as hosts for pathogens I. ricinus and B. burgdorferi s.l. in a previously unoccupied and for arthropod vectors, although their ecological role area. Major land use changes, the increased abundance of can vary according to the diseases. wildlife populations, as well as a general climate warming Data collection on small rodents was used to study the in the Mediterranean area, might be interacting factors eco-epidemiology of two tick-borne pathogens, Rickettsia affecting the altitudinal range expansion of I. ricinus in the slovaca (agent of tick-borne lymphadenopathy, trans- Northern Apennines, and in Italy in general. mitted by Dermacentor marginatus)andBorrelia burgdorferi sensu lato (agent of Lyme Borreliosis, transmitted by Authors’ details Ixodes ricinus), in the Apennine mountains, Tuscany 1Dipartimento di Scienze Veterinarie, University of Torino, Grugliasco, Italy. (Italy), where human cases of tick-borne diseases were 2Ufficio Territoriale per la Biodiversità, Corpo Forestale dello Stato, Lucca, 3 reported. Small rodents are preferential hosts for the Italy. Osservatorio Permanente per Patologie a trasmissione Vettoriale, ASL2, Lucca, Italy. immature stages of the two tick vectors and are involved in the transmission cycle of both diseases. Published: 1 April 2014 In the summers from 2009 to 2012, we live trapped Apodemus spp. and Myodes glareolus from 1100 to 1650 m above the sea level (a.s.l.). Rodents were found infested doi:10.1186/1756-3305-7-S1-O36 by immature I. ricinus and D. marginatus. The monthly Cite this article as: Martello et al.: Use of small rodents for the surveillance of agents and vectors of tick-borne zoonoses in the activities of these two tick species on the same hosts were northern Apennines, Italy. Parasites & Vectors 2014 7(Suppl 1):O36. different, reflecting differences in their life cycles. Although few individuals were co-infested, both tick species tended to aggregate on the same Apodemus spp. males. R. slovaca and B. burgdorferi s.l. were detected in rodent ear biopsies and attached ticks up to 1650 m a.s.l. In our study area, rodents might play a role as amplifiers of R. slovaca infection; even in the absence of the host’s systemic infection, tick aggregation on the same individuals might favour the transmission of the pathogen

* Correspondence: [email protected] 1Dipartimento di Scienze Veterinarie, University of Torino, Grugliasco, Italy Full list of author information is available at the end of the article © 2014 Martello et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sonnleitner et al. Parasites & Vectors 2014, 7(Suppl 1):O37 http://www.parasitesandvectors.com/content/7/S1/O37

ORALPRESENTATION Open Access Are Babesia a risk factor for blood products in an alpine area?

ST Sonnleitner1, R Baumgartner1, R Edelhofer2, H Schennach3, M Bednarska4, K Pister5, G Walder1,6* From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

After malaria, babesiosis is the second most common Authors’ details 1Dr. Gernot Walder GmbH, Außervillgraten, Austria. 2Institute of Parasitology, transfusion-transmitted vector-borne disease. This study University of Veterinary Medicine, Vienna, Austria. 3Central Institute for Blood investigates seroprevalence rates to Babesia divergens Transfusion and Immunology, Innsbruck Medical University, Innsbruck, 4 and B. microti in the Tyrol and assesses the risk of Austria. Department of Parasitology, University of Warsaw, Warsaw, Poland. 5Comparative Tropical Medicine and Parasitology, University of Munich, blood products being contaminated by either agent. Munich, Germany. 6Department of Hygiene, Medical Microbiology and Social The area of investigation comprises the Austrian part Medicine, Innsbruck Medical University, Innsbruck, Austria. of Tyrol. A number of 988 sera were tested for IgG anti- Published: 1 April 2014 bodies against B. divergens and B. microti by in-house immunofluorescence assays (IFA). IFA-slides were tested by using commercially available hyperimmunesera. doi:10.1186/1756-3305-7-S1-O37 Collection of questing ticks was performed in summer Cite this article as: Sonnleitner et al.: Are Babesia a risk factor for blood 2009 by about 120 volunteers among hunters at 25 sam- products in an alpine area? Parasites & Vectors 2014 7(Suppl 1):O37. pling sites over a period of three months by flagging. Of 988 sera, 21 (2.1%) were positive in IFA against the B. divergens-complex at titres of 1:128 or higher and 5 (0.5%) were positive in IFA against B. microti. Under the presumption of a long-lasting immune response we can expect 0.5 (±0.2, 95%) seroconversions against B. divergens per 10.000 persons per year. For B. microti the same calculation results in 0.1 (±0.08, 95%) seroconversions per 10.000 persons per year. B. divergens The risk of a blood donation being contaminated by B. divergens or B. microti is estimated to be 24.2 and 5.8 per 100,000 blood donations. The present study shows that the local population Submit your next manuscript to BioMed Central comes into seroreactive contact with at least one member and take full advantage of: of the B. divergens-complex and - to a lesser extent - B. microti. To our knowledge, it is the first demonstration of • Convenient online submission B. venatorum in the Tyrols. Thus, and as vector-borne dis- • Thorough peer review eases are subjected to dynamic changes, we recommend • No space constraints or color ﬁgure charges re-assessment of the risk of transfusion-mediated infec- • Immediate publication on acceptance tions on a regular basis and to introduce PRT for blood • Inclusion in PubMed, CAS, Scopus and Google Scholar components like platelets. • Research which is freely available for redistribution

* Correspondence: [email protected] Submit your manuscript at 1Dr. Gernot Walder GmbH, Außervillgraten, Austria www.biomedcentral.com/submit Full list of author information is available at the end of the article © 2014 Sonnleitner et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Dumitrache et al. Parasites & Vectors 2014, 7(Suppl 1):P1 http://www.parasitesandvectors.com/content/7/S1/P1

POSTERPRESENTATION Open Access Ixodid ticks in red foxes (Vulpes vulpes) from Romania

MO Dumitrache1,GD’Amico1*, IA Matei1, A Ionică1, CM Gherman1, S Sikó Barabási2, DT Ionescu3, M Oltean1, A Balea1, IC Ilea4, AD Sándor1, AD Mihalca1 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Among wildlife, the red foxes (Vulpes vulpes)areoneof health, through the emergence of tick-borne diseases. the most adapted wild species to anthropic ecosystems. Moreover, foxes represent a good model of sentinel They are well recognized as important reservoirs for a species. large number of zoonotic agents in Europe, including ticks This research was performed as part of project IDEI and tick-borne pathogens. Currently, there are few avail- PCE 236/2011. able data on the importance of red foxes in the ecoepidemiology of vector-borne diseases. Therefore, the aim of Authors’ details this study was to screen the dynamics of tick infestation in 1Department of Parasitology and Parasitic Diseases, Faculty of Veterinary 357 red foxes from 12 Romanian counties. Tick identifica- Medicine, University of Agricultural Sciences and Veterinary Medicine, Cluj- 2 tion was performed using the morphological keys. The Napoca, România. Department of Environmental Science, Faculty of Environmental Science, Babeș-Bolyai University, Sfântu Gheorghe, România. overall prevalence of tick infestation was 43.7%. The 5753 3Department of Game and Wildlife, Faculty of Silviculture and Forestry collected ticks belonged to five species: Ixodes hexagonus Engineering, Transilvania University, Brașov, România. 4Department of (on 113 out of 156 foxes; prevalence 72.44%), I. ricinus Congress Publication Coordinator, XPE Pharma&Science, Cluj-Napoca, România. (28.84%), I. crenulatus (7.7%), Dermacentor marginatus (7.05%) and Haemaphysalis punctata (0.64%). Coinfesta- Published: 1 April 2014 tion occurred in 24 foxes (22 with 2 tick species; 2 with 3 tick species) with the following associations: I. ricinus + I. hexagonus (n = 10), I. hexagonus + D. marginatus (n = 5), doi:10.1186/1756-3305-7-S1-P1 I. ricinus + I. crenulatus (n = 4), I. ricinus + D. marginatus Cite this article as: Dumitrache et al.: Ixodid ticks in red foxes (Vulpes vulpes) from Romania. Parasites & Vectors 2014 7(Suppl 1):P1. (n = 2), I. hexagonus + I. crenulatus (n = 1), D. marginatus +I.hexagonus+I.ricinus(n = 1), and H. punctata + I. hexagonus + I. ricinus (n = 1). This study indicates that foxes are hosts to a relevant number of tick species with recognized vectorial role. The provided information can Submit your next manuscript to BioMed Central facilitate the understanding of the ecology of ticks and can and take full advantage of: be the basis for studies on the epidemiology of tick-borne diseases. The high prevalence of tick infestation in red • Convenient online submission foxes, coupled with the increasing presence of this species • Thorough peer review in synanthropic environments and a more nature-oriented • No space constraints or color ﬁgure charges lifestyle of people, can pose a significant threat to human • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar * Correspondence: [email protected] • Research which is freely available for redistribution 1Department of Parasitology and Parasitic Diseases, Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine, Cluj- Submit your manuscript at Napoca, România www.biomedcentral.com/submit Full list of author information is available at the end of the article © 2014 Dumitrache et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Mircean et al. Parasites & Vectors 2014, 7(Suppl 1):P2 http://www.parasitesandvectors.com/content/7/S1/P2

POSTERPRESENTATION Open Access The role of cervids (Cervus elaphus) in the ecobiology of some tick- borne diseases V Mircean1, Z Kalmar1, M Mircean2, A Györke1, E Vitos1, MO Dumitrache1* From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

The maintenance of tick-borne pathogens in nature fol- Anaplasma spp. (6.5%), Ehrlichia spp. (2%), Theileria spp. lows a variety of patterns that are always involving ticks, (0.8%), Babesia spp. (0.6%) and Borrelia spp. (0.3%). From reservoir hosts and pathogens. The red deer (Cervus both heart and spleen tissue samples 3 pathogens were elaphus) is the fourth-largest deer species and inhabits diagnosed: Anaplasma spp. (21%), Theileria spp. (15.8%), most of Europe in a wide variety of habitats. In the last Ehrlichia spp. (5.3%) and Anaplasma spp. (16.7%), 50 years the red deer (Cervus elaphus)population Theileria spp. (16.7%), Babesia spp. (8.3%), respectively. remained constant in Romania, with around 30,000 indi- Only Theileria spp. (9.5%) and Ehrlichia spp. (4.8%) were viduals and no significant fluctuations registered. These present in liver samples. To our knowledge this is the first animals are one of the preferred hosts for Ixodes ricinus, molecular study that evaluates the presence of tick-borne a tick with vectorial capacity for a wide range of patho- pathogens in ticks and tissues collected from Cervus ela- gens; they are reservoir hosts for many tick-borne diseases; phus in Romania, offering important eco-epidemiological due to their movement from sylvatic to synantropic envir- data from public health perspective, stressing out the onment and back they can influence the spreading of both necessity of permanent surveillance. ticks and pathogens. In Romania, studies about the epide- This research was partially supported by grant CNCSIS miology of tick-borne diseases in wild animals, eminently IDEI PCCE 7/2010. in red deer are limited. In this frame, our study aimed to describe the diversity of ticks that are parasitizing Cervus Authors’ details elaphus and to evaluate the molecular prevalence of 1Department of Parasitology and Parasitic Diseases, University of Agricultural Borrelia spp., Babesia spp., Theileria spp., Anaplasma spp. Sciences and Veterinary Medicine Cluj-Napoca, Calea Mănăştur 3-5 2 and Ehrlichia spp. in ticks (I. ricinus) and tissues (heart, Cluj-Napoca 400372, Romania. Department of Internal Medicine, University of Agricultural Sciences and Veterinary Medicine, Calea Mănăştur 3-5, spleen, liver) by PCR. In September and October 2012 Cluj-Napoca, 400372, Cluj, Romania. samples from 53 animals (24 bred in captivity; 29 wild) were collected. All animals originated from Harghita Published: 1 April 2014 County that harbours 10% of red deer population from Romania. Ixodes ricinus was the dominant tick species (99.7%). Only three specimens of Dermacentor marginatus doi:10.1186/1756-3305-7-S1-P2 Cite this article as: Mircean et al.: The role of cervids (Cervus elaphus)in were identified. Samples (1007 ticks, 19 heart tissues, 12 the ecobiology of some tick- borne diseases. Parasites & Vectors 2014 spleen tissues and 21 liver tissues) were used for molecular 7(Suppl 1):P2. diagnosis. Genomic DNA extraction was performed individually on all ticks and tissue samples, followed by PCR. In ticks samples, all investigated pathogens were present:

* Correspondence: [email protected] 1Department of Parasitology and Parasitic Diseases, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Calea Mănăştur 3-5 Cluj-Napoca 400372, Romania Full list of author information is available at the end of the article © 2014 Mircean et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lupșe et al. Parasites & Vectors 2014, 7(Suppl 1):P3 http://www.parasitesandvectors.com/content/7/S1/P3

POSTERPRESENTATION Open Access Recurrent subcutaneous human Dirofilariasis due to Dirofilaria repens after surgical removal of the worm and anthelmintic treatment M Lupșe1*, V Mircean2, A Cavasi1, AD Mihalca2 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

The genus Dirofilaria includes various species that are nat- northern part of the country and, to our knowledge, the ural parasites of dogs, cats, foxes, and wild mammals, first case of recurrent subcutaneous human dirofilariasis. transmitted by mosquito vectors. Dirofilaria repens is com- The recurrence in this case is most probably the result of monly encountered in the subcutaneous tissue of dogs, infection with multiple larvae, of which at least two devel- foxes, and cats. Due to recent increase of human Dirofi- oped into nodules at four month interval, rather than a laria repens it is considered an emerging zoonosis. In repeated infection. We recommend the treatment with Romania, there are only few reports regarding the infection ivermectin after surgical excision to avoid recurrences in with D. repens in dogs and in humans. Patients usually pre- dirofilariasis. sent single migratory submucosal or subcutaneous nodule which may or may not be tender. Surgical excision of Authors’ details lesions and affected areas is the treatment of choice for dir- 1University of Medicine and Pharmacy “Iuliu Haţieganu” Cluj-Napoca, ofilariasis. Anthelmintics are not usually recommended. Romania. 2University of Agricultural Sciences and Veterinary Medicine, Cluj- We describe the case of a 65 years old, retired woman, Napoca, Romania. without history of traveling in the past years, living in the Published: 1 April 2014 Oradea (north-west part of Romania), in an urban area, who presented a left breast subcutaneous nodule with fever and eosinophilia in August 2012. After surgical exci- doi:10.1186/1756-3305-7-S1-P3 sion, histopathology and PCR assay (Isolate Genomic Cite this article as: Lupșe et al.: Recurrent subcutaneous human DNA Kit, Bioline, UK using 12S ADNr gene (250 bp) the Dirofilariasis due to Dirofilaria repens after surgical removal of the worm and anthelmintic treatment. Parasites & Vectors 2014 7(Suppl 1):P3. diagnosis of D. repens infection was established. Patient received Albendazole 400 mg/day for 7 days after surgery. The eosinophil count became normal and no symptoms were noticed till December 2012 when another nodular Submit your next manuscript to BioMed Central lesion developed in the left breast, accompanied by eosino- and take full advantage of: philia. After the surgical removal of the second nodule, another larval worm was identified as D. repens.The • Convenient online submission patient was treated with 150 μg/kg BW of ivermectin and • Thorough peer review no other nodules appeared. Eosinophiles level remained • No space constraints or color ﬁgure charges normal during one year follow-up. This is the first report • Immediate publication on acceptance D. repens of autochthonous infectioninhumansinthe • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution * Correspondence: [email protected] 1 University of Medicine and Pharmacy “Iuliu Haţieganu” Cluj-Napoca, Submit your manuscript at Romania www.biomedcentral.com/submit Full list of author information is available at the end of the article © 2014 Lupûe et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Morozov et al. Parasites & Vectors 2014, 7(Suppl 1):P4 http://www.parasitesandvectors.com/content/7/S1/P4

POSTERPRESENTATION Open Access Prevalence of tick-borne pathogens in ticks from migratory birds in republic of Moldova AC Morozov1*, AA Tischenkov2, AA Proka1, C Silaghi3, IK Toderas1, AA Movila1, S Poppert4,5 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Migratory birds are often implicated in transporting ticks, typhi, 2 for Neoehrlichia mikurensis, 2 for Babesia microti which can carry pathogenic agents of several human and and 2 for Borrelia miyamotoi. These included 12 cases domestic animals diseases. By themselves ticks are not with a mix of the two pathogens and 2 cases with three highly mobile, and the only ways to expand the habitat are pathogens. hosts.Birds,duetotheabilitytofly,havethegreatestinflu- The presence of the above pathogens in ticks collected ence on the resettlement of ticks. As was documented by from passerine birds corroborates the role of these verte- many authors birds can transport ticks harboring Borrelia brates in the epidemiology and dispersion of tick-borne spp., Babesia spp., Anaplasma spp. and Rickettsia spp. diseases. Some of the birds are considered as migratory or throughout many parts of Europe. partial migratory. In addition these birds share the ecolo- Migratory birds were caught in ornithological nets gic niche with the others migratory birds which migrate from March 2012 till October 2013 at natural forest for long distances from Africa to the Europe. Our data reservations (Yagorlik, Prutul de Jos, Padurea Dom- suggest the involvement of ticks parasitic on birds in the neasca), and urbanocenoses of Chisinau and suburbs. All cycle of human and cattle tick-borne diseases. birds were identified to species level, and their sex. Birds were placed in bags made of thick fabric, right after birds Authors’ details were examined in the camp for the presence of ticks. 1Institute of Zoology, Academy of Science of Moldova, Chiûinău, Republic of After the examination, birds were released without being Moldova. 2Dniester State University named T.G. Shevchenko, Tiraspol, DMR, 3 harmed. Tick species were detached from birds and Republic of Moldova. Comparative Tropical Medicine and Parasitology, Ludwig-Maximilians-Universität, Munich, Germany. 4Bernhard Nocht Institute stored individually in 70% alcohol. Total DNA was For Tropical Medicine, Hamburg, Germany. 5Justus Liebig University, Giessen, extracted using QIAamp DNA Mini Kit. Specific PCRs Germany. were carried using DNA to detect Borrelia, Anaplasma, Published: 1 April 2014 Rickettsia, Neoehrlichia, Brucella species. Total of 131 ticks relating to the 3 ticks species (Ixodes ricinus n=124,I. frontalis n=6,Haemaphysalis punctata doi:10.1186/1756-3305-7-S1-P4 n =1) were collected from 45, tick-infested birds relating Cite this article as: Morozov et al.: Prevalence of tick-borne pathogens to the 7 avian species. The most common host was Turdus in ticks from migratory birds in republic of Moldova. Parasites & Vectors merula. From 131 ticks in extracted DNA of 77 one or 2014 7(Suppl 1):P4. more of the following pathogens was detected: 25 ticks were positive for Anaplasma phagocytophilum,18were positive for Borrelia garinii, 1 was positive for B. lusitaniae,1forB. valaisiana,9forRickettsia monacensis,2 ticks were positive for Rickettsia helvetica, 1 for Rickettsia

