Xenomonitoring of Mosquitoes (Diptera: Culicidae) for the Presence of Filarioid Helminths in Eastern Austria.
In Europe, filarioid helminths of veterinary and/or medical relevance have mainly been documented in Mediterranean regions, but increasingly these pathogens are being reported in temperate climate zones in Central and Northern Europe as well [1-3].
The most important filarioid helminths in Europe are Dirofilaria immitis and D. repens, causing canine pulmonary (D. immitis), subcutaneous (D. repens), and ocular (mainly D. repens) dirofilariosis . Both, D. immitis and D. repens, are zoonotic parasites . The first Central European discoveries of D. immitis were confirmed in Switzerland, in 1995 and 1998 [5, 6]. Since then, both parasites, D. immitis and D. repens, have been described in humans (accidental hosts), dogs (definite hosts), and mosquitoes (vectors) in many Central European countries. Both filarioid species have now been shown to be present in all countries neighbouring Austria except for Liechtenstein, namely, Switzerland, Italy, Slovenia, Hungary, Slovakia, the Czech Republic (D. repens only), and Germany [1, 3, 4, 7-11]. D. repens was documented in most Central European countries prior to D. immitis.
Mosquito-borne filarioid helminths of the genus Setaria mainly parasitize in the abdominal cavities of artiodactyls, hyracoids, and equines. Mosquitoes of the genus Aedes are thought to be the main vectors of these parasites (e.g., Ae. vexans for S. labiatopapillosa) [12,13]. Setaria tundra is a parasite of roe deer documented in several European countries such as Austria, Switzerland, Germany, France, Italy, Hungary, Poland, Spain, and Denmark (summarized in Enemark et al. ). In Northern Europe, S. tundra can also be found in domestic reindeer, wild forest reindeer, and moose . This species is associated with climate changes and causes severe outbreaks of periodontitis in semidomestic reindeer in Finland .
The aim of this study was to xenomonitor Eastern Austrian mosquitoes for the presence of DNA of filarioid helminths--with the main focus on Dirofilaria repens and D. immitis but also on Setaria tundra and other mosquito-borne filarioid helminths.
2. Materials and Methods
The present study combines the analysis for filarioid helminth DNA in mosquitoes sampled in two independent mosquito monitoring programs using two different storage conditions (dry and -80[degrees]C) conducted between 2013 and 2015.
2.1. Mosquito Sampling Method 1. In 2013 and 2014, adult female mosquitoes were trapped at three locations in Vienna using new standard miniature light traps (John W. Hook Company, Gainesville, Florida) baited with C[O.sub.2]. Collection was carried out on a daily basis for 24 hours from March to October. Mosquitoes were killed using the insecticide dichlorvos as soon as they entered the trap. Once a week, the traps were emptied, and Culicidae were dried and stored at room temperature until further processing .
2.2. Mosquito Sampling Method 2. Mosquitoes were monitored across three provinces of Eastern Austria (Burgenland, Lower Austria, and Vienna) at 35 permanent and 23 nonpermanent trapping sites. At permanent sampling sites, mosquitoes were monitored on a regular basis every second week for a 24-hour time period from April to October 20142015 using Biogents Sentinel Traps (Regensburg, Germany) equipped with carbon dioxide as attractant. Nonpermanent sampling sites were investigated at least once and up to six times during the summer months using Biogents Sentinel Traps (Regensburg, Germany) or exhausters. All mosquitoes were stored at -80[degrees]C until further processing .
Mosquitoes were identified morphologically using the identification key of Becker et al.  and pooled by species, collection site, and date, with a maximum number of 50 individuals per pool. To each pool, 400 [micro]l of DNA/RNA lysis buffer (Zymo Research Corp., USA) and two ceramic beads (Precellys Ceramic Beads, Peqlab Biotechnologie GmbH) were added, and the samples were homogenized in a TissueLyser II (Qiagen, Germany). Approximately 350 [micro]l of the homogenized pulp was loaded onto a QIAshredder (Qiagen, Germany). The filled QIAshredders were centrifuged for two minutes at 13,000 rpm to filter the samples (solid components remained on the column). In the next step, DNA was extracted using a ZR-Duet[TM] DNA/RNA MiniPrep kit (Zymo Research Corp., USA) according to the manufacturer's instructions.
