BACKGROUND: Despite long-term research on dirofilariosis in Slovakia, little attention has thus far been paid to Dirofilaria vectors. The particular aim of the present study was molecular screening for filarioid parasites in two different habitats of Bratislava, the capital city of Slovakia. In addition, the effect of urbanisation on mosquito species abundance and composition, associated with the risk of mosquito-borne infections, was studied and discussed. METHODS: Mosquitoes were identified by morphological features, and molecular methods were also used for determination of selected individuals belonging to cryptic species from the Anopheles maculipennis and Culex pipiens complexes. The presence of filarioid DNA (Dirofilaria repens, Dirofilaria immitis and Setaria spp.) was detected using standard PCR approaches and sequencing. RESULTS: A total of 6957 female mosquitoes were collected for the study. Overall, the most abundant mosquito species was Aedes vexans, closely followed by unidentified members of the Cx. pipiens complex and the less numerous but still plentiful Ochlerotatus sticticus species. Further investigation of mosquito material revealed 4.26% relative prevalence of Dirofilaria spp., whereby both species, D. repens and D. immitis, were identified. The majority of positive mosquito pools had their origin in a floodplain area on the outskirts of the city, with a relative prevalence of 5.32%; only two mosquito pools (1.26%) were shown to be positive in the residential zone of Bratislava. Setaria spp. DNA was not detected in mosquitoes within this study. CONCLUSIONS: The study presented herein represents initial research focused on molecular mosquito screening for filarioid parasites in urban and urban-fringe habitats of Bratislava, Slovakia. Molecular analyses within the Cx. pipiens complex identified two biotypes: Cx. pipiens biotype pipiens and Cx. pipiens biotype molestus. To our knowledge, Dirofilaria spp. were detected for the first time in Slovakia in mosquitoes other than Ae. vexans, i.e. D. repens in Anopheles messeae and unidentified members of An. maculipennis and Cx. pipiens complexes, and D. immitis in Coquillettidia richiardii and Cx. pipiens biotype pipiens. Both dirofilarial species were found in Och. sticticus. The suitable conditions for the vectors' biology would represent the main risk factor for dirofilariosis transmission.

Department of Zoology Faculty of Natural Sciences Comenius University Mlynská dolina B 1 SK 842 15 Bratislava Slovakia

Institute of Parasitology Slovak Academy of Sciences Hlinkova 3 040 01 Košice Slovakia

Institute of Vertebrate Biology v v i Czech Academy of Sciences Květná 8 603 65 Brno Czech Republic

State Veterinary and Food Institute Botanická 15 842 52 Bratislava Slovakia

See more in PubMed

Medlock JM, Hansford KM, Schaffner F, Versteirt V, Hendrickx G, Zeller H, Van Bortel W. A review of the invasive mosquitoes in Europe: ecology, public health risks, and control options. Vector Borne Zoonotic Dis. 2012;12:435–447. doi: 10.1089/vbz.2011.0814. PubMed DOI PMC

Becker N, Petric D, Zgomba M, Boase C, Madon M, Dahl C, Kaiser A. Mosquitoes and their control. 2. Berlin: Springer; 2010.

Mahakalkar AL, Sapkal HP, Baig MM. Report of high genetic diversity of filarial worm, Wuchereria bancrofti from endemic region of eastern Maharashtra (India). Helminthologia. 2017;54:292–7.

Pampiglione S, Rivasi F. Human dirofilariasis to Dirofilaria (Nochtiella) repens: an update of world literature from 1995–2000. In: Genchi C, Rinaldi L, Cringoli G, editors. Dirofilaria immitis and D. repens in dog and cat and human infections. Naples: Rolando Editore; 2007. pp. 83–116.

Eldridge BF, Edman JD. Medical entomology. Dordrecht: Springer; 2000.

