Introduction

Globalization and its side effects appear to be causing profound, sometimes unpredictable changes in the ecological, biological, and social conditions that shape the burden of infectious diseases in many populations. Changes in these conditions have led to alterations in the prevalence, spread, and geographical range of many infections, particularly those transmitted by vectors (Saker et al. 2004). The climatic conditions which facilitate the extrinsic incubation for Dirofilaria species extend the geographical area of these parasites towards Northern-Europe (Genchi et al. 2011). Other factors, such as human activities (e.g., dog import) may also support the introduction of these parasites into novel territories within Europe (Sævik et al. 2014). In addition, the aggressive spread of the invasive Aedes albopictus mosquito (Schaffner and Mathis, 2014) along with its potential vector capacity even for Dirofilaria species (Licitra et al. 2010), broadens the dispersion potential for Dirofilaria parasites. Such natural hosts as wild carnivores, e.g., red foxes (Vulpes vulpes), grey wolf (Canis lupus lupus), or golden jackals (Canis aureus) are also able to facilitate the spread of these parasites into new areas with sufficient climatic conditions, although natural reservoirs are mainly domestic dogs. Beyond having a serious impact on veterinary health, Dirofilaria species are also able to cause serious infections in humans, which emphasizes their importance as human zoonotic pathogens (Széll et al. 2013; Tasić-Otašević et al. 2015; Tolnai et al. 2015).

Dirofilaria repens and recently Dirofilaria immitis are known to be circulating in Serbia, verified among different canine species and even humans (Tasić-Otašević et al. 2015). Hence, several studies published data regarding the infection of different carnivore hosts from Serbia and surrounding countries (Penezić et al. 2014; Gavrilović et al. 2014; Tolnai et al. 2014). Regarding studies related to mosquito vector species, D. immitis and D. repens have only been detected in the area from the surrounding territory of Hungary (Zittra et al. 2015), and further from Southwest Hungary where only D. repens was detected from mosquitoes (Kemenesi et al. 2015). Although the environmental factors and mosquito abundance are suitable for the transmission of infection (Tasić-Otašević et al. 2015), mosquito-related survey was not conducted in Serbia before.

Data on transmitting mosquito species and their potential role in local transmission cycles are still scanty; hence, we conducted a PCR-based molecular detection of Dirofilaria nematodes to reveal potential mosquito vector species of dirofilariosis in Serbia. Altogether, 6369 female mosquito samples were retrospectively tested, collected from 13 localities in Vojvodina province, Serbia, from April to October 2013. Although our study does not focus on clarifying whether the collected mosquitoes were competent or noncompetent vectors, still it outlines potential vector species to be investigated in the future and provides sequence data on locally circulating Dirofilaria species among mosquitoes. Such sequence data from multiple geographic regions are essential for future in silico analyses and primer design (Masny et al. 2016).

Materials and methods

Mosquito collection, species identification, and sample homogenization were conducted as described previously (Kemenesi et al. 2014). Since mosquitoes were determined by species according to their taxonomic keys (Becker et al. 2010), female mosquitoes were pooled by collection site, date, and species, with a maximum of 50 individuals per pool. Nucleic acid was extracted from fresh mosquito pool homogenates with DiaExtract DNA Mini Kit (Diagon Ltd., Hungary) following the protocol provided by the manufacturer. A generic filarial real-time PCR targeting a 94 bp long fragment of the 12S ribosomal RNA (rRNA) gene from the mitochondrial genome was performed using the primers FILA-F, FILA-R, and probe FILA-P as previously described (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 (Krueger et al. 2007). Sequencing and sequence identification was performed as published previously (Kemenesi et al. 2015).

Results

A total of 6369 female mosquitoes (combined in 180 pools) representing 11 species were tested in this study. The most abundant mosquito species was Culex pipiens (n = 5568), followed by Aedes vexans (n = 405), Ochlerotatus caspius (n = 195), and Ochlerotatus sticticus (n = 120) (Table 1). Altogether, 8.33 % of tested mosquito pools were positive for Dirofilaria parasites, composed of five mosquito species: Culex pipiens, Aedes vexans, Ochlerotatus caspius, Ochlerotatus sticticus, and Coquillettidia richiardii (Table 1).

