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Circulation of Dirofilaria repens, Setaria tundra, and Onchocercidae species in Hungary during the period 2011–2013
Veterinary Parasitology 214 (2015) 108–113
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Circulation of Dirofilaria repens, Setaria tundra, and Onchocercidae species in Hungary during the period 2011–2013 Gábor Kemenesi a,b , Kornélia Kurucz b , Anett Kepner a,b , Bianka Dallos a,b , Miklós Oldal a,b , Róbert Herczeg c , Péter Vajdovics d , Krisztián Bányai e , Ferenc Jakab a,b,∗ a
Virological Research Group, Szentágothai Research Centre, University of Pécs, Pécs, Hungary Institute of Biology, Faculty of Sciences, University of Pécs, Pécs, Hungary c Seqomics Ltd., Szeged, Hungary d Department of Clinical Pathology and Oncology, Faculty of Veterinary Science, Szent István University, Budapest, Hungary e Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Hungary b
a r t i c l e
i n f o
Article history: Received 19 May 2015 Received in revised form 28 August 2015 Accepted 5 September 2015 Keywords: Mosquito Dirofilaria repens Setaria tundra Filariosis Onchocercidae sp. Surveillance Dirofilaria immitis
a b s t r a c t Dirofilaria repens and recently Dirofilaria immitis are known to be endemic in Hungary. Since there is no related research on Dirofilaria carrier mosquito species from Hungary, we conducted a three-year mosquito surveillance study between 2011 and 2013. During the study period we examined 23,139 female mosquitoes with a generic filaria-specific TaqMan PCR assay, and characterized them by sequencing a 500 bp segment of 12S rRNA. An important result of our study was the detection of Setaria tundra and D. repens along with an unidentified Onchocercidae nematode. D. repens is known to be endemic in Hungary, however, the detection of S. tundra in all sampling sites throughout the study period indicates for the first time the endemicity of this parasite in Hungary. The Onchocercidae sp. nematode showed 95% nucleotide identity with previously detected unidentified nematodes from Germany, indicating a broader geographical distribution of this nematode in Europe. D. immitis specific DNA was not detected among the screened mosquitoes in this study. Here we report 11 mosquito species as potential vector organisms for local filarial infections, including Aedes vexans, Ochlerotatus annulipes, Ochlerotatus sticticus, Coquillettidia richiardii, Anopheles hyrcanus and Ochlerotatus rusticus. Dirofilaria development unit was calculated and the potential transmission period was estimated, which ranged between 65 and 113 days between sampling seasons. A relatively high infection rate (36.8%) was identified, which is a notable finding for veterinary and human health professionals. Moreover, the results of our study widen the group of possible mosquito vector species for D. repens and S. tundra in Central Europe. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Filarial species of the order Spirurida represent a serious healthcare problem worldwide, mainly in the tropical and Mediterranean areas (Morales-Hojas, 2009). Lymphatic filariosis caused by Wuchereria bancrofti and onchocercosis (Onchocerca volvulus) continue to cause serious diseases in many parts of the world, with approximately 40 million people infected from 73 countries in South Asia and Africa (Simonsen et al., 2009). Dirofilaria species are considered as emerging zoonotic parasites in Europe. Their original distribution territory includes mainly the Mediterranean region
∗ Corresponding author at: Virological Research Group, Szentágothai Research Centre, University of Pécs, Ifjúság út 20., H-7624 Pécs, Hungary. E-mail address: [email protected] (F. Jakab). http://dx.doi.org/10.1016/j.vetpar.2015.09.010 0304-4017/© 2015 Elsevier B.V. All rights reserved.
(Morchón et al., 2007; Cancrini et al., 2007) and even further southern territories such as the Canary Islands (Morchón et al., 2011). The changing climatic conditions and other factors which facilitate the extrinsic incubation for Dirofilaria species extend the geographical area of these parasites towards Northern-Europe (Genchi et al., 2011). Dirofilaria repens and Dirofilaria immitis represent the most significant problem with both veterinary and human health relevance in Europe. Although dirofilariosis has been diagnosed in Hungary both in humans and dogs (Szénási et al., 2008; Fodor et al., 2009; Tolnai et al., 2014), data on natural carrier mosquito species are scanty (Zittra et al., 2015). D. repens is the causative agent of canine and feline subcutaneous and ocular filariasis. Furthermore it can also cause serious infection in humans as subcutaneous or subconjuctival nodules ´ (Tasic-Otaˇ sevic´ et al., 2015). The first autochthonous case of D. repens was reported in 1998 from Hungary in a dog (Fok et al., 1998).
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Table 1 List of mosquito species collected in Southwestern Hungary between 2011 and 2013, along with IR (infection rate) values, positive pools and the total number of mosquitoes tested. Values represent the whole sampling period. CI (confidence intervals 95%) IR. Species
IR
CI lower limit
CI upper limit
Number of pools
Positive pools
Number of specimens
Aedes cinereus Aedes rossicus Aedes vexans Anopheles algeriensis Anopheles claviger Anopheles hyrcanus Anopheles maculipennis Anopheles plumbeus Coquillettidia richiardii Culex martinii Culex modestus Culex pipiens Culex territans Culex torrentium Culiseta alaskaensis Ochlerotatus annulipes Ochlerotatus cantans Ochlerotatus dorsalis Ochlerotatus geniculatus Ochlerotatus pulcripalpis Ochlerotatus refiki Ochlerotatus rusticus Ochlerotatus sticticus Uranotaenia unguiculata
3.07 2.01 15.44 0.00 0.00 14.91 92.31 0.00 15.61 0.00 5.83 21.42 0.00 0.00 0.00 15.28 0.00 22.85 0.00 0.00 0.00 3.26 11.38 0.00
1.37 0.12 12.69 0.00 0.00 7.77 26.89 0.00 8.15 0.00 1.17 5.00 0.00 0.00 0.00 6.84 0.00 1.53 0.00 0.00 0.00 0.59 8.81 0.00
6.08 9.69 18.69 199.17 134.36 27.15 393.36 88.49 28.39 260.73 18.60 73.08 270.53 178.93 793.45 31.51 51.97 121.34 219.68 793.45 499.14 10.93 14.55 48.45
56 19 200 4 3 24 7 6 26 3 13 5 1 1 1 19 5 3 7 1 2 21 150 2
7 1 105 0 0 10 4 0 10 0 2 2 0 0 0 7 0 1 0 0 0 2 62 0
2399 484 9545 11 13 876 113 27 835 7 323 87 5 8 1 603 45 41 12 1 3 642 7024 34
Since its first detection, several human cases have been reported, a finding which not only reflects the importance of this parasite in veterinary health but also sheds light on the public health risks (Pampiglione et al., 1999; Elek et al., 2000; Pónyai et al., 2006; Szénási et al., 2008; Fodor et al., 2009). D. immitis as the causative agent of hearthworm filariosis is a major veterinary health threat in Europe. Since 2007, when the first autochthonous canine infection was confirmed, Hungary has been considered an endemic country for this parasite (Tolnai et al., 2014). Setaria tundra was recently detected in Hungary for the first time (Zittra et al., 2015). This parasite has a significant veterinary health importance in boreal regions of Europe as the causative agent of setariosis in cervids (Rangifer tarandus, Capreolus capreolus, Alces alces etc.) (Laaksonen et al., 2007). Its significance as a human parasite is still unknown. In Finland, S. tundra caused severe outbreaks among semi-domestic reindeer in the last couple of years (Laaksonen et al., 2009). As there
are no veterinary health data available about the Hungarian cases of S. tundra, the most efficient method for estimating the risk for infection is the screening of mosquito vector population. As a vector-borne infection, filarial parasites are transmitted by various haematophagous arthropod vectors, mostly mosquitoes (Culicidae) or arachnids (Acari) (Anderson, 2000). The main mosquito vector species in Europe belong to the Aedes, Anopheles, Ochlerotatus, and Culex genera (Czajka et al., 2012). Climatic and ecological factors may affect the life cycle of both the mosquito vector and the filarial parasites. Thus far a total of 50 mosquito species have been detected in Hungary, representing eight genera (Anopheles, Aedes, Ochlerotatus, Coquillettidia, Culex, Culiseta, Orthopodomyia, and Uranotaenia). Most frequent species are Ochlerotatus annulipes, Culex pipiens pipiens, Aedes vexans, Coquillettidia richiardii, Ochlerotatus sticticus, Culiseta annulata (Kenyeres and Tóth, 2012).
