Prevalence of selected tick-borne pathogens in wild ungulates and ticks in southern Spain

1 Original article 1 Prevalence of selected tick-borne pathogens in wild ungulates and ticks in southern 2 Spain 3 Running head: Tick-borne pathogens in wild ungulates and ticks in Spain 4 5 José Manuel Díaz-Cao1†, Łukasz Adaszek2†, Beata Dzięgiel2, Jorge Paniagua1, Javier 6 Caballero-Gómez1,3, Stanislaw Winiarczyk2, Dagmara Winiarczyk2, David Cano-Terriza1*, 7 Ignacio García-Bocanegra1 8 9 10 1 Animal Health and Zoonosis Research Group (GISAZ), Department of Animal Health, Faculty 11 of Veterinary Medicine, University of Cordoba, Campus de Rabanales, 14014 Cordoba, 12 Spain. 13 2Department of Epizootiology and Infectious Diseases, Faculty of Veterinary Medicine, 14 University of Life Sciences in Lublin, 30 Głęboka St. 20-612 Lublin, Poland. 15 3Infectious Diseases Unit, Clinical Virology and Zoonoses research group, Hospital 16 Universitario Reina Sofía de Córdoba, Instituto Maimonides de Investigación Biomédica de 17 Córdoba (IMIBIC), University of Cordoba, 14006 Cordoba, Spain. 18 †These authors contributed equally to this work. 19 20 *Correspondence: Corresponding author: Dr. Cano-Terriza. Department of Animal Health, 21 University of Cordoba, Campus Universitario Rabanales, 14014 Córdoba, Spain. Tel.: +34 22 957218718; E-mail: [email protected] 23 24 2 Summary 25 A survey study was carried out to assess the occurrence of selected tick-borne pathogens (TBP) 26 in wild ungulates in Mediterranean ecosystems in southern Spain. Spleen samples were 27 collected from 1,132 wild ungulates, including 578 red deer, 269 wild boar, 135 mouflon, 121 28 fallow deer and 29 roe deer, between 2009 and 2015. Eighty-nine ticks collected from TBP29 positive animals were also analyzed. Samples were tested by PCR and sequenced whenever 30 possible. TBP DNA was detected in 127 of 863 wild ruminants (14.7%; 95%CI: 12.4-17.3) 31 including: Anaplasma phagocytophilum (9.2%), Babesia divergens (2.9%), Theileria sp. OT3 32 (1.7%), Borrelia afzelii (0.7%) and Theileria capreoli (0.2%); but no positive samples were 33 detected in wild boar (0/269). All the strains from mouflon were identified as Theileria sp. OT3, 34 while B. divergens and T. capreoli were mainly found in red deer. Co-infection with A. 35 phagocytophilum and B. divergens, and A. phagocytophilum and Theileria spp. were detected 36 in red deer and mouflon, respectively. The risk factor analysis showed that the prevalences of 37 A. phagocytophilum and piroplasms were species-related. Eighty-nine tick specimens collected 38 from ungulates found to be infected with the selected TBP were identified as Hyalomma 39 lusitanicum (95.5%) and Ixodes ricinus (4.5%). Thirty ticks were positive for 40 Anaplasma/Ehrlichia spp. (33.7%), 25 for Babesia/Theileria (28.1%) and two for B. 41 burgdorferi s.l. (2.3%). Eleven specimens showed co-infections with Anaplasma/Ehrlichia and 42 Babesia/Theileria (10.1%) or Anaplasma/Ehrlichia and B. burgdorferi s.l. (2.3%). The 43 estimated prevalences obtained in the present study suggest the possible contribution of wild 44 ruminants to the maintenance of some selected TBP in Mediterranean ecosystems in southern 45 Spain, while the role of wild boar in the epidemiology of these pathogens seems to be limited 46 in this region. 47 48 Keywords: Vector-borne disease; wild ruminants; public health; tick-borne pathogens; Spain 49 3 Introduction 50 Tick-borne diseases (TBD) constitute a diversified group of diseases of increasing importance 51 in human and veterinary medicine (Vayssier-Taussat et al., 2015). Among the high number of 52 tick-borne pathogens (TBP) of human and animal health concern, infections caused by the 53 obligate intracellular parasites Anaplasma/Ehrlichia spp., Babesia/Theileria spp. and Borrelia 54 spp. are especially noteworthy. They cause severe economic losses in the livestock industry 55 (Bock, Jackson, de vos, & Jorgensen, 2004; Kocan, de la Fuente, & Cabezas-Cruz, 2015; 56 Morrison, 2015) and include some species that are well known zoonotic pathogens causing 57 diseases with an increasing incidence in Europe (Kocan et al., 2015; Stanek, Wormser, Gray, 58 & Strle, 2012). 