First report of Setaria tundra in roe deer (Capreolus capreolus) from the Iberian Peninsula inferred from molecular data: epidemiological implications
- Samer Angelone-Alasaad†1, 2Email authorView ORCID ID profile,
- Michael J. Jowers†3, 4,
- Rosario Panadero5,
- Ana Pérez-Creo5,
- Gerardo Pajares5,
- Pablo Díez-Baños5,
- Ramón C. Soriguer1 and
- Patrocinio Morrondo5
© The Author(s). 2016
Received: 25 July 2016
Accepted: 6 September 2016
Published: 29 September 2016
Filarioid nematode parasites are major health hazards with important medical, veterinary and economic implications. Recently, they have been considered as indicators of climate change.
In this paper, we report the first record of Setaria tundra in roe deer from the Iberian Peninsula. Adult S. tundra were collected from the peritoneal cavity during the post-mortem examination of a 2 year-old male roe deer, which belonged to a private fenced estate in La Alcarria (Guadalajara, Spain). Since 2012, the area has suffered a high roe deer decline rate (75 %), for unknown reasons. Aiming to support the morphological identification and to determine the phylogenetic position of S. tundra recovered from the roe deer, a fragment of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene from the two morphologically identified parasites was amplified, sequenced and compared with corresponding sequences of other filarioid nematode species. Phylogenetic analyses revealed that the isolate of S. tundra recovered was basal to all other formely reported Setaria tundra sequences. The presence of all other haplotypes in Northern Europe may be indicative of a South to North outbreak in Europe.
This is the first report of S. tundra in roe deer from the Iberian Peninsula, with interesting phylogenetic results, which may have further implications in the epidemiological and genetic studies of these filarioid parasites. More studies are needed to explore the reasons and dynamics behind the rapid host/geographic expansion of the filarioid parasites in Europe.
KeywordsFilarioid Mitochondrial cytochrome c oxidase subunit 1 (cox1) Phylogenetics Epidemiology Climate change Bayesian inference analysis
Filarioid nematode parasites are major health hazards with significant medical, veterinary and economic implications, with millions of people and animals globally affected . Haematophagous arthropods are the vectors of these parasites , which have recently been considered as indicators of climate change [3, 4]. Filarioid parasites are difficult to control due to vectors’ mobility and/or the risk of resistance to drugs .
The phylogenetic relationships of filarioid parasites have been assessed on morphological characters [6, 7]. Nevertheless, molecular analyses are needed to confirm their morphological descriptions or taxonomic position and classification, and to improve our understanding of the species epidemiology [5, 8].
In Europe, Setaria tundra has been reported from Germany [9, 10], Bulgaria , Sweden , Norway , the Baikal area , Italy , Poland  and Finland . Setaria tundra is considered as a common parasite in the northern hemisphere, especially in Finland, where mass infection has occurred . Here there seems to be a correlation of infection to wetland areas, where S. tundra vectors (mosquitoes) are at their optimal microclimate condition and reindeer herds are present , with 30–40 % of all S. tundra outbreaks in Finland.
The aims of the present study were to: (i) Report for the first time S. tundra in the roe deer Capreolus capreolus from the Iberian Peninsula, and (ii) examine the phylogenetic position of the newly-found S. tundra, based on sequences of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene.
Case report, sample collection and morphological examination
During the last 3 years, a private fenced estate in La Alcarria (Guadalajara, Spain) has suffered from a decline of 75 % of roe deer population for unknown reasons. Moreover, lower body weight and reduction of trophy size in males have been noted. Roe deer in this fenced estate are native animals (the density before decline was c.30 animals per 100 ha). In the study area, there are no domestic animals. The sympatric wild animals are the wild boar Sus scrofa, the red fox Vulpes vulpes and the European badger Meles meles, together with different bird species, which interact with roe deer, namely the golden eagle Aquila chrysaetos, the griffon vulture Gyps fulvus and the cinereous vulture Aegypius monachus.
Genomic DNA was extracted from each of the two S. tundra specimens using DNeasy Blood & Tissue Kit (Quigen, Hilden, Germany). DNA extractions were carried out in a sterilized laboratory dedicated exclusively for low DNA concentration samples. Two blanks (reagents only) were included in each extraction to monitor for contamination.