* Correspondence: [email protected] 1Institute of Zoology, Academy of Science of Moldova, Chiûinău, Republic of Moldova Full list of author information is available at the end of the article © 2014 Morozov et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Gomes et al. Parasites & Vectors 2014, 7(Suppl 1):P5 http://www.parasitesandvectors.com/content/7/S1/P5

POSTERPRESENTATION Open Access Development and field trial of a Tams1-targeted isothermal DNA amplification (Tams1-lamp) assay for detection of Theileria annulata in cattle J Gomes1, A Amaro1*, G Santos-Gomes2, I Pereira da Fonseca3, J Inácio1 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Mediterranean Theileriosis is frequently diagnosed in cattle total of 67% infected animals with very low parasitaemia in southern Portugal. This tick-borne hemoprotozoan that usually is characteristics of chronic infections. disease, caused by the apicomplexan parasite Theileria Tams1-LAMP identified 66% infected animals from the 14 annulata, poses important health problems in cattle in farms. Cohen’s test was used to evaluate the agreement addition to the economic losses caused by decrease of pro- between methods and the k value found (0.98) is indicative ductivity in chronic infected animals. Integrated control of an excellent agreement for this group of samples. These strategies for this disease will then benefit to the detection results permitted to conclude that Tams1-LAMP is a use- of cattle with chronic infection that usually acts as parasite ful molecular technique to be applicable for detecting reservoirs constituting a risk to susceptible animals. The Theileria annulata in cattle with chronic infections. highly sensitive and specific molecular methods for T. annulata detection are suitable for the identification of Authors’ details 1 such animals often with very low parasitaemia. The iso- Instituto Nacional de Investigação Agrária e Veterinária, I.P. Lisbon, Portugal. 2Instituto de Higiene e Medicina Tropical, Lisbon, Portugal. 3Faculdade de thermal amplification methods proved particularly useful Medicina Veterinária, Universidade Técnica de Lisboa, Portugal. for diagnostic laboratories with fewer resources since it does not require the use of expensive and sophisticated Published: 1 April 2014 equipment such as thermal cyclers. In the present study we develop an isothermal amplification technique targeting doi:10.1186/1756-3305-7-S1-P5 Tams1 gene (Tams1-LAMP) for detection of T. annulata Cite this article as: Gomes et al.: Development and field trial of a in cattle blood samples and compared this technique with Tams1-targeted isothermal DNA amplification (Tams1-lamp) assay for detection of Theileria annulata in cattle. Parasites & Vectors 2014 real time PCR for assessing its applicability for diagnosis. 7(Suppl 1):P5. One hundred blood samples from 16 farms were collected and analyzed. Genomic DNA extracted from blood was used as template in real time PCR and LAMP reactions for Submit your next manuscript to BioMed Central Tams1 gene amplification. A DNA sample extracted from and take full advantage of: T. annulata cell culture and one from an animal with 0.03% parasitaemia were used as positive controls. DNA • Convenient online submission samples from other closely related parasites were used as • Thorough peer review negative controls. The LAMP technique detection limit has • No space constraints or color ﬁgure charges been determined to correspond to a parasitaemia of • Immediate publication on acceptance approximately 0.008% and a specificity of 100%. Real time • Inclusion in PubMed, CAS, Scopus and Google Scholar PCR positive samples were detected in 14 farms with a • Research which is freely available for redistribution

* Correspondence: [email protected] Submit your manuscript at 1Instituto Nacional de Investigação Agrária e Veterinária, I.P. Lisbon, Portugal www.biomedcentral.com/submit Full list of author information is available at the end of the article © 2014 Gomes et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kalmár et al. Parasites & Vectors 2014, 7(Suppl 1):P6 http://www.parasitesandvectors.com/content/7/S1/P6

POSTERPRESENTATION Open Access Borrelia turcica in Hyalomma aegyptium ticks in Romania

Z Kalmár*,GD’Amico, IA Matei, AI Paștiu, DI Mărcuţan, MO Dumitrache, AD Mihalca From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Testudo graeca tortoises are distributed in the south- Published: 1 April 2014 eastern part (Dobrogea region) of Romania. T. graeca is a potential host for the three-host ticks, Hyalomma doi:10.1186/1756-3305-7-S1-P6 aegyptium. H. aegyptium ticks are important from epide- Cite this article as: Kalmár et al.: Borrelia turcica in Hyalomma aegyptium ticks in Romania. Parasites & Vectors 7 miological point of view as they constitute potential reser- 2014 (Suppl 1):P6. voirs for numerous zoonotic bacterial pathogens (Anaplasma phagocytophilum, Ehrlichia canis, Coxiella burnetii). However, H. aegyptium wasreportedtohostless studied bacteria, non-Lyme members of genus Borrelia. Despite its relatively wide distribution range, the extent of co-distribution of ticks with these bacteria was not investigated in detail. The aim of the present study was to evaluate H. aegyptium engorged ticks collected from tortoises insouth-easternRomaniaforthepresenceofnon-Lyme Borrelia. Between 2008 and 2013, 448 H. aegyptium ticks were collected from 45 Tgraecatortoises located in Dobrogea region in Romania. DNA extraction was performed individually from each tick using a commercial kit. For the total 78 (17.4%) Borrelia spp. positive ticks, PCR analysis targeting the intergenic spacer 5S-23S region, glpQ, respectively gyrB genes, and further sequencing was performed for the further identification. Sequences of gyrB and glpQ genes showed 99%-100% similarities with rep- tile-associated Borrelia turcica. The most frequently infected stages were males (10.7% of the total males exam- Submit your next manuscript to BioMed Central ined or 61.5% from the total infected ticks) followed by and take full advantage of: females (5.36% of the total females examined or 31% from the total infected ticks) and nymphs (1.34% of the total • Convenient online submission nymphs examined or 7.7% from the total infected ticks). • Thorough peer review This is the first report of Borrelia turcica in Romania. • No space constraints or color ﬁgure charges This research was supported by grant CNCSIS IDEI • Immediate publication on acceptance PCCE 7/2010 and IDEI PCE 236/2011. • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution

* Correspondence: [email protected] Submit your manuscript at Department of Parasitology and Parasitic Diseases, University of Agricultural www.biomedcentral.com/submit Sciences and Veterinary Medicine Cluj-Napoca, Cluj-Napoca, Romania © 2014 Kalmár et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Achterberg et al. Parasites & Vectors 2014, 7(Suppl 1):P7 http://www.parasitesandvectors.com/content/7/S1/P7

POSTERPRESENTATION Open Access Bead-based suspension array for detection and identification of tick-borne Borrelia species RP Achterberg1, CB Van Solt-Smits1, KE Hovius2, H Sprong3, M Fonville3, FM De Bree1, FJ Van der Wal1* From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

In the Netherlands, screening for tick-borne pathogens To evaluate the suspension array, DNA of nearly 1000 is performed using real time PCR on DNA extracted ticks was isolated and used to generate amplicons with a from ticks, followed by sequencing of positive samples, universal PCR targeting the 23S-5S IGS of Borrelia spp. thereby disallowing identification in case of double- All PCR reactions were then used in a direct hybridiza- infections. Until recently, testing was performed with tionassayinpresenceofamixtureofprobe-containing several reverse line blots (RLBs), each containing ~10 beads. Out of 981 ticks, ~20% were found to carry one or different probes. Advantages of RLBs are the capabilities more Borrelia species (resp. 15 and 5%). The most preva- to differentiate between bacterial species and to detect lent species was B. afzelii, followed by B. burgdorferi double infections, disadvantages are that RLBs are fasti- sensu stricto, B. garinii, B. sensu lato (unidentified) and dious, elaborate, and time-consuming. B. valaisiana. These results show the viability of bead- The aim of this work was to ‘test drive’ bead-based sus- based suspension arrays for (relatively) rapid detection pension arrays for the rapid detection and identification and identification of multiple species, but at this of tick-borne Borrelia species. The platform used is the stage need verification by a ‘gold standard’.Currently, flow cytometry-based xMAP technology of Luminex, in confirmation of these results is being performed by next which fluorescent nanoparticles (beads) are used to build generation sequencing. multiplex assays simply by mixing different bead sets that are covered with specific probes. Each bead set contains Funding a different ratio of red and infrared fluorophores, which This work was partly funded by the European Union enables identification of beads that pick up a third fluoro- Seventh Framework Programme (FP7/2007-2013) under phore by hybridization to a labelled target molecule. Grant Agreement Number 222633 (WildTech). Using standard chemistry, aminated oligonucleotides were covalently linked to carboxylated Luminex beads, Authors’ details resulting in bead sets with general and specific probes for 1Infection Biology, Central Veterinary Institute, Lelystad, the Netherlands. the 23S-5S intergenic spacer (IGS) of Borrelia species. 2Companion Animal Hospital ‘t Heike, Veldhoven, the Netherlands. 3 Presence of probes on the beads was verified by hybridi- Laboratory for Zoonoses and Environmental Microbiology, National Institute of Public Health and Environment (RIVM), Bilthoven, the Netherlands. zation of complementary biotinylated oligonucleotides in presence of a mixture of 1000 beads per bead set, using a Published: 1 April 2014 30 minute incubation at 37°C in a NaCl/Tris buffer, followed by labelling with streptavidin-phycoerythrin. Of each mixture, a minumum of 100 beads per bead set doi:10.1186/1756-3305-7-S1-P7 were investigated by flow cytometry on a dedicated Cite this article as: Achterberg et al.: Bead-based suspension array for detection and identification of tick-borne Borrelia species. Parasites & Luminex instrument. Vectors 2014 7(Suppl 1):P7.

* Correspondence: [email protected] 1Infection Biology, Central Veterinary Institute, Lelystad, the Netherlands Full list of author information is available at the end of the article © 2014 Achterberg et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Rinaldi et al. Parasites & Vectors 2014, 7(Suppl 1):P8 http://www.parasitesandvectors.com/content/7/S1/P8

POSTERPRESENTATION Open Access Ixodidae ticks in sheep and cattle in the Basilicata region (southern Italy) L Rinaldi1,3, ME Morgoglione1, E Noviello1, A Bosco1, G Prestera2, G Cringoli1,3* From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Ixodidae are an important health problem for domestic (9.7%), I. ricinus (9.7%), Ixodes gibbosus (6.5%) and and wild animals for direct damages caused by these Hyalomma detritum (3.2%). ticks, but mostly because they are involved in the trans- The results of the present cross-sectional survey confirm mission of many diseases (tick borne diseases - TBDs). that the environmental and climatic conditions of south- Therefore, the surveys on the presence and geographical ernItalyaresuitablefordifferent tick species infecting distribution of ticks in domestic ruminants need to be domestic ruminants that are therefore exposed to different constantly updated. TBDs. The aim of the present study was to update the data on the presence and distribution of Ixodidae ticks in Authors’ details cattle and sheep bred in the Basilicata region (southern 1Department of Veterinary Medicine and Animal Productions, University of Italy), an area with a Mediterranean climate favourable Naples Federico II, Regional Center for Monitoring Parasitic Infections 2 to the biology and ecology of ticks. In this region, the (CREMOPAR, Regione Campania), Naples, Italy. Regional Association of Farmers, Basilicata, Italy. 3CIRPAR, Italy. breeding of pasturing cattle and sheep is a widespread reality very important from an economical point of Published: 1 April 2014 view, especially for the natural vocation of animals in using marginal hilly and mountainous pastures. From May to September 2013, sheep and cattle farms doi:10.1186/1756-3305-7-S1-P8 (n = 82 and 31, respectively) were visited and ticks were Cite this article as: Rinaldi et al.: Ixodidae ticks in sheep and cattle in the Basilicata region (southern Italy). Parasites & Vectors 2014 collected from 20 animals per farm, preserved in ethanol 7(Suppl 1):P8. 70% and then identified at species level using the mor- phometric keys present in the literature. A total of 2,179 ticks were collected. The following species were identified in the sheep farms (total = 1594 adult ticks collected): Rhipicephalus bursa (67.1%), R. sanguineus (34.1%), R. turanicus (25.6%), Dermacentor marginatus Submit your next manuscript to BioMed Central (6.1%), Hyalomma detritum (3.7%), Ixodes ricinus (3.7%) and take full advantage of: and Hyalomma marginatum (1.2%). In the cattle farms, a total of 585 adult ticks were collected and the following • Convenient online submission species identified: R. bursa (61.3%), H.marginatum (29.0%), • Thorough peer review R. turanicus (25.8%) sanguineus (25.8%), D. marginatus • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar * Correspondence: [email protected] • Research which is freely available for redistribution 1Department of Veterinary Medicine and Animal Productions, University of Naples Federico II, Regional Center for Monitoring Parasitic Infections Submit your manuscript at (CREMOPAR, Regione Campania), Naples, Italy www.biomedcentral.com/submit Full list of author information is available at the end of the article © 2014 Rinaldi et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kalan et al. Parasites & Vectors 2014, 7(Suppl 1):P9 http://www.parasitesandvectors.com/content/7/S1/P9

POSTERPRESENTATION Open Access Distribution of two invasive mosquito species in Slovenia in 2013

K Kalan1, VE Buzan1,2, V Ivović1,2* From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

In the recent years there has been growing interest in the diseases. Therefore an effective national monitoring establishment and spread of invasive mosquito species in program of invasive mosquito species is highly needed. Europe. Apart from the most dispersed, Aedes albopictus, With well planned, long term strategy we will be able there are five other alien aedine mosquito species spread- to detect and control their occurrence, monitor their ing in Europe. Their spreading represents a considerable pathogens and prevent the establishment of new foci threatforpublichealthastheycanserveasvectorsof of mosquito-borne diseases. mosquito-borne diseases.