35.0
100 90
30.0
80 70 60
20.0
50 15.0
40 30
10.0
Infecon Rate
Temperature (Co)
25.0
Tmin (C°) Tmax (C°) IR
20 5.0
10
0.0
2011
July
June
May
July
2012
August
June
May
September
August
July
May
June
0
2013
Fig. 1. IR (Infection Rate) value changes in parallel with temperature data along the whole sampling period. IR value represents all mosquito species.
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Table 2 Positive mosquito species listed by detected filarial nematodes and the number of positive pools per sampling year. Sampling year
Parasite
Mosquito species
No. of positive pools
2011
Dirofilaria repens
Aedes vexans Ochlerotatus annulipes Aedes cinereus Ochlerotatus sticticus Coquillettidia richiardii Anopheles maculipennis Anopheles hyrcanus Aedes vexans Culex pipiens
39 4 6 8 4 3 6 5 1
Aedes vexans Ochlerotatus annulipes Aedes cinereus Ochlerotatus sticticus Coquillettidia richiardii Anopheles maculipennis Anopheles hyrcanus Culex modestus Aedes vexans Coquillettidia richiardii
47 2 1 37 5 1 3 1 2 1
Aedes vexans Ochlerotatus sticticus Ochlerotatus rusticus Ochlerotatus dorsalis Ochlerotatus annulipes Aedes rossicus
13 19 2 1 1 1
Setaria tundra Onchocercidae sp. 2012
Dirofilaria repens
Setaria tundra 2013
Dirofilaria repens
Setaria tundra
Although the transmission cycle of many filarial species (e.g. D. repens or D. immitis) are well described, the lack of sequence data and information on the competent vector species could lead to the detection of unidentified filarial species (Czajka et al., 2012). Therefore, novel molecular detection techniques are efficient and important tools for surveillance of vector-borne nematodes. The examination of the arthropod vectors are useful for measuring the risk for human and animal health in a territory and also for gaining insight on the role of different vector species in the transmission cycle of these parasites. Although, D. repens and also D. immitis are endemic in Hungary, there is only one recently published report on potential mosquito vector species (Zittra et al., 2015). Moreover, the recent detection of S. tundra and the rapid expansion of other filarial nematodes require more related studies in the future about the Hungarian mosquito fauna. To determine the transmission patterns of filarial nematodes in Southwestern Hungary, we retrospectively analyzed over twenty thousand female mosquitoes collected in the mosquito breeding season of three consecutive years as part of a national West Nile virus surveillance program. Additionally, our study represents the first dataset on filarial transmission dynamics from Hungary. 2. Methods 2.1. Mosquito collection Mosquitoes were collected with the human-landing collection method and updraft box traps equipped with white light attractant (Hall-Mendelin et al., 2010) during the mosquito breeding season (May–September) limited by field conditions (floods, impassable roads). Thus the sampling period for each year was as follows: May–September in 2011; May–August in 2012 and May–July in 2013. The study area included four sampling points within Baranya county, Southwest-Hungary: marshlands near the city of Pécs; bird ringing station in Sumony; dead channels of the Danube river near Mohács; floodplains of Drava river near Drávaszabolcs border crossing point. Mosquito identification was performed according to the taxonomic keys of Beckeret al., (2003). Positive mosquito pools were further verified to species level by barcoding (COI gene) as
described previously (Kumar et al., 2007). Specimens were grouped by species, collection site and date, and finally pooled with a maximum size of 50 individuals. After sample homogenization DNA was extracted with DiaExtract DNA Mini Kit (Diagon Ltd., Hungary) according to the manufacturer’s recommendations. 2.2. PCR screening, sequencing and data analyses The gereric filarial real-time PCR targeting a 94 bp long fragment of the 12S rRNA gene from the mitochondrial genome was performed using the primers FILA-F, FILA-R and probe FILA-P as previously described by Czajka et al. (2012). TaqMan PCR assay was performed with GoTaq® G2 Flexi DNA Polymerase PCR kit (Promega) according to the manufacturer’s protocol on LineGene 9600 platform (Bioer). Real-time PCR-positive samples were further examined by a conventional PCR targeting the 500 bp long fragment of 12S rRNA (ribosomal RNA) (Krueger et al., 2007). After optimization, the annealing temperature for PCR was modified to 55 ◦ C. PCR products were purified with Gel/PCR DNA Fragments Extraction Kit (Geneaid) and were bidirectionally sequenced (BigDye Terminator v1.1Cycle Sequencing Kit, ABI Prism 310 DNA Sequencer-Applied Biosystems). Nucleic acid sequences of the detected filarial nematodes were identified preliminarily by GenBank BLAST searches based on the greatest homology. Representative sample sequences were deposited in the GenBank (KR676613, KR676614, and KR676615). 2.3. Statistical analysis Differences in mosquito abundance among species were analyzed using linear model (lm) implemented in the ‘nlme’ package for R (Pinheiro et al., 2014), performed with R 3.1.0. software (R Development Core Team 2014). The infection rate (IR) of mosquitoes was estimated by the Mosquito Surveillance Software of CDC (CDC, 2013). Dirofilaria Development Units (DDU) was calculated as described previously (Fortin and Slocombe, 1981). For estimating the hypothetical period in which infective mosquito vectors may occure, DDU30 was also calculated as described elsewhere (Ferreira et al., 2015).
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111
96 KM205377 Dirofilaria repens 67
KC953031 D. repens
99
D. repens Hungary GQ292761 D. repens EU182328 Dirofilaria immitis
54
AB518879 Onchocerca sp. AM779812 Onchocerca suzukii DQ523747 Onchocerca flexuosa
88
DQ523745 Onchocerca jakutensis 54
DQ523737 Onchocerca ramachandrini AJ544841 Foleyella furcata AM779794 Cercopithifilaria japonica FR827903 Aproctella sp. HM773029 Chandlerella quiscali JX870434 Filaroidea sp. Onchocercidae sp. Hungary
58
JN228381 Onchocercidae sp.
100 99
JN228379 Onchocercidae sp.