DNA extracted from female mosquito pools was examined for the presence of genomic material of filarioid helminths using primers and PCR conditions published elsewhere . The primers used target a 724 bp fragment of the mitochondrial cytochrome oxidase subunit I gene and are specific for various filarioid helminths (e.g., Dirofilaria, Wuchereria, Brugia, Onchocerca, Setaria, and Acanthocheilonema). PCR products were separated by electrophoresis in 2% agarose gels stained with Midori Green Advance DNA stain (Nippon Genetics Europe, Germany). Finally, purified PCR products were sequenced by a commercial company (LGC Genomics GmbH, Germany). Sequences thus obtained were compared for similarity to sequences available in GenBank[R] database (http://www.ncbi.nlm.nih.gov/BLAST).
3. Results and Discussion
45,848 mosquitoes representing 25 mosquito species were analysed for the presence of filarioid DNA in this xenomonitoring survey (Table 1), resulting in the identification of DNA from D. repens, S. tundra, and two unknown filarioid helminths in 20 of the mosquito pools (Table 2; Figure 1).
DNA of D. repens was only found in 2015 in a single Anopheles plumbeus mosquito in Marchegg (Lower Austria) close to the Slovakian border. Although several mosquito species of different genera are proven as potential vectors of D. repens , DNA of this parasite has so far only been detected in other Anopheles species (An. algeriensis and An. maculipennis complex) in Austria .
To date, all D. repens positive mosquitoes have been collected in close proximity to the Slovakian (this study) and the Hungarian borders . In both Slovakia and Hungary, D. repens is known to be endemic with a prevalence above 10% in dogs in the Bratislava area, close to the Austrian border [22, 23]. Previous metadata analysis has shown that most reported but also potential autochthonous findings in dogs were in Eastern Austria . Furthermore, Duscher et al.  described the examination of D. repens positive dogs in the same districts where positive mosquitoes were documented (Ganserndorf and Neusiedl am See). This indicates that D. repens might be endemic with low prevalence in this area. Simon et al.  postulated that two preconditions are required for a successful establishment of D. repens and D. immitis in a novel area: (i) the presence of competent mosquito vectors, which is the case in Austria, and (ii) a certain number of positive dogs shedding microfilaria. The second precondition seems to limit the distribution of D. repens (but also D. immitis) because there are almost no stray dogs, and kennel holding is not common in Austria.
Dirofilaria immitis was not identified in the present large-scale survey, confirming previous results that this parasite has not yet established itself in Eastern Austria . This pathogen has however been confirmed in dogs [22,23,25] and in mosquitoes [26, 27] in Slovakia and Hungary in the vicinity of our study area.
The most commonly found filarioid helminth within the present study area in Eastern Austria was S. tundra, with most occurrences of S. tundra DNA in mosquitoes of the genus Aedes, especially Ae. vexans. Similarly, prevalences of up to 12.3% have been reported in roe deer in Central Europe (e.g., northern Bavaria ). This parasite has also been recorded in Ae. vexans in studies in Germany and Hungary [1,27,29,30], suggesting that S. tundra is a common parasite of roe deer in Eastern Austria.
The discovery of DNA of unknown filarioid helminths in ornithophilic Culex mosquitoes (Cx. modestus and Cx. pipiens complex) is not surprising because several avian filarioid helminths (with low pathogenicity for bird hosts) are present in Central Europe .
This xenomonitoring survey confirms the presence of DNA of certain filarioid helminths in mosquitoes in Eastern Austria and indicates possible vector competence of select mosquito species. However, comparison of the two sampling techniques and storage schemes used here suggests that storage of dried mosquitoes at room temperature increases the number of false negative pools because of a decrease of DNA quality, a circumstance that has also been noticed during analyses of mosquito DNA itself . Moreover, different techniques for mosquito sampling (certain mosquito species are attracted by certain traps) and the use of different PCR protocols also influence the outcome of xenomonitoring studies . Nevertheless, xenomonitoring is an effective tool to examine if certain pathogens are present in an area (e.g., ). It can be concluded that D. repens, S. tundra, and unknown filarioid helminths (most probably avian parasites) are present in Eastern Austria. Further studies are needed to monitor in more detail the situation of D. repens and D. immitis in Austria and neighbouring countries.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
The authors thank all citizen scientists who helped in mosquito sampling within this study. Parts of this research were funded by the ERA-Net BiodivERsA, with the national funders FWF I-1437, ANR-13-EBID-0007-01, and DFG BiodivERsA KL 2087/6-1 as part of the 2012-13 BiodivERsA call for research proposals.