A survey in whole Slovenia was performed in 2013 to Authors’ details determine the distribution of invasive mosquitoes. The 1Institute for Biodiversity studies, Science and research centre, University of study was based on the search for invasive mosquito larvae Primorska, Koper, Slovenia. 2Faculty of Mathematics, Natural Sciences and in artificial water containers at the cemeteries, around Information Technologies, University of Primorska, Koper, Slovenia. human dwellings and in used tires at vulcanizing compa- Published: 1 April 2014 nies. Additionally, a call for citizens to report any nuisance from “unusual” mosquitoes was published in local media. With the study we have revealed the presence of two doi:10.1186/1756-3305-7-S1-P9 invasive mosquito species in Slovenia, Ae. albopictus Cite this article as: Kalan et al.: Distribution of two invasive mosquito and Ae. japonicus. Altogether we caught 494 larvae of species in Slovenia in 2013. Parasites & Vectors 2014 7(Suppl 1):P9. Ae. albopictus and 1318 larvae of Ae. japonicus.The results show that Ae. albopictus is present mostly in Southwestern and central part of the country, with some isolated locations in other parts of Slovenia. Nevertheless, Ae. japonicus was found in a large part of the investigated area. This species was previously collected from a small area near the Austrian border in 2011, and in only two years it has colonized the majority of Northeastern part Submit your next manuscript to BioMed Central of Slovenia. and take full advantage of: In Slovenia no systematic mosquito surveillance or control is currently done. There are only some minor projects, • Convenient online submission which are carried out by local institutions at a regional • Thorough peer review Ae. albopictus Ae. japonicus level. and are spreading fast • No space constraints or color ﬁgure charges and they both present high nuisance for the humans, as • Immediate publication on acceptance well as a public health risk since transmitting vector borne • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution * Correspondence: [email protected] 1 Institute for Biodiversity studies, Science and research centre, University of Submit your manuscript at Primorska, Koper, Slovenia www.biomedcentral.com/submit Full list of author information is available at the end of the article © 2014 Kalan et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bencurova et al. Parasites & Vectors 2014, 7(Suppl 1):P10 http://www.parasitesandvectors.com/content/7/S1/P10

POSTERPRESENTATION Open Access Effect of PilE4 of Francisella tularensis subsp. holarctica (live vaccine strain) to brain endothelium

E Bencurova1*, R Mucha2, L Pulzova1, M Bhide1,2 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Francisella are rare, highly infectious vector-borne bac- endothelium, that offers new insight into the pathobiology teria, which infect more than 250 host species, including of neuroinvasion of Francisella. humans. Francisella areabletoinvademultipleorgans in the host body, such a skin, liver, lung and central ner- Acknowledgements vous system. To invade various organs, Francisella have This work was supported by APVV-0036/10, VEGA-1/0054/12 and structural funds for centers of excellence-26220120002 (INFEKTZOON). E.B and L.B. are to cross the cell linings like endothelial barriers. Interac- funding for post-doctoral studies from ITMS 26220220185. tion with endothelial cells is multistage process, which Authors’ details includes adhesion and activation of signaling events. 1 Francisella tularensis Laboratory of biomedical microbiology and immunology, University of Our previous work has shown that veterinary medicine and pharmacy in Kosice, Kosice, Slovakia. 2Institute of subsp. holarctica Live Vaccine Strain (LVS) interacts Neuroimmunology, Slovak Academy of Science, Bratislava, Slovakia. and adhere to endothelial cells through PilE4 and Inter- Published: 1 April 2014 cellular adhesion molecule 1(ICAM-1) interaction. To corroborate consequences of PilE4 adhesion to endothelial cells, we incubated brain microvascular endothelial cells doi:10.1186/1756-3305-7-S1-P10 with recombinant PilE4 protein and assessed the regula- Cite this article as: Bencurova et al.: Effect of PilE4 of Francisella tion of several genes with qRT-PCR. Results showed that tularensis subsp. holarctica (live vaccine strain) to brain endothelium. PilE4 upregulated the expression of adhesion molecules Parasites & Vectors 2014 7(Suppl 1):P10. (ICAM-1 and PECAM-1), matrix metalloproteases (MMP 1, MMP 3 and MMP 9) and molecules that are involved in pathogen recognition (TLR-6, MyD88, IRAK-1 and TRAF- 6) in brain endothelium. On the other hand, we found down regulation of interleukins and NF-kB. Inhibition of Francisella NF-kB indicates that might use NF-kB subver- Submit your next manuscript to BioMed Central sion mechanism to evade immune response. This mechan- and take full advantage of: ism is described in other pathogens such as Listeria and Yersinia. Previous studies have showed that matrix-metal- • Convenient online submission loprotases, mainly MMP-9, is crucial to disrupt cytoskele- • Thorough peer review ton of brain endothelium to increase the permeability • No space constraints or color ﬁgure charges of BBB. In summary, work reveal hidden aspects of • Immediate publication on acceptance Francisella invasion and translocation of across the brain • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution * Correspondence: [email protected] 1 Laboratory of biomedical microbiology and immunology, University of Submit your manuscript at veterinary medicine and pharmacy in Kosice, Kosice, Slovakia www.biomedcentral.com/submit Full list of author information is available at the end of the article © 2014 Bencurova et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Chochlakis et al. Parasites & Vectors 2014, 7(Suppl 1):P11 http://www.parasitesandvectors.com/content/7/S1/P11

POSTERPRESENTATION Open Access Rhipicephalus turanicus: from low numbers to complete establishment in Cyprus. Its possible role as a bridge-vector D Chochlakis1*, I Ioannou2, B Papadopoulos3, Y Tselentis1,3, A Psaroulaki3 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

We describe herein the abundance of Rhipicephalus tur- first time in Cyprus and was identified in R. turanicus only anicus in Cyprus, and its potential role as bridge vector disregarding the animal host. Of the 805 ticks, 107 carrying tick-borne pathogens among various hosts. (IR: 13.3%) were tested positive for C. burnetii,and16 Following the first recording (1970-72), at low numbers, (IR: 2%) were positive for Anaplasma species. Five ticks of R. turanicus in Cyprus, two studies took place (March harboured both Rickettsia species and C. burnetii. 1999 - March 2001, January 2004 - December 2006), Rhipicephalus turanicus,largelydistributedinthe during which, ticks were collected from ruminants Mediterranean sub-region, Africa and Asia, is flexible (goats, sheep, bovine), dogs and wild mammals (foxes, and adaptable and can be found in a rich variety of hosts wild-rabbits and mouflons) from different sites of the (domestic and wild). It has well adapted and spread over island. All ticks were morphologically identified to the spe- a great variety of animal species in Cyprus during the last cies level, washed in 70% alcohol, rinsed in sterile water, 40 years and seems to play an importance role as a dried on sterile filter paper and triturated individually. bridge-vector of tick-borne pathogens. The changing Following DNA extraction [QIAampTissue Kit (QIAGEN, climate, geographical position of Cyprus (wintering area Germany)] all ticks were tested for Rickettsia species and for migratory birds), make the island an important cross- Coxiella burnetii by Real-time PCR targeting the gltA gene over for possible dispersal of ticks and their tick-borne and IS1111 insertion sequence, respectively, and for pathogens from East to the West. Anaplasma species by PCR targeting the 16s rRNA gene.

Samples positive for Rickettsia species were further ampli- Authors’ details fied by PCR targeting the ompA and ompB genes. 1Regional Laboratory of Public Health of Crete, Heraklion, Crete, Greece. Of the 3950 ticks collected, 805 (20.4%) were identified 2Veterinary Services, Nicosia, Cyprus. 3Laboratory of Clinical Bacteriology, as R. turanicus; this species was the only one, amongst the Parasitology, Zoonoses and Geographical Medicine, University of Crete, Heraklion, Crete, Greece. rest of the ticks (R. bursa, R. sanguineus, Hyalomma anatolicum excavatum, H. marginatum, H. m. rufipes, Ixodes Published: 1 April 2014 gibbosus, I. ventalloi, Haemaphysalis sulcata, H. punctata), that was collected from every host. Of the ticks tested, 125/805 (IR: 15.5%) were tested positive for Rickettsia doi:10.1186/1756-3305-7-S1-P11 species [R. massilliae (83/125), “Candidatus Rickettsia Cite this article as: Chochlakis et al.: Rhipicephalus turanicus: from low “ “ numbers to complete establishment in Cyprus. Its possible role as a barbariae (5/125), Candidatus Rickettsia barbariae geno- bridge-vector. Parasites & Vectors 2014 7(Suppl 1):P11. type Cretocypriensis” (26/125) and “Candidatus Rickettsia tselentii“ (11/125)]. The latter Rickettsia species showed a close relationship with R. africae,wasdescribedforthe

* Correspondence: [email protected] 1Regional Laboratory of Public Health of Crete, Heraklion, Crete, Greece Full list of author information is available at the end of the article © 2014 Chochlakis et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Christova et al. Parasites & Vectors 2014, 7(Suppl 1):P12 http://www.parasitesandvectors.com/content/7/S1/P12

POSTERPRESENTATION Open Access Rodents as reservoirs of human pathogens in Bulgaria I Christova1*, I Trifonova1, N Kalvatchev1, H Dimitrov2, V Mitkovska2, T Gladnishka1, V Ivanova1 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Small mammals are reservoirs of various human patho- detected also in A. agrarius mice. Medical authorities gens. The aim of this work was to investigate infections should be aware of the risk for humans. with human pathogens in rodents trapped in different regions of Bulgaria. A total of 284 rodents were investi- Authors’ details gated by PCR for detection of the flagellin gene of borre- 1Department of Microbiology, National Center of Infectious and Parasitic liae within Borrelia burgdorferi sensu lato complex, ankA Diseases, Sofia, Bulgaria. 2Department of Zoology, University of Plovdiv, gene of Anaplasma phagocytophilum and nucleoprotein Plovdiv, Bulgaria. gene of hantavirus Dobrava - conventional nested Published: 1 April 2014 RT-PCR and Real Time PCR with TaqMan probe. B. burgdorferi was detected in 64/284 (22.5%) of the investigated rodents by PCR. Of them, 41 samples originated from doi:10.1186/1756-3305-7-S1-P12 Apodemus flavicollis,20fromA. agrarius,and3fromA. Cite this article as: Christova et al.: Rodents as reservoirs of human sylvaticus. Overall, 33 of the investigated 284 rodents were pathogens in Bulgaria. Parasites & Vectors 2014 7(Suppl 1):P12. infected with Anaplasma phagocytophilum (11.6%) - 11 Apodemus flavicollis (infectivity rate 8.6% of the 128 investigated), 13 Apodemus agrarius (infectivity rate 13.5% of the 96 tested), and 1 Apodemus sylvaticus (infectivity rate 9% of the 11 tested). Hantavirus RNA was detected in 9 of therodents.OnlyDobrava-BelgradevirusbutnotPuu- mala or Saaremaa virus was detected. Almost all infected rodents were A. flavicollis (8/9 PCR-positive rodents). Rodents are important reservoirs of human pathogens. In this study, active infection in rodents was confirmed by detection of microorganism’s genome. Remarkably, a high number of rodents from genus Apodemus were Submit your next manuscript to BioMed Central infected with borreliae. The high rate of detection of and take full advantage of: A. phagocytophilum in rodents from A. agrarius species suggested that this species might serve as major reservoir • Convenient online submission of human anaplasmosis in Bulgaria. Mainly infected with • Thorough peer review hantaviruses were A. flavicollis mice, known as reservoir • No space constraints or color ﬁgure charges of Dobrava hantavirus but hantavirus infections were • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution * Correspondence: [email protected] 1 Department of Microbiology, National Center of Infectious and Parasitic Submit your manuscript at Diseases, Sofia, Bulgaria www.biomedcentral.com/submit Full list of author information is available at the end of the article © 2014 Christova et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Duh et al. Parasites & Vectors 2014, 7(Suppl 1):P13 http://www.parasitesandvectors.com/content/7/S1/P13

POSTERPRESENTATION Open Access Flavivirus genome detected in Mus and Apodemus species sampled in illegal waste sites D Duh1*, E Varljen-Buzan1,2, U Kunej2, S Hasic2 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Tick-borne encephalitis virus (TBEV), a zoonotic flavi- genome. Six amplicons of correct size were seen on virus that occurs on the Eurasian continent and causes agarose gel and are in the process of sequencing to reveal tick-borne encephalitis (TBE) in humans, is considered the identity of detected flavivirus. medically the most important arthropod vector transmitted virus in Europe. As such, TBEV is of course not Authors’ details a neglected pathogen. However, the knowledge about 1Science and Research Centre, University of Primorska, Koper - Capodistria, TBEV could importantly contribute to the research of Slovenia. 2Faculty of Mathematics, Natural Sciences and Information other neglected vector borne pathogens. In nature, Technologies University of Primorska, Koper - Capodistria, Slovenia. TBEV is transmitted from tick to tick via vertebrates in Published: 1 April 2014 a process named co-feeding which is independent of a systemic viraemia in vertebrates. Therefore the ticks are the reservoirs as well as the vectors for TBEV, while the doi:10.1186/1756-3305-7-S1-P13 vertebrate is considered the transient bridge in transmis- Cite this article as: Duh et al.: Flavivirus genome detected in Mus and sion and maintenance of TBEV. Apodemus species sampled in illegal waste sites. Parasites & Vectors 2014 7(Suppl 1):P13. Slovenia is an endemic country for TBEV with approximately 300 cases reported annually and incidence around 14 per 100,000 inhabitants. It was shown, that TBEV in Slovenia is maintained in tick-rodent-deer cycle. TBEV viraemic rodents of Myodes and Apodemus species were described previously in Slovenia. We were interested whether TBEV could be present in Mus species which share a living habitat with Apodemus mice in illegal waste sites. Thus we collected 83 samples of Mus and Apodemus species from different dump sites in Slovenia and Croatia. The presence of TBEV was determined using molecular Submit your next manuscript to BioMed Central techniques. Total RNA was extracted from spleen samples and take full advantage of: using RTP Pathogen Kit (Invitek-Stratec) and tested with real time RT-PCR specific for TBEV-Eur subtype. All sam- • Convenient online submission ples were negative which could be explained by the fact • Thorough peer review that testing area was in the part of Slovenia with low inci- • No space constraints or color ﬁgure charges dence of TBE. However, distinctive samples were also • Immediate publication on acceptance tested with universal primers for amplification of flavivirus • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution * Correspondence: [email protected] 1 Science and Research Centre, University of Primorska, Koper - Capodistria, Submit your manuscript at Slovenia www.biomedcentral.com/submit Full list of author information is available at the end of the article © 2014 Duh et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Huemer et al. Parasites & Vectors 2014, 7(Suppl 1):P14 http://www.parasitesandvectors.com/content/7/S1/P14

POSTERPRESENTATION Open Access Bunyaviruses in human, animal and mosquito samples from southeast Austria

HP Huemer1,3*, B Seidel2, P Hufnagl3, A Deutz4, A Posautz5, S Dowall6, R Hewson6, Z Hubalek7, F Allerberger3 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

In Austria occurrence of 40 different species of mosqui- Authors’ details 1Dpt. Hygiene, Microbiology & Social Medicine, Medical University toes belonging to 6 genera has been described. AGES in Innsbruck, Austria. 2Ecology and Landscape Assessment, Persenbeug, Austria. 2011 initiated a nation-wide mosquitoe-surveillance pro- 3Austrian Agency for Health and Food Safety (AGES), Vienna, Austria. 4Styrian 5 gram to identify invading species. As part of this project, Public Health Authority, BH Murau, Austria. Inst.Wildlife Ecology, University for Veterinary Medicine, Vienna, Austria. 6Public Health England, Microbiol. pools of Culex, Aedes, Anopheles and Culisetta species Services, Porton Down, UK. 7Inst. Vertebrate Biology, Academy of Sciences, collected in different parts of the country were analyzed by Brno, Czech Republic.

PCR analysis; in addition seroepidemiological testing for Published: 1 April 2014 bunyaviruses was performed in selected regions. In order to be able to detect also new strains, a broad spectrum CODEHOP (Consensus-Degenerate Hybrid Oligonucleo- doi:10.1186/1756-3305-7-S1-P14 tide Primer) approach was used initially; presumably due Cite this article as: Huemer et al.: Bunyaviruses in human, animal and to the low sensitivity of degenerated primer designs and mosquito samples from southeast Austria. Parasites & Vectors 2014 due to dilution in insect pools, we did not pick up any 7(Suppl 1):P14. new isolates so far using Orthobunyavirus, Phlebovirus or Nairovirus group-specific primers. Two bunyaviruses were found in Culex pipiens from the southern province Carynthia in 2012, detected with non-degenerate multiplex PCR, the sequences highly homologous to Italian Tahynavirus (TAHV) isolates. 245 sera collected for a study of zoonotic infections in hunters, veterinarians, farmers, and abattoir workers (conducted by the Styrian health authorities in 2003), were tested by ELISA tests, using inactivated Crimean Congo virus (CCHF) from Bulgaria as well as recombinant CCHF nucleoprotein produced in baculovirus. In addition, cell culture derived Submit your next manuscript to BioMed Central TAHV lysates as well as TAHV infected cells were used as and take full advantage of: antigens in ELISA and immunofluorescence (IF) under an experimental setup. No seropositivity for CCHF or related • Convenient online submission nairoviruses was found in these Austrian human sera • Thorough peer review originating from risk groups for zoonotic infection. • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution * Correspondence: [email protected] 1 Dpt. Hygiene, Microbiology & Social Medicine, Medical University Submit your manuscript at Innsbruck, Austria www.biomedcentral.com/submit Full list of author information is available at the end of the article © 2014 Huemer et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ivănescu et al. Parasites & Vectors 2014, 7(Suppl 1):P15 http://www.parasitesandvectors.com/content/7/S1/P15

POSTERPRESENTATION Open Access Anopheles maculipennis complex-responsible for the re-emergence of malaria in Romania? ML Ivănescu*, D Acatrinei, L Miron From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Between 2010 and 2012, in Iaûi City of Romania 5 species of the African continent, the human reservoir of Plasmo- belonging to the complex Anopheles maculipennis dium will be permanently maintained. Correlating the were identified by PCR: A. melanoon, A. labranchiae, three factors implicated in the transmission of malaria: A. atroparvus, A. messeaeand A. maculipennis sp. The last favourable environmental conditions, presence of the vec- three species were involved in the transmission of tor and of the human reservoir, we could highlight the malaria in Romania between 1892 and 1961. The species growing risk of the re-emergence of malaria in Romania. A. labranchiae was signalled for the first time in Romania, being considered as the main vector of malaria in Europe. Published: 1 April 2014 The identified samples of Anopheles labranchiae were two stage IV larvae, concluding with the adaptation of the species to the climate of Iaûi City. Using a new mathematical doi:10.1186/1756-3305-7-S1-P15 Cite this article as: Ivănescu et al.: Anopheles maculipennis complex- model realized and implemented by ourselves, based on responsible for the re-emergence of malaria in Romania? Parasites & the construction of a function of interpolation of Lagrange Vectors 2014 7(Suppl 1):P15. polynomial type, we realized an extrapolation of the evolution of temperatures for the year 2030, demonstrating the existence of favourable conditions of development of both the vector and the malaria parasite. Therefore, it was a temperature raise by 0.72°C in the whole country, as compared to the period of malaria eradication in Romania, and the extrapolation of the evolution of temperatures in the year 2030 showed a raise by 0.8°C, result which coincides with the prognosis made by prestigious research institutes: NIES, CSIRO, HCCPR, MPIM and NCAR, which show a raise by 0.8-1.7° in 2030. In Iaûi City, the prognosis made for 2030 shows an average of 24°C for this year in the Submit your next manuscript to BioMed Central spring-summer months, temperature which is ideal for the and take full advantage of: development of the life cycle of mosquitoes, but also for the development of the parasite inside the vector. • Convenient online submission Taking into account the fact that Iasi, as a cultural • Thorough peer review city, hosts many foreign students who are carriers of • No space constraints or color ﬁgure charges Plasmodium , coming from malaria endemic areas, and • Immediate publication on acceptance the immigration of the population for work in countries • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution * Correspondence: [email protected] Department of Clinics, Faculty of Veterinary Medicine, “Ion Ionescu de la Submit your manuscript at Brad” University of Agricultural Sciences and Veterinary Medicine, Iasi, www.biomedcentral.com/submit Romania © 2014 Ivănescu et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Mărcuţan et al. Parasites & Vectors 2014, 7(Suppl 1):P16 http://www.parasitesandvectors.com/content/7/S1/P16