Simón F, Gonzáles-Miguel J, Diosdado A, Gómez PJ, Morchón R, Kartashev V. The complexity of zoonotic filariasis episystem and its consequences: a multidisciplinary view. Biomed Res Int. 2017;2017:6436130. PubMed PMC

Ferraguti M, Martínez-de la Puente J, Roiz D, Ruiz S, Soriguer R, Figuerola J. Effects of landscape anthropization on mosquito community composition and abundance. Sci Rep. 2016;6:29002. doi: 10.1038/srep29002. PubMed DOI PMC

Miterpáková M, Antolová D, Hurníková Z, Dubinský P. Dirofilariosis in Slovakia - a new endemic area in central Europe. Helminthologia. 2008;4:20–23.

Miterpáková M, Iglódyová A, Čabanová V, Stloukal E, Miklisová D. Canine dirofilariosis endemic in Central Europe - 10 years of epidemiological study in Slovakia. Parasitol Res. 2016;11:2389–2395. doi: 10.1007/s00436-016-4989-2. PubMed DOI

Miterpáková M, Antolová D, Ondriska F, Gál V. Human Dirofilaria repens infections diagnosed in Slovakia in the last 10 years (2007–2017). Wien Klin Wochenschr. 2017;12:634–41. PubMed

Bocková E, Kočišová A, Letková V. First record of Aedes albopictus in Slovakia. Acta Parasitol. 2013;58:603–606. doi: 10.2478/s11686-013-0158-2. PubMed DOI

Bocková E, Iglódyová A, Kočišová A. Potential mosquito (Diptera: Culicidae) vector of Dirofilaria repens and Dirofilaria immitis in urban areas of eastern Slovakia. Parasitol Res. 2015;114:4487–4492. doi: 10.1007/s00436-015-4692-8. PubMed DOI

Reháčková T, Lehotská B, Nevřelová M, Pauditšová E, Ružičková J. Forest fragments in built-up territory of Bratislava. Bratislava: Cicero; 2007. (In Slovak).

Šeffer J, Stanová V. Morava River floodplain meadows - importance, restoration and management. Bratislava: DAPHNE-Centre for Applied Ecology; 1999.

Natura 2000 . State Nature Conservancy of the Slovak Republic. 2000.

Bohuš M, Ružičková J, Lehotská B. Danube river, its ecosystems and human activity. Bratislava: Univerzita Komenského v Bratislave; 2011. (In Slovak).

Kumar NP, Rajavel AR, Natarajan R, Jambulingam P. DNA barcodes can distinguish species of Indian mosquitoes (Diptera: Culicidae) J Med Entomol. 2007;44:1–7. doi: 10.1093/jmedent/41.5.01. PubMed DOI

Smith J, Fonseca DM. Rapid assays for identification of members of the Culex (Culex) pipiens complex, their hybrids, and other sibling species (Diptera: Culicidae) Am J Trop Med Hyg. 2004;70:339–345. PubMed

Zittra C, Flechl E, Kothmayer M, Vitecek S, Rossiter H, Zechmeister T, Fuehrer HP. Ecological characterization and molecular differentiation of Culex pipiens complex taxa and Culex torrentium in eastern Austria. Parasit Vectors. 2016;9:197. doi: 10.1186/s13071-016-1495-4. PubMed DOI PMC

Bahnck C, Fonseca DM. Rapid assay to identify the two genetic forms of Culex (Culex) pipiens L. (Diptera: Culicidae) and hybrid populations. Am J Trop Med Hyg. 2006;75:251–255. doi: 10.4269/ajtmh.2006.75.251. PubMed DOI

Blažejová H, Šebesta O, Rettich F, Mendel J, Čabanová V, Miterpáková M, et al. Cryptic species Anopheles daciae (Diptera: Culicidae) found in the Czech Republic and Slovakia. Parasitol Res. 2018;117:315–321. doi: 10.1007/s00436-017-5670-0. PubMed DOI