PCR and sequencing of the mitochondrial cox1 gene
The methods of Casiraghi et al.  for PCR amplification of the partial cox1 gene, were followed. The 30-μl PCR mixture contained 2 μl of template DNA, 0.25 μM of the primers cox1intF (5′-TGA TTG GTG GTT TTG GTA A-3′) and cox1intR (5′-ATA AGT ACG AGT ATC AAT ATC-3′), 0.12 mM of each dNTP, 3 μl of PCR buffer (Bioline, Sydney, Australia), 1.5 mM MgCl2, 0.4 % BSA, 1.5 μl DMSO, and 0.2 μl (0.2 U/reaction) Taqpolymerase (Bioline). The following thermal profile for amplification in a PTC0200 thermal cycler (Bio-Rad) was used: 4 min at 94 °C (initial denaturation), followed by 30 cycles of three steps of 1 min at 94 °C (denaturation), 1 min at 52 °C (annealing), and 50 s at 72 °C (extension), before a final elongation of 5 min at 72 °C. PCR blanks (reagents only) were included.
Sequencing was carried out in both directions using the BigDye® Terminator v1.1 cycle sequencing kit (Applied Biosystems) according to the manufacturer’s instructions. Labelled fragments were resolved on an automated A3130xl genetic analyser (Applied Biosystems).
Amplicons were sequenced on both strands, and the complementary reads were used to resolve rare, ambiguous base-calls in Sequencher v.4.9. Additionally, BLAST searches were conducted in GenBank and matches with high genetic affinity were downloaded and included in the alignment, with a cut-off point at 91 % genetic similarity. Higher genetic divergence recovered from other homologous sequences from Setaria spp. were downloaded and included in the alignment. The selected outgroup was Thelazia callipaeda. Sequences were aligned in SeaView v.4.2.11  under ClustalW2  default settings. The most appropriate substitution model for the Bayesian Inference (BI) analysis was determined by the Bayesian Information Criterion (BIC) in jModeltest v.2 . MrBayes v.3.2.6  was used with default priors and Markov chain settings, and with random starting trees. Each run consisted of four chains of 20,000,000 generations, sampled each 10,000 generations and posterior distributions of parameter estimates were visually inspected in Tracer v1.5 . A plateau was reached after few generations with 25 % of the trees resulting from the analyses discarded as ‘burn-in’. Phylogenetic relationships among haplotypes were estimated using a Maximum Likelihood (ML) approach, as implemented in the software RAxML v7.0.4 , using the default settings. The 50 % bootstrap consensus tree was built in PAUP 4 . All analyses were performed through the CIPRES platform .
Results and discussion
In this paper, we report the first record of Setaria tundra in roe deer from the Iberian Peninsula. The parasites were identified morphologically and genetically, based on the sequences of a fragment of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. The newly-obtained sequences were submitted to GenBank (KX599455–KX599456). The alignment length was 637 bp. GenBank blast matched Setaria tundra, with 99 % similarity and a highest divergence (similarity of 98 %) to the isolate 71YT MNHN (KP760209) with 10 bp substitutions. The closest match of the present isolate was to isolates 6615 (KF692104) and 5808 (KF692103) from Germany, with only two bp substitutions.
The recovered phylogenetic tree showing two monophyletic clades of unidentified nematodes within Setaria may suggest paraphyly of the genus (Fig. 2), which demands further taxonomic assessment within Setaria. The basal positioning of Setaria tundra recovered from this study suggests ancestry to all known European S. tundra haplotypes. Thus, although ancestral, its presence may have been undetected until now by limited case studies such as this one. The presence of all other haplotypes in northern Europe may be indicative of a South to North European outbreak route rather than the opposite. Further molecular taxonomic identification is needed to assess the extent of the parasite range in the Iberian Peninsula, the host species and parasite genetic diversity. Through such sampling the ancestral and descendent populations could elucidate important information regarding intermediate populations likely to be infected by Setaria that are currently unrecorded.
Other studies have found C. capreolus to be the host for S. tundra in Germany , Bulgaria , Italy , Finland  and Poland . Our study thus increases the host infective range from Northern to Southern Europe, as well as the parasite range. Several are the candidate insect vectors for Setaria spp. infections [17, 31]. Mammophilic mosquitoes, Aedes spp. and Anopheles spp., have shown to have an important role in the transmission of S. tundra in Finland, which suggests that this parasite is probably not vector-specific, and this consequently enhances the ability of S. tundra to expand geographically. Infections with S. tundra in reindeer herds in Finland are associated to wetlands, with fresh food pastures and drinking water, where mosquito populations are at their optimum microclimate condition to thrive . The presence of S. tundra here seems to be related to herd migrations, with a decline in the presence of S. tundra in hosts or mosquitoes in the Upper Lapland [17, 32]. These data are key to assess the likely areas to conduct surveys in the Iberian Peninsula and to establish molecular screening protocols for possible vectors and hosts. The ecological and topographical conditions at the La Alcarria conform to the vectors’ optimal environmental conditions. Furthermore, the presence of permanent water and the closure of the herd prevent animals to avoid possible high-risk infectious areas by migration. The Scandinavian (1973) S. tundra outbreak was associated to an exceptional warm period and high numbers of possible vectors such as mosquitoes and gnats . Thus, further studies are needed to assess the possible impact of climate change on transmission dynamics of Setaria spp. in Europe .