POSTERPRESENTATION Open Access Prevalence of Anaplasma phagocytophilum in ticks collected from migratory birds in Danube Delta, Romania ID Mărcuţan*, AD Sándor, AD Mihalca, CM Gherman, Z Kalmár, G D’Amico, MO Dumitrache, V Cozma From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Wild birds are hosts for several species of ticks, contribut- The bird species that carried ticks infected with A. pha- ing to the maintenance of their local populations in delim- gocytophilum were Turdus merula, Erithacus rubecula and ited geographic areas. migratory birds play important roles Fringilla coelebs. Several other studies have demonstrated as distributors of ticks within and between distant terri- the presence of A. phagocytophilum in ticks collected from tories, including continents. Ticks collected from birds are birds. Comparing our data with these results, we confirm responsible for hosting a significant number of human the low prevalence of A. phagocytophilum,showingthat pathogens. The extensive wetland complex of the Danube migratory birds, despite being without reservoir com- Delta provides an internationally important stopover site petencemight be important hosts for the dispersal of for millions of birds, belonging to 300 different species, infected ticks. travelling annually. The aim of this study was to detect Anaplasma phagocytophilum in feeding ticks collected Funding from migratory birds along four migratory seasons. Ticks This research was supported by grant CNCSIS IDEI were collected from the birds with forceps and preserved PCCE 7/2010 and IDEI PCE 236/2011. in 96% ethanol for later examination using a separate vial for each bird. A total of 1436 birds in 56 species Published: 1 April 2014 (15 families) of Passeriformes and 3 non-Passeriformes bird species were captured. A total of 400 ticks were collected and identified as larvae (n = 191; 47.75%), doi:10.1186/1756-3305-7-S1-P16 nymphs (n = 201; 50.25%) or adult females (n = 8; 2%). Cite this article as: Mărcuţan et al.: Prevalence of Anaplasma phagocytophilum in ticks collected from migratory birds in Danube No adult males were found. The ticks belonged to four Delta, Romania. Parasites & Vectors 2014 7(Suppl 1):P16. species (Ixodes ricinus, I. arboricola, I. redikorzevi and Haemaphysalis punctata). Ixodes ricinus was the most common tick (369/400 ticks, Submit your next manuscript to BioMed Central 92.25% of the total collected), with a total of 181 larvae, and take full advantage of: 180 nymphs and 8 females. For detection of Anaplasma phagocytophilum all 400 ticks were examined by PCR, • Convenient online submission targeting the msp2 gene. Anaplasma phagocytophilum • Thorough peer review specific DNA was detected in 2 larvae and 2 nymphs of, • No space constraints or color ﬁgure charges I. ricinus, from 3 birds prevalence 1.08-%. The infection • Immediate publication on acceptance was absent from the other species of collected ticks • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution

* Correspondence: [email protected] Submit your manuscript at Department of Parasitology and Parasitic Diseases, University of Agricultural www.biomedcentral.com/submit Sciences and Veterinary Medicine, Cluj-Napoca, Romania © 2014 Mărcuţan et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Mihailescu et al. Parasites & Vectors 2014, 7(Suppl 1):P17 http://www.parasitesandvectors.com/content/7/S1/P17

POSTERPRESENTATION Open Access Diagnosis and management of human borreliosis in Romania P Mihailescu1*, D Predeteanu2,3, A Craciunoiu2,4, G Ceaus1, C Istrate1, C-M Cretu1,2,5 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Borrelia burgdorferi sensu lato (s.l.) represents a category and Borrelia garinii (11). Confirmed co-infections (using of bacteria, the etiologic agent of borreliosis, well known IIFT/ELISA/WB techniques) included: Bartonella quin- as Lyme Disease (LD). On our territory, B. burgdorferi s.l. tana/henselae (19), Coxakie virus (2), Rickettsia spp. (2), is transmitted by the bite of infected ticks belonging to a Chlamydia pneumoniae (11), Mycoplasma pneumoniae few species of genus Ixodes. The most important species (10), Ehrlichia spp. (2), Babesia spp. (1), Yersinia enteroco- responsible for LD are Borrelia burgdorferi sensu stricto litica (3). Co-morbidities were also evaluated for the differ- (s.s.), Borrelia afzelii and Borrelia garinii. entials and the presence of multiple sclerosis, SLE, The objectives of this study were to define the best way rheumatoid arthritis or ankylosing spondylitis was noticed. to confirm and consider Lyme disease using commercial All patients received antibiotics, supplements and sugar serological tests: screening (indirect immunofluorescence restricted diet. Cases with confirmed associated diseases test - IIFT) and confirmatory (DNA and Western Blotting received concomitant specific therapy. Combinations of - WB), according to the epidemiological, professional antibiotics were in accordance with LD and documented risks, animal contact and clinical signs/symptoms. The co-infections. samples were collected from patients supposed with LD, According to our results existence of LD in some parts of referred to Eco-Para-Diagnostic Clinic, between October Romania was emphasized. The most common confirmed 2010 and December 2013. Borrelia burgdorferi s.l. species were Borrelia afzelii and A number of 1661 patients were referred to the clinic Borrelia burgdorferi s.s. Early diagnosis and appropriate with LD suspicion and they were tested according to our treatment are associated with a good evolution and prog- protocol (serology, clinical, epidemiological). A number of nosis. Chronic cases with multiple co-infections required 488 samples were tested using IIFT, 1086 with WB and 87 longer treatment and monitoring. Multidisciplinary team by DNA detection. Potential co-infections were also ana- works increase the accuracy of the diagnosis. Public health lyzed according to the history and clinical signs. Out of the information and prophylactic measures should be correctly 1661 suspected patients, 80 cases (study group) were con- provided for the population, in different manners. firmed by IIFT, WB, PCR, treated and monitored. Tick bite was recognized by 35 patients: 23 presented neurological Authors’ details symptoms, 10 cardiovascular signs, 20 rheumatologic signs, 1Eco-Para-Diagnostic - Parasitology Department, Bucharest, Romania. 12 dermatological signs (mostly EM) and one case pre- 2University of Medicine and Pharmacy “Carol Davila”- Parasitology 3 sented ocular damage. The infection with a single Borrelia Department, Bucharest, Romania. “Saint Mary” Hospital - Reumatology Department, Bucharest, Romania. 4University Teaching Emergency burgdorferi s.l. species was found in 56 cases, with two spe- Hospital - Neurology Department, Bucharest, Romania. 5Colentina cies in 22 cases and more than 2 species in 2 cases. Most Teaching Hospital - Parasitology Department, Bucharest, Romania. common documented species (using the confirmation tests Published: 1 April 2014 Western Blot and Borrelia DNA presence when it was possible): Borrelia afzelii (26) and Borrelia burgdorferi s.s. (19)

doi:10.1186/1756-3305-7-S1-P17 * Correspondence: [email protected] Cite this article as: et al 1 Mihailescu .: Diagnosis and management of Eco-Para-Diagnostic - Parasitology Department, Bucharest, Romania human borreliosis in Romania. Parasites & Vectors 2014 7(Suppl 1):P17. Full list of author information is available at the end of the article © 2014 Mihailescu et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Potkonjak et al. Parasites & Vectors 2014, 7(Suppl 1):P18 http://www.parasitesandvectors.com/content/7/S1/P18

POSTERPRESENTATION Open Access Molecular characterization of Borrelia strains isolated from ticks in Vojvodina

A Potkonjak1*, S Savić2, E Ruzić-Sabljić3, V Vračar1, B Lako1, A Jurišić4, A Petrović4, D Rajković4 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

Borrelia burgdorferi sensu lato (s.l.) complex represents the tick species I. ricinus from the territory of Vojvodina a group of different types of spirochete that are present were identified as Borrelia afzelii by applying molecular globally, which causes Lyme borreliosis. The total num- methods (MluI-LRFP and real-time PCR for hbb gene). By ber of types is still not final because new genetic and applying MluI-LRFP all three isolated strains of B. afzelii antigenic isolates are still being described. The clinical were characterized as a subtype Mlal. In this research, like pictureofLymeborreliosisin people is polymorphic in the previous researches, we have not proved the and is characterized with symptoms similar to flu syn- presence of a pathogenic species Borrelia spielmanii in dromes together with erythema migrans, rheumatologic, ticks of the species I. ricinus. cardiologic and neurological complications. Apart from people, dogs, horses, bovines and sheep can suffer from Funding Lyme borreliosis. In Europe, the most important vector This research was supported by Provincial Secretariat for transmitting Lyme disease is the tick Ixodes ricinus. Science and Technological Development, Autonomous A description of different species of Borrelia in ticks has Province of Vojvodina, Republic of Serbia (grant numbers: opened a completely new field of investigating the ecol- 114-451-3406/2013-04 and 114-451-3638/2012-01). ogy of Lyme borreliosis. It is especially important to investigate a connection between different types of Authors’ details Borrelia, ticks as vectors and vertebrates as the reser- 1Department of Veterinary Medicine, Faculty of Agriculture, University of voirs on various geographical localities. Novi Sad, Novi Sad, Serbia. 2Scientific Veterinary Institute “Novi Sad”, Novi 3 The aim of the work is to isolate Borrelia species from Sad, Serbia. Institut of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia. 4Department of Environmental the collected ticks I. ricinus from the geographical territory and Plant Protection, Faculty of Agriculture, University of Novi Sad, Novi Sad, of Vojvodina, and to do a molecular characterization of Serbia. the isolated strains of Borrelia. Published: 1 April 2014 A total of 12 tick pools of the type I. ricinus were cul- tivated in Barbour-Stoenner-Kelly-H medium with additional antibiotics and subcultivated into a modified doi:10.1186/1756-3305-7-S1-P18 Kelly-Pettenkofer/Preac-Mursic medium, until a clear Cite this article as: Potkonjak et al.: Molecular characterization of Borrelia culture is reached. For the molecular characterization of strains isolated from ticks in Vojvodina. Parasites & Vectors 2014 isolated strains of Borrelia, molecular methods MluI-LRFP 7(Suppl 1):P18. and real-time PCR for hbb gene were used. In this research, out of 12 pools of ticks species I. ricinus 3strainsofBorrelia from the B. burgdorferi s.l. complex were isolated. All three isolates of Borrelia from ticks of

* Correspondence: [email protected] 1Department of Veterinary Medicine, Faculty of Agriculture, University of Novi Sad, Novi Sad, Serbia Full list of author information is available at the end of the article © 2014 Potkonjak et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ćirović et al. Parasites & Vectors 2014, 7(Suppl 1):P19 http://www.parasitesandvectors.com/content/7/S1/P19

POSTERPRESENTATION Open Access Jackals as carriers of Leishmania and Brucella species in Serbia

D Ćirović1, D Chochlakis2, S Tomanović3*, R Sukara3, A Penezić1, Y Tselentis2,4, A Psaroulaki4 From The 1st Conference on Neglected Vectors and Vector-Borne Diseases (EurNegVec): with Management Committee and Working Group Meetings of the COST Action TD1303 Cluj-Napoca, Romania. 8-11 April 2014

The golden jackal Canis aureus occurs in south-eastern Jackals are known omnivores and scavengers and they Europe, Asia, the Middle East, the Caucasus and Africa usually prey on small mammals, on vegetables, fruit and as well as in countries of the European continent, garbage. Their ability to adapt to novel habitats both on including Serbia. The scope of the current survey was to rural and urban areas allows them to come into contact study the presence of two zoonotic agents, Leishmania with animals that live in close proximity to humans, such species (vector transmitted) and Brucella species, in the as dogs, hence their ability to host a number of zoonotic, jackal population in Serbia. including vector-borne, pathogens. An increase in jackal Animal samples were collected over a three-year period population has lately been observed in Serbia, while com- (01/2010 - 02/2013) from 48 localities corresponding to plaints about damages that jackals may cause to livestock 12 regions of Serbia. Sampling took place from dead ani- mean that their wildlife cycle may well interfere with mals brought at the laboratory of Department for Animal areas of natural human activity. Although further studies Ecology and Geography, Faculty of Biology, University of are required to discern the potential epidemiologic role Belgrade, by hunters. Data regarding location were of the golden jackal in spreading and transmitting the recorded. At necropsy, samples from different tissues above studied pathogens, local awareness of residents, were collected (spleen, liver, hart); spleen was chosen as veterinarians, and health professionals would prove of the tissue to work with. Samples were homogenised with great importance in order to efficiently monitor these micropastles (Eppendorf). DNA extraction took place and other zoonotic diseases. from a small portion of smashed tissue using the Gene Jet Genomic DNA Purification Kit, (Fermentas, Thermo Brucella Authors’ details Scientific). For the detection of species, a multi- 1Department for Animal Ecology and Geography, Faculty of Biology, plex Real-time PCR protocol was used, targeting the University of Belgrade, Serbia. 2Regional Laboratory of Public Health of Crete, bcsp31 alkB BMEI1162 Brucella Heraklion, Crete, Greece. 3Laboratory for medical Entomology, Institute for , and genes of species, 4 B. abortus B. melitensis B. canis medical Research, University of Belgrade, Serbia. Laboratory of Clinical and ; was detected using Bacteriology, Parasitology, Zoonoses and Geographical Medicine, University a PCR protocol targeting the omp2B gene. For the detec- of Crete, Heraklion, Crete, Greece. Leishmania tion of species a Real-time PCR protocol Published: 1 April 2014 targeting the SSU rRNA gene was used. A total of 216 spleen samples were tested. Most samples (196/90.7%) were collected during the hunting period doi:10.1186/1756-3305-7-S1-P19 (November-February). Of the samples tested, 108 animals Cite this article as: Ćirović et al.: Jackals as carriers of Leishmania and were male and 108 were female. Fifteen (6.9%) were posi- Brucella species in Serbia. Parasites & Vectors 2014 7(Suppl 1):P19. tive for Leishmania species, while four (1.9%) were positive for B. canis.

* Correspondence: [email protected] 3Laboratory for medical Entomology, Institute for medical Research, University of Belgrade, Serbia Full list of author information is available at the end of the article © 2014 Čirović et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. SECTION 2

Abstracts published only in the Abstract Book

Abstract Book

Q FEVER IN BELGIUM: PREVALENCE, MOLECULAR TYPING AND VACCINATION

Boarbi S, Michel P, Escobar L, Desqueper D, Bakinahe R, Marin M, Fretin D, Mori M

Veterinary and Agrochemical Research Centre, VAR-CODA-CERVA, Brussels, Belgium. Correspondence: [email protected]

Q fever is a bacterial zoonosis caused by Coxiella burnetii. Domestic ruminants (sheep and goats) are recognized as the main sources of human infection, and inhalation of contaminated aerosols as the primary route. Concerns with Q fever were brought back following a large outbreak occurring in the Netherlands between 2007 and 2011, which affected more than 4,000 people. Because of the C. burnetii high zoonotic potential and because Belgium borders the affected area, since 2009 a surveillance programme for Q fever was implemented in Belgium. Here we present results of this programme together with the effect of vaccination so as applied in the field setting and in a murine experimental model of infection. The goat herd prevalence calculated from analyses of bulk tank milks from December 2009 to March 2013 was estimated to range between 6.3 and 12.1%. We identified 3 single-nucleotide-polymorphism genotypes (1, 2 and 6) among the goat isolated strains corresponding to three main MLVA profiles. Genotype 1 strain (named CbNL01-like) was identical to the CbNL01 isolate, which was the emerging C. burnetii clone identified as responsible for the Dutch human Q fever cluster cases. Bacterial load in milk, indicative of the profile of shedding, was either continuous (type I) and associated to the CbNL01-like genotype or intermittent (type II) and linked to other genotypes. Vaccination of positive farms (mandatory for Belgian authorities since June 2011) with Coxevac® (prepared from the Nine Mile strain isolated from ticks, U.S.) has led to a reduction in shedding during the first year after vaccination but this effect was not maintained in the following months. These field observation raised question on the efficacy of the current vaccine. We have previously established a murine model of infection to study C. burnetii potential of infection in vivo. We used this model to study the efficacy of Coxevac (Nine mile strain isolated from tick, U.S.) together with vaccine derived from our field strains (bovine and goat). We evaluated the bacterial load in various organs (spleen, liver and lungs) and demonstrate that field strains were more protective than the current vaccine isolated from ticks. Whether our observation in mice could be extrapolated to goats will require further investigation.

