title: Molecular detection of Dirofilaria immitis, Dirofilaria repens and Setaria tundra in mosquitoes from Germany.
abstract: BACKGROUND
  As a result of globalization and climate change, Dirofilaria immitis and Dirofilaria repens, the causative agents of dirofilariosis in Europe, continue to spread from endemic areas in the Mediterranean to northern and northeastern regions of Europe where autochthonous cases of dirofilarial infections have increasingly been observed in dogs and humans. Whilst D. repens was recently reported from mosquitoes in putatively non-endemic areas, D. immitis has never been demonstrated in mosquitoes from Europe outside the Mediterranean.
  
  
  METHODS
  From 2011 to 2013, mosquitoes collected within the framework of a German national mosquito monitoring programme were screened for filarial nematodes using a newly designed filarioid-specific real-time PCR assay. Positive samples were further processed by conventional PCR amplification of the cytochrome c oxidase subunit I (COI) gene, amplicons were sequenced and sequences blasted against GenBank.
  
  
  RESULTS
  Approximately 17,000 female mosquitoes were subjected to filarial screening. Out of 955 pools examined, nine tested positive for filariae. Two of the COI sequences indicated D. immitis, one D. repens and four Setaria tundra. Two sequences could not be assigned to a known species due to a lack of similar GenBank entries. Whilst D. immitis and the unknown parasites were detected in Culex pipiens/torrentium, D. repens was found in a single Anopheles daciae and all S. tundra were demonstrated in Aedes vexans. All positive mosquitoes were collected between mid-June and early September.
  
  
  CONCLUSIONS
  The finding of dirofilariae in German mosquitoes implies the possibility of a local natural transmission cycle. While the routes of introduction to Germany and the origin of the filariae cannot be determined retrospectively, potential culicid vectors and reservoir hosts must prospectively be identified and awareness among physicians, veterinarians and public health personnel be created. The health impact of S. tundra on the indigenous cervid fauna needs further investigation.
authors.forename: Mandy
authors.initials: M
authors.lastname: Kronefeld
authors.affiliation: Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Greifswald-Insel Riems, Germany. helge.kampen@fli.bund.de.
authors.forename: Helge
authors.initials: H
authors.lastname: Kampen
authors.forename: Reinhold
authors.initials: R
authors.lastname: Sassnau
authors.forename: Doreen
authors.initials: D
authors.lastname: Werner
mesh_terms: D000818:Animals; D009033:Culicidae / Q000469:parasitology*; D004183:Dirofilaria immitis / Q000235:genetics / Q000302:isolation & purification*; D060348:Dirofilaria repens / Q000235:genetics / Q000302:isolation & purification*; D005260:Female; D017238:Genes, Helminth; D005843:Geography; D005858:Germany; D008969:Molecular Sequence Data