77 JN228380 Onchocercidae sp.
AJ544843 Brugia malayi AJ544835 Setaria equina
54
JN228376 Setaria tundra
57 100
S. tundra Hungary AM779828 S. tundra AJ544855 Filaria martes
0.02
Fig. 2. Phylogenetic classification of Setaria tundra, Dirofilaria repens and Onchocercidae sp. samples compared with other filarial nematode species. Taxon identification tree based on partial 400 bp long fragment of 12S rRNA sequences (neighbor-joining algorithm with Maximum Composite Likelihood parameter, bootstrap replication value was 1000). Representative samples derived from our study are marked with a black triangle (KR676613, KR676614, and KR676615).
Regional meteorological data was collected from the National Meteorological database (NMD, 2015). 2.4. Phylogenetic analyses Phylogenetic characterization was carried out by using cognate sequences available in public databases. Basic sequence manipulation and verification were performed using GeneDoc v2.7 software and nucleotide sequences were aligned by ClustalX v2.0 software. Phylogenetic trees were constructed with MEGA v6.0 software using neighbor-joining algorithm with Maximum Composite Likelihood parameter model based on nucleic acid sequences of a 400 bp-long region of 12S mRNA gene. The number of bootstrap replications was 1000. 3. Results A total of 23,139 female mosquitoes were collected during the mosquito breeding seasons between 2011 and 2013. Twenty-four mosquito species belonging to the genera Aedes, Anopheles, Coquillettidia, Culex, Ochlerotatus and Uranotaenia have been collected and tested in this study. The most frequent species was A. vexans (n = 9545) followed by O. sticticus (n = 7024) and A. cinereus (n = 2399). Relative abundance of mosquito species differed significantly (F28,559 = 22,62, p < 0.001). Five of the most abundant
mosquito species with the highest IR value were: A. vexans, A. hyrcanus, C. richiardii, O. annulipes and O. sticticus. A noticeable decrease of average IR value in august along the whole study was observed. All collected mosquito species along with IR values are shown in Table 1. Variations of IR depending on temperature value changes along the whole sampling period are shown in Fig. 1. The overall infection rate regarding all tested nematode species among all samples for the three-year sampling period was 36.8% with 213 positive pools out of the 579 tested (Table 2). The DNA of D. repens, S. tundra, has been detected along with the DNA of an unidentified nematode with high similarity to a previously published Onchocercidae sp. nematode from Germany (Genbank JN228379–JN228381). D. repens was detected in all four sampling locations during the whole study period. Altogether 198 pools were infected with D. repens, which is 92.9% of all positive pools. D. repens has been detected in eight mosquito species (A. vexans, A. cinereus, A. hyrcanus, A. maculipennis, C. richiardii, C. modestus, O. sticticus, O. dorsalis). S. tundra has been detected in all sampling years during the study. A total of 10 pools from four mosquito species (A. vexans, A. rossicus, C. richiardii, O. sticticus) were positive, each one being potential vectors for S. tundra. The unidentified Onchocercidae filaria has been detected in a single pool of C. pipiens, collected in June of 2011. D. immitis was not detected during this study. The calculation of the DDU30 for the studied periods showed that there were at least 113 days in 2011, 92 days in 2012 and 65 days
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in 2013 with suitable conditions for the completion of the extrinsic development of D. repens., and consequently, for its transmission to the vertebrate hosts. According to the phylogenetic tree, novel D. repens sequence clustered with other D. repens sequences from Italy and Russia. Moreover, S. tundra also grouped together with related S. tundra sequences from Europe. The unidentified Onchocercidae sp. sequence clustered with related sequences from Germany (Fig. 2).
4. Discussion The study reported here provides the first large-scale surveillance data from Hungary regarding the detection of multiple filarial nematode species from various mosquito species. Results may indicate several mosquito species as potential vector organisms for D. repens and S. tundra, but further studies are necessary to confirm their role as competent vector organisms. Interestingly, D. immitis was not identified in the study area. This is particularly interesting in light of recent findings that demonstrated the occurrence of this parasite in O. caspius mosquitoes from South Hungary (Zittra et al., 2015). Since Hungary is an endemic region for the parasite (Tolnai et al., 2014), further investigations are needed to estimate the risk in Southwest Hungary. The most frequent filarial parasite was D. repens, representing the majority of the positive samples (n = 202). The human and veterinarian health risk posed by this parasite is well known with confirmed clinical cases documented in Hungary (Pampiglione et al., 1999; Elek et al., 2000; Pónyai et al., 2006; Szénási et al., 2008; Fodor et al., 2009). The high prevalence of D. repens in the area could enhance the risk for the exportation of the disease via pet dogs to previously not endemic regions, which scenario has been described before (Pantchev et al., 2011; Sævik et al., 2014). The expanding range of wild carnivores, such as golden jackal may also facilitate the persistence of D. repens and could naturally widen the distribution area of the parasite (Tóth et al., 2007). Human cases are also known from Hungary and other D. repens endemic regions, which occasionally could lead to serious infections for instance with ocular, subcutaneous or even lymphatic localization (Szénási et al., 2008). Seroepidemiological studies are needed to estimate the impact of the parasite on local human population as well as in herds of domestic livestock, pets and potential wild animal reservoirs. Previously, S. tundra was identified (Zittra et al., 2015) in Szeged, therefore the second detection of this parasite in Hungary has confirmed its wider distribution area in the country. Furthermore, its emergence in Hungary may indicate a spread to southern areas from the Scandinavian region, where this parasite is endemic and poses veterinary health threat (Laaksonen et al., 2007, 2009). Detailed studies regarding its abundance and distribution in areas south of Scandinavia are missing. Although S. tundra has been described in Germany, Poland and Slovakia (Kronefeld et al., 2014; Czajka et al., 2012; Kowal et al., 2013; Rudolf et al., 2014), the information on veterinary importance for these countries is unknown. The recent detection of this parasite in Hungary (Zittra et al., 2015) along with the result of this study added Hungary to the list of potentially endemic countries. Identification of S. tundra in Ae. vexans mosquitoes suggests the potential role of this species as a vector for setariosis in the Central European region (Rudolf et al., 2014). Additionally, positive mosquito pools (A. rossicus, C. richiardii and O. annulipes) further widen the group of potential vector species in Hungary. Since we successfully confirmed the presence of S. tundra in all sampling seasons, it implies the endemic nature of the parasite in Hungary and extends its estimated geographic distribution in Europe. Furthermore, an unidentified filarial species was detected with high similarity to a previously described filarial nematode from C. pipiens mosquitoes in Germany (Czajka et al.,
