Cited by 107Parasitic Diseases Research and TreatmentResearch on Leishmaniasis StudiesHelminth infection and control

Molecular detection of Dirofilaria immitis, Dirofilaria repens and Setaria tundra in mosquitoes from Germany

Open access
Kronefeld, Mandy, Kampen, Helge, Sassnau, Reinhold, Werner, Doreen·2014·Parasites & Vectors

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. CONCLUSION: 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.

AI snapshot

Written from the paper’s full text.

TL;DR

Screening of approximately 17,000 female mosquitoes from Germany by pan-filarioid real-time PCR and COI sequencing detected Dirofilaria immitis, D. repens, Setaria tundra, and two unassigned filarioid sequences in 9 of 955 pools. The authors report the first detection of D. immitis in German mosquitoes, implying possible local natural transmission.

Key findings

  • Of 16,878 female mosquitoes representing 16 species or species complexes in six genera, 955 pools were screened; 9 pools (0.94%) tested positive for filarioid DNA.
  • Identified sequences included two D. immitis (100% and 99% identity), one D. repens (99% identity), four S. tundra (99% identity), and two unassigned filarioid sequences (89% and 92% maximum identity to GenBank entries).
  • D. immitis and the unknown filarioid sequences were found in Cx. pipiens/torrentium, D. repens in a single An. daciae, and all S. tundra in Ae. vexans; all positive mosquitoes were collected between mid-June and early September in 2011, 2012, and 2013 in four German federal states.
  • The paper states this is the first report from Germany of D. immitis apparently acquired locally, and notes that D. repens was detected in An. daciae, a species with unknown vector potential.

Methods

  • Adult mosquitoes were collected all over Germany from 2011 to 2013 using BG sentinel traps with BG Lure and CO2 or by hand, identified morphologically or genetically, and pooled by species, collection site, and date with up to 25 specimens per pool.
  • Pools were homogenized and extracted for RNA/DNA, then screened with a newly designed filarioid-specific real-time PCR targeting a 90 bp fragment of the mitochondrial 16S rRNA gene; positive samples were processed by conventional PCR amplifying about 650 bp of the COI gene, sequenced, and compared with GenBank.
  • COI sequences of identified worms were deposited in GenBank under accession numbers KF692100–KF692106.

Limitations

  • The paper states that the routes of introduction to Germany and the origin of the filariae cannot be determined retrospectively, and that these must remain speculative.
  • The paper notes that pooling prevented molecular differentiation between morphologically indistinguishable Cx. pipiens and Cx. torrentium females, and that the real-time PCR was intended for screening only, possibly allowing false negatives but not false positives.
  • The paper states that no information exists on possible spread and prevalence increase of S. tundra in Central Europe because respective studies are missing; evident from the text, the study also does not demonstrate vector competence or local transmission directly, and two filarioid sequences could not be assigned to known species due to insufficient GenBank matches.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1186/1756-3305-7-30

Cites

45 of 45 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 10 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Two COI sequences from the positive pools showed 100% and 99% identity to D. immitis.

    “Of the sequences obtained, two showed 100% and 99% identity to D. immitis (Table 2) while presenting two nucleotide differences in direct comparison.”
    • Also stated in: § Results · hedged
      “Two of the COI sequences indicated D. immitis”
    Quote in the paperNot a substantial claim
  • Findingown data

    All filariae-carrying culicids were collected between mid-June and early September in 2011, 2012 and 2013 in four German federal states.

    “All filariae-carrying culicids had been collected between mid-June and early September in 2011, 2012 and 2013 in four German federal states (Table 2, Figure 1).”
    • Also stated in: § Results · plain
      “All positive mosquitoes were collected between mid-June and early September.”
    Quote in the paperNot a substantial claim
  • Findingcitation

    The D. repens-positive Anopheles daciae was collected in the same area where D. repens had been isolated from dogs in 2007.

    “it was collected in the same area where D. repens had been isolated from dogs in 2007 [12].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [12] Pantchev N, Norden N, Lorentzen L, Rossi M, Rossi U, Brand B, Dyachenko V: Current surveys on the prevalence and distribution of Dirofilaria spp. in dogs in Germany. Parasitol Res. 2009, 105: S63-S74. 10.1007/s00436-009-1497-7. source

      Not checked in the free run.

  • Findingown data

    Two positive samples could not be assigned to a known species due to insufficient identities to GenBank entries (89 and 92% maximum).

    “Two positive samples, displaying 91% homology in direct comparison, could not be assigned to a species due to insufficient identities to GenBank entries (89 and 92% maximum; Table 2).”
    • Also stated in: § Results · plain
      “Two sequences could not be assigned to a known species due to a lack of similar GenBank entries.”
    Quote in the paperNot a substantial claim
  • Findingown data

    One COI sequence from the positive pools displayed 99% identity to D. repens.