The 1st Conference on Neglected Vectors and Vector-Borne Diseases with MC and WG Meetings of the COST Action TD1303 | 1

Abstract Book

RICKETTSIAL INFECTIONS IN HUMANS IN GREECE: SEARCHING FOR RARE CASES

Chochlakis D1, Germanakis A2, Kokkini S3, Makridaki E3, Tselentis Y1,3, Psaroulaki A3

1Regional Laboratory of Public Health of Crete, Heraklion, Crete, Greece; 2Department of Internal Medicine, Regional Hospital of Ag. Nikolaos, Agios Nikolaos, Crete, Greece; 3Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, University of Crete, Heraklion, Greece. Correspondence: [email protected]

Up until 2002 only Rickettsia typhi and R. conorii were considered to cause human infection in Greece and were routinely tested by IFA. The description of R. sibirica mongolotimonae and R. aeschlimanii in humans and of R. slovaca and R. massiliae in ticks, urged us to set up a protocol testing for another four Rickettsia species under the suspicion that other mis-/under-diagnosed cases existed. From September of 2011 we introduced the routine testing of R. slovaca, R. sibirica mongolotimonae, R. felis and R. massiliae, together with R. typhi and R. conorii, by IFA at the Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine (University of Crete), Reference Center for tick-borne diseases in Greece, in conjunction with the Regional Laboratory of Public Health of Crete. Until December 2013, 404 samples were tested for the above Rickettsia species. In seven cases a second sample was sent for confirmation. Only 66 whole blood samples were sent as well, which were tested for Rickettsia species by Real-time PCR targeting the gltA gene. Of the 404 tested samples, 67 were positive for IgG and/or IgM (29 for R. typhi, 23 for R. conorii, four for R. slovaca, seven for R. sibirica mongolotimonae, three for R. felis and one for R. massiliae). Eight samples were tested positive based on IgG only, 34 on IgM only and 25 on both IgG and IgM. Eight (8/66) whole blood samples were tested positive by Real-time PCR without antibody titers on their accompanying sera. Eleven samples fulfilling epidemiological (tick-bite, occupation with animals of veterinary importance) and clinical (fever, rash, eschar, increased transaminases) signs revealed no antibodies against the tested Rickettsia species. In the absence the above protocol, 15 human cases would have been missed. Since signs and symptoms are not generally specific, rickettsial infection does not constitute the first diagnosis of choice for most clinicians. Thereafter, samples (sera and whole blood) are tested days after the onset of symptoms and, in the vast majority of cases, following the administration of antibiotic therapy, minimizing therefore the possibility to detect antibodies of acute infection and/or a positive PCR. Serological methods may lag behind PCR amplification and culture, however various conditions may not always favor their implementation. Up until now, six Rickettsia species (R. slovaca, R. sibirica mongolotimonae, R. massiliae, R. typhi and R. conorii) had been described in Greece, either in ticks and/or in humans; perhaps many more are out there.

The 1st Conference on Neglected Vectors and Vector-Borne Diseases with MC and WG Meetings of the COST Action TD1303 | 2

Abstract Book

ASSESSEMENT OF SPATIAL VARIABILITY OF BORRELIA BURGDORFERI SENSU LATO USING THE 5S-23S RDNA SPACER REGION

Coipan EC1,2, Fonville M1, Tijsse-Klasen E1, van der Giessen JWB1, Takken W2, Sprong H1,2, Takumi K1

1Centre for Infectious Disease Control Netherlands, National Institute for Public Health and Environment (RIVM), Postbus 1, 3720 BA, Bilthoven, The Netherlands; 2Laboratory of Entomology, Wageningen University, P.O. Box 8031, 6700 EH, Wageningen, The Netherlands. Correspondence:[email protected]

Lyme borreliosis is the predominant tick-borne disease in the Northern hemisphere, with considerable heterogeneity in clinical manifestations. Here, we evaluated one genetic marker for its use in population genetic based analysis. For that, we collected molecular and epidemiological records of Borrelia burgdorferi sensu lato isolates from ticks, animals and humans at various sites in The Netherlands and worldwide. The 5S-23S rDNA (rrfA-rrlB) intergenic spacer region (IGS) from 291 Dutch Borrelia positive ticks was sequenced and compared to Borrelia sequences from GenBank. We estimated several population genetic measures to test the neutrality of the marker. We also assessed the ability of this marker to discriminate between Eurasian Borrelia’s at a finer geographical resolution, and to detect population expansion per genospecies. The most prevalent genospecies in The Netherlands was B. afzelii, whereas B. garinii, B. burgdorferi senso stricto, B. spielmanii and B. valaisiana were found less frequently. The result of the Ewens- Watterson-Slatkin test was consistent with neutral selection of IGS region. Estimated pairwise Fixation indices (Fst) were significantly different from zero between The Netherlands, the rest of Europe, Russia and Asia for B. afzelii and B. garinii. Estimated Fu’s Fs were significantly negative for B. afzelii and B. garinii. At least seven B. burgdorferi s.l. genospecies circulate in the Ixodes ricinus population in The Netherlands. The population genetic analyses of the IGS region can resolve subpopulations within a genospecies and detect a large excess of rare genetic variants at the genospecies level. A genetic trace of population expansion for B. afzelii and B. garinii is consistent with the reported increase in Lyme borreliosis incidence in European countries.

The 1st Conference on Neglected Vectors and Vector-Borne Diseases with MC and WG Meetings of the COST Action TD1303 | 3

Abstract Book

CONSIDERATIONS ON THE RECENT OUTBREAK OF LEISHMANIASIS IN SOUTHWESTERN MADRID, SPAIN

Cruz I1,2, Molina R1,3, Jiménez M1,3, Nieto J1,2, Chicharro C1,2, Llanes-Acevedo IP1,2, Carrillo E1,2, Moreno J1,2

1WHO Collaborating Centre for Leishmaniasis; 2Leishmaniasis and Chagas Disease Unit; 3Medical Entomology Unit. Servicio de Parasitología, Centro Nacional de Microbiología, Instituto de Salud Carlos III. Majadahonda, Madrid Spain. Correspondence: [email protected]

The epidemiology of leishmaniasis in Europe is dynamic. In the last decades HIV/AIDS epidemic caused a large increase of cases. Spain was particularly affected, accounting for most of them and peaking in 1996-1998. In recent years, there has been increased attention to the northward spread of leishmaniasis. The recently reported presence of sandfly vectors of Leishmania infantum and canine leishmaniasis cases in the northernmost area of Spain is an example. Early in the 2000s, WHO identified urbanization and environmental changes as important driving factors for the spread of zoonotic visceral leishmaniasis in Europe. In 2009, an urban outbreak of leishmaniasis occurred in the southwest of Madrid Autonomous Community, Spain. It caused 446 human cases up to December 2012, rising the incidence from 0.5 to 22.2 cases per 100,000 inhabitants. Certain features were present in this outbreak: i) all age groups were affected, and children younger than 14 years accounted for less than 15% of the cases; ii) no significant association with immunosuppression was found (this condition was present in only 15% of the cases); and iii) studies on different dog populations recorded seroprevalences below 4%. A raised awareness of the clinicians identified a high proportion of cutaneous (64.1%) versus visceral (35.9%) leishmaniasis. The investigation on alternative reservoirs found that 30% of hares from a nearby green park were infected by L. infantum. Entomological studies indicated a high density of Phlebotomus perniciosus in the outbreak area (143.8/m2) compared to the mean of Madrid region (30/m2); and direct xenodiagnosis proved the capability of infected hares to transmit the parasite to P. perniciosus. The same L. infantum genotype was common in the studied hares, sand flies, and most of the human cases. The epidemiological picture of this outbreak is consistent with focal transmission of the parasite in a population without previous, or little, immunity against Leishmania. The most likely explanation of its origin is the environmental changes which recently converted an agricultural land into a green recreation park adjacent to the urban area, which brought a wild transmission cycle into contact with people.

The 1st Conference on Neglected Vectors and Vector-Borne Diseases with MC and WG Meetings of the COST Action TD1303 | 4

Abstract Book

EXPANDING DIVERSITY OF BORRELIAE IN AFRICA – FORGOTTEN TO THE EMERGING

Cutler SJ

School of Health, Sport & Bioscience, University of East London, London, UK. Correspondence: [email protected]

Africa is home to a diverse range of Borrelial species and associated arthropod vectors. Classically spirochaete species attribution utilised tick vector species, however with the more recent advent of molecular approaches, we need to revisit correlation of spirochaetes with particular arthropod species. Early descriptions of Borrelia were of the louse-borne epidemic borreliosis, louse-borne relapsing fever (LBRF). Once worldwide, this now only persists in Africa where unlike the other borreliae, it is transmitted between humans via the clothing louse, Pediculus humanus humanus. Following this, description of other forms of relapsing fever, this time vectored by soft ticks were described notably following the voyages of Livingstone. The role of Ornithodoros ticks in transmission of B. duttonii in East Africa, and B. crocidurae in West Africa soon became apparent. Meanwhile, a recurrent febrile illness was described affecting cattle, this time associated with the bite of hard ticks (mixed pool of Amblyomma and Rhipicephalus (Boophilus) spp.). The causative Borrelia, B. theileri was described, but has attracted little subsequent research. Also of veterinary importance is the type species of this genus, B. anserina which like the relapsing fever borreliae, is transmitted by soft ticks and can cause overwhelming infestations and be associated with significant reduction of productivity among poultry and in consequence impact upon poverty among African agropastoralists. The discovery of the Lyme associated borreliae drew the world’s attention away from relapsing fever borreliae. Surprisingly, Lyme associated borreliae have only been described from North Africa. Application of molecular diagnostics has enabled us to revisit the diversity of African borreliae. Indeed, this has revealed that the epidemic LBRF Borrelia is a louse-adapted variant of the tick-borne B. duttonii. Furthermore, O. moubata tick vectors have been found to harbour an additional Borrelia, (B. mvumi). Whether this has pathogenic potential has yet to be established. B. crocidurae has been found to be a heterogeneous species comprised of several genotypes, though correlation with tick genotypes was not apparent. Descriptions of new borreliae have also been made, though their significance remains to be determined. These have been found in Amblyomma ticks from Senegal and from Carios spp. ticks and their preferred penguin hosts. In summary, our understanding of Borrelia and their vector associations has been largely neglected, but molecular diagnostics have furnished us with the tools to start to address these complex interactions and to decipher the transmission and potential impact of old and newly emerging borreliae.

The 1st Conference on Neglected Vectors and Vector-Borne Diseases with MC and WG Meetings of the COST Action TD1303 | 5

Abstract Book

RICKETTSIA HOOGSTRAALII-LIKE ORGANISMS IN SOFT TICKS

Cutler SJ

School of Health, Sport & Bioscience, University of East London, London, UK. Correspondence: [email protected]

Soft ticks belonging to Ornithodoros moubata collected from Tanzania and Argas persicus from Ethiopia, were both found to carry rickettsiae with 44 of 77 A. persicus pools (57%) being positive. Others have similarly assessed soft ticks from bats and seabirds, also reporting the presence of rickettsiae. Meanwhile, a highly similar species has been detected in European hard tick Haemaphysalis sulcata and the name Rickettsia hoogstraalii proposed, though this still remains a Candidatus species. Using shell vial tissue culture methods, researchers in Portugal have successfully obtained a closely related isolate from Ornithodoros erraticus ticks, and named this Rickettsia lusitaniae. Phylogenetic studies of this rickettsial complex clusters them within the spotted fever group of rickettsiae, however they cluster to the periphery of this group sitting closely with R. akari and R. felis. In an attempt to decipher the identity and phylogenetic relationship of this group of rickettsiae with other members of the genus, a range of phylogenetically established outer membrane proteins and house-keeping genes were amplified and sequenced. Collectively, these results show these strains to be highly related. A similar approach was used to characterise plasmid-borne genes (hsp2), however these grouped distantly from R. hoogstraalii in a separate clade. This intriguing finding could be an artefact resulting from the rather limited current database that exists for plasmid-borne rickettsial genes, or might be evidence of lateral gene transfer from other species. The possibility of lateral gene transfer among the rickettsiae has not been previously documented. These organisms have been believed to have little opportunity for such exchanges as a result of their obligate intracellular lifestyle. It must however be remembered that ticks serve as both reservoir and vector for these organisms whereby the Rickettsia is only one member of the milieu of tick microbial flora, thus might be in a permissive environment within the tick for lateral gene transfer.Whether this group of rickettsiae have any clinical relevance remains to be established. Interestingly, ticks carrying significant burdens of infection do not necessarily result in seroconversion in their vertebrate hosts as seen with the lack of detectable antibodies from which Amblyomma ticks carrying infection levels in excess of 90% with R. africae. Conversely, infection of ticks with one species of Rickettsia can exclude transmission of other rickettsial species. African populations generally have evidence of rickettsial exposure, but this could be a consequence of exposure to one of many species endemic to this area.

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Abstract Book

SCHMALLENBERG VIRUS MONITORING IN CULICOIDES TRAPPED DURING 2011 AND 2012 IN BELGIUM: A USEFUL TOOL TO STUDY EPIDEMIOLOGICAL ASPECTS RELATED TO THE SCHMALLENBERG VIRUS EMERGENCE AND INDICATIONS OF VECTOR COMPETENCE OF INDIGENOUS AVARITIA MIDGES.

De Regge N1, Deblauwe I2, Madder M2, De Deken R2, Fassotte C3, Losson B4, Cay B1

1Unit of Enzootic and (Re)merging Viral Diseases, CODA-CERVA, Brussel, Belgium; 2Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium; 3Plant Protection and Ecotoxicology Unit, Centre Wallon de Recherches Agronomiques, Gembloux, Belgium; 4Department of Infectious and Parasitic Diseases, Université de Liège, Liège, Belgium. Correspondence: [email protected]

Schmallenberg virus (SBV) was first identified in November 2011 by the FLI (Germany), and has since then been detected in many European countries. To examine the potential role of midges in the rapid expansion of the virus and SBV circulation in Belgium, midges caught with OVI traps from July to October 2011 and 2012 at 4 different regions in Belgium (Antwerp (north-east), Liège (east), Gembloux (centre), Libramont (south)) were screened by rRT-PCR (S segment, protocol FLI) for the presence of SBV. In 2011, pools consisting of heads (≤ 20/pool) of parous females morphologically identified upon species level were analyzed. Pools of the Obsoletus complex, C. dewulfi and C. chiopterus were found positive indicating that these species might play a role in the transmission and spread of SBV. The first SBV positive midges were found in August 2011 and depending on time and place, a high percentage (up to 30%) of pools were found positive. No positive pools were found in the south of Belgium, correlating with a low seroprevalence rate in sheep and cows at the end of the first vector season (end 2011) in that region. In 2012, pools of whole parous females (≤ 20/pool) identified at subgenus level (Avaritia, Culicoides, Monoculicoides) were screened. All pools from July remained negative while positive pools were found at every location in August. In the south (Libramont), more than 50% of the pools from August were SBV positive, indicating virus replication and spread in the (partially) non-protected host population. Interestingly, pools collected in September and October at all four locations tested negative, indicating a narrow timeframe of SBV circulation in 2012. Furthermore, a frequency analysis of the Ct values obtained for 31 SBV-S segment positive pools of Avaritia midges showed a clear bimodal distribution with peaks of Ct values between 21-24 and 33-36. This closely resembles the laboratory results obtained for SBV infection of C. sonorensis (Veronesi et al, 2013) and implicates indigenous midges belonging to the subgenus Avaritia as competent vectors for SBV. In conclusion, these studies allowed identifying several Culicoides species as putative vectors of the recently emerged Schmallenberg virus, confirmed the recirculation of the virus in Belgium in 2012, and provide strong indications of vector competence of indigenous Avaritia midges for Schmallenberg virus.

The 1st Conference on Neglected Vectors and Vector-Borne Diseases with MC and WG Meetings of the COST Action TD1303 | 7

Abstract Book

AN INTEGRATED GEOGRAPHICAL DATABASE ON TICKS AND TICK-BORNE ZOONOSES IN THE (SUB)TROPICS

Vesco U, De Meneghi D

Department of Veterinary Sciences, University of Turin, Grugliasco-Turin, Italy. Correspondence: [email protected]

Tick-borne zoonoses (TBZ) pose a serious public health threat in both Southern and Northern hemispheres. A geographical database on TBZ in the (sub)tropics was developed with the aim of producing updated TBZ distribution maps, constructing ecological niches models for vectors/zoonoses and drawing risk maps. The database (db) includes data on TBZ within the tropical and subtropical belt (35°S-35°N). Data/records have been collected by a multidisciplinary team through an extensive bibliographic search of scientific papers, conference proceedings, unpublished material, reports from national/international organisations and info from relevant experts. Data on pathogens, animal and human hosts and vectors were recorded. As spatial information is the core component of the db, each record is geo-referenced: geographical coordinates, when not provided by the authors, were extracted from web- gazetteers. Accuracy of such records varies depending on the administrative level of the location cited (e.g. village/district). As coordinate precision level is a key issue for modelling purposes, the db accounts for such uncertainties: a quantitative estimate of the mean error of village-level coordinates from gazetteer was carried out by comparing some hundred records of coordinates provided by the authors to those of the same locations obtained from gazetteer, thus resulting about 10 km. So far, from a total of 524 relevant publications reviewed and entered in the database, 705 geo-referenced TBZ records (out of 1,446) have been extracted. About 347 records are geo-referenced at village level, and 43 have coordinates provided by authors. Thanks to the participation of key collaborators from Latin America, the richest dataset is about SFG Rickettsia species in the Neotropics. A web site (http://www.tickbornezoonoses.org) was created to allow researchers/collaborators to have access (after registration) to data collected, to download a blank db copy for providing new data/info on TBZ to the web manager (via e-mail). After assessment of data consistency, the new data/info provided were uploaded on the webpage, and made freely available (after registration). In order to improve the efficacy/efficiency of data collection and management, the webpage is presently being completely re-designed, and updated. A web-form to feed data directly at the website level is being implemented; such interface, expected to be available soon, is meant to simplify and speed-up the process of data submitting, being much quicker than the system so far used, besides being a platform-independent approach. Funding: db was developed as a research activity under the Doctorate (PhD) in Veterinary Sciences, University of Turin, and facilitated through the ICTTD-3 project (6th FP UE).