Kronefeld M, Werner D, Kampen H. Molecular confirmation of the occurrence in Germany of Anopheles daciae (Diptera, Culicidae) Parasit Vectors. 2014;5:250. doi: 10.1186/1756-3305-5-250. PubMed DOI PMC

Casiraghi M, Anderson TJC, Bandi C, Bazzocchi C, Genchi CA. phylogenetic analysis of filarial nematodes: comparison with the phylogeny of Wolbachia endosymbiont. Parasitology. 2001;122:93–103. doi: 10.1017/S0031182000007149. PubMed DOI

Rishniw M, Barr SC, Simpson KW, Frongillo M, Franz M, Dominquez AJL. Discrimination between six species of canine microfilariae by a single polymerase chain reaction. Vet Parasitol. 2006;135:303–314. doi: 10.1016/j.vetpar.2005.10.013. PubMed DOI

Laaksonen S, Kuusela J, Nikander S, Nylund M, Oksanen A. Outbreak of parasitic peritonitis in reindeer in Finland. Vet Rec. 2007;160:835–841. doi: 10.1136/vr.160.24.835. PubMed DOI

Simón F, Siles-Lucas M, Morchón R, González-Miquel J, Mellado I, Carretón E, Montoya-Alonso JA. Human and animal dirofilariasis: the emergence of a zoonotic mosaic. Clin Microbiol Rev. 2012;25:507–544. doi: 10.1128/CMR.00012-12. PubMed DOI PMC

Tomasello D, Schlagenhauf P. Chikungunya and dengue autochtonmous cases in Europe, 2007–2012. Travel Med Infect Dis. 2013;11:274–284. doi: 10.1016/j.tmaid.2013.07.006. PubMed DOI

Badieritakis E, Papachristos D, Larinpoulos D, Stefopoulou A, Kolimenakis A, Bithas K, et al. Aedes albopictus (Skuse, 1895) (Diptera: Culicidae) in Greece: 13 years of living with the Asian tiger mosquito. Parasitol Res. 2018;117:453–460. doi: 10.1007/s00436-017-5721-6. PubMed DOI

Kwon YS, Bae MJ, Chung N, Lee YR, Hwang S, Kim SA, et al. Modelling occurrence of urban mosquitos based on land use types and meteorological factors in Korea. Int J Environ Res Publ Health. 2015;12:13131–13147. doi: 10.3390/ijerph121013131. PubMed DOI PMC

Fitz O. Mosquito calamities caused by flooding activity - report. Regional Public Health Authority Bratislava. 2013:2. (In Slovak). http://www.archiv.karlovaves.sk/sites/default/files/OZNAMENIE_Regionalny_urad_verejneho_zdravotnictva.pdf. 23 Jan 2018.

Tomšík K. Summary report of the flooding activity, its consequences and prevention. Magistrát hlavného mesta Slovenskej Republiky Bratislavy, Bratislava. 2013;44. (In Slovak). http://www.bratislava.sk/MsZ/Archiv/MsZ_13_09_25_26/MsZ_body_25_260913_anonym/49_Povodne_sprava.pdf. 23 Jan 2018.

Zittra C, Vitecek S, Obwaller AG, Rossiter H, Eigner B, Zechmeister T, et al. Landscape structure affects distribution of potential disease vectors (Diptera: Culicidae) Parasit Vectors. 2017;10:205. doi: 10.1186/s13071-017-2140-6. PubMed DOI PMC

Rudolf I, Šebesta O, Mendel J, Betášová L, Bocková E, Jedličková P, et al. Zoonotic Dirofilaria repens (Nematoda: Filarioidea) in Aedes vexans mosquitoes, Czech Republic. Parasitol Res. 2014;13:4663–4667. doi: 10.1007/s00436-014-4191-3. PubMed DOI