2012). Although the recent study by Czajka et al. (2012) suggests an avian origin for this parasite, this study can only confirm the wider distribution of this undefined parasite in Europe. Since there is a notable lack of scientific data about the avian nematode infections in Hungary, it narrows the possibility to estimate potential vertebrate host organisms. Based on the feeding preferences of C. pipiens (Rizzoli et al., 2015) this study support the previous hypothesis surmised by Czajka et al. (2012) on the avian origin. A BLAST search of GenBank database retrieves the highest nucleotide sequence identity (86%) with Onchocerca suzukii parasite. Based on the phylogenetic tree, the novel Hungarian sequence cannot be assigned to any of the known species, moreover clustered separately from O. suzukii. Further examinations, involving potential vertebrate hosts and morphological identification are needed to reveal the origin of this unidentified parasite. We revealed a notable decrease in IR values in both sampling months of august during the study period which was followed by an increase in september. Unfortunately, unequal sampling periods between sampling years do not provide sufficient data. The temperature limitation for development of L3 larvae in mosquitoes could be an explanation for the above mentioned IR value decrease. Since the weather data is regional and the local microclimatic conditions can differ, the exact limiting temperature was unmeasurable for evaluating the possible temperature limit which may endorse the abortation of L3 larvae stage development. Based on these results, mosquito surveillance could be an efficient method for measuring the persistence of these parasites in nature. The disadvantage of mosquito surveillance is the lack of information on local vertebrate hosts, hence with other study design; blood meal analyses of mosquitoes could be feasible. Therefore we can only assume the vertebrate hosts based on local wildlife data. Main vertebrate hosts for D. repens are dogs, cats and other wild carnivores such as red fox (Vulpes vulpes) or golden jackal (Canis aureus) which are all present in the sampling area. In the case of S. tundra, the common roe-deer (C. capreolus), red-deer (Cervus elaphus) and fallow-deer (Dama dama) are presented locally with known susceptibility for the parasite (IWC, 2015). Since the identification of the unidentified Onchocercidae species failed, the potential vertebrate hosts can only be speculated. A clear feeding preference of C. pipiens for the common blackbird (Turdus merula) was recently demonstrated in Italy (Rizzoli et al., 2015). Considering T. merula as the most abundant species in rural and also in suburban territories at the study area (Kurucz et al., 2012), assume this bird as a potential host animal. These results are also remarkable in mosquito control aspects, since several different mosquito species were tested positive. Further vector competence studies are needed. The fluctuating water level at all sampling sites assists the breeding of species preferring inundated breeding areas (e.g. Ae. vexans and O. sticticus as the most abundant species). These species are able to migrate up to 15 km (approximately 1 km/night), therefore posing a potential risk of disease transmission at the inhabited areas (Becker et al., 2003). Hence the expanding of mosquito control areas may be relevant near human inhabited areas in order to lower the risk of filarial infections. To our knowledge, the current study is the first large-scale PCR screening of filarial nematodes in a variety of mosquito species from Hungary. Although several potential mosquito vector species have been described, vector competence studies are needed in the future. Furthermore, the possible endemicity of S. tundra has been suggested, which may potentially affect the health of local wildlife populations. The high prevalence of these parasites is also notable for local clinicians and veterinary professionals, since increased attention should be taken for filarial disease symptoms.
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Acknowledgements Research activity of Gábor Kemenesi and Ferenc Jakab was supported by the TÁMOP 4.2.4. A/2-11-1-2012 0001—National Excellence Program Elaborating and operating an inland student and researcher personal support system. The project was subsidized by the European Union and co-financed by the European Social Fund. Krisztián Bányai was supported by the “Momentum program”. References Anderson, R.C., 2000. Nematode Parasites of Vertebrates: Their Development and Transmission. CABI Publishing, Wallingford. Becker, N., Petric, D., Zgomba, M., Boase, C., Madon, M., Dahl, C., Kaiser, A., 2003. Mosquitoes and Their Control. Kluwer Academic/Plenum Publisher, New York. Cancrini, G., Scaramozzino, P., Gabrielli, S., Di Paolo, M., Toma, L., Romi, R., 2007. Aedes albopictus and Culex pipiens implicated as natural vectors of Dirofilaria repens in central Italy. J. Med. Entomol. 44, 1064–1066. CDC, (Centers for Disease Control and Prevention) 2013. http://www.cdc.gov/ westnile/resourcepages/mosqSurvSoft.html. Czajka, C., Becker, N., Poppert, S., Jöst, H., Schmidt-Chanasit, J., Krüger, A., 2012. Molecular detection of Setaria tundra (Nematoda: Filarioidea) and an unidentified filarial species in mosquitoes in Germany. Parasit. Vectors 11 (5), 14. Elek, G., Minik, K., Pajor, L., Parlagi, G., Varga, I., Vetési, F., Zombori, J., 2000. New human Dirofilarioses in Hungary. Pathol. Oncol. Res. 6, 141–145. Ferreira, C.A., de Pinho Mixão, V., Novo, M.T., Calado, M.M., Gonc¸alves, L.A., Belo, S.M., de Almeida, A.P., 2015. First molecular identification of mosquito vectors of Dirofilaria immitis in continental Portugal. Parasit. Vectors 8, 139. Fodor, E., Fok, E., Maka, E., Lukáts, O., Tóth, J., 2009. Recently recognized cases of ophthalmofilariasis in Hungary. Eur. J. Ophthalmol. 19, 675–678. Fok, É., Szabó, Z., Farkas, R., 1998. Dirofilaria repens fert"ozöttség els"o hazai diagnosztizálása kutyában sebészeti beavatkozás során. Kisállatorvoslás. 4, 218–219. Fortin, J.F., Slocombe, J.O.D., 1981. Temperature requirements for the development of Dirofilaria immitis in Aedes triseriatus and Ae. vexans. Mosq. News 41, 625–633. Genchi, C., Kramer, L.H., Rivasi, F., 2011. Dirofilarial infections in Europe. Vector Borne Zoonotic Dis. 11, 1307–1317. Hall-Mendelin, S., Ritchie, S.A., Johansen, C.A., Zborowski, P., Cortise, G., Dandridgef, S., Halla, R.A., Hurk, A.F., 2010. Exploiting mosquito sugar feeding to detect mosquito-borne pathogens. PNAS 107, 11255–11259. IWC (Institute for Wildlife Conservation), National Game Management Database, Hungary. http://www.ova.info.hu/. Kenyeres, Z., Tóth, S., 2012. Landscape-structure determined mosquito diversity in Hungary (Central Europe). J. Mosq. Res. 5, 32–38. Kowal, J., Korna´s, S., Nosal, P., Basiaga, M., Lesiak, M., 2013. Setaria tundra in roe deer (C. capreolus)—new findings in Poland. Ann. Parasitol. 59, 179–182. Kronefeld, M., Kampen, H., Sassnau, R., Werner, D., 2014. Molecular detection of Dirofilaria immitis, Dirofilaria repens and Setaria tundra in mosquitoes from Germany. Parasit. Vectors 16 (7), 30. Krueger, A., Fischer, P., Morales-Hojas, R., 2007. Molecular phylogeny of the filaria genus Onchocerca with special emphasis on Afrotropical human and bovine parasites. Acta Trop. 101, 1–14. Kumar, N.P., Rajavel, A.R., Natarajan, R., Jambulingam, P., 2007. DNA barcodes can distinguish species of Indian mosquitoes (Diptera: Culicidae). J. Med. Entomol. 44, 1–7. Kurucz, K., Bertalan, L., Purger, J.J., 2012. Survival of blackbird (Turdus merula) clutches in an urban environment: experiment with real and artificial nests North-Western. J. Zoology 8, 362–364.