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Abstract Book

IDENTIFICATION OF TICK GENES DIFFERENTIALLY EXPRESSED IN RESPONSE TO INFECTION TOWARDS VACCINE DEVELOPMENT AGAINST PARASITE DISEASES

Antunes S1, Merino O2, Mosqueda J3, Moreno-Cid JA2, Domingos A1,4, de la Fuente J2,5

1Instituto de Higiene e Medicina Tropical, Lisboa, Portugal; 2SaBio. Instituto de Investigación en Recursos Cinegéticos UCLM-JCCM, Ciudad Real, Spain; 3Facultad de Ciencias Naturales, Universidad Autónoma de Querétaro, Campus Juriquilla, Querétaro, Mexico; 4Centro de Malária e Outras Doenças Tropicais, Instituto de Higiene e Medicina Tropical, Lisboa, Portugal; 5Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, USA. Correspondence: [email protected]

Ticks while obligate hematophagous ectoparasites of animals and Man can be vectors of neglected human diseases. Tick-borne diseases are increasingly becoming an important public health issue. Babesiosis, caused Babesia species is one of the most important diseases vectored by ticks with a world-wide distribution affecting many species of mammals with a major economic impact on cattle industry. The objective of this study was to identify Rhipicephalus annulatus tick genes differentially expressed in response to infection with B. bigemina and evaluate its potential use in the development of a vaccine targeting both the vector and pathogen. We used a suppression subtractive hybridization library which allowed obtain differences in relative abundance of transcripts between populations retaining differentially expressed transcripts. Eight hundred randomly selected clones were obtained and further sequenced and analysed. Differentially expressed genes with putative functions in tick-pathogen interactions were selected for validation by real-time RT-PCR. Functional analysis of six genes was further performed by dsRNA-mediated RNAi showing that knockdown allowed to reduce pathogen infection levels, tick weight and oviposition, when compared to controls. According to gene knockdown results, genes were chosen based on tick phenotype and on their potential role on tick-pathogen interactions and their putative function and further expressed in E. coli recombinant system. Antibodies against those proteins were produced and their protection effect was evaluated by capillary feeding assays on infected and non-infected ticks. Our results indicate that the use of in vitro tick capillary feeding together with RNAi functional studies can be a useful tool to support antigens validation for in vivo vaccine trials. The efficacy of cattle vaccination with tick recombinant proteins was evaluated and we found a positive correlation between antigen-specific antibody titers and reduction of tick infestations and pathogen DNA levels strongly suggesting that the effect of the vaccine was the result of the antibody response in vaccinated cattle suggesting that these proteins can be included in a vaccine targeting both arthropods and multiplet pathogens greatly contributing as well for other vectors and parasites control. In order to obtain additional information, a transcriptomic analysis, using a Illumina platform was also conducted and data are being currently analysed.

The 1st Conference on Neglected Vectors and Vector-Borne Diseases with MC and WG Meetings of the COST Action TD1303 | 9

Abstract Book

IDENTIFICATION OF PHLEBOTOMINE SAND FLIES (DIPTERA: PSYCHODIDAE) BY MATRIX-ASSISTED LASER DESORPTION/IONIZATION TIME OF FLIGHT MASS SPECTROMETRY

Dvorak V1, Halada P2, Hlavackova K1, Dokianakis E3, Antoniou M3, Volf P1

1Department of Parasitology, Faculty of Science, Charles University in Prague, Prague, Czech Republic; 2Laboratory of Molecular Structure Characterization, Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic; 3Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, Faculty of Medicine, University of Crete, Crete, Greece. Correspondence: [email protected]

Phlebotomine sand flies transmit causative agents of several human and veterinary diseases, most importantly leishmaniases. To evaluate the significance of particular sand fly species in transmission cycle of a disease, their proper identification is profound. Since traditional morphological determination relies mainly on minute characteristics on their head and genitalia, which require laborious mounting and certain expertise and some populations can exhibit intermediate, there is a demand for novel rapid, simple and cost-effective molecular methods of species identification. Protein profiling using the matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) was established as a routine method to identify and classify organisms upon unique protein patterns. For the first time, this method was applied on phlebotomine sand flies originating from field collections and laboratory-reared colonies. Several species of subgenera (Phlebotomus, Paraphlebotomus, Larroussius, Adlerius) were used to evaluate the discriminatory power MALDI-TOF MS protein profiling. Various storage conditions and methods of samples preparation were tested on specimens of both sexes. Specimens of all analyzed sand fly species produced informative, reproducible and species-specific protein spectra that enabled their 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. Tested conditions of specimen storage and sample preparation give ground to a standard protocol that is generally applicable on analyzed sand fly specimens and represents a first step towards the establishment of a protein spectra database that would enable quick and reliable species identification of sand flies. The results demonstrate that species identification of sand flies by MALDI-TOF MS is feasible and gives a novel promising tool to improve biological and epidemiological studies on these medically important insects. Funding: This study was funded by EU grant FP7-261504 EDENext, Institutional Research Project of the Institute of Microbiology (RVO61388971) and Grant Agency of Charles University (GAUK 9108/2013). The research was conducted within the "Prague Infrastructure for Structure Biology and Metabolomics" which has been built up by financial support of the Operational Program Prague – Competitiveness (Project No.: CZ.2.16/3.1.00/24023).

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Abstract Book

DISCOVERING AND UNCOVERING PATHOGENS BY HIGH THROUGHPUT SEQUENCING: APPLICABILITY TO TICK BORNE DISEASES

Vayssier-Taussat M1, Moutailler S1, Dumarest M2, Sauvage V3, Cheval J3, Gratigny M3, Hébert C3, Gagnieur L2, Cabannes E3, Eloit M2, 3

1UMR Bipar ENVA, Anses, USC INRA, Maisons-Alfort, France; 2Institut Pasteur, Laboratory of Pathogen Discovery, Department of Virology, 28 rue du Docteur Roux, F-75724 Paris, France; 3PathoQuest, Bâtiment François Jacob, 28 rue du Dr Roux, 75015, Paris, France. Correspondence: [email protected]

Despite extensive microbiological investigations and the use of the most advanced molecular biology-based assays, a significant number of patients affected by vector-borne diseases remain without an etiological diagnosis. In Europe, ticks are among the most important arthropod vectors of disease agents to humans and domestic animals and the incidence of tick-borne diseases is increasing worldwide. New species, strains or genetic variants of tick-borne microorganisms are constantly being detected, resulting in an ever-increasing list of new pathogens infecting animals and humans. In Europe, the most prevalent tick-borne zoonoses are Lyme borreliosis and tick borne encephalitis, with over 65,000 and 30,000 cases respectively, each year rising concern to European public health. Patients bitten by ticks can also be infected by many other pathogens some of them being little known or unknown, and it is estimated that half of all human tick-borne disease has an unknown origin. In the recent years, unexplained syndromes occurring after tick bite has become a very important issue. Using High Throughput Sequencing, a powerful technique for discovering or uncovering pathogens, we tested tick samples from various areas in the world as well as samples from patients suffering for encephalitis that scored PCR negative against a large set of putative causative agents. We will present the procedures used for our analysis together with results of identification of infectious agents in samples from ticks and human patients. Briefly, we have identified known but unexpected bacterial species. We have also identified new or poorly known viral species within genus known to contain arboviruses: Bunyaviridae (Nairovirus and Phlebovirus); Rhabdoviridae (Vesiculovirus) and Reoviridae (Coltivirus).

The 1st Conference on Neglected Vectors and Vector-Borne Diseases with MC and WG Meetings of the COST Action TD1303 | 11

Abstract Book

ONCHOCERCA LUPI: CUTANEOUS DISTRIBUTION AND CIRCADIAN RHYTHM OF MICROFILARIAE IN DOGS

Giannelli A1, Dantas-Torres F1,2, Abramo F3, Ignjatović Ćupina A4, Petrić D4, Cardoso L5, Mutafchiev Y6, Cortes H7, Otranto D1

1Department of Veterinary Medicine, University of Bari, Bari, Italy; 2Department of Immunology, Aggeu Magalãhes Research Institute, Oswaldo Cruz Foundation, Recife, Brazil; 3Department of Animal Pathology, University of Pisa, Pisa, Italy; 4Faculty of Agriculture, University of Novi Sad, Novi Sad, Serbia; 5Department of Veterinary Sciences, University of Trás-os-Montes e Alto Douro, Vila Real, and Instituto de Biologia Molecular e Celular, Universidade do Porto, Oporto, Portugal; 6Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, Sofia, Bulgaria; 7Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Universidade de Évora, Évora, Portugal. Correspondence: [email protected]

Onchocerca lupi (Spirurida, Onchocercidae) is a neglected canine filarioid of increasing zoonotic concern. This nematode has been sporadically reported in symptomatic animals (wolves, dogs and cats) from Europe (Hungary, Greece, Germany and Portugal) and from the United States. In dogs, O. lupi may cause ocular alterations, ranging from non-specific conditions up to blindness, following the onset of subconjunctival granulomas encysting the adult worms. Nonetheless, canine onchocercosis may also undergo totally asymptomatic, with dogs playing as carriers and reservoirs of the infection, and increase the risk for the Public Health. Indeed, O. lupi has been only recently recognized as a zoonotic helminth infesting the human eye, with six cases reported worldwide. In order to enhance the knowledge on the biology of this filarioid, the distribution and periodicity of O. lupi microfilariae were assessed in naturally infested dogs. Skin samples were collected with a biopsy punch from animals without overt clinical signs. In particular, skin samples were collected from 18 anatomical regions of a necropsied animal (dog 1), while single biopsies were performed from the forehead, inter-scapular and lumbar regions of five dogs (dogs 2-6) at different time points (i.e., in the morning at 10h00, late afternoon at 18h00, and during the night at 23h00). The sediment of each sample was microscopically observed, microfilariae counted and their identity morphologically and molecularly confirmed through microscopy and sequencing, respectively. Most of microfilariae retrieved from dog 1 were observed in the head (i.e., ears, nose, and forehead) and inter-scapular regions. Accordingly, the overall mean number of microfilariae in dogs 2-6 was larger on the head (n = 122.8), followed by the inter-scapular (n = 119.0) and lumbar (n = 12.8) regions and increased in the afternoon sampling (n = 153.4), followed by night (n = 75.4) and morning (n = 25.8). Results indicate that O. lupi microfilariae tend to concentrate in specific body regions, being featured by a moderate circadian periodicity, and may be useful for diagnostic purposes. Indeed, veterinary practitioners living in endemic areas should be aware about the best body sites and period of the day for performing the skin biopsy. In addition, the pattern of microfilariae of O. lupi and their concentration in specific body regions is most likely a result of the co-evolution with the vector and may be useful to investigate its identity, which is still unknown.

The 1st Conference on Neglected Vectors and Vector-Borne Diseases with MC and WG Meetings of the COST Action TD1303 | 12

Abstract Book

CERCOPITHIFILARIA BAINAE: A LITTLE-KNOWN CANINE FILARIOID

Giannelli A1, Ramos RA1, Brianti E2, Dantas-Torres F1,3, Otranto D1

1Department of Veterinary Medicine, University of Bari, Bari, Italy; 2Dipartimento di Scienze Veterinarie, Università degli Studi di Messina, Messina, Italy; 3Department of Immunology, Aggeu Magalãhes Research Institute, Oswaldo Cruz Foundation, Recife, Brazil ;Correspondence: [email protected]

The genus Cercopithifilaria (Spirurida, Onchocercidae) includes 28 filarioid species, which parasitize mammals and are transmitted by hard ticks. Adults live in the subcutaneous tissues of the definitive vertebrate hosts and, after mating, females release dermal microfilariae, which are ingested by ticks (competent vectors) during the blood meal. Three Cercopithifilaria species have been reported in dogs, namely Cercopithifilaria grassii, Cercopithifilaria bainae and, a yet undescribed Cercopithifilaria sp. II. Although C. bainae is probably the best-known species, information about its pathogenic role and biology is still unclear. Cercopithifilaria bainae was reported for the first time in South America by two Brazilian researchers in 1984, but only recently it has been recognized as a widespread filarioid in Europe. Indeed, following the assessment of ticks belonging to the Rhipicephalus sanguineus group as competent vectors, this parasite has been diagnosed in several regions within the Mediterranean basin (Italy, Spain, Greece, and Portugal). The overall prevalence of the infection in the animal populations exposed to ticks may reach 21.6% and 45.5% in Spain, by microscopical examination and PCR, respectively. In addition, dogs may be co-infested by different Cercopithifilaria species. The examination of sediment after skin snip soaking is the gold standard for the diagnosis of these filarioids and should be performed on specific body regions of dogs in order to enhance the possibility to detect microfilariae. Indeed, microfilariae of C. bainae are unevenly distributed on the body, with higher frequencies on the interscapular region and on the head, which are also among the most common tick attachment sites. Although this filarioid is regarded as minimally pathogenic, the detection of dermal microfilariae provides evidence for a previous tick infestation and should stimulate practitioners investigate infections with other tick-borne pathogens. However, whether C. bainae infection adversely affects the dog’s immune response to other tick-borne pathogens, or vice versa, deserves to be studied.

The 1st Conference on Neglected Vectors and Vector-Borne Diseases with MC and WG Meetings of the COST Action TD1303 | 13

Abstract Book

CO-INFECTION ANALYSIS OF BARTONELLA VARIANTS IN WILD RODENTS AND THEIR FLEAS USING A 454-PYROSEQUENCING ASSAY

Gutiérrez R1, Morick D1, Cohen C2, Hawlena H2, Harrus S1

1Koret School of Veterinary Medicine, The Hebrew University of Jerusalem, Rehovot, Israel; 2Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel. Correspondence: [email protected]

Infection with more than one Bartonella species or variant is a known phenomenon in rodents and fleas. Due to the fastidious nature of these bacteria, the determination of co-existing Bartonella variants, their organization and distribution within rodents and fleas are challenging tasks. Moreover, an apparent incongruity has been reported between the Bartonella variants detected in rodents and those identified in fleas they hosted, through the use of common laboratory techniques. The aim of this study was to broadly characterize the composition of Bartonella infections in wild Gerbillus andersoni rodents and their hosted Synosternus cleopatrae fleas from the Negev desert, Israel. Rodents were captured twice during the study, four months apart (capture re-capture method). Blood samples and fleas were collected and processed from each gerbil captured at each collection period. Samples were initially screened for Bartonella-DNA by conventional PCR targeting a Bartonella-citrate synthase gene (gltA) fragment. Positive samples were further characterized using a next generation sequencing assay (454-pyrosequencing) targeting a gltA-sequence. The Bartonella infection composition was evaluated and compared across the samples. Results indicated a great diversity of co-infecting Bartonella variants. The bartonellae variants were distributed within rodent and flea samples in an expanded repertoire of infection compositions. Moreover, a filtering role of rodents was suggested by the results, favoring the dominance of certain variants. Our findings suggest that traditional diagnostic methods could bias the “real picture” of Bartonella infection structures in reservoir rodents and vectors, and co-infection represent a more common event than reported to date.

The 1st Conference on Neglected Vectors and Vector-Borne Diseases with MC and WG Meetings of the COST Action TD1303 | 14

Abstract Book

TRANSMISSION ROUTES OF BARTONELLA IN FLEAS AND JIRDS FROM ISRAEL

Harrus S1, Morick D1, Khokhlova IS2, Gutiérrez R1, Gottlieb Y1, Krasnov BR3

1Koret School of Veterinary Medicine, The Hebrew University of Jerusalem, Rehovot, Israel; 2Wyler Department of Dryland Agriculture, Ben-Gurion University of the Negev, Sede-Boqer, Israel. 3Mitrani Department of Desert Ecology, Ben-Gurion University of the Negev, Sede-Boqer, Israel. Correspondence: [email protected]

Bartonellae are small gram-negative bacteria, parasitizing mammalian erythrocytes and endothelial cells. They are transmitted by a wide range of blood-feeding arthropods. Rodents and fleas were found to have a high prevalence of Bartonella infection worldwide, with a high diversity of Bartonella spp., genotypes and strains. Although the high prevalence is noticed, the transmission routes of bartonellae are not fully understood. Bartonellae were shown to use diverse pathways to infect host populations by horizontal and/or vertical routes. Horizontal transmission of bartonellae by hematophagous vectors is well known. Vertical transmission of Bartonella species was also suggested to occur but its routes remain to be unveiled. In a series of studies, using Bartonella sp. OE 1-1, Meriones crassus jirds and Xenopsylla ramesis fleas, we showed that no transovarial transmission of Bartonella occurred in X. ramesis fleas, and that fleas feeding on Bartonella-positive jirds produced Bartonella-positive gut voids. In addition, we demonstrated that Bartonella-positive flea feces and gut voids are proper infection source for flea larvae, and that vertical non-transovarial transmission of Bartonella occurs in fleas. We also demonstrated that maternal transmission may occur in rodents however in a low efficiency. These studies broaden our understanding of Bartonella transmission routes in flea vectors and rodents and enlighten pathways of bartonellae transmission in the wild.