Kemenesi G, Kurucz K, Kepner A, Dallos B, Oldel M, Herczeg R, et al. Circulation of Dirofilaria repens, Setaria tundra, and Onchocercidae species in Hungary during the period 2011-2013. Vet Parasitol. 2015;214:108–113. doi: 10.1016/j.vetpar.2015.09.010. PubMed DOI

Rudolf M, Czajka C, Bӧrstker J, Melaun C, Jӧst H, von Thein H, et al. First nationwide surveillance of Culex pipiens complex and Culex torrentium mosquitoes demonstrated the presence of Culex pipiens biotype pipiens/molestus hybrids in Germany. PLoS One. 2013;8:71832. doi: 10.1371/journal.pone.0071832. PubMed DOI PMC

Sambri V, Capobianchi M, Charrel R, Fyodorova M, Gaibani P, Gould E, et al. West Nile virus in Europe: emergence, epidemiology, diagnosis, treatment, and prevention. Clin Microbiol Infect. 2013;19:699–704. doi: 10.1111/1469-0691.12211. PubMed DOI

Fonseca DM, Keyghobadi N, Malcolm CA, Mehmet C, Schaffner F, Mogi M, et al. Emerging vectors in the Culex pipiens complex. Science. 2004;303:1535–1538. doi: 10.1126/science.1094247. PubMed DOI

Ratovonjato J, Rajerison M, Rahelinirina S, Boyer S. Serologic surveillance for West Nile Virus in Dogs, Africa. Emerg Infect Dis. 2014;20:1415–1417. doi: 10.3201/eid2008.130629. PubMed DOI PMC

Csank T, Bhide K, Bencúrová E, Dolinská S, Dzewnioková P, Major P, et al. Detection of West Nile virus and tick-borne encephalitis virus in birds in Slovakia, using a universal primer set. Arch Virol. 2016;161:1679–1683. doi: 10.1007/s00705-016-2828-5. PubMed DOI

Strelková L, Halgoš J. Mosquitoes (Diptera, Culicidae) of the Morava River floodplain, Slovakia. Cent Eur J Biol. 2012;7:917–926.

Okáli I, Labuda M. History of malaria in Slovakia. Entomol Probl. 1988;18:233–251.

Halgoš J, Benková I. First record of Anopheles hyrcanus (Diptera: Culicidae) from Slovakia. Biológia. 2004;59:68.

Šebesta O, Rettich F, Minár J, Halouzka J, Hubálek Z, Juřicová Z, et al. Presence of the mosquito Anopheles hyrcanus in South Morava, Czech Republic. Med Vet Entomol. 2009;23:284–286. doi: 10.1111/j.1365-2915.2009.00810.x. PubMed DOI

Tóth S. Mosquito fauna (Diptera: Culicidae) of Sopron and its environs. Fol Hist Nat Mus Matr. 2003;27:327–32. (In Hungarian).

Seidel B, Silbermayr K, Kolodziejek J, Indra A, Nowotny N, Allerberger F. Detection of Plasmodium sp.-infested Anopheles hyrcanus (Pallas, 1771) (Diptera: Culicidae) in Austria, 2012. Wien Klin Wochenschr. 2013;125:139–43. PubMed

Townroe S, Callaghan A. British container breeding mosquitoes: the impact of urbanisation and climate change on community composition and phenology. PLoS One. 2014;9:95325. doi: 10.1371/journal.pone.0095325. PubMed DOI PMC

Schaffner F, Thiéry I, Kaufmann C, Zettor A, Lengeler C, Mathis A, Bourgouin C. Anopheles plumbeus (Diptera: Culicidae) in Europe: a mere nuisance mosquito or potential malaria vector? Malaria J. 2012;11:393. doi: 10.1186/1475-2875-11-393. PubMed DOI PMC

Čabanová V, Pantchev N, Hurníková Z, Miterpáková M. Recent study on canine vector-borne zoonoses in southern Slovakia - serologic survey. Acta Parasitol. 2015;60:749–758. PubMed