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Sarah Susanne Ubleis, (1) Claudia Cuk, (1) Michaela Nawratil, (1) Julia Butter, (1) Ellen Schoener, (1) Adelheid G. Obwaller, (2) Thomas Zechmeister, (3) Georg G. Duscher, (1) Franz Rubel, (4) Karin Lebl, (4) Carina Zittra, (1) and Hans-Peter Fuehrer (1)
(1) Institute of Parasitology, Department of Pathobiology, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria
(2) Division of Science, Research and Development, Federal Ministry of Defence and Sports, Vienna, Austria
(3) Biological Station Lake Neusiedl, Burgenland, Austria
(4) Institute for Veterinary Public Health, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria
Correspondence should be addressed to Hans-Peter Fuehrer; firstname.lastname@example.org
Received 24 November 2017; Accepted 14 February 2018; Published 15 March 2018
Academic Editor: Jose A. Oteo
Caption: Figure 1: Geographic distribution of mosquito pools positive for filarioid helminths in Eastern Austria.
Table 1: Number of mosquitoes and species collected in Eastern Austria included within this study. Mosquito species 2013 HC 2014 HC 2014 Biodiversa Aedes cinereus/geminus 6 6 299 Aedes vexans 1718 1847 4417 Anopheles algeriensis 4 -- -- Anopheles claviger -- -- -- Anopheles hyrcanus 145 63 -- Anopheles maculipennis complex 14 2 13 Anopheles plumbeus 9 26 150 Coquillettidia richiardii 2169 4333 1287 Culex martinii -- -- 66 Culex modestus 31 8 -- Culex pipiens complex/Cx. torrentium 2707 2090 2118 Culex territans -- 8 -- Culiseta annulata 4 7 -- Ochlerotatus cantans -- -- 1 Ochlerotatus caspius -- 51 17 Ochlerotatus cataphyla -- -- 7 Ochlerotatus communis -- -- 22 Ochlerotatus flavescens -- -- 1 Ochlerotatus geniculatus 4 41 20 Ochlerotatus intrudens -- -- 24 Ochlerotatus japonicus japonicus -- -- -- Ochlerotatus leucomelas -- -- -- Ochlerotatus rusticus -- -- 4 Ochlerotatus sticticus 559 202 1113 Uranotaenia unguiculata -- -- -- Aedes/Ochlerotatus sp. (a) (a) 735 Anopheles sp. (a) (a) 22 Culex sp. (a) (a) 131 Total 7370 8684 10447 Mosquito species 2015 Total Biodiversa Aedes cinereus/geminus 33 344 Aedes vexans 1179 9161 Anopheles algeriensis 3 7 Anopheles claviger 13 13 Anopheles hyrcanus 241 449 Anopheles maculipennis complex 41 70 Anopheles plumbeus 196 381 Coquillettidia richiardii 8034 15823 Culex martinii 996 1062 Culex modestus 61 100 Culex pipiens complex/Cx. torrentium 7124 14039 Culex territans -- 8 Culiseta annulata 33 44 Ochlerotatus cantans 1 2 Ochlerotatus caspius 4 72 Ochlerotatus cataphyla 5 12 Ochlerotatus communis -- 22 Ochlerotatus flavescens -- 1 Ochlerotatus geniculatus 6 71 Ochlerotatus intrudens -- 24 Ochlerotatus japonicus japonicus 6 6 Ochlerotatus leucomelas 1 1 Ochlerotatus rusticus -- 4 Ochlerotatus sticticus 499 2373 Uranotaenia unguiculata 10 10 Aedes/Ochlerotatus sp. 217 952 Anopheles sp. 387 409 Culex sp. 257 388 Total 19347 45848 HC, Hook Company Traps; Biodiversa, combination of various traps including BG-Sentinel Traps, HC, and aspirators; (a) mosquitoes specified to genus level were not included. Table 2: Filarioid