Parasitology Research https://doi.org/10.1007/s00436-019-06590-6 HELMINTHOLOGY - ORIGINAL PAPER Occurrence, prevalence, and explanatory environmental variables of Spirocerca vulpis infestation in the foxes of western Spain M. Martín-Pérez1 & J.M. Lobo2 & J.E. Pérez-Martín1 & D. Bravo-Barriga1 & J. Galapero3 & E. Frontera1 Received: 2 May 2019 /Accepted: 22 December 2019 # Springer-Verlag GmbH Germany, part of Springer Nature 2020 Abstract The main aim of this study was to not only establish the prevalence of the recently described Spirocerca vulpis parasite in the wild-life cycle of carnivores in western Spain but to also elaborate a model to explain the risk of infestation based on 16 topo- climatic and habitat variables. During the period from June 2016 to November 2017, 1644 carcasses of red foxes (Vulpes vulpes) and another 105 wild mammals, legally hunted or killed in car accidents, were analyzed. Parasitic nodules of Spirocerca were found in 6% of the foxes, and the molecular analyses established a homology of our samples with the species S. vulpis. There were no differences in the occurrence of the infestation between sexes, but there were differences in terms of age, such that infestation was proportionally more frequent among young individuals. In terms of temporality, a higher percentage of positive cases was observed during the late-autumn and winter months, especially between December and February. This study provides new data on the factors that predispose S. vulpis infection in the red fox. Model results indicate that a spatial pattern exists in the occurrence and prevalence of this species in the studied area (higher probabilities to the west), and that this pattern seems to mainly be associated with topo-climatic variables. Keywords Spirocerca vulpis . Red fox . Explanatory factors . Dung beetles . Topo-climatic factors . Habitat factors Introduction Spirocerca vulpis (Rojas et al. 2018a), was described from red foxes in Europe, which is morphologically and genetically Spirocerca lupi (Rudolphi 1809) is a nematode that affects different from S. lupi and produces stomach nodules in these animals of the Canidae family (Bailey et al. 1963; Lobetti hosts (Rojas et al. 2018a). Adults of Spirocerca are usually 2000). Most research on this parasitosis has focused on the located in nodules in the thoracic wall of the esophagus. The dog as the definitive host. However, there is a great lack of females expel embryonated eggs that leave with the feces into information on the epidemiology and pathogenicity of S. lupi the environment. These eggs are then ingested by dung beetles in wild hosts, specifically in foxes. Recently, a new species, (Coleoptera Scarabaeidae and Geotrupidae), where they de- velop into infectious third-stage larvae (L3) until they are ingested by the definitive host (Bailey et al. 1963; Section Editor: Domenico Otranto Mukaratirwa et al. 2010). Other transport or paratenic hosts Electronic supplementary material The online version of this article sometimes participate in the cycle (Chhabra and Singh, 1972). (https://doi.org/10.1007/s00436-019-06590-6) contains supplementary Cases of infestation by Spirocerca have been reported material, which is available to authorized users. worldwide (Bailey 1972; Van der Merwe et al. 2008; Dantas-Torres and Otranto 2014; Ferrantelli et al. 2010; * E. Frontera Szafrańska et al. 2007;Popiołek et al. 2011). In Spain, previ- [email protected] ous studies have shown that Spirocerca is present in foxes and 1 Department of Animal Health, Veterinary Faculty, University of dogs with prevalence of up to 22.5% depending on the prov- Extremadura, Cáceres, Spain ince (Criado-Fornelio et al. 2000; Martínez-Carrasco et al. 2 Department of Biogeography and Global Change, Museo Nacional 2007; Sanchis-Monsonís et al. 2019). In general, a greater de Ciencias Naturales (CSIC), Madrid, Spain incidence has been described in warm climates, leading us to 3 Department of Animal Medicine, Veterinary Faculty, University of suspect that certain topo-climatic and habitat factors may pre- Extremadura, Cáceres, Spain dispose hosts to these parasites. We examined a large number Parasitol Res of fox carcasses from western-central Spain. The objective date of the corpse from the most probable date of birth taking was to describe the occurrence and prevalence of spirocercosis into account that foxes in Spain have a unique breeding season in this area, to discriminate the species responsible, as well as during the spring (Voigt and MacDonald 1984; Zapata et al. to estimate climatic and habitat-associated variables to deter- 1997), and the external appearance of the fox based on re- mine the most likely factors conditioning the progress of this searcher experience. parasitism in Iberian wild foxes. The corpses (n =105)ofnineadditionalmammalspecies killed in traffic accidents were also examined (see Table 1). Materials and methods Determining Spirocerca infestation Study area The presence of parasitic nodules in the gastric wall or other locations during necropsy was considered the parameter of The study was conducted in 202 municipalities in five prov- positivity of infection. No other parameter was considered to inces of western Spain (Cáceres, Salamanca, Zamora, Ávila, establish positivity, considering that coprological methods and Valladolid). The total area covered by these municipalities have not been highly effective (Al-Sabi et al. 2014). is about 19,851 km2, ranging geographically from 49.336 and The molecular study was carried out from a group of rep- 39.945 in latitude to − 8.842 and − 4.891 in longitude (Fig. 1). resentative samples from the different geographical areas. DNA was extracted from individual specimens of worms cor- Animal collection responding to 31 Spirocerca spp.