Filarioid infections in wild carnivores: a multispecies survey in Romania

Filarioids are vector-borne parasitic nematodes of vertebrates. In Europe, eight species of filarioids, including zoonotic species, have been reported mainly in domestic dogs, and occasionally in wild carnivores. In Romania, infections with Dirofilaria spp. and Acanthocheilonema reconditum are endemic in domestic dogs. Despite the abundant populations of wild carnivores in the country, their role in the epidemiology of filarioid parasites remains largely unknown. The aim of the present study was to assess the host range, prevalence and distribution of filarioid infections in wild carnivores present in Romania. Between May 2014 and February 2016, 432 spleen samples originating from 14 species of wild carnivores have been tested for the presence of DNA of three species of filarioids (D. immitis, D. repens and A. reconditum). Overall 14 samples (3.24%) were molecularly positive. The most prevalent species was D. immitis (1.62%), accounting for 50% (n = 7) of the positive animals. The prevalence of D. repens was 1.39%, while that of A. reconditum was 0.23%. No co-infections were detected. Dirofilaria immitis DNA was detected in five golden jackals, Canis aureus (7.58%), one red fox, Vulpes vulpes (0.33%), and one wildcat, Felis silvestris (10%). The presence of D. repens DNA was detected in two red foxes (0.66%), two golden jackals (3.03%), one grey wolf (7.14%), and one least weasel, Mustela nivalis (33.33%). Acanthocheilonema reconditum DNA was found only in one red fox (0.33%). The present study provides molecular evidence of filarial infections in wild carnivore species in Romania, suggesting their potential epidemiological role and reports a new host species for D. repens.


Background
Filarioids (Spirurida, Onchocercidae) are vector-borne parasitic nematodes that reside in various tissues of vertebrates [1]. In Europe, eight species of filarioids of four genera (Dirofilaria: 2 species; Acanthocheilonema: 2 species; Cercopithifilaria: 3 species; and Onchocerca lupi) have been reported in domestic dogs. Among these, most of the research has been focused on the zoonotic species, namely D. immitis, which causes a severe and life-threatening cardio-pulmonary disease in dogs [2], D. repens, which resides in the subcutaneous tissues of the canine host and is associated with a variety of dermatological conditions [3,4] and, more recently, O. lupi, which is localized in the connective tissue of the sclera or in the retrobulbar regions of the eye of dogs [5]. Acanthocheilonema spp. and Cercopithifilaria spp. have a less known ecology, as they seem to be nonpathogenic, and have a minimal clinical importance [6]. Globally, there are several records of free-roaming wild carnivores being naturally infected with filarioid helminths that typically parasitize domestic dogs . However, in Europe, only a few extensive studies assessing the prevalence, distribution and mainly patency (i.e. presence of circulating microfilariae) of filarial infections in wild carnivores have been published [7][8][9][10][11][12][13][14][15][16][17][18]21].
Dirofilaria spp. and A. reconditum are distributed in dog populations throughout the country [33], while other species (C. bainae, O. lupi) have been reported only locally [34,35]. Romania is mostly a rural country, having an extended wildlife-domestic animal interface, which may facilitate the spreading of canine parasites to wild carnivores, which in turn may act as natural reservoirs. The country is characterized by a high diversity of habitats and biodiversity, having a rich wild carnivore fauna comprising 18 species belonging to 5 families: Mustelidae (10 species), Canidae (4 species), Felidae (2 species), Ursidae (1 species) and Phocidae (1 species) [36]. However, despite this large diversity and abundance of wild carnivores present in the country, their role in the epidemiology of filarioid parasites remains unknown. The aim of the present study was to assess the prevalence and distribution of filarioid infections in wild carnivores present in Romania.

Methods
Between May 2014 and February 2016, a total of 432 spleen samples originating from 14 species of wild carnivores have been tested ( Table 1). The animals were legally hunted, road-killed, or found dead due to natural causes at various locations. For each animal, species, sex, estimated age (juvenile or adult, according to dentition) and collection site were recorded. Collection of samples took place either directly on the field (performed by hunters), or during necropsy. All necropsies were performed at the Department of Parasitology and Parasitic Diseases within the University of Agricultural Sciences and Veterinary Medicine of Cluj-Napoca (Romania). When available, the heart and pulmonary arteries were dissected in order to assess the presence of adults of D. immitis. Samples were labelled and stored at -20°C until further processing.
Genomic DNA was extracted individually from up to 20 mg of splenic tissue using a commercial kit (Isolate II Genomic DNA Kit, Bioline, London, UK) according to the manufacturer's instructions. The detection of filarioid DNA was performed by means of multiplex PCR discriminating three species of filarioids commonly present in Europe (D. immitis, D. repens and A. reconditum), using primers and protocols available in literature [37]. PCR products were visualised under UV light after electrophoresis in a 2% agarose gel stained with Red-Safe™ 20,000× Nucleic Acid Staining Solution (Chembio, St Albans, UK). The size of the attained bands was assessed by comparison to a molecular marker (O'Gen-eRuler™ 100 bp DNA Ladder, Thermo Fisher Scientific Inc., Waltham, MA, USA).
The frequency and prevalence of infection and their 95% confidence intervals (95% CI) were established using EpiInfo™ 7 software (CDC, USA).