This is the first report of Setaria tundra in roe deer from the Iberian Peninsula, with interesting phylogenetic results, which may have further implications in the epidemiological and genetic studies of filarioid nematode parasites. More studies are needed to explore the reasons and dynamics behind the rapid host/geographical expansion of filarioid parasites in Europe.
This work was supported by the Programme for Consolidating and Structuring Competitive Research Groups (GRC2015/003, Xunta de Galicia). Molecular analyses were carried out in the LEM of EBD, CSIC and funded by RNM 118; Junta Andalucia. The authors express their gratitude to the Asociación del Corzo Español (ACE), which facilitated sample collection of the animals.
Availability of data and material
The datasets supporting the conclusions of this article are included within the article. Sequences are submitted to the GenBank database under accession numbers KX599455–KX599456.
RP, APC, GP, PDB, and PM performed the necropsy and the morphological identification of the parasites. SAA, MJJ and RCS did the genetic characterization and phylogenetic analyses. SAA, MJJ, RP, APC, GP, PDB, RCS, and PM discussed and wrote the paper. All authors read and approved the final version of the manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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.
- World Health Organization. Global programme to eliminate lymphatic filariasis: annual report on lymphatic filariasis 2006. Wkly Epidemiol Record. 2007;82:361–80.Google Scholar
- Anderson RC. The superfamily Filaroidea. In: In nematode parasites of vertebrates. Their development and transmission. 2nd ed. New York: CABI Publishing; 2000. p. 467–529.View ArticleGoogle Scholar
- Genchi C, Rinaldi L, Mortarino M, Genchi M, Cringoli G. Climate and Dirofilaria infection in Europe. Vet Parasitol. 2009;163:286–92.View ArticlePubMedGoogle Scholar
- Laaksonen S, Pusenius J, Kumpula J, Venäläinen A, Kortet R, Oksanen HE. Climate change promotes the emergence of serious disease outbreaks for Filarioid nematodes. Ecohealth. 2010;1:7–13.View ArticleGoogle Scholar
- Yatawara L, Wickramasinghe S, Nagataki M, Rajapakse RPVJ, Agatsuma T. Molecular characterization and phylogenetic analysis of Setaria digitata of Sri Lanka based on CO1 and 12S rDNA genes. Vet Parasitol. 2007;148:161–5.View ArticlePubMedGoogle Scholar
- Chabaud AG, Bain O. The evolutionary expansion of the Spirurida. Int J Parasitol. 1994;24:1179–201.View ArticlePubMedGoogle Scholar
- Bain O. Evolutionary relationships among filarial nematodes. In: Chabaud AG, Bain O, editors. 1976. La lignée Dipetalonema. Nouvel essai de classification. Ann Parasitol Hum Comp, vol. 51. 2002. p. 365–97.Google Scholar
- Alasaad S, Pascucci I, Jowers MJ, Soriguer RC, Zhu XQ, Rossi L. Phylogenetic study of Setaria cervi based on mitochondrial cox1 gene sequences. Parasitol Res. 2012;110:281–5.View ArticlePubMedGoogle Scholar
- Rehbein S, Lutz W, Visser M, Winter R. BeiträgezurKenntnis der Parasitenfauna des Wildes in Nordrhein-Westfalen.1. Der Endoparasitenbefall des Rehwildes. Z Jagdwiss. 2000;46:248–69.Google Scholar
- Czajka C, Becker N, Poppert S, Jöst H, Schmidt-Chanasit J, Andreas KA. Molecular detection of Setaria tundra (Nematoda: Filarioidea) and an unidentified filarial species in mosquitoes in Germany. Parasit Vectors. 2012;5, e14.View ArticleGoogle Scholar
- Yanchev Y. The helminth fauna of roe deer (Capreolus capreolus) in Bulgaria. 3. Material on helminth fauna in roe deer (Capreolus capreolus L.) in the mountains of southern Bulgaria. Izv Tsentr Khelmintol Lab. 1973;16:205–20 (In Bulgarian).Google Scholar
- Rehbinder C, Christensson D, Glatthard V. Parasitic granulomas in reindeer. A histopathological, parasitological and bacteriological study. Nord Vet Med. 1975;27:499–507.PubMedGoogle Scholar