-positive red foxes using the kit Exgene Tissue SV (GeneAll, Seoul, Korea) following the From June 2016 to November 2017, a total of 1644 carcasses manufacturer’s instructions. PCR amplification was carried of red foxes (Vulpes vulpes) were subjected to necropsies out as described previously for Spirurida (Casiraghi et al. where they were collected or, alternatively, at the parasitology 2001), where a 689 bp-long region of the cox1 gene was laboratories of the Faculty of Veterinary Medicine at the amplified using the primer set NTF (5′-TGAT University of Extremadura. The sampled foxes came from TGGTGGTTTTGGTAA-3′)andNTR(5′-ATAA government-authorized hunting within legal guidelines and GTACGAGTATCAATATC-3′). All the amplicons were se- for recreational or commercial purposes or from deaths in quenced by Macrogen Sequencing Service (https://dna. traffic accidents. Of the 1644 analyzed foxes, 52.2% were macrogen.com), where for automated directing sequencing, males (n = 859), 47.4% were females (n = 779) and 6 could the PCR products were amplified using the internal forward not be sexed. The approximate age of each of the foxes was and reverse primer. Direct PCR sequencing reaction was determined according to the analysis of the dental formula performed using a PRISM BigDye Terminator v3.1 Cycle (Sáenz de Buruaga et al. 2001), differences in the collection Sequencing Kit. The DNA samples containing the extension products were added to Hi-Di formamide (Applied Biosystems, Foster City, CA). The mixture was incubated at 95 °C for 5 min, followed by 5 min on ice and then analyzed Table 1 Number and taxonomic identity of the studied animals No. of animals of sampling Negative red foxes sampled (Vulpes vulpes)1544 Positive Spirocerca vulpis red foxes (Vulpes vulpes)100 Genet (Genetta genetta)9 European badger (Meles meles)22 Marten (Martes foina)29 Wild cat (Felis silvestris silvestris)11 European polecat (Mustela putorius)9 Hedgehog (Erinaceus europaeus)4 Fig. 1 Variation in the prevalence of Spirocerca vulpis derived from the Egyptian mongoose (Herpestes ichneumon)7 foxes examined in 202 municipalities (green polygons) of peninsular Eurasian otter (Lutra lutra)3 Spain. Prevalence values varied from 0 (small yellow circles) to 1 (big red circles). The lower right map indicates the location of the Spanish Wild boar (Sus scrofa)11 provinces of Zamora (Z), Valladolid (V), Salamanca (S), Ávila (A), and Total 1749 Cáceres (C) Parasitol Res by ABI Prism 3730XL DNA analyzer (Applied Biosystems, municipality using GIS software (TerrSet 18.21; Clark Labs, Foster City, CA). Sequences were edited in Chromas Lite 2.1. Clark University). 1 (Technelysium Pty Ltd) and consensus sequences for each forward/reverse pair were created in BioEdit (Hall 1999). The Statistical analyses identity at the species level was assessed based on the analysis of the generated sequences, taking into consideration the The 1644 cases of foxes were first analyzed to examine if sex higher results of homology searches using the sequences de- and age factors are associated with the occurrence of S. vulpis. posited at GenBank (http://www.ncbi.nlm.nih.gov/genbank/). A Chi-square test was used to estimate the association be- tween the two binary variables (male/female sex and Origin of environmental data positive/negative infestation). In the case of age, 1-year clas- ses were established, and the Wald-Wolfowitz non-parametric Two types of environmental data, topo-climatic and habitat test (WW test) was calculated to compare the distribution of variables, were used, and their predictive capacity accounting the number of observations in these age classes among fox for the occurrence and prevalence of S. vulpis infestations in specimens with and without S. vulpis infestation. The WW- the studied municipalities was assessed. In total, 11 topo- test was used because it is not only based on the comparison of climatic variables were considered to measure the probable mean values, but also on the shape of the complete distribution environmental degree of association of S. vulpis with climatic of these values (Sprent and Smeeton 2007). and topographic characteristics. Data on current climate came Subsequently, all the available data were used to determine from the University of Extremadura (see methodology in the presence-absence of S. vulpis in each of the 202 munici- Felicísimo et al.