Latrofa MS, Montarsi F, Ciochetta S, Annoscia G, Dantas-Torres F, Ravagan S, et al. Molecular xenomonitoring of Dirofilaria immitis and Dirofilaria repens in mosquitoes from north-eastern Italy by real-time PCR coupled with melting curve analysis. Parasit Vectors. 2012;5:76. doi: 10.1186/1756-3305-5-76. PubMed DOI PMC

Kronefeld M, Kampen H, Sassnau R, Werner D. Molecular detection of Dirofilaria repens and Setaria tundra in mosquitoes from Germany. Parasit Vectors. 2014;7:30. doi: 10.1186/1756-3305-7-30. PubMed DOI PMC

Rudolf I, Bakonyi T, Šebesta O, Mendel J, Peško J, Betášová L, et al. West Nile virus lineage 2 isolated from Culex modestus mosquitoes in the Czech Republic, 2013: expansion of the European WNV endemic area to the North? Euro Surveill. 2014;19:20867. doi: 10.2807/1560-7917.ES2014.19.31.20867. PubMed DOI

Kurucz K, Kepner A, Krtinic B, Zana B, Földes F, Bányai K, et al. First molecular identification of Dirofilaria spp. (Ochocercidae) in mosquitoes from Serbia. Parasitol Res. 2016;115:3257–3260. doi: 10.1007/s00436-016-5126-y. PubMed DOI

Ionică AM, Zittra C, Wimmer V, Leitner N, Votýpka J, Modrý D, et al. Mosquitoes in the Danube Delta: searching for vectors of filarioid helminths and avian malaria. Parasit Vectors. 2017;10:324. doi: 10.1186/s13071-017-2264-8. PubMed DOI PMC

Bravo-Barriga D, Parreira R, Almeida PG, Calado M, Blanco-Ciudad J, Serrano-Aguilera FJ, et al. Culex pipiens as a potential vector for transmission of Dirofilaria immitis and other unclassified Filarioidea in Southwest Spain. Vet Parasitol. 2016;15:173–80. PubMed

Morchón R, Carretón E, Gonzáles-Miquel J, Mellado-Hernández I. Heartworm disease (Dirofilaria immitis) and their vectors in Europe - new distribution trends. Front Physiol. 2012;3:196. doi: 10.3389/fphys.2012.00196. PubMed DOI PMC

Cancrini G, Gabrielli S. Vectors of Dirofilaria nematodes: biology, behaviour and host/parasite relationships. In: Genchi C, Rinaldi L, Cringoli G, editors. Dirofilaria immitis and D. repens in dog and cat and human infections. Naples: Rolando Editore; 2007.

Morchón R, Bargues MD, Latorre JM, Melero-Alcíbar R, Pou-Barreto C, Mas-Coma S, Simón F. Haplotype H1 of Culex pipiens implicated as natural vector of Dirofilaria immitis in an endemic area of western Spain. Vector Borne Zoonotic Dis. 2007;7:653–8. PubMed

Silbermayr K, Eigner B, Joachim A, Duscher GG, Seidel B, Allerberger F, et al. Autochtonous Dirofilaria repens in Austria. Parasit Vectors. 2014;7:226. doi: 10.1186/1756-3305-7-226. PubMed DOI PMC

Masny A, Salamatin R, Rozej-Bielicka W, Golab E. Is molecular xenomonitoring of mosquitoes for Dirofilaria repens suitable for dirofilariosis surveillance in endemic region? Parasitol Res. 2016;115:511–525. doi: 10.1007/s00436-015-4767-6. PubMed DOI

Trájer A, Rengei A, Farkas-Iványi K, Bede. Fazekas Á. Impacts of urbanisation level and distance from potential natural mosquito breeding habitats on the abundance of canine dirofilariosis. Acta Vet Hung. 2016;64:340–359. doi: 10.1556/004.2016.032. PubMed DOI

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