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Laaksonen, S., Kuusela, J., Nikander, S., Nylund, M., Oksanen, A., 2007. Outbreak of parasitic peritonitis in reindeer in Finland. Vet. Rec. 160, 835–841. Laaksonen, S., Solismaa, M., Kortet, R., Kuusela, J., Oksanen, A., 2009. Vectors and transmission dynamics for Setaria tundra (Filarioidea; Onchocercidae), a parasite of reindeer in Finland. Parasit. Vectors 6 (2), 3. Morales-Hojas, R., 2009. Molecular systematics of filarial parasites, with an emphasis on groups of medical and veterinary importance, and its relevance for epidemiology. Infect. Genet. Evol. 9, 748–759. Morchón, R., Bargues, M.D., Latorre, J.M., Melero-Alcíbar, R., Pou-Barreto, C., Mas-Coma, S., Simón, F., 2007. Haplotype H1 of Culex pipiens implicated as natural vector of Dirofilaria immitis in an endemic area of Western Spain. Vector Borne Zoonotic Dis. 7, 653–658. Morchón, R., Bargues, M.D., Latorre-Estivalis, J.M., Pou-Barreto, C., Melero-Alcibar, R., Moreno, M., Valladares, B., Molina, R., Montoya-Alonso, J.A., Mass-Coma, S., Simón, F., 2011. Molecular characterization of Culex Theileri from Canary Islands Spain, a potential vector of Dirofilaria Immitis. J. Clinic. Experiment. Pathol. S3, 001. NMD (National Meteorological Database), Hungary http://www.metnet.hu/. Pampiglione, S., Elek, G., Pálfi, P., Vetési, F., Varga, I., 1999. Human Dirofilaria repens infection in Hungary: a case in the spermatic cord and a review of the literature. Acta. Vet. Hung. 47, 77–83. Pantchev, N., Etzold, M., Daugschies, A., Dyachenko, V., 2011. Diagnosis of imported canine filarial infections in Germany 2008–2010. Parasitol. Res. 109, 61–76. Pinheiro, J., Bates, D., DebRoy, S., Sarkar, D., R Core Team., 2014. nlme:Linear and Nonlinear Mixed Effects Models. R package version 3. 1-117,URL: http://CRAN. R-project.org/package=nlme. Pónyai, K., Wikonkál, N., Bottlik, G., Hársing, J., Kucsera, I., Horváth, A., Kárpáti, S., 2006. Dirofilaria repens infection case in Hungary: a case report. J. Dtsch. Dermatol. Ges. 4, 1051–1053. R Development Core Team, 2014. A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria http:// www.R-project.org/. Rizzoli, A., Bolzoni, L., Chadwick, E.A., Capelli, G., Montarsi, F., Grisenti, M., Puente, ˜ J., Figuerola, J., Soriguer, R., Anfora, G., Di Luca, M., Rosà, R., 2015. J.M., Munoz, Understanding West Nile virus ecology in Europe: Culex pipiens host feeding preference in a hotspot of virus emergence. Parasit. Vectors 8 (1), 213. ˇ Rudolf, I., Sebesta, O., Mendel, J., Betáˇsová, L., Bocková, E., Jedliˇcková, P., Venclíková, ˇ S., Hubálek, Z., 2014. Zoonotic Dirofilaria repens K., Blaˇzejová, H., Sikutová, (Nematoda: Filarioidea) in Aedes vexans mosquitoes, Czech Republic. Parasitol. Res. 113, 4663–4667. Sævik, B.K., Jörundsson, E., Stachurska-Hagen, T., Tysnes, K., Brun-Hansen, H., Wikström, H.C., Robertson, L.J., 2014. Dirofilaria repens infection in a dog imported to Norway. Acta. Vet Scand. 21, 6. Simonsen, P.E., Filariases, Cook, G.C., Zumla, A.I. (Eds.), 2009. Elsevier, China, p. 1477. Szénási, Z., Kovács, A.H., Pampiglione, S., Fioravanti, M.L., Kucsera, I., Tánczos, B., Tiszlavicz, L., 2008. Human dirofilariosis in Hungary: an emerging zoonosis in central Europe. Wien. Klin. Wochenschr. 120, 96–102. ´ ´ S.A., Trenkic-Boˇ ´ ´ M.S., Gabrielli, S.V., Genchi, C., 2015. Canine Tasic-Otaˇ sevic, zinovic, and human Dirofilaria infections in the Balkan Peninsula, Vet. Parasitol., pii: S0304-4017(15) 72-2. Tolnai, Z., Széll, Z., Sproch, Á., Szeredi, L., Sréter, T., 2014. Dirofilaria immitis: an emerging parasite in dogs: red foxes and golden jackals in Hungary. Vet. Parasitol. 14, 339–342. Tóth, T., Krecsák, L., Sz"ucs, E., Heltai, M., Huszár, Gy., 2009. Records of the golden jackal (Canis aureus Linnaeus, 1758) in Hungary from 1800 until 2007, based on a literature survey. North-Western J. Zool. 5, 357–363. Zittra, C., Kocziha, Z., Pinnyei, S., Harl, J., Kieser, K., Laciny, A., Eigner, B., Silbermayr, K., Duscher, G.G., Fok, É., Fuehrer, H.P., 2015. Screening blood-fed mosquitoes for the diagnosis of filarioid helminths and avian malaria. Parasit. Vectors. 13 (8(1)), 16.
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Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Circulation of Dirofilaria repens, Setaria tundra, and Onchocercidae species in Hungary during the period 2011–2013 Gábor Kemenesi a,b , Kornélia Kurucz b , Anett Kepner a,b , Bianka Dallos a,b , Miklós Oldal a,b , Róbert Herczeg c , Péter Vajdovics d , Krisztián Bányai e , Ferenc Jakab a,b,∗ a
Virological Research Group, Szentágothai Research Centre, University of Pécs, Pécs, Hungary Institute of Biology, Faculty of Sciences, University of Pécs, Pécs, Hungary c Seqomics Ltd., Szeged, Hungary d Department of Clinical Pathology and Oncology, Faculty of Veterinary Science, Szent István University, Budapest, Hungary e Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Hungary b
a r t i c l e
i n f o
Article history: Received 19 May 2015 Received in revised form 28 August 2015 Accepted 5 September 2015 Keywords: Mosquito Dirofilaria repens Setaria tundra Filariosis Onchocercidae sp. Surveillance Dirofilaria immitis
a b s t r a c t Dirofilaria repens and recently Dirofilaria immitis are known to be endemic in Hungary. Since there is no related research on Dirofilaria carrier mosquito species from Hungary, we conducted a three-year mosquito surveillance study between 2011 and 2013. During the study period we examined 23,139 female mosquitoes with a generic filaria-specific TaqMan PCR assay, and characterized them by sequencing a 500 bp segment of 12S rRNA. An important result of our study was the detection of Setaria tundra and D. repens along with an unidentified Onchocercidae nematode. D. repens is known to be endemic in Hungary, however, the detection of S. tundra in all sampling sites throughout the study period indicates for the first time the endemicity of this parasite in Hungary. The Onchocercidae sp. nematode showed 95% nucleotide identity with previously detected unidentified nematodes from Germany, indicating a broader geographical distribution of this nematode in Europe. D. immitis specific DNA was not detected among the screened mosquitoes in this study. Here we report 11 mosquito species as potential vector organisms for local filarial infections, including Aedes vexans, Ochlerotatus annulipes, Ochlerotatus sticticus, Coquillettidia richiardii, Anopheles hyrcanus and Ochlerotatus rusticus. Dirofilaria development unit was calculated and the potential transmission period was estimated, which ranged between 65 and 113 days between sampling seasons. A relatively high infection rate (36.8%) was identified, which is a notable finding for veterinary and human health professionals. Moreover, the results of our study widen the group of possible mosquito vector species for D. repens and S. tundra in Central Europe. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Filarial species of the order Spirurida represent a serious healthcare problem worldwide, mainly in the tropical and Mediterranean areas (Morales-Hojas, 2009). Lymphatic filariosis caused by Wuchereria bancrofti and onchocercosis (Onchocerca volvulus) continue to cause serious diseases in many parts of the world, with approximately 40 million people infected from 73 countries in South Asia and Africa (Simonsen et al., 2009). Dirofilaria species are considered as emerging zoonotic parasites in Europe. Their original distribution territory includes mainly the Mediterranean region
∗ Corresponding author at: Virological Research Group, Szentágothai Research Centre, University of Pécs, Ifjúság út 20., H-7624 Pécs, Hungary. E-mail address: [email protected] (F. Jakab). http://dx.doi.org/10.1016/j.vetpar.2015.09.010 0304-4017/© 2015 Elsevier B.V. All rights reserved.