helminths in mosquitoes in Eastern Austria: Vienna, Lower Austria (LA), and Burgenland (B). Filarioid species Mosquito species Collection site Dirofilaria repens Anopheles plumbeus Marchegg (LA) Setaria tundra Aedes cinereus/geminus Lobau (Vienna) Setaria tundra Aedes vexans Marchegg (LA) Setaria tundra Aedes sp. Lobau (Vienna) Setaria tundra Aedes vexans Lobau (Vienna) Setaria tundra Coquillettidia richiardii Lobau (Vienna) Setaria tundra Aedes vexans Vienna Setaria tundra Aedes vexans Marchegg (LA) Setaria tundra Aedes cinereus/geminus Eckartsau (LA) Setaria tundra Aedes sp. Klosterneuburg (LA) Setaria tundra Culex pipiens complex Bruckneudorf (B) Setaria tundra Aedes sp. Gotzendorf (LA) Setaria tundra Aedes vexans Gotzendorf (LA) Setaria tundra Aedes vexans Gootzendorf (LA) Setaria tundra Aedes vexans Jennersdorf (B) Setaria tundra Aedes sp. Bruckneudorf (B) Setaria tundra Aedes vexans Bruckneudorf (B) Setaria tundra Culex pipiens complex Vienna Filarioidea Culex modestus Vienna Filarioidea Culex pipiens complex Vienna Filarioid species Sampling Collection date Pool GenBank method size entry Dirofilaria repens BG-Sentinel August 2015 1 MF695085 Setaria tundra HC (a) July 2013 1 MF695086 Setaria tundra BG-Sentinel July 2014 25 MF695087 Setaria tundra HC (a) August2014 9 MF695088 Setaria tundra HC (a) August2014 42 MF695089 Setaria tundra HC (a) August2014 50 MF695090 Setaria tundra Aspirator August 2014 1 MF695091 Setaria tundra BG-Sentinel August2014 3 nd (c) Setaria tundra BG-Sentinel June 2015 1 MF695096 Setaria tundra BG-Sentinel June 2015 25 nd (c) Setaria tundra BG-Sentinel July 2015 50 nd (c) Setaria tundra BG-Sentinel July 2015 6 MF695092 Setaria tundra BG-Sentinel July 2015 50 MF695093 Setaria tundra BG-Sentinel July 2015 50 nd (c) Setaria tundra BG-Sentinel July 2015 1 MF695094 Setaria tundra BG-Sentinel July 2015 4 nd (c) Setaria tundra BG-Sentinel July 2015 26 MF695095 Setaria tundra BG-Sentinel August 2015 50 nd (c) Filarioidea HC (a) June 2014 1 nd (c) Filarioidea HC (a) September 2014 1 nd (c) Filarioid species Maximum % identity to GenBank entries (b) Dirofilaria repens 100% Setaria tundra 100% Setaria tundra >99% Setaria tundra >99% Setaria tundra 100% Setaria tundra >99% Setaria tundra 100% Setaria tundra 100% Setaria tundra >99% Setaria tundra 100% Setaria tundra 100% Setaria tundra 99% Setaria tundra 100% Setaria tundra 100% Setaria tundra 100% Setaria tundra 100% Setaria tundra 100% Setaria tundra 100% Filarioidea 93% Filarioidea 95% (a) Hook Company C[O.sub.2] baited mosquito traps; (b) analysis of maximum identity to GenBank Entries was performed on August 4, 2017; (c) sequences were not uploaded to GenBank (e.g., short sequences or poor sequence quality).
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|Title Annotation:||Research Article|
|Author:||Ubleis, Sarah Susanne; Cuk, Claudia; Nawratil, Michaela; Butter, Julia; Schoener, Ellen; Obwaller, A|
|Publication:||Canadian Journal of Infectious Diseases and Medical Microbiology|
|Date:||Jan 1, 2018|
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