1 Occurrence, Prevalence and Explanatory Environmental Variables of Spirocerca Vulpis

1 Occurrence, prevalence and explanatory environmental variables of Spirocerca vulpis 2 infestation in the foxes of western Spain. 3 4 M. Martín-Pérez1 ꞏ J.M. Lobo2 ꞏ J.E. Pérez-Martín1 ꞏ D. Bravo-Barriga1 ꞏ J. Galapero3 ꞏ E. 5 Frontera1 * 6 7 1 Department of Animal Health, Veterinary Faculty, University of Extremadura, Cáceres, Spain 8 2 Department of Biogeography and Global Change, Museo Nacional de Ciencias Naturales, 9 CSIC, Madrid, Spain 10 3 Department of Animal Medicine, Veterinary Faculty, University of Extremadura, Cáceres, 11 Spain 12 13 *Corresponding author: [email protected] (E. Frontera) 1 14 ABSTRACT 15 The main aim of this study was to not only establish the prevalence of the recently described 16 Spirocerca vulpis parasite in the wild life cycle of carnivores in western Spain, but to also 17 elaborate a model to explain the risk of infestation based on 16 topo-climatic and habitat 18 variables. During the period from June 2016 to November 2017, 1644 carcasses of red foxes 19 (Vulpes vulpes) and another 105 wild mammals, legally hunted or killed in car accidents, were 20 analyzed. Parasitic nodules of Spirocerca were found in 6% of the foxes and the molecular 21 analyses established a homology of our samples with the species Spirocerca vulpis. There were 22 no differences in the occurrence of the infestation between sexes, but there were differences in 23 terms of age, such that infestation was proportionally more frequent among young individuals. 24 In terms of temporality, a higher percentage of positive cases was observed during the late- 25 autumn and winter months, especially between December and February. This is the first study 26 of these characteristics that is carried out for S. vulpis since this species was described. Model 27 results indicate that a spatial pattern exists in the occurrence and prevalence of this species in 28 the studied area (higher probabilities to the west), and that this pattern seems to mainly be 29 associated with topo-climatic variables. 30 31 Keywords spirocercosis ꞏ red fox ꞏ explanatory factors ꞏ dung beetles ꞏ topo-climatic factors ꞏ 32 habitat factors 33 2 34 Introduction 35 Spirocerca lupi (Order Spirurida, Railliet and Henry 1911) is a nematode that affects animals of 36 the Canidae family (Bailey et al. 1963; Lobetti 2000). Most research on this parasitosis has 37 focused on the dog as the definitive host. However, there is a great lack of information on the 38 epidemiology and pathogenicity of S. lupi in wild hosts, specifically in foxes. Recently, a new 39 species, Spirocerca vulpis (Rojas et al. 2018a), was described from red foxes in Europe, which 40 is morphologically and genetically different from S. lupi and produces stomach nodules in these 41 hosts (Rojas et al. 2018a). Adults of Spirocerca are usually located in nodules in the thoracic 42 wall of the esophagus. The females expel embryonated eggs that leave with the feces into the 43 environment. These eggs are then ingested by dung beetles (Coleoptera Scarabaeidae and 44 Geotrupidae), where they develop into infectious third stage larvae (L3) until they are ingested 45 by the definitive host (Bailey et al. 1963; Mukaratirwa et al. 2010). Other transport or paratenic 46 hosts sometimes participate in the cycle (Chhabra and Singh, 1972). 47 Cases of infestation by Spirocerca have been reported worldwide (Bailey 1972; Van der 48 Merwe et al. 2008; Dantas-Torres and Otranto 2014; Ferrantelli et al. 2010; Szafrańska et al. 49 2007; Popiołek et al. 2011). In Spain, previous studies have shown that S. lupi is present in 50 foxes and dogs with prevalence of up to 22.5% depending on the province (Criado-Fornelio et 51 al. 2000; Martínez-Carrasco et al. 2007; Sanchis-Monsonís 2015). In general, a greater 52 incidence has been described in warm climates, leading us to suspect that certain topo-climatic 53 and habitat factors may predispose hosts to this parasite. We examined a large number of fox 54 carcasses from western-central Spain. The objective was to describe the occurrence and 55 prevalence of spirocercosis in this area, to discriminate the species responsible, as well as to 56 estimate climatic and habitat-associated variables to determine the most likely factors 57 conditioning the progress of this parasitism in Iberian wild foxes. 58 59 Materials and methods 60 3 61 Study area 62 The study was conducted in 202 municipalities in five provinces of western Spain (Cáceres, 63 Salamanca, Zamora, Ávila and Valladolid). The total area covered by these municipalities is 64 about 19,851 km2, ranging geographically from 49,336 and 39,945 in latitude to -8,842 and - 65 4,891 in longitude (Fig. 1). 