First report of Filaria martis Gmelin, 1790 in the European mink, Mustela lutreola (Linnaeus, 1761)

The riparian European mink (Mustela lutreola), currently surviving in only three unconnected sites in Europe, is now listed as a critically endangered species according to the IUCN. Habitat loss and degradation, anthropic mortality, interaction with the feral American mink (Neovison vison), and infectious diseases are among the principal causes of its decline. Surveys of helminth parasites of this host that also include focus on subcutaneous potentially pathogenic helminths such as those belonging to the genus Filaria are very scarce. We report here the presence of specimens of Filaria martis in the subcutaneous connective tissues of three M. lutreola individuals from Spain. This is the first finding of a subcutaneous nematode in a representative of the genus Mustela. The report also enlarges the known range of the definitive hosts of this nematode. These worms were mainly located in the dorsal region of mink and more rarely in the knees, elbows, and hips. Skin sloughing was only observed in one M. lutreola with both septicaemia and an associated high burden of F. martis. Therefore, more attention should be paid to potentially pathogenic helminths when designing conservation programs dedicated to M. lutreola.

The highest density of M. lutreola in its western population was detected in 2004 in Navarre (Spain), where a health survey was thus initiated by the government, including necropsies on animals found dead in the natural environment (Ceña et al. 2005;Fournier-Chambrillon and Fournier 2013). Earlier parasitological surveys have allowed a thorough characterization of the helminth fauna of M. lutreola in Europe (Shimalov et al. 1993;Sidorovich and Anisimova 1993;Torres et al. 2003Torres et al. , 2008 but most of the individuals surveyed were not examined for the presence of subcutaneous potentially pathogenic helminths such as those belonging to the genus Filaria Gmelin, 1790. Therefore, the results here presented provide novel information about the presence of these scantly known nematodes parasitizing mustelids in Europe.

Materials and methods
Carcasses of 91 M. lutreola specimens were collected between 1999 and 2013 in the Spanish Foral Community of Navarre ( Fig. 1). They were kept frozen and then necropsied by trained veterinarians in order to determine the cause of death of each animal. A range of samples was taken at this time for genetic, virological, bacteriologic and histological studies. When it was possible the skin of several specimens was completely removed to enable examination for any subcutaneous lesions, but given the major traumas to which the animals were subjected by road collision (major cause of mortality) only 25 specimens could be completely examined. All the nematodes found in the subcutaneous tissues were removed and transferred to vials containing 70 % ethanol. Some individuals were later processed using conventional techniques for nematodes (cleared in Amman's lactophenol) allowing their specific identification according to previous descriptions of F. martis given in Anderson (1960) andChabaud andMohammad (1989). Later this identification was molecularly corroborated.
Genomic DNA was isolated from an individual worm stored in ethanol using a QIAamp DNA Mini Kit and a Qiacube (Qiagen Inc, Hilden, Germany), according to manufacturer's instructions with the following minor modifications: one single elution of 50 µl was performed with an incubation time of 3 min. A partial sequence of the mitochondrial cytochrome oxidase I (COI) gene was amplified using the primers

Results
The presence of several specimens of F. martis in the subcutaneous connective tissue was observed in three (one male and two females) adult European minks that represent a prevalence of 12.0 % (Fig. 1). The morphological and morphometric data of our Filaria specimens are consistent with those previously reported for this species in other hosts by Anderson (1960), Chabaud and Mohammad (1989) and Otranto et al. (2007). The molecular analysis (Fig. 2) (Zimmerli 1981) and Spain (Sospedra 2000) but no consistent information is available about its prevalence in any host under natural conditions throughout its distribution range except for Italy (Otranto et al. 2007 in Spain, so far no ticks and fleas have been detected, most likely because of the time interval between host death and subsequent discovery of the carcass. Little information is currently available about the pathology that F. martis can produce in its hosts, but it is known that these infections usually do not evoke a significant inflammatory response (Anderson 1960). In our study, in the most infected specimen of M. lutreola, on which septicaemia was detected, two nematodes were apparently trapped in subcutaneous membranous capsules, and skin sloughing was also observed in this individual.
In a broader context it can be pointed out that nematodes of the genus Filaria cause a poorly known subcutaneous filariasis in mustelids and no robust data are available yet about the extent of their occurrence. However, the present report enlarges the known range of the definitive hosts of F. martis including now a representative of the genus Mustela (M. lutreola), which paradoxically is the more endangered and has the lowest population density of all representatives of the genus in Europe.