59 Transmission of TBP depends upon a complex arrangement of factors including the presence 60 and abundance of competent vectors, density of competent hosts, climatic factors (temperature, 61 humidity and rainfall) and landscape structure (Matei et al., 2019; Pfäffle, Littwin, Muders, & 62 Petney, 2013). Wild ungulates have been shown to be important contributors to the maintenance 63 of TBP by hosting ticks and favoring their abundance (Hofmeester et al., 2017; Kilpatrick, 64 Labonte & Stafford III, 2014), as well as acting as natural reservoirs for some of these pathogens 65 (Kauffman et al., 2017; Kazimírová et al., 2018). Their importance in the epidemiology of TBD 66 is currently rising due to the increasing density and abundance of these species in some 67 European regions in recent decades, particularly red deer (Cervus elaphus), roe deer (Capreolus 68 capreolus) and wild boar (Sus scrofa) (Apollonio, Andersen, & Putman; 2010; Martin, 69 Chamaillé-Jammes, & Waller, 2020; Massei et al., 2015). This has also led to the frequent 70 sharing of natural resources with livestock and humans in certain areas, which enhances the risk 71 of interspecies transmission of TBP (Gortázar, Acevedo, Ruiz-Fons, & Vicente, 2006). 72 Infections with TBP species have been reported in different regions in Spain, in both domestic 73 (Calleja-Bueno et al., 2017; Habela et al., 1999; Nagore et al., 2004; Ros-García et al., 2013) 74 4 and wild ungulates (de la Fuente et al., 2005; García-Pérez et al., 2016; García-Sanmartín et al., 75 2007; Remesar et al., 2019) with outbreaks of clinical disease and economic loss associated 76 with TBP observed in livestock (García-Pérez et al., 2003; Hurtado et al., 2015; Lacasta et al., 77 2020). Furthermore, B. burgdorferi s.l. is endemic in humans in northern Spain (Portillo, 78 Santibáñez & Oteo, 2014; Vázquez et al., 2015) and human cases of anaplasmosis and 79 babesiosis have also been reported in this country (García, Núñez, Portillo & Oteo, 2015; 80 Guerrero-Espejo, Muñoz-Parada & Tomás-Dols, 2017). 81 The Mediterranean ecosystems of southern Spain present specific characteristics that may 82 influence the epidemiology of tick-borne disease (TBD). First, there is a variety and abundance 83 of those populations of wild ungulate species, such as red deer, fallow deer (Dama dama), roe 84 deer, mouflon (Ovis aries musimon) and wild boar (Palomo, Gisbert, & Blanco, 2007), that can 85 act as potential hosts of different TBP. Second, these wildlife species frequently share habitats 86 with certain tick species and livestock, which is favored by the livestock production system in 87 southern Spain, characterized by the prevalence of extensive farming (European Parliament, 88 2016). Finally, the climatic conditions of this region, with low precipitation and dry summers, 89 influence the composition of tick populations and hence the epidemiology of TBD. In this 90 regard, the countries of the Mediterranean Basin lie along the limits of the distribution range of 91 a number of tick species such as I. ricinus which are competent vectors for different TBP and 92 are highly prevalent in northern Spain and several European regions (EFSA, 2010). In addition, 93 these regions are considered to be especially sensitive to climate change (Lionello & Scarascia, 94 2018), which may affect future trends in the epidemiology of TBD (Semenza & Suk, 2018). 95 Despite this, the available information about the role of wild ungulates in the maintenance of 96 TBP in Spanish Mediterranean ecosystems is limited, and mainly focused on central and 97 northern regions (García-Pérez et al., 2016; García-Sanmartín et al., 2007). The lack of reports 98 is an important limitation for TBP such as Anaplasma/Ehrlichia spp., Babesia/Theileria spp. 99 5 and B. burgdorferi sensu lato (s.l.), since high rates of infection with some of these have been 100 reported in both domestic and wild ungulates in neighboring areas of northern Spain and 101 Portugal (García-Pérez, Barandika, Oporto, Povedano, & Juste, 2003; García-Pérez et al., 2016; 102 García-Sanmartín et al., 2007; Nagore, García-Sanmartín, García-Pérez, Juste & Hurtado, 103 2004; Naranjo et al., 2006; Pereira et al., 2016; Silva, Marques & Oliva, 2010). Therefore, the 104 aims of the present study were: 1) to determine the occurrence of selected TBP 105 (Anaplasma/Ehrlichia spp., Babesia/Theileria spp. and B. burgdorferi s.l.) in wild ungulates, 106 and 2) to assess the presence of these pathogens in ticks removed from TPB-infected animals 107 in Mediterranean ecosystems in southern Spain. 