    “One sequence displayed 99% identity to D. repens”
    • Also stated in: § Results · plain
      “one D. repens”
    Quote in the paperNot a substantial claim
  • The quote only partly states this claim. 1
  • May combine two claims. 6
  • Classified as probably not a substantial claim. 4
  • The quote only partly states this claim.

    Findingown data

    From 2011 to 2013, 16,878 female mosquitoes representing 16 species or species complexes in six genera were processed.

    “16,878 female mosquitoes representing 16 species or species complexes in six genera were processed, with Cx. pipiens/torrentium (73%), Anopheles plumbeus (11%) and Aedes vexans (8%) being the most frequent mosquito taxa examined (Table 1).”
    • Also stated in: § Results · hedged
      “Approximately 17,000 female mosquitoes were subjected to filarial screening.”
    Quote in the paperNot a substantial claim
  • May combine two claims.

    Findingown data

    The D. immitis-positive pools and the pools with unknown filarioid DNA sequences were composed of Culex pipiens/torrentium specimens.

    “The D. immitis-positive pools as well as those with unknown filarioid DNA sequences were composed of Cx. pipiens/torrentium specimens”
    • Also stated in: § Results · plain
      “Whilst D. immitis and the unknown parasites were detected in Culex pipiens/torrentium”
    Quote in the paperNot a substantial claim
  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Genchi and colleagues considered both Baden-Wurttemberg and Brandenburg as climatically suitable for dirofilarial development in mosquitoes and assigned to these regions a risk of stable endemicity.

    “Genchi and colleagues [5,44] considered both Baden-Wurttemberg and Brandenburg as climatically suitable for dirofilarial development in mosquitoes and assigned to these regions a risk of stable endemicity.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [5] Genchi C, Kramer LH, Rivasi F: Dirofilarial infections in Europe. Vector-borne Zoon Dis. 2011, 11: 1307-1317. 10.1089/vbz.2010.0247. source

      Not checked in the free run.

    • cites [44] Sassnau R, Genchi C: Qualitative risk assessment for the endemisation of Dirofilaria repens in the state of Brandenburg (Germany) based on temperature-dependent vector competence. Parasitol Res. 2013, 112: 2647-2652. 10.1007/s00436-013-3431-2. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Simón et al. calculated a transmission period of 3-4 months in Central Europe for both D. repens and D. immitis.

    “Simón et al.[1] calculated a transmission period of 3–4 months in Central Europe for both D. repens and D. immitis, taking into account the extrinsic incubation periods of the worms.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [1] Simón F, Siles-Lucas M, Morchón R, González-Miguel 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. 10.1128/CMR.00012-12. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    Setaria tundra seems to be more common in Germany than generally assumed, as it had been detected microscopically or by PCR on several occasions in the past.

    “The third mosquito-borne filarial nematode described, S. tundra, seems to be more common in Germany than generally assumed, as it had been detected microscopically or by PCR on several occasions in the past [22,43].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [22] Rehbein S, Lutz W, Visser M, Winter R: Beiträge zur Kenntnis der Parasitenfauna des Wildes in Nordrhein-Westfalen. 1. Der Endoparasitenbefall des Rehwildes. Z Jagdwiss. 2000, 4: 248-269.

      Not checked in the free run.

    • cites [43] Czajka C, Becker N, Poppert S, Jöst H, Schmidt-Chanasit J, Krüger A: Molecular detection of Setaria tundra (Nematoda: Filarioidea) and an unidentified filarial species in mosquitoes in Germany. Parasit Vectors. 2012, 5: 14-10.1186/1756-3305-5-14. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    At a mean temperature of 18°C, the extrinsic incubation periods of D. repens and D. immitis last about 29 days; at 22°C they last 16-20 days.

    “At a mean temperature of 18°C, for example, these will last about 29 days, at 22°C still 16–20 days.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [1] Simón F, Siles-Lucas M, Morchón R, González-Miguel 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. 10.1128/CMR.00012-12. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    As hot summer periods are predicted to become more frequent and longer as a result of climate change, mosquito-borne filarioses will probably become a growing problem to veterinary and human health in Central and eastern Europe in the future.

    “However, as hot summer periods are predicted to become more frequent and longer as a result of climate change, mosquito-borne filarioses will probably become a growing problem to veterinary and human health in Central and eastern Europe in the future.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Hot summer periods are predicted to become more frequent and longer as a result of climate change.
  • May combine two claims.