66 67 Animal collection 68 From June 2016 to November 2017, a total of 1644 carcasses of red foxes (Vulpes vulpes) were 69 subjected to necropsies where they were collected or, alternatively, at the parasitology 70 laboratories of the Faculty of Veterinary Medicine at the University of Extremadura. The 71 sampled foxes came from government-authorized hunting within legal guidelines and for 72 recreational or commercial purposes, or from deaths in traffic accidents. Of the 1644 analyzed 73 foxes, 52.2% were males (n = 859) and 47.4% were females (n = 779) and 6 could not be sexed. 74 The approximate age of each of the foxes was determined according to the analysis of the dental 75 formula (Sáenz de Buruaga et al. 1991), differences in the collection date of the corpse from the 76 most probable date of birth taking into account that foxes in Spain have a unique breeding 77 season during the spring (Voigt and McDonald 1984; Zapata et al. 1997), and the external 78 appearance of the fox based on researcher experience. 79 The corpses (n = 105) of nine additional mammal species killed in traffic accidents were 80 also examined (see Table 1). 81 82 Determining Spirocerca infestation 83 The presence of parasitic nodules in the gastric wall or other locations during necropsy was 84 considered the parameter of positivity of infection. No other parameter was considered to 85 establish positivity, considering that coprological methods have not been highly effective (Al- 86 Sabi et al. 2014). 87 The molecular study was carried out from a group of representative samples from the 88 different geographical areas. Genomic DNA (gDNA) was extracted from individual specimens 4 89 of worms corresponding to 31 Spirocerca spp. positive red-foxes using the kit Exgene Tissue 90 SV (GeneAll, Seoul, Korea) following the manufacturer’s instructions. PCR amplification was 91 carried out as described previously for Spirurida (Casiraghi et al., 2001), where a 689 bp-long 92 region of the cox1 gene was amplified using the primer set NTF (5′- 93 TGATTGGTGGTTTTGGTAA-3′) and NTR (5′-ATAAGTACGAGTATCAATATC-3′). All the 94 amplicons were sequenced by Macrogen sequencing service (https://dna.macrogen.com), where 95 for automated directing sequencing, the PCR products were amplified using the internal forward 96 and reverse primer. Direct PCR sequencing reaction were performed using a PRISM BigDye 97 Terminator v3.1 Cycle sequencing Kit. The DNA samples containing the extension products 98 were added to Hi-Di formamide (Applied Biosystems, Foster City, CA). The mixture was 99 incubated at 95 °C for 5 min, followed by 5 min on ice and then analyzed by ABI Prism 100 3730XL DNA analyzer (Applied Biosystems, Foster City, CA). Sequences were edited in 101 Chromas Lite 2.1.1 (Technelysium Pty Ltd) and consensus sequences for each forward/reverse 102 pair were created in BioEdit (Hall, 1999). The identity at the species level was assessed based 103 on the analysis of the generated sequences, taking into consideration the higher results of 104 homology searches using the sequences deposited at GenBank 105 (http://www.ncbi.nlm.nih.gov/genbank/). 106 107 Origin of environmental data 108 Two types of environmental data, topo-climatic and habitat variables, were used and their 109 predictive capacity accounting for the occurrence and prevalence of S. vulpis infestations in the 110 studied municipalities was assessed. In total, 11 topo-climatic variables were considered to 111 measure the probable environmental degree of association of S. vulpis with climatic and 112 topographic characteristics. Data on current climate came from the University of Extremadura 113 (see methodology in Felicísimo et al. 2011), which include average figures about temperature 114 and precipitation during the period from 1950 to 2007 for the complete Iberian Peninsula (see 115 http://ide.unex.es/conocimiento/). Using this primary source of climatic information at a 116 resolution of 1 km2 UTM grid cells and the formulas of Valencia-Barrera et al. (2002) and 5 117 López Fernández and López (2008), nine bioclimatic variables were built: mean monthly 118 precipitation, minimum monthly precipitation, maximum monthly precipitation, total annual 119 precipitation, minimum monthly temperature, maximum monthly temperature, mean monthly 120 temperature, continentality and aridity. Topographic data came from a digital elevation model of 121 the Iberian Peninsula at a resolution of 90 meters downloaded from the same web page of the 122 University of Extremadura. This topographic information was used to derive two variables: 123 mean elevation and elevation range (difference between the minimum and maximum elevation 124 in each municipality). 125 Habitat variables come from the CORINE Land Cover project for the year 2011 126 (www.eea.europa.eu) at a resolution of 100 m2, using three of the five recognized land uses of 127 level 1: natural (forest and semi-natural areas), cultivated (agricultural areas) and urban areas 128 (artificial surfaces). Human population density of each municipality was also included as a 129 habitat variable according to the data provided by the Instituto Nacional de Estadística 130 (http://www.ine.es/).

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