108 Materials and Methods 109 Sampling and data collection 110 A total of 1,132 spleen samples from wild ungulate species, including red deer (n = 578), wild 111 boar (n = 269), mouflon (n = 135), fallow deer) (n = 121) and roe deer (n = 29) were collected 112 on 53 hunting estates in southern Spain (Figure 1). The animals were legally hunted during the 113 hunting seasons (October to March) from 2009 to 2015. Harvested animals were collected 114 together at the same location on each of the hunting estates where visual inspection and 115 sampling were carried out. Data on species, sex age, hunting estate sampled and sampling year 116 were recorded for each animal, whenever possible. Determination of age (yearlings: < 1 year 117 old; sub-adults: between 1 and 3 years old; adults: > 3 years old) was performed according to 118 the method described by Sáenz de Buruaga, Lucio-Calero & Purroy-Iraizoz (2001). Sampled 119 animals were inspected in situ for the presence of ticks. A thorough examination of the skin of 120 the animals was carried out, paying special attention to the preferred feeding sites of ticks round 121 the ears, head, neck and ventral surface of the sampled animals. Ticks were kept in plastic tubes 122 permeable to air until their arrival in the laboratory. Spleen samples and ticks were stored at - 123 20 ºC until further analysis. The ticks were identified on the basis of morphology using 124 6 taxonomic keys (Estrada-Peña, Bouattour, Camicas & Walker, 2004). The feeding status of 125 collected ticks was unknown. An epidemiological questionnaire was completed via on-site 126 interviews with gamekeepers in each hunting estate to assess the influence of different 127 explanatory variables including presence of fences, presence of livestock (goats, cattle, sheep, 128 horses and swine), presence of river, stagnant waters, repopulations of wild ungulate species in 129 the last two years, presence of Mediterranean forest, pinewood and mountain pastures, 130 estimated density of red deer and wild ruminants (high: > 30 individuals/km2; medium: between 131 20 and 30 individuals/km2 and low: < 20 individuals/km2). Density cut-offs were set in 132 accordance with the optimal density values recommended for the Mediterranean ecosystems of 133 southern Spain to ensure sustainability of game species and conservation of biological diversity 134 (Perea, Girardello & San Miguel, 2014). In addition, climatological variables (continuous 135 variable) including average temperature (categorized using Jenk’s natural breaks), relative 136 humidity (categorized using Jenks natural breaks) and rainfall (categorized by terciles) of the 137 season in which each sampling was carried out were retrieved from public databases (RIA, 138 2020). The set of variables collected is showed in Supplementary Table 1. 139 DNA extraction and PCR amplification 140 DNA from spleen samples was extracted to determine the presence of TBP. Ticks collected 141 from TBP-positive animals were also analyzed to study the relationship between TBP status in 142 host and potential vectors. DNA was extracted using the commercial DNA Genomic kit (A&A 143 Biotechnology Gdańsk, Poland), following the manufacturer’s instructions. PCR analyses were 144 performed to detect infection with Anaplasma/Ehrlichia spp., Babesia/Theileria spp. and B. 145 burgdorferi s.l. The PCR protocols were carried out as described previously (Supplementary 146 Table 2). A. phagocytophilum and B. burgdorferi s.l. DNA obtained from the National 147 Reference Center for Borrelia of the Max von Pettenkofer Institute, as well as Babesia canis 148 EU622792 DNA and Babesia/Theileria DNA obtained from a previous study (Adaszek et al