- Kummeneje K. Diseases in reindeer in northern Norway. In: Proceedings of the Symposium of the Second International Reindeer – Caribou Symposium in Roros 17–21 September Part B, Norway. 1979. 1980. p. 456–8.Google Scholar
- Shagraev MA, Zhaltsanova DSD. Fauna I resursy pozvonochnykh basseina ozera Baikal. In: A study of the helminth fauna of domestic deer in the Buryat ASSR. 1980. p. 128–30. In Russian with English abstract.Google Scholar
- Favia G, Cancrini G, Ferroglio E, Casiraghi M, Ricci I, Rossi L. Molecular assays for the identification of Setaria tundra. Vet Parasitol. 2003;117:139–45.View ArticlePubMedGoogle Scholar
- Kowal J, Kornaś S, Nosal P, Basiaga M, Lesiak M. Setaria tundra in roe deer (Capreolus capreolus) new findings in Poland. Ann Parasitol. 2013;59:179–82.PubMedGoogle Scholar
- Laaksonen S, Solismaa M, Kortet R, Kuusela J, Oksanen A. Vectors and transmission dynamics for Setaria tundra (Filarioidea; Onchocercidae), a parasite of reindeer in Finland. Parasit Vectors. 2009;2, e3.View ArticleGoogle Scholar
- Anderson JR, Nilssen A. Do reindeer aggregate on snow patches to reduce harassment by parasitic flies or thermoregulate? Rangifer. 2008;18:3–17.View ArticleGoogle Scholar
- Nikander S, Laaksonen S, Saari S, Oksanen A. The morphology of the filaroid nematode Setaria tundra, the cause of peritonitis in reindeer Rangifer tarandus. J Helminth. 2007;81:49–55.View ArticlePubMedGoogle Scholar
- Casiraghi M, Anderson TJC, Bandi C, Bazzocchi C, Genchi C. A phylogenetic analysis of filarial nematodes: comparison with the phylogeny of Wolbachia endosymbionts. Parasitology. 2001;122:93–103.View ArticlePubMedGoogle Scholar
- Gouy M, Guindon S, Gascuel O. SeaView version 4. A multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol Phylogenet Evol. 2010;27:221–4.View ArticleGoogle Scholar
- Larkin MA, Backshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. Clustal W and Clustal X version 2.0. Bioinfomatics Application Note. 2007;23:2947–8.View ArticleGoogle Scholar
- Posada D. jModelTest: Phylogenetic model averaging. Mol Biol Evol. 2008;25:1253–6.View ArticlePubMedGoogle Scholar
- Ronquist F, Huelsenbeck JP. MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics. 2003;19:1572–4.View ArticlePubMedGoogle Scholar
- Rambaut A, Drummond AJ. Tracer v1.5. Available at: http://beast.bio.ed.ac.uk/Tracer 2007
- Silvestro D, Michalak I. A user friendly graphical front-end for phylogenetic analyses using RAxML (Stamatakis, 2006). Org Divers Evol. 2010;12:335–7.View ArticleGoogle Scholar
- Swofford DL. PAUP. Phylogenetic Analysis Using Parsimony (and Other Methods). Version 4. Sunderland: Sinauer Associates; 2002.Google Scholar
- Miller MA, Pfeiffer W, Schwartz T. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. New Orleans: Proceedings of the Gateway Computing Environments Workshop (GCE); 2010. p. 1–8.Google Scholar
- Buttner K. A study on parasites of roe deer in Steigerwald. Waldhygiene. 1975;11:83–100.Google Scholar
- Laaksonen S, Solismaa M, Orro T, Kuusela J, Saari S, Kortet R, et al. A: Setaria tundra microfilariae in reindeer and other cervids in Finland. Parsitol Res. 2009;104:257–65.View ArticleGoogle Scholar
- Laaksonen S, Oksanen S. Status and review of the vector-borne nematode Setaria tundra in Finnish cervids. Alces. 2009;45:81–4.Google Scholar
- Kumpula J, Colpaert A, Nieminen M. Suomen poronhoitoalueen kesälaidunvarat. The summer pasture recourses of the Finnish reindeer management area. In: Kala-ja riistaraportteja nro 152. Kaamanen: Riistan-ja kalantutkimus; 1999. p. 40–6. In Finnish, English abstract.Google Scholar
- Rehbinder C. Some vector borne parasites in Swedish reindeer (Rangifer tarandus tarandus). Rangifer. 1990;10:67–73.View ArticleGoogle Scholar