Table 1 List of mosquito species, number of collected specimens, and the number of pools tested for the presence of Dirofilaria nematodes
Full size table

From the 13 sampled localities in Vojvodina, 6 were found to be positive, comprising both urbanized and rural areas. Dirofilaria positivity was confirmed during the whole period of mosquito breeding season, from May to August with a maximum rate in July, 60 % of the positive pools are derived from July (Table 2).

Table 2 The occurrence of Dirofilaria nematodes in sampled mosquito species from Vojvodina province, Serbia, April–October 2013 (for detailed description of mosquito sampling location sites and collection dates see Kemenesi et al. 2014)
Full size table

Based on Sanger sequencing data of PCR products derived from positive mosquito pools, we separated D. immitis and D. repens nematodes. D. immitis positivity represented 80 % of the 15 infected mosquito pools, including Cx. pipiens, Cq. richiardii, and Oc. caspius species, while D. repens positivity (20 %) was confirmed in Ae. vexans, Cx. pipiens, and Ox. sticticus mosquito pools (Table 2).

Highest nucleotide sequence similarity (99 %) of the Serbian D. repens sequences was shared with a Hungarian sequence (GenBank: KR676613) retrieved from mosquitoes. In the case of Serbian D. immitis sequences, they shared the highest (99 %) identity with a D. immitis sequence, identified in a dog from Tunisia (GenBank: KP898738).

The consensus sequences (454 bp and 456 bp) derived from 12S rRNA gene of D. repens and D. immitis, retrospectively, were deposited in GenBank®: KU885998; KU885999.

Discussion

The results of this preliminary study represent the first molecular evidence of D. immitis and D. repens nematodes from mosquito samples in Serbia. Such data from this geographic region, regarding potential mosquito vector species, are available only from the northern adjacent country, Hungary (Kemenesi et al. 2015; Zittra et al. 2015).

Aedes vexans mosquitoes were previously reported with D. repens positivity from Slovakia (Rudolf et al. 2014; Bocková et al. 2015) and Hungary (Kemenesi et al. 2015). The potential involvement of Oc. sticticus mosquitoes in natural transmission cycle of the parasite was also indicated recently from Hungary (Kemenesi et al. 2015). Whereas the role of Cx. pipiens mosquitoes as a potential vector species for D. repens nematode is indicated in this study for the first time in the Balkans, although a study from Italy reported such positivity (Latrofa et al. 2012).

Dirofilaria immitis positivity was previously confirmed in Cx. pipiens mosquitoes from Germany and Hungary (Kronefeld et al. 2014; Zittra et al. 2015) and further southern territories in Italy (Latrofa et al. 2012). Regarding Oc. caspius mosquitoes, D. immitis positivity was reported from adjacent territories of Hungary (Zittra et al. 2015) and from Italy (Latrofa et al. 2012). Molecular evidence for D. immitis in Cq. richiardii is reported here for the first time, from the Central European and the Balkans.

Though our study design was not investigating if the collected mosquitoes were competent vectors, since filarial nematodes can be detected in both competent and noncompetent mosquito vectors (Erickson et al. 2009), our study also provides evidence for local transmission of Dirofilaria nematodes and outlines mosquito species for future vector competent studies.

The most abundant mosquito species in the study area were Cx. pipiens and Ae. vexans; furthermore, Dirofilaria positivity was also confirmed mainly from these mosquito species. Both mosquito species have many of the attributes of an ideal vector. They are widespread in the Holarctic region and are found throughout Europe. Both Cx. pipens and Ae. vexans can become very abundant as they are able to reproduce in a wide quality range of water sources, frequently in human-related water bodies such as flooded cellars, construction sites, and containers in gardens (Becker et al. 2010). Since natural and rural territories were found to be positive, our results need to be concerned by veterinary and human health professionals as well. Nucleotide sequences of D. immitis and D. repens provided by this study facilitate the susceptibility of future in silico analyses or primer designs by extending the available sequence data in GenBank database.