    Interpretationcitation

    The finding of two unknown filarial species in Culex pipiens/torrentium mosquito pools suggests avian bloodmeal sources of the mosquitoes and therefore avian nematode species, one of them possibly being Cardiofilaria pavlovskyi.

    “The finding of two unknown filarial species in Cx. pipiens/torrentium mosquito pools suggests avian bloodmeal sources of the mosquitoes due to the feeding preferences within this group of culicids, and therefore avian nematode species, one of them possibly being Cardiofilaria pavlovskyi, as discussed by Cjaka et al. [43].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [43] Czajka C, Becker N, Poppert S, Jöst H, Schmidt-Chanasit J, Krüger A: Molecular detection of Setaria tundra (Nematoda: Filarioidea) and an unidentified filarial species in mosquitoes in Germany. Parasit Vectors. 2012, 5: 14-10.1186/1756-3305-5-14. source

      Not checked in the free run.

    • Rests on an assumption: Culex pipiens/torrentium mosquitoes have feeding preferences for avian bloodmeal sources.
  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductreasoning

    A more in-depth testing of the real-time PCR was not considered necessary as the PCR was meant for sample screening only, thus possibly allowing false negative but not false positive results.

    “A more in-depth testing was not considered necessary as the PCR was meant for sample screening only, thus possibly allowing false negative but not false positive results.”
    Quote in the paperSubstantial claim
9 of 9
  • The paper relies on this without support.

    Dirofilaria immitis had never been demonstrated in mosquitoes from Europe outside the Mediterranean before this study.

    “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.”
  • The paper relies on this without support.

    Culex pipiens/torrentium mosquitoes have feeding preferences for avian bloodmeal sources.

    “due to the feeding preferences within this group of culicids”
  • The paper relies on this without support.

    Hot summer periods are predicted to become more frequent and longer as a result of climate change.

    “as hot summer periods are predicted to become more frequent and longer as a result of climate change”
  • The paper relies on this without support.

    The lifespan of a mosquito is a few weeks at its best.

    “Considering the lifespan of a mosquito of a few weeks at its best”
  • The paper relies on this without support.

    Globalization and climate change will continue to progress.

    “With progressing globalization and climate change”
5 of 5

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Eight species listed as nine

    Minor

    The primer-design description says the alignment included nine filarial nematode species, but the parenthetical list names only eight.

1 of 1

Cites

Flagged first, then by citations

Research works printed but not linked: 2

  1. Świątalska A, Demiaszkiewicz AW: First autochthonous case of Dirofilaria immitis invasion in dog in Poland. Žycie Weterynaryjne. 2012, 87: 685-686. [Article in Polish]
  2. Schaffner F, Angel G, Geoffroy B, Hervy JP, Rhaiem A, Brunhes J: The Mosquitoes of Europe (CD-ROM). 2001, Montpellier, France: IRD Éditions & EID Méditerrannée

Cited by

Sorted by citations

Authors
Kronefeld, M., Kampen, H., Sassnau, R., & Werner, D.
Affiliations
  • Friedrich-Loeffler-Institut (DE)
  • Leibniz Centre for Agricultural Landscape Research (DE)
Corresponding author
Kampen, Helge
Venue
Parasites & Vectors
Published
2014-01-16
Volume
7
Issue
1
Pages
30-30
ISSN
1756-3305 (ISSN-L)
OpenAlex
W2122202607
Cited by
107
References
45
Citations by year
2026: 1 · 2025: 4 · 2024: 9 · 2023: 7 · 2022: 8 · 2021: 7 · 2020: 6 · 2019: 10 · 2018: 11 · 2017: 5 · 2016: 13 · 2015: 16 · 2014: 10
FWCI
10.31
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Parasites & Vectors (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology
  • University of Oxford
  • Rheinische Friedrich-Wilhelms-Universität Bonn
  • Bundesamt für Landwirtschaft
  • Bundesministerium für Verbraucherschutz, Ernährung und Landwirtschaft
  • Robert Koch Institut
  • Bundesanstalt für Landwirtschaft und Ernährung
Topics
  • Health Sciences › Medicine › Infectious Diseases › Parasitic Diseases Research and Treatment
  • Health Sciences › Medicine › Public Health, Environmental and Occupational Health › Research on Leishmaniasis Studies
  • Health Sciences › Veterinary › Small Animals › Helminth infection and control
Keywords
Dirofilaria immitis, Dirofilaria repens, dirofilariasis, Culex pipiens, Aedes vexans
MeSH terms
Animals, Dirofilaria immitis, Female, Geography, Germany, Molecular Sequence Data, Culicidae, Genes, Helminth, Dirofilaria repens

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