The 1st Conference on Neglected Vectors and Vector-Borne Diseases with MC and WG Meetings of the COST Action TD1303 | 15

Abstract Book

THE EFFECT OF TEMPERATURE ON LEISHMANIA DEVELOPMENT IN SAND FLIES

Hlavacova J1, Votypka J1, Volf P1

1Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic. Correspondence: [email protected]

Recent findings of new leishmaniases foci in Latin America and Europe urge for more data on the development of Leishmania in their vectors. In several countries in the New and Old World climate changes were suggested as an important factor affecting the spread of the disease. Ambient temperature is known to affect the metabolism of the sand fly vector, but little is known about its effect on Leishmania development in sand flies. In our study we therefore compared development of three important Leishmania species (L. infantum, L. braziliensis and L. peruviana) in two sand fly vectors (Phlebotomus perniciosus and Lutzomyia longipalpis) at different ambient temperatures. Sand fly females were membrane-fed on a suspension of Leishmania and rabbit blood, fed females were maintained at 20°C or 26°C. On various intervals post blood meal females were examined under a light microscope or used for DNA isolation and quantification of Leishmania by real-time PCR. To study defecation times, fully blood-fed females were individually placed in small glass vials, maintained at 20°C or 26°C and checked twice a day under a binocular microscope for defecation. Leishmania peruviana thrived well in L. longipalpis females kept at 20°C, while at 26°C almost 100% of infections were lost during the defecation. We studied the timing of L. longipalpis defecation and found that the lower temperature tested delayed defecation for about 3 days. This provides to Leishmania promastigotes more time to establish before defecation. Due to the fact that L. peruviana is a geographically-restricted mountain species, we suppose that it is adapted to the slower metabolism of sand flies living in lower ambient temperatures. On the contrary, L. braziliensis and L. infantum developed well in sand fly females kept at both temperatures tested. These results show the ability of L. infantum and L. braziliensis to thrive in sand flies at 20°C. The ability of L. infantum and L. braziliensis to develop heavy late-stage infections in sand flies even at ambient temperatures around 20°C could be an important factor enabling the spread of leishmaniases into new areas. Therefore, the risk of spread of these two Leishmania species to higher latitudes and altitudes should not be neglected. Funding: The study was partially supported by grants GAUK 426111/2011 and by EU grants 2011-261504 EDENext.

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MOLECULAR EVIDENCE FOR THE SPOTTED FEVER GROUP RICKETTSIAE AND OTHER ZOONOTIC PATHOGENS IN TICKS (ACARI: IXODIDAE) FROM ROMANIA

Ionita M1, Mitrea IL1, Pfister K2, Hamel D2, Silaghi C2

1Department of Parasitology and Parasitic Diseases, Faculty of Veterinary Medicine, University of Agronomical Sciences and Veterinary Medicine of Bucharest, Romania; 2Comparative Tropical Medicine and Parasitology, Ludwig- Maximilians-Universität München, Germany. Correspondence: [email protected]

Ticks serve as vectors for many pathogens of veterinary but also zoonotic importance. So far, there is limited information on the occurrence of tick borne pathogens in Romania. Therefore, the aim of the present study was to provide a preliminary insight into the diversity of tick-borne pathogens circulating at the domestic host–tick interface in Romania, using molecular techniques. A total of 382 ticks were analyzed, including questing (n=40) and feeding (n=342) adult ticks collected from naturally infested cattle, sheep, goats, horses or dogs, in eight different geographic locations in Romania. Tick species were identified as Hyalomma marginatum (n=135), Ixodes ricinus (n=134), Rhipicephalus sanguineus (n=50), Dermacentor marginatus (n=48), and Rhipicephalus bursa (n=15. All ticks were screened for Rickettsia spp. and Babesia spp. with conventional PCR followed by sequencing. I. ricinus ticks were additionally screened for Anaplasma phagocytophilum and Borrelia spp., and R. sanguineus for Ehrlichia canis and Anaplasma platys, all with real-time PCR. Four rickettsiae of the spotted fever group of zoonotic concern were identified: Rickettsia monacensis and Rickettsia helvetica in I. ricinus, and Rickettsia slovaca and Rickettsia raoultii in D. marginatus. Other zoonotic pathogens such as A. phagocytophilum, Borrelia afzelii, and Babesia microti were found in I. ricinus. Pathogens of veterinary importance were also identified, including Theileria equi in H. marginatum, Babesia occultans in D. marginatus and H. marginatum, Theileria orientalis/sergenti/buffeli-group in I. ricinus and in H. marginatum and E. canis in R. sanguineus. These findings show a wide distribution of very diverse bacterial and protozoan pathogens in the Romanian tick fauna, with the potential of causing both human and animal diseases. All this data emphasize the need for further in-depth study in both ticks and animals.

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Abstract Book

CLIMATE AND ENVIRONMENTAL CHANGE DRIVES IXODES RICINUS GEOGRAPHICAL EXPANSION AT THE NORTHERN RANGE MARGIN

Jore S1, Vanwambeke SO2, Viljugrein H1,3, Isaksen K4, Kristoffersen AB1,5, Woldehiwet Z6, Johansen B7, Brun E1, Brun-Hansen H 8, Westermann S9, Larsen IL1, Ytrehus B1, Hofshagen M1

1Norwegian Veterinary Institute, Ullevålsveien 68, P.O.Box 750 Sentrum 0106 Oslo, Norway; 2Georges Lemaître Centre for Earth and Climate Research, Earth & Life Institute, Université Catholique de Louvain, Place Louis Pasteur 3, B1348 Louvain-la-Neuve, Belgium; 3Centre for Ecological and Evolutionary Synthesis (CEES), Department of Bioscience, University of Oslo, P.O.Box 1066, Blindern, 0316 Oslo, Norway; 4The Norwegian Meteorological Institute, Research and Development Department, Division for Model and Climate Analysis, P.O.Box 43 Blindern, 0313 Oslo, Norway; 5Department of Informatics, University of Oslo, P.O.Box 1080, Blindern, 0316 Oslo, Norway; 6Department of Infection Biology, Institute of Infection & Global Health, University of Liverpool, Leahurst Campus, Chester High Road, Neston, Wirral CH64 7TE, United Kingdom; 7Northern Research Institute, P.o.b 6434 Forskningsparken, 9294 Tromsø, Norway; 8Norwegian School of Veterinary Science, Ullevålsveien 72, P.O.Box 8146 Dep., 0033 Oslo, Norway; 9Department of Geosciences, University of Oslo, P.O.Box 1066, Blindern, 0316 Oslo, Norway. Correspondence: [email protected]

Global environmental change is causing spatial and temporal shifts in the distribution of species and the associated diseases of humans, domesticated animals and wildlife. In the on-going debate on the influence of climate change on vectors and vector-borne diseases, there is a lack of a comprehensive interdisciplinary multi-factorial approach utilizing high quality spatial and temporal data. We explored biotic and abiotic factors associated with the latitudinal and altitudinal shifts in the distribution of Ixodes ricinus observed during the last three decades in Norway using antibodies against Anaplasma phagocytophilum in sheep as indicators for tick presence. Samples obtained from 2963 sheep from 90 farms in 3 ecologically different districts during 1978 – 2008 were analysed. We modelled the presence of antibodies against A. phagocytophilum to climatic-, environmental and demographic variables, and abundance of wild cervids and domestic animals, using mixed effect logistic regressions. Significant predictors were large diurnal fluctuations in ground surface temperature, spring precipitation, duration of snow cover, abundance of red deer and farm animals and bush encroachment/ecotones. The length of the growth season, mean temperature and the abundance of roe deer were not significant in the model. Our results highlight the need to consider climatic variables year-round to disentangle important seasonal variation, climatic threshold changes, climate variability and to consider the broader environmental change, including abiotic and biotic factors. The results offer novel insight in how tick and tick-borne disease distribution might be modified by future climate and environmental change.

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HUMAN DIROFILARIASIS (DIROFILARIA REPENS): IS LATVIA A NEW TERRITORY?

Kirjušina M1, Krūmiņa A2

1Laboratory of Parasitology and Histology, Institute of Systematic Biology, Daugavpils University, Daugavpils. Latvia; 2Department of Infectology and Dermatology, Medical Faculty, Riga Stradiņš University, Riga, Latvia. Correspondence: [email protected]

Global warming and climate change are usually discussed as a reason of the spread of organisms to new territories where they had never been registered before. This phenomenon has become a recurrent topic in parasite distribution, especially for human zoonotic parasites (e.g. Dirofilaria repens). The question of primordial parasite distribution is still open due to continued scientific debate on the impact of climate change on the distribution of hosts. The definitive hosts of Dirofilaria are dogs which have lived in proximity with humans for a long time. Parasite transmission occurs by mosquitoes of genera Anopheles (4 species), Aedes (19) and Culex (3), serving as vectors and intermediate hosts. Several potential vector species are widespread throughout the territory of Latvia. The infection of Dirofilaria spp. has been recorded in dogs since 2008. Thereby, through mosquito bite, dirofilariasis can be transmitted from dog to humans. There were at least six clinical cases of dirofilariasis in humans between 2009 and 2014 in urban territories of Latvia. For clinicians it is complicated to pronounce the differential diagnosis because filariasis can manifest itself in a variety of acute clinical, subclinical conditions and chronic sequelae. Diagnostics of dirofilariasis in humans is difficult due to the absence of microfilariae in blood. The clinical symptomatology of the disease is unspecific and the diagnosis is based on parasite species determination by morphological characteristics (in all six cases – D. repens) of the discharged parasite. Out of the six patients, granuloma and lymphadenitis were observed in three of them; clinically severe arthritis and nephritis for the other two. As for the last patient, it was one of the most complicated cases of human dirofilariasis on the territory of Latvia caused by D. repens as this patient developed unusual and severe clinical manifestations that mimicked different pathological conditions, such as Crohn disease, deep venous thrombosis, arthritis, weakness and depression. However, a more in-depth epidemiological and parasitological research is necessary to establish how dirofilariasis came on the territory of Latvia.

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Abstract Book

SEARCHING FOR GENERAL RULES IN THE ECOLOGY OF ARTHROPOD ECTOPARASITES

Krasnov BR

Mitrani Department of Desert Ecology, Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Sede-Boqer Campus, 84990 Midreshet Ben-Gurion, Israel. Correspondence: [email protected]

Any scientific study revealing some patterns or processes still should answer the question: how general are these patterns and processes? The findings of a particular study should invariably be validated by studies in other geographic locations or on other taxa. Here, I present comparison of several studies that examined various ecological patterns of different taxa of arthropod ectoparasites (fleas, gamasid mites and ixodid ticks) exploiting small mammals as well as a few studies of helminths parasitic on fish and carried out in different geographic regions (Middle East, Central Europe, South Africa, Western Siberia, Canada, Argentina). I consider ecological patterns at three hierarchical levels (parasite populations, parasite communities and host-parasite networks. In particular, I present the following questions. At ectoparasite population level, I will ask: (1) How are ectoparasites distributed among their hosts? (2) Is abundance a true species attribute of an ectoparasite species? and (3) May prevalence of an ectoparasite be predicted from abundance data? At ectoparasite community level, I will ask: (1) Is diversity of ectoparasites true species attributes of a host species? (2) What are host characters that affect the diversity of their ectoparasite communities; and (3) Do ectoparasite species co-occur on the same host individual more or less often than expected by chance? At network level, I will consider both ecto- and endoparasites (due to lack of studies in ectoparasite taxa other than fleas and ask: (1) Is distribution of specialization in parasite-host networks random, symmetric or asymmetric?; and (2) Does phylogeny drive architecture of a parasite-host network? I conclude that general laws apply well to the lowest (populations) and the highest (networks) levels of ectoparasite ecological studies, while most patterns observed at the parasite community level are highly contingent and far from universal.

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A RETROSPECTIVE SEROLOGICAL SURVEY ON HUMAN BABESIOSIS IN BELGIUM

Lempereur L1,2, Shiels B2, Heyman P3, Moreau E4, Saegerman C5, Losson B1, Malandrin L4

1Laboratory of Parasitology and Parasitic Diseases, Department of Infectious and Parasitic Diseases, Faculty of Veterinary Medicine, University of Liège, B-4000 Liège, Belgium; 2Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary & Life Sciences, University of Glasgow, 464 Bearsden Road, Glasgow G61 1QH, Scotland; 3Research Laboratory for Vector-borne Diseases, Queen Astrid Military Hospital, B- 1120 Brussels, Belgium; 4INRA, UMR BioEpAR1300, Biology, Epidemiology and Risk Analysis, F-44307 Nantes (France). Oniris, UMR BioEpAR1300, Biology, Epidemiology and Risk Analysis, F-44307 Nantes, France; 5Research Unit in Epidemiology and Risk Analysis Applied to the Veterinary Sciences (UREAR-ULg), Department of Infectious and Parasitic Diseases, Faculty of Veterinary Medicine, University of Liège, B-4000 Liège, Belgium. Correspondence: [email protected]

Babesiosis is a tick-borne disease caused by different species of intraerythrocytic protozoa classified within the genus Babesia. Interest in Babesia species with the potential to cause zoonotic disease has increased recently. In humans, clinical symptoms that develop early or during a mild Babesia sp. infection can be confused with malaria, showing high fever, headaches and myalgia. In acute clinical infection, anaemia, icterus and haemoglobinuria are conspicuous and this condition can be fatal. In Europe, most clinical babesiosis cases have been attributed to Babesia divergens and Babesia sp. EU1. Babesia microti occurs mainly in the USA although a case of autochthonous B. microti infection and serological evidence of human infections in different European countries have been reported. In Belgium, B. divergens is known to be present in cattle in the south of the country, but the disease is considered to be absent from other parts of the country. Because of the recent first report of potentially zoonotic Babesia species in the Ixodes ricinus tick together with an older report of a human clinical case in Belgium, a retrospective serological survey was performed. IFAT was used to screen 200 sera from anonymous Belgian patients with history of tick bite and clinical symptoms compatible with a tick-borne disease. The serological screening detected positive reactivity in 9% (18), 38.5% (77) and 42% (84) of the samples against B. microti, B. divergens or Babesia sp. EU1, respectively. Presence of the three potentially zoonotic species of Babesia has been confirmed together with strong evidence for infection of humans reported in Belgium. Preventive actions, such as improved awareness of physicians and development of better diagnostic tools should be helpful to prevent clinical cases and assess the true risk to public health.

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TICK SPECIES COLLECTED FROM SHEEP AND GOATS FROM NORTHERN GREECE

Papadopoulos E1, Farkas R2

1School of Veterinary Medicine, Aristotle University of Thessaloniki, Greece; 2Faculty of Veterinary Science, Szent Istvan University, Budapest, Hungary. Correspondence: [email protected]

Ticks are important disease vectors transmitting a range of pathogens to livestock and in heavy infestations can cause anaemia and local injury, which may predispose to secondary bacterial infection and myiasis. In certain cases, ticks may infest humans resulting to significant problems. The aim of our study was to collect ticks from sheep and goats from Northern Greece in order to identify them and investigate their potential vector capacity. Towards this end, ticks were collected from a total of 224 animals (91 sheep and 133 goats) coming from 21 infested sheep flocks (7) and goat herds (14). All animals were kept under the semi extensive system grazing or browsing lowland or mountainous areas of Northern Greece, varying from 18 to 760 m from sea level. The size of the flocks/herds varied from 60 to 800 head. None of them received any recent acaricide treatment. Each animal was individually inspected to collect all the ticks into a vial containing ethanol. Also, from each animal blood samples were collected, with and without anticoagulant. A total of 662 ticks were collected, 365 male and 297 female. Ticks were identified morphologically to be Rhipicephalus bursa 92.9%, Ixodes ricinus 5.1%, Hyalomma excavatum 1.5% and H. marginatum 0.5%. Results indicate that R. bursa is the most common tick species infesting small ruminants in Northern Greece. This tick is able to transmit important pathogens, including Babesia spp., Theileria spp., Anaplasma spp. Ehrlichia spp. and it has been associated with other diseases like tick paralysis and Crimean-Congo haemorrhagic fever. Finally, samples were tested using PCR to detect piroplasms, which are being sequenced.

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ERADICATION OF HEAVY FLEA INFESTATION IN A DAIRY SHEEP FARM

Papadopoulos E, Diakou A, Kodonas A

School of Veterinary Medicine, Aristotle University of Thessaloniki, Greece. Correspondence: [email protected]

Fleas pose one of the most significant problems of the small ruminant farms during the last years in Greece, since they attack both animals and humans. In all the cases of severe infestation, fleas cause extreme nuisance, anaemia, mortality, morbidity and disease transfer. Animal production and welfare are largely lowered. Same time, farmers or animal personnel suffer from irritation, allergic reactions and discomfort due to the flea bites. Control is difficult, because fleas spend much time off the host. Furthermore, insecticide residues in milk are a restraining factor. In most cases, flea control is not fully achieved or lasts very short periods, due to the incomplete eradication, cost and residues constrains. Herein is presented one case of successful long term flea eradication in a dairy sheep farm. The farm had 2000 head of dairy breeds (Lacaune, Assaf, Friesland and their cross-breeds) kept under the intensive management system. Animals were heavily infested with fleas presenting severe clinical signs. The fleas were identified as Pulex irritans (81.7%) and Ctenocephalides felis (18.3%). The control included: i) pour on administration of deltamethrin (Butox®, MSD) individually on all animals, ii) manure removal from the indoor animal area and thorough cleaning and iii) spraying the premises with pyriproxyfen, an Insect Growth Regulator, combined with deltamethrin, repeated twice per week. The dogs of the farm were also treated against ectoparasites. The great advantage of deltamethrin (Butox®, MSD) is the very short withdrawal period in milk (12 hours) making the treatment practically possible at any time of animal production. The farm is being monitored for the last 8 months and no flea or any kind of relevant clinical sign is present. However, the above control scheme will be repeated every year in order to maintain the successful eradication. This report provides useful information as very limited data exist in the scientific literature regarding effective flea control on livestock.