(Morchón et al., 2007; Cancrini et al., 2007) and even further southern territories such as the Canary Islands (Morchón et al., 2011). The changing climatic conditions and other factors which facilitate the extrinsic incubation for Dirofilaria species extend the geographical area of these parasites towards Northern-Europe (Genchi et al., 2011). Dirofilaria repens and Dirofilaria immitis represent the most significant problem with both veterinary and human health relevance in Europe. Although dirofilariosis has been diagnosed in Hungary both in humans and dogs (Szénási et al., 2008; Fodor et al., 2009; Tolnai et al., 2014), data on natural carrier mosquito species are scanty (Zittra et al., 2015). D. repens is the causative agent of canine and feline subcutaneous and ocular filariasis. Furthermore it can also cause serious infection in humans as subcutaneous or subconjuctival nodules ´ (Tasic-Otaˇ sevic´ et al., 2015). The first autochthonous case of D. repens was reported in 1998 from Hungary in a dog (Fok et al., 1998).
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Table 1 List of mosquito species collected in Southwestern Hungary between 2011 and 2013, along with IR (infection rate) values, positive pools and the total number of mosquitoes tested. Values represent the whole sampling period. CI (confidence intervals 95%) IR. Species
IR
CI lower limit
CI upper limit
Number of pools
Positive pools
Number of specimens
Aedes cinereus Aedes rossicus Aedes vexans Anopheles algeriensis Anopheles claviger Anopheles hyrcanus Anopheles maculipennis Anopheles plumbeus Coquillettidia richiardii Culex martinii Culex modestus Culex pipiens Culex territans Culex torrentium Culiseta alaskaensis Ochlerotatus annulipes Ochlerotatus cantans Ochlerotatus dorsalis Ochlerotatus geniculatus Ochlerotatus pulcripalpis Ochlerotatus refiki Ochlerotatus rusticus Ochlerotatus sticticus Uranotaenia unguiculata
3.07 2.01 15.44 0.00 0.00 14.91 92.31 0.00 15.61 0.00 5.83 21.42 0.00 0.00 0.00 15.28 0.00 22.85 0.00 0.00 0.00 3.26 11.38 0.00
1.37 0.12 12.69 0.00 0.00 7.77 26.89 0.00 8.15 0.00 1.17 5.00 0.00 0.00 0.00 6.84 0.00 1.53 0.00 0.00 0.00 0.59 8.81 0.00
6.08 9.69 18.69 199.17 134.36 27.15 393.36 88.49 28.39 260.73 18.60 73.08 270.53 178.93 793.45 31.51 51.97 121.34 219.68 793.45 499.14 10.93 14.55 48.45
56 19 200 4 3 24 7 6 26 3 13 5 1 1 1 19 5 3 7 1 2 21 150 2
7 1 105 0 0 10 4 0 10 0 2 2 0 0 0 7 0 1 0 0 0 2 62 0
2399 484 9545 11 13 876 113 27 835 7 323 87 5 8 1 603 45 41 12 1 3 642 7024 34
Since its first detection, several human cases have been reported, a finding which not only reflects the importance of this parasite in veterinary health but also sheds light on the public health risks (Pampiglione et al., 1999; Elek et al., 2000; Pónyai et al., 2006; Szénási et al., 2008; Fodor et al., 2009). D. immitis as the causative agent of hearthworm filariosis is a major veterinary health threat in Europe. Since 2007, when the first autochthonous canine infection was confirmed, Hungary has been considered an endemic country for this parasite (Tolnai et al., 2014). Setaria tundra was recently detected in Hungary for the first time (Zittra et al., 2015). This parasite has a significant veterinary health importance in boreal regions of Europe as the causative agent of setariosis in cervids (Rangifer tarandus, Capreolus capreolus, Alces alces etc.) (Laaksonen et al., 2007). Its significance as a human parasite is still unknown. In Finland, S. tundra caused severe outbreaks among semi-domestic reindeer in the last couple of years (Laaksonen et al., 2009). As there
are no veterinary health data available about the Hungarian cases of S. tundra, the most efficient method for estimating the risk for infection is the screening of mosquito vector population. As a vector-borne infection, filarial parasites are transmitted by various haematophagous arthropod vectors, mostly mosquitoes (Culicidae) or arachnids (Acari) (Anderson, 2000). The main mosquito vector species in Europe belong to the Aedes, Anopheles, Ochlerotatus, and Culex genera (Czajka et al., 2012). Climatic and ecological factors may affect the life cycle of both the mosquito vector and the filarial parasites. Thus far a total of 50 mosquito species have been detected in Hungary, representing eight genera (Anopheles, Aedes, Ochlerotatus, Coquillettidia, Culex, Culiseta, Orthopodomyia, and Uranotaenia). Most frequent species are Ochlerotatus annulipes, Culex pipiens pipiens, Aedes vexans, Coquillettidia richiardii, Ochlerotatus sticticus, Culiseta annulata (Kenyeres and Tóth, 2012).
35.0
100 90
30.0
80 70 60
20.0
50 15.0
40 30
10.0
Infecon Rate
Temperature (Co)
25.0
Tmin (C°) Tmax (C°) IR
20 5.0
10
0.0
2011
July
June
May
July
2012
August
June
May
September
August
July
May
June
0
2013
Fig. 1. IR (Infection Rate) value changes in parallel with temperature data along the whole sampling period. IR value represents all mosquito species.