Results
Overall, 432 spleen samples were tested for the presence of DNA of three filarioid species. A total of 14 samples were positive (3.24%; 95% CI: 1.94-5.37%). Dirofilaria immitis DNA was detected in the spleen of five golden jackals, Canis aureus, one red fox, Vulpes vulpes, and one wildcat, Felis silvestris (Table  1). Additionally, heartworms were also present in the right ventricle or pulmonary arteries of one Eurasian   Table 2). The presence of D. repens DNA was detected in splenic tissue of two golden jackals, two red foxes, one grey wolf, C. lupus, and one least weasel, Mustela nivalis (Table 1). Acanthocheilonema reconditum DNA was found only in one sample, originating from a red fox (Table 1). No co-infections were detected. The geographical distribution of the positive animals is shown in Fig. 1.

Discussion
The present study reports the presence of filarioid DNA in spleen samples collected from various species of wild carnivores throughout Romania. For Dirofilaria spp. the distribution pattern in wild carnivores in Romania is similar to that recorded in domestic dogs [33,[38][39][40][41]. Moreover, most positive animals originated from the south and southeast of the country, where the prevalence of infection in dogs is the highest, with values of up to 26% [33]. As wild carnivores are susceptible hosts, infections occur most often as an epi-phenomenon of dog infection, particularly in overlapping territories [42]. However, infected microfilaremic carnivores may, in the presence of competent vector species, also act as reservoir hosts. In Europe, D. immitis infections have been previously reported from several species of wild carnivores, but patency of infection was rarely evaluated (Table 3). Overall, in the present study, heartworm infections were detected in the case of one Eurasian otter, ten golden jackals, one wild cat and one red fox. However, DNA of D. immitis was not detected in all spleen samples, a fact indicating that the molecular positivity may be related to the presence of microfilariae, therefore, the occurrence of a patent infection. The lack of microfilaremia may be related to several factors, including unisexual infections, prepatency, or the hosts' immune response leading to the clearance of microfilariae [43]. The molecularly negative animals were either harbouring nematodes of the same sex, or were at juvenile age (under one year old) and had died during the winter. This would correspond to a relatively recent (prepatent) infection, considering that the prepatency period ranges between six and  Fig. 1 Geographical origin of the samples examined in the country nine months [44]. This represents the second record of D. immitis infection in two European species, otter and wild cat. Among mustelids, the reservoir status has been demonstrated experimentally for the domesticated form of the ferret (Mustela putorius furo) [45]. In the case of otters, the present study provides further evidence of the possibility of infection to occur. Apart from a single case, data regarding D. immitis infection in wild cats is currently absent, but their situation is probably similar to that of the domestic ones, which play a minimal epidemiological role, because they generally have a low worm burden and display low levels and a short duration of microfilaremia [46]. In red foxes, the recorded prevalence is considerably lower compared with those in neighbouring countries, such as Bulgaria or Hungary (Table 3). However, in most of these studies, the authors only reported the presence of adult nematodes and microfilaremia was not assessed. Similarly, in Italy the prevalence of adult heartworms in red foxes was of 9.56% (n = 50), while microfilaremia was recorded in only 0.38% (n = 2) of cases, indicating that red foxes may not be suitable reservoir hosts [7]. The low prevalence in our study may further support this theory. So far, European records of D. repens in wildlife include only a handful of cases apart from red foxes (Table  3). To our knowledge, we provide the first evidence for a new host species, the least weasel, Mustela nivalis. Given that studies on D. repens infection in wild carnivores are mostly limited to singular cases, it is difficult to estimate their role in the epidemiology of this parasite. More comprehensive studies were performed on red foxes in Italy and revealed a relatively low prevalence of microfilaremia ( Table 3). The prevalence recorded in foxes in the present study (0.66%) has a similar low value. These data may indicate that red foxes are not efficient reservoir hosts.
Acanthocheilonema reconditum is a largely neglected and poorly known species of filarioid. Microfilariae develop and become infective in fleas or lice [47] and require proximity between infected and uninfected hosts [48]. So far, in Europe, among wild carnivores, this parasite has been documented only in red foxes from Italy, with relatively high prevalence values, suggesting their reservoir competence [7,13]. In the present study a single red fox (0.33%) was positive. In Romania, in dogs, this species seems to be adapted to higher altitudes and to have a relatively large distribution area, despite the low prevalence [33]. However, neither data regarding the climatic requirements for development, nor full distribution maps are currently available for this species.

Conclusion
The present study provides molecular evidence for filarial infections in wild carnivore species present in Romania, suggesting a potential epidemiological role and demonstrates a new host species for D. repens.