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RODENT-ASSOCIATED BARTONELLA SPECIES IN FLEAS AND THEIR SMALL MAMMAL HOSTS IN THE METROPOLITAN AREA OF LEIPZIG, GERMANY

Silaghi C1, Woll D2, Kiefer D1, Kiefer M3, Pfister K1, Pfeffer M2

1Comparative Tropical Medicine and Parasitology, Ludwig-Maximilians-Universität München, Munich, Germany; 2Institute for Animal Hygiene and Veterinary Public Health, University of Leipzig, Germany; 3Bavarian State Collection of Zoology, Munich, Germany. Correspondence: [email protected]

Bartonella spp. are facultative intracellular bacteria infecting erythrocytes and endothelial cells of their hosts. The life cycle of these zoonotic pathogens includes different reservoir hosts such as small mammals and hematophagous arthropods such as fleas acting as vectors. Several rodent-associated Bartonella species have been linked to human disease, for example, B. grahamii and B. elizabethae. As data on the occurrence of Bartonella species in small mammals is scarce in Germany, DNA-extracts of spleen samples of 79 small mammals, mainly bank voles (Myodes glareolus) and yellow-necked mice (Apodemus flavicollis), and their fleas (n=135; from 43 individuals) from the metropolitan area of Leipzig, Germany, were screened with a conventional PCR for Bartonella spp. followed by sequencing. Overall, more than 65% of the small mammals contained DNA of Bartonella: B. grahamii, B. taylorii, B. doshiae and DNA similar to some other Bartonella species. Double infections in individual animals were also detected. A total of five flea species was found on the small mammals: Megabothris turbidus, M. walkerii, Ctenocephalus agyrtes, Doratopsylla dasycnema, and Nosopsylla fasciatus. Nearly 60% of the fleas were positive for Bartonella spp. Sequencing revealed the Bartonella species B. taylorii, B. grahamii, B. doshiae, B. elizabethae and Bartonella spp. The public health impact of this finding of zoonotic Bartonella species in an urban as well as recreational area requires further investigation.

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Abstract Book

TORTOISE TICKS AS CARRIERS OF CRIMEAN-CONGO HAEMORRHAGIC FEVER VIRUS

Široký P1,2, Bělohlávek T1, Papoušek I1, Jandzik D3,4, Mikulíček P3, Kautman M1, Zdražilová- Dubská L5

1Department of Biology and Wildlife Diseases, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences, Palackého tř. 1/3, 612 42 Brno, Czech Republic; 2CEITEC-Central European Institute of Technology, University of Veterinary and Pharmaceutical Sciences Brno, Palackého tř. 1/3, 612 42 Brno, Czech Republic; 3Department of Zoology, Comenius University in Bratislava, Mlynská dolina B-1, 84215 Bratislava, Slovakia; 4Department of Ecology and Evolutionary Biology (EBIO), University of Colorado, Boulder, Ramaley N122, Campus Box 334, Boulder, CO 80309-0334, USA; 5Department od Laboratory Medicine, Masaryk Memorial Cancer Institute, Žlutý kopec 7, 65653 Brno, Czech Republic. Correspondence: [email protected]

Crimean-Congo haemorrhagic fever (CCHF) possesses the largest geographical area among medically important tick- borne viral zoonotic diseases. Small mammals are considered to be usual reservoirs in endemic areas, while migratory birds can spread CCHF virus (CCHFV) over long distances. Ticks of the genus Hyalomma, particularly H. marginatum, are considered principal vectors of CCHFV. Hyalomma aegyptium, a three-host tick species, represents a single exception within the genus Hyalomma, since the adults are host-specific, feeding on tortoises of the genus Testudo. Nevertheless, larvae and nymphs are less host-specific, commonly infesting various reptiles, small mammals and birds, thus having potential for transmitting pathogenic agent among various vertebrate groups. However, ticks parasitizing reptiles have traditionally been considered epidemiologically less important, and as a result they have been understudied and their vector capability has often been overlooked. To check importance of H. aegyptium in epidemiology of CCHF, we took an advantage of previous sampling of this tick species from areas in southern Turkey and in north-western Syria – a region so far not screened for CCHFV. We have analyzed 245 adult H. aegyptium collected from 38 spur- thighed tortoises (Testudo graeca) during field trips carried out between April 2005 and June 2007. RNA isolation and RT-PCR amplifying S-segment of CCHFV followed the standard procedures with following modifications: decreased total volume of 25 µl for each of the two PCR rounds, reverse transcription for 60 min and increased annealing temperature at 47 °C. We sequenced 22 of the obtained PCR products. All newly produced sequences were identical differing in a single nucleotide from CCHFV strain IbAr10200 from Nigeria. Bayesian inference and maximum likelihood analysis also confirmed that our isolates belong to the African clade. We have revealed 30.2% (74/245) prevalence of CCHFV in studied H. aegyptium. Despite these results, we cannot confirm right now whether tortoises serve as a reservoir of CCHFV or only can carry infected ticks. Funding: This study was supported by the project “CEITEC – Central European Institute of Technology” (CZ.1.05/1.1.00/02.0068) from the European Regional Development Fund.

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MONITORING OF THE MOSQUITOES IN FYR-MACEDONIA IN THE PAST AND CURRENT ACTIVITIES

Sokolovska N1, Lazarevska S2, Arsenievski Z1

1P.H.O.Center for public health-Skopje, R. Macedonia; 2Faculty of Agricultural Sciences and Food-University "Ss.Cyril and Methodius" - Skopje-R.M. Correspondence: [email protected]

With the global warming and climate changes, R. Macedonia with its geographical location and mild climate is facing the threat of a possible outbreak of already eradicated tropical and subtropical diseases (malaria), which are vector- transmitted (mosquitoes). In December 19th 1973, the WHO has issued a certificate to Yugoslavia (with R. Macedonia as part of that country) for a malaria eradication achieved by working together. The importance of monitoring mosquito was one of the key points in the process. Since 1970 continuous entomological research was done, with the following species discovered: Anopheles maculipennis var. typicus, A. superpictus, A. saharovi. In the latest monitoring from 2010 that was initiated and financially supported by the WHO-Office in Skopje the following 7 species were identified: Anopheles maculipennis, Ochlerotatus hungaricus, O. geniculatus, Culex mimeticus, C. triteniorhynchus, C. martinii, Orthopodomyia pulchripalpis. These results were not verified. After the eradication of malaria the monitoring process took another direction. The success continues and it is to be maintained. Our view is that the necessary entomological exploration (May to September) is needed to acknowledge the qualitative and quantitative aspects of the mosquitoes, with a special emphasis on the access points to avoid the possible import of invasive species from neighboring countries. Entomological research will initially be carried out by setting the bait for mosquitoes in the Skopje area, with ambitions to further expand research across R. Macedonia including the entry points.

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Abstract Book

CIRCULATION OF ANAPLASMA PHAGOCYTOPHILUM ECOTYPES IN TICKS AND WILDLIFE

Jahfari S1, Coipan EC1,6, Fonville M1, van Leeuwen AD1, Hengeveld P1, Heylen D2, Heyman P3, Tack W4, Rijks JM5, Takken W6, van der Giessen J1, Takumi K1 , Sprong H1,6

1Laboratory for Zoonoses and Environmental Microbiology, National Institute for Public Health and Environment (RIVM), Antonie van Leeuwenhoeklaan 9, P.O. Box 1, Bilthoven; 2 Ecology Group, University of Antwerp, Antwerpen, Belgium; 3 Research Laboratory for Vector Borne Diseases, Queen Astrid Military Hospital, Brussels, Belgium; 4 Laboratory of Forestry, Department of Forest and Water Management, Ghent University, Ghent, Belgium; 5Dutch Wildlife Health Centre, Utrecht university, Utrecht, the Netherlands; 6Laboratory of Entomology, Wageningen University, Wageningen, the Netherlands. Correspondence: [email protected]

Anaplasma phagocytophilum is the etiological agent of granulocytic anaplasmosis in humans and animals. Wild animals and ticks play key roles in the enzootic cycles of the pathogen. Potential ecotypes of Anaplasma phagocytophilum have been characterized genetically. The host range, zoonotic potential and demography of these ecotypes in the Netherlands and Belgium were investigated. The presence of Anaplasma phagocytophilum DNA was determined in more than 5500 ixodid ticks collected from the vegetation and wildlife, as well as in 264 spleen samples from wildlife, originating from various geographic locations. Infection rates and their confidence intervals were estimated using Fisher's exact tests. A fragment of the groEL-gene was amplified and sequenced from the qPCR-positive lysates. Additional groEL sequences of ticks and animals in other European countries were obtained from GenBank, all known human cases were obtained through a literature search. Statistical analyses were performed to identify ecotypes and to investigate their host range and zoonotic potential. The demographic expansion of Anaplasma ecotypes was investigated using population genetics analyses. DNA of Anaplasma phagocytophilum was present in all stages of questing and feeding Ixodes ricinus, host- derived I. hexagonus and one female I. trianguliceps, but was absent in I. arboricola, and Dermacentor reticulatus. DNA of Anaplasma phagocytophilum was present in engorged ticks, deer keds and spleens from large wild vertebrates, including roe deer, foxes, wild boar and sheep, but almost completely absent from rodents, lizards and birds. Three Anaplasma phagocytophilum ecotypes were identified, that had different host ranges. All sequences of human cases belonged only to one of these ecotypes. Based on population genetic parameters, the potentially zoonotic ecotype showed significant demographic expansion. Three ecotypes of Anaplasma phagocytophilum with enzootic cycles were identified in the Netherlands and Belgium. The zoonotic ecotype has the broadest range of wildlife hosts. The demographic expansion of the zoonotic Anaplasma phagocytophilum ecotype is indicative for a recent increase of the acarological risk of exposure of humans and animals.

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BABESIA BIGEMINA AND ANAPLASMA MARGINALE IN DOMESTIC RUMINANTS IN SLOVENIA – PRELIMINARY RESULTS

Starič J, Ježek J, Klinkon M, Nemec M

Clinic for ruminants, Veterinary faculty, University of Ljubljana, Ljubljana, Slovenia. Correspondence: [email protected]

Diseases of domestic ruminants associated with haemotropic microorganism are prevalent in certain areas of Slovenia. Especially babesiosis in cattle is also clinically diagnosed frequently and successfully treated. Babesia divergens in cattle was also confirmed by laboratory methods. Anaplasma phagocytophilum was confirmed in many game species in Slovenia in a recent study. It looks like the disease is widespread among game. So far no studies about it prevalence were performed in domestic ruminants. Babesia bigemina, Anaplasma marginale and Theileria spp. have also not been studied in domestic ruminants yet. The objective of this study was to investigate if Babesia bigemina and Anaplasma marginale are present in domestic ruminants in Slovenia. Sixteen domestic ruminant herds that graze during pasture season and with history of tick infestations from different areas of Slovenia were included in the study. Grazing sheep, goats and cattle were serologically checked for specific antibodies against Babesia bigemina and Anaplasma marginale with ELISA test (Svanova, Biotech AB, Sweden). Additionally blood smears stained corresponding to Pappenheim (Hemacolor, Merck, Germany) were microscopically examined in seropositive and suspicious animals. All small ruminant samples (38 sheep and 39 goats) were serologically negative for both pathogens. Two (5%) out of 40 cattle samples were positive on A. marginale. Nine (22.5%) reacted positively to B. bigemina and 12 (30%) with suspicious reaction. Additionally inclusions suspicious for B. bigemina in erythrocytes were noted in 10 seropositive and suspicious animals and one of A. marginale. High prevalence of both pathogens in cattle was surprising to us. Further, confirmed high prevalence of A. phagocytophilum in game warrants more detailed investigation of haemotropic microorganism in domestic ruminants. It appears that diseases caused by haemotropic microorganism in domestic ruminants could be underdiagnosed and could cause much more loses than we anticipate in Slovenia.

The 1st Conference on Neglected Vectors and Vector-Borne Diseases with MC and WG Meetings of the COST Action TD1303 | 28

Abstract Book

RICKETTSIA AESCHLIMANNII IN HYALOMMA TICKS COLLECTED FROM HERDS IN THE SOMALI REGION OF ETHIOPIA AND IN BURKINA FASO.

Tomassone L1, Grego E1, Chiavassa E1, Adakal H2, Rodighiero P3,4, Pressi G3,4, Callà G1, Gebre S5, Zeleke B4, De Meneghi D1

1Dipartimento di Scienze Veterinarie, University of Torino, Grugliasco, Italy; 2Centre International de Recherche- Développement sur l’Elevage en zone Subhumide, Bobo Dioulasso, Burkina Faso; 3AVEC PVS, www.avec-pvs.org, Torino, Italy; 4Comitato di Collaborazione Medica, Torino, Italy; 5National Animal Health Diagnostic and Investigation Center, Sebeta, Ethiopia. Correspondence: [email protected]

Tick-borne rickettsiae are emerging pathogens belonging to the Spotted Fever Group. Among them, Rickettsia aeschlimannii is characterized by a broad geographical distribution. Indeed, it was detected in Hyalomma, Rhipicephalus and Haemaphysalis ticks from southern Europe, north and sub-Saharan Africa, southwestern Russia. R. aeschlimannii-like strains were reported from South America. Recently, it was also detected in Germany, in a tick feeding on a migratory bird. Ticks dissemination through migratory birds can indeed be a cause of the heterogeneous distribution of this pathogen. R. aeschlimannii causes symptoms similar to Mediterranean Spotted Fever, which have been reported so far in patients in Africa or travelling from Africa. In the framework of two development/research projects in Ethiopia and Burkina Faso, we developed a quantitative PCR assay to detect R. aeschlimannii OmpA gene. This rickettsia was detected in Hyalomma rufipes, H. truncatum and H. impeltatum randomly collected from nomadic herds (cattle, sheep, goats and camels) in villages located in Filtu and Dollo districts, Libaan zone, Somali region of Ethiopia; the overall prevalence was 80% (95% confidence intervals, IC: 56.3-94.3). Preliminary results show the presence of R. aeschlimannii in 50% (95% CI: 24.6-75.3) H. truncatum and H. rufipes collected from cattle in different provinces of Burkina Faso. This is the first detection of R. aeschlimannii in Burkina Faso and in the Somali Region of Ethiopia. As recorded in other African countries, R. aeschlimannii shows a high prevalence in Hyalomma spp. ticks, which are its main vectors and reservoir. Further studies are needed to assess the zoonotic risk for local populations, who can have high contact rates with ticks and their vertebrate hosts.

The 1st Conference on Neglected Vectors and Vector-Borne Diseases with MC and WG Meetings of the COST Action TD1303 | 29

Abstract Book

LEISHMANIA – SAND FLY INTERACTION

Volf P

Department of Parasitology, Fac. Sci., Charles University, Prague, Czech Republic. Correspondence: [email protected]

Leishmania development in the vector is confined to the digestive tract. During the first few days after bloodfeeding, natural barriers to Leishmania development include secreted proteolytic enzymes, the peritrophic matrix surrounding the ingested blood meal and sand fly immune reactions. As the blood digestion proceeds, parasites need to bind to the midgut epithelium to avoid being excreted with the blood remnant. While the attachment in specific vectors (P. papatasi, P. duboscqi and P. sergenti) involves lipophosphoglycan, this Leishmania molecule is not required for parasite attachment in other sand fly species experimentally permissive for various Leishmania. During late-stage infections, large numbers of parasites accumulate in the anterior midgut and produce filamentous proteophosphoglycan creating a gel-like plug physically obstructing the gut. The parasites attached to stomodeal valve cause damage to the chitin lining and epithelial cells of the valve, interfering with its function and facilitating reflux of parasites from the midgut. Leishmania parasites are inoculated into the host skin together with sand fly saliva. In naive host, immunomodulatory effect of sand fly salivary molecules enhances the parasite infection. On the other hand, multiple exposures to sand fly bites induce an immune reaction that protects the host against Leishmania parasites co-injected with sand fly saliva or transmitted by sand fly vector. In bitten hosts, sand fly saliva also elicits specific antibody response which reflects the intensity of sand fly exposure and, therefore, the risk of Leishmania transmission. As screening of specific anti-saliva antibodies is limited by the availability of salivary gland homogenate, the utilization of recombinant sand fly salivary proteins in such studies is promising.

The 1st Conference on Neglected Vectors and Vector-Borne Diseases with MC and WG Meetings of the COST Action TD1303 | 30

Abstract Book

MOLECULAR CHARACTERIZATION OF ANAPLASMA PHAGOCYTOPHILUM STRAINS USING MULTILOCUS SEQUENCE AND ankA GENE BASED-TYPING

Huhn C, Winter C, Wolfsperger T, Wüppenhorst N, von Loewenich FD

Institute of Medical Microbiology, University of Freiburg, Freiburg, Germany. Correspondence: [email protected]

Anaplasma phagocytophilum is a Gram-negative obligate intracellular bacterium that is transmitted via tick-bite. It replicates in neutrophil granulocytes and causes febrile disease in humans and domestic animals such as sheep, cattle, horses, dogs, and cats. Evidence for strain variation within the species A. phagocytophilum comes from cross-infection experiments showing that A. phagocytophilum isolates from humans and animals differ in their pathogenicity for heterologous hosts. Further, A. phagocytophilum is thought to depend on reservoir hosts to complete its life cycle, because it is not transmitted transovarially in Ixodes spp. ticks. In order to explain host preference and to elucidate potential reservoir hosts, we used multi locus sequence (MLST) and ankA gene-based typing for the molecular characterization of A. phagocytophilum strains. Almost 400 strains from humans, animals and Ixodes ricinus ticks from Germany and other European countries were analyzed. MLST and ankA gene-based typing revealed that A. phagocytophilum strains from humans, dogs and horses were homologous, because they were part of the same clonal complex and the same ankA gene cluster. However, strains from sheep and cattle were found to be more diverse belonging to different ankA gene clusters. Further, wild boars and hedgehogs may serve as reservoir hosts for granulocytic anaplasmosis in humans, dogs and horses, because their strains were members to the same clonal complex and the same ankA gene cluster. In contrast, A. phagocytophilum strains from roe deer as well as from voles and shrews belonged to different MLST- and ankA-gene clusters as all other strains. It is therefore unlikely that roe deer, voles and shrews harbor A. phagocytophilum strains infectious for humans, domestic or farm animals.

The 1st Conference on Neglected Vectors and Vector-Borne Diseases with MC and WG Meetings of the COST Action TD1303 | 31