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Table 2 Positive mosquito species listed by detected filarial nematodes and the number of positive pools per sampling year. Sampling year
Parasite
Mosquito species
No. of positive pools
2011
Dirofilaria repens
Aedes vexans Ochlerotatus annulipes Aedes cinereus Ochlerotatus sticticus Coquillettidia richiardii Anopheles maculipennis Anopheles hyrcanus Aedes vexans Culex pipiens
39 4 6 8 4 3 6 5 1
Aedes vexans Ochlerotatus annulipes Aedes cinereus Ochlerotatus sticticus Coquillettidia richiardii Anopheles maculipennis Anopheles hyrcanus Culex modestus Aedes vexans Coquillettidia richiardii
47 2 1 37 5 1 3 1 2 1
Aedes vexans Ochlerotatus sticticus Ochlerotatus rusticus Ochlerotatus dorsalis Ochlerotatus annulipes Aedes rossicus
13 19 2 1 1 1
Setaria tundra Onchocercidae sp. 2012
Dirofilaria repens
Setaria tundra 2013
Dirofilaria repens
Setaria tundra
Although the transmission cycle of many filarial species (e.g. D. repens or D. immitis) are well described, the lack of sequence data and information on the competent vector species could lead to the detection of unidentified filarial species (Czajka et al., 2012). Therefore, novel molecular detection techniques are efficient and important tools for surveillance of vector-borne nematodes. The examination of the arthropod vectors are useful for measuring the risk for human and animal health in a territory and also for gaining insight on the role of different vector species in the transmission cycle of these parasites. Although, D. repens and also D. immitis are endemic in Hungary, there is only one recently published report on potential mosquito vector species (Zittra et al., 2015). Moreover, the recent detection of S. tundra and the rapid expansion of other filarial nematodes require more related studies in the future about the Hungarian mosquito fauna. To determine the transmission patterns of filarial nematodes in Southwestern Hungary, we retrospectively analyzed over twenty thousand female mosquitoes collected in the mosquito breeding season of three consecutive years as part of a national West Nile virus surveillance program. Additionally, our study represents the first dataset on filarial transmission dynamics from Hungary. 2. Methods 2.1. Mosquito collection Mosquitoes were collected with the human-landing collection method and updraft box traps equipped with white light attractant (Hall-Mendelin et al., 2010) during the mosquito breeding season (May–September) limited by field conditions (floods, impassable roads). Thus the sampling period for each year was as follows: May–September in 2011; May–August in 2012 and May–July in 2013. The study area included four sampling points within Baranya county, Southwest-Hungary: marshlands near the city of Pécs; bird ringing station in Sumony; dead channels of the Danube river near Mohács; floodplains of Drava river near Drávaszabolcs border crossing point. Mosquito identification was performed according to the taxonomic keys of Beckeret al., (2003). Positive mosquito pools were further verified to species level by barcoding (COI gene) as
described previously (Kumar et al., 2007). Specimens were grouped by species, collection site and date, and finally pooled with a maximum size of 50 individuals. After sample homogenization DNA was extracted with DiaExtract DNA Mini Kit (Diagon Ltd., Hungary) according to the manufacturer’s recommendations. 2.2. PCR screening, sequencing and data analyses The gereric filarial real-time PCR targeting a 94 bp long fragment of the 12S rRNA gene from the mitochondrial genome was performed using the primers FILA-F, FILA-R and probe FILA-P as previously described by Czajka et al. (2012). TaqMan PCR assay was performed with GoTaq® G2 Flexi DNA Polymerase PCR kit (Promega) according to the manufacturer’s protocol on LineGene 9600 platform (Bioer). Real-time PCR-positive samples were further examined by a conventional PCR targeting the 500 bp long fragment of 12S rRNA (ribosomal RNA) (Krueger et al., 2007). After optimization, the annealing temperature for PCR was modified to 55 ◦ C. PCR products were purified with Gel/PCR DNA Fragments Extraction Kit (Geneaid) and were bidirectionally sequenced (BigDye Terminator v1.1Cycle Sequencing Kit, ABI Prism 310 DNA Sequencer-Applied Biosystems). Nucleic acid sequences of the detected filarial nematodes were identified preliminarily by GenBank BLAST searches based on the greatest homology. Representative sample sequences were deposited in the GenBank (KR676613, KR676614, and KR676615). 2.3. Statistical analysis Differences in mosquito abundance among species were analyzed using linear model (lm) implemented in the ‘nlme’ package for R (Pinheiro et al., 2014), performed with R 3.1.0. software (R Development Core Team 2014). The infection rate (IR) of mosquitoes was estimated by the Mosquito Surveillance Software of CDC (CDC, 2013). Dirofilaria Development Units (DDU) was calculated as described previously (Fortin and Slocombe, 1981). For estimating the hypothetical period in which infective mosquito vectors may occure, DDU30 was also calculated as described elsewhere (Ferreira et al., 2015).
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111
96 KM205377 Dirofilaria repens 67
KC953031 D. repens
99
D. repens Hungary GQ292761 D. repens EU182328 Dirofilaria immitis
54
AB518879 Onchocerca sp. AM779812 Onchocerca suzukii DQ523747 Onchocerca flexuosa
88
DQ523745 Onchocerca jakutensis 54
DQ523737 Onchocerca ramachandrini AJ544841 Foleyella furcata AM779794 Cercopithifilaria japonica FR827903 Aproctella sp. HM773029 Chandlerella quiscali JX870434 Filaroidea sp. Onchocercidae sp. Hungary
58
JN228381 Onchocercidae sp.
100 99
JN228379 Onchocercidae sp.
77 JN228380 Onchocercidae sp.
AJ544843 Brugia malayi AJ544835 Setaria equina
54
JN228376 Setaria tundra
57 100
S. tundra Hungary AM779828 S. tundra AJ544855 Filaria martes
0.02
Fig. 2. Phylogenetic classification of Setaria tundra, Dirofilaria repens and Onchocercidae sp. samples compared with other filarial nematode species. Taxon identification tree based on partial 400 bp long fragment of 12S rRNA sequences (neighbor-joining algorithm with Maximum Composite Likelihood parameter, bootstrap replication value was 1000). Representative samples derived from our study are marked with a black triangle (KR676613, KR676614, and KR676615).
Regional meteorological data was collected from the National Meteorological database (NMD, 2015). 2.4. Phylogenetic analyses Phylogenetic characterization was carried out by using cognate sequences available in public databases. Basic sequence manipulation and verification were performed using GeneDoc v2.7 software and nucleotide sequences were aligned by ClustalX v2.0 software. Phylogenetic trees were constructed with MEGA v6.0 software using neighbor-joining algorithm with Maximum Composite Likelihood parameter model based on nucleic acid sequences of a 400 bp-long region of 12S mRNA gene. The number of bootstrap replications was 1000. 3. Results A total of 23,139 female mosquitoes were collected during the mosquito breeding seasons between 2011 and 2013. Twenty-four mosquito species belonging to the genera Aedes, Anopheles, Coquillettidia, Culex, Ochlerotatus and Uranotaenia have been collected and tested in this study. The most frequent species was A. vexans (n = 9545) followed by O. sticticus (n = 7024) and A. cinereus (n = 2399). Relative abundance of mosquito species differed significantly (F28,559 = 22,62, p < 0.001). Five of the most abundant
mosquito species with the highest IR value were: A. vexans, A. hyrcanus, C. richiardii, O. annulipes and O. sticticus. A noticeable decrease of average IR value in august along the whole study was observed. All collected mosquito species along with IR values are shown in Table 1. Variations of IR depending on temperature value changes along the whole sampling period are shown in Fig. 1. The overall infection rate regarding all tested nematode species among all samples for the three-year sampling period was 36.8% with 213 positive pools out of the 579 tested (Table 2). The DNA of D. repens, S. tundra, has been detected along with the DNA of an unidentified nematode with high similarity to a previously published Onchocercidae sp. nematode from Germany (Genbank JN228379–JN228381). D. repens was detected in all four sampling locations during the whole study period. Altogether 198 pools were infected with D. repens, which is 92.9% of all positive pools. D. repens has been detected in eight mosquito species (A. vexans, A. cinereus, A. hyrcanus, A. maculipennis, C. richiardii, C. modestus, O. sticticus, O. dorsalis). S. tundra has been detected in all sampling years during the study. A total of 10 pools from four mosquito species (A. vexans, A. rossicus, C. richiardii, O. sticticus) were positive, each one being potential vectors for S. tundra. The unidentified Onchocercidae filaria has been detected in a single pool of C. pipiens, collected in June of 2011. D. immitis was not detected during this study. The calculation of the DDU30 for the studied periods showed that there were at least 113 days in 2011, 92 days in 2012 and 65 days
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in 2013 with suitable conditions for the completion of the extrinsic development of D. repens., and consequently, for its transmission to the vertebrate hosts. According to the phylogenetic tree, novel D. repens sequence clustered with other D. repens sequences from Italy and Russia. Moreover, S. tundra also grouped together with related S. tundra sequences from Europe. The unidentified Onchocercidae sp. sequence clustered with related sequences from Germany (Fig. 2).
4. Discussion The study reported here provides the first large-scale surveillance data from Hungary regarding the detection of multiple filarial nematode species from various mosquito species. Results may indicate several mosquito species as potential vector organisms for D. repens and S. tundra, but further studies are necessary to confirm their role as competent vector organisms. Interestingly, D. immitis was not identified in the study area. This is particularly interesting in light of recent findings that demonstrated the occurrence of this parasite in O. caspius mosquitoes from South Hungary (Zittra et al., 2015). Since Hungary is an endemic region for the parasite (Tolnai et al., 2014), further investigations are needed to estimate the risk in Southwest Hungary. The most frequent filarial parasite was D. repens, representing the majority of the positive samples (n = 202). The human and veterinarian health risk posed by this parasite is well known with confirmed clinical cases documented in Hungary (Pampiglione et al., 1999; Elek et al., 2000; Pónyai et al., 2006; Szénási et al., 2008; Fodor et al., 2009). The high prevalence of D. repens in the area could enhance the risk for the exportation of the disease via pet dogs to previously not endemic regions, which scenario has been described before (Pantchev et al., 2011; Sævik et al., 2014). The expanding range of wild carnivores, such as golden jackal may also facilitate the persistence of D. repens and could naturally widen the distribution area of the parasite (Tóth et al., 2007). Human cases are also known from Hungary and other D. repens endemic regions, which occasionally could lead to serious infections for instance with ocular, subcutaneous or even lymphatic localization (Szénási et al., 2008). Seroepidemiological studies are needed to estimate the impact of the parasite on local human population as well as in herds of domestic livestock, pets and potential wild animal reservoirs. Previously, S. tundra was identified (Zittra et al., 2015) in Szeged, therefore the second detection of this parasite in Hungary has confirmed its wider distribution area in the country. Furthermore, its emergence in Hungary may indicate a spread to southern areas from the Scandinavian region, where this parasite is endemic and poses veterinary health threat (Laaksonen et al., 2007, 2009). Detailed studies regarding its abundance and distribution in areas south of Scandinavia are missing. Although S. tundra has been described in Germany, Poland and Slovakia (Kronefeld et al., 2014; Czajka et al., 2012; Kowal et al., 2013; Rudolf et al., 2014), the information on veterinary importance for these countries is unknown. The recent detection of this parasite in Hungary (Zittra et al., 2015) along with the result of this study added Hungary to the list of potentially endemic countries. Identification of S. tundra in Ae. vexans mosquitoes suggests the potential role of this species as a vector for setariosis in the Central European region (Rudolf et al., 2014). Additionally, positive mosquito pools (A. rossicus, C. richiardii and O. annulipes) further widen the group of potential vector species in Hungary. Since we successfully confirmed the presence of S. tundra in all sampling seasons, it implies the endemic nature of the parasite in Hungary and extends its estimated geographic distribution in Europe. Furthermore, an unidentified filarial species was detected with high similarity to a previously described filarial nematode from C. pipiens mosquitoes in Germany (Czajka et al.,
2012). Although the recent study by Czajka et al. (2012) suggests an avian origin for this parasite, this study can only confirm the wider distribution of this undefined parasite in Europe. Since there is a notable lack of scientific data about the avian nematode infections in Hungary, it narrows the possibility to estimate potential vertebrate host organisms. Based on the feeding preferences of C. pipiens (Rizzoli et al., 2015) this study support the previous hypothesis surmised by Czajka et al. (2012) on the avian origin. A BLAST search of GenBank database retrieves the highest nucleotide sequence identity (86%) with Onchocerca suzukii parasite. Based on the phylogenetic tree, the novel Hungarian sequence cannot be assigned to any of the known species, moreover clustered separately from O. suzukii. Further examinations, involving potential vertebrate hosts and morphological identification are needed to reveal the origin of this unidentified parasite. We revealed a notable decrease in IR values in both sampling months of august during the study period which was followed by an increase in september. Unfortunately, unequal sampling periods between sampling years do not provide sufficient data. The temperature limitation for development of L3 larvae in mosquitoes could be an explanation for the above mentioned IR value decrease. Since the weather data is regional and the local microclimatic conditions can differ, the exact limiting temperature was unmeasurable for evaluating the possible temperature limit which may endorse the abortation of L3 larvae stage development. Based on these results, mosquito surveillance could be an efficient method for measuring the persistence of these parasites in nature. The disadvantage of mosquito surveillance is the lack of information on local vertebrate hosts, hence with other study design; blood meal analyses of mosquitoes could be feasible. Therefore we can only assume the vertebrate hosts based on local wildlife data. Main vertebrate hosts for D. repens are dogs, cats and other wild carnivores such as red fox (Vulpes vulpes) or golden jackal (Canis aureus) which are all present in the sampling area. In the case of S. tundra, the common roe-deer (C. capreolus), red-deer (Cervus elaphus) and fallow-deer (Dama dama) are presented locally with known susceptibility for the parasite (IWC, 2015). Since the identification of the unidentified Onchocercidae species failed, the potential vertebrate hosts can only be speculated. A clear feeding preference of C. pipiens for the common blackbird (Turdus merula) was recently demonstrated in Italy (Rizzoli et al., 2015). Considering T. merula as the most abundant species in rural and also in suburban territories at the study area (Kurucz et al., 2012), assume this bird as a potential host animal. These results are also remarkable in mosquito control aspects, since several different mosquito species were tested positive. Further vector competence studies are needed. The fluctuating water level at all sampling sites assists the breeding of species preferring inundated breeding areas (e.g. Ae. vexans and O. sticticus as the most abundant species). These species are able to migrate up to 15 km (approximately 1 km/night), therefore posing a potential risk of disease transmission at the inhabited areas (Becker et al., 2003). Hence the expanding of mosquito control areas may be relevant near human inhabited areas in order to lower the risk of filarial infections. To our knowledge, the current study is the first large-scale PCR screening of filarial nematodes in a variety of mosquito species from Hungary. Although several potential mosquito vector species have been described, vector competence studies are needed in the future. Furthermore, the possible endemicity of S. tundra has been suggested, which may potentially affect the health of local wildlife populations. The high prevalence of these parasites is also notable for local clinicians and veterinary professionals, since increased attention should be taken for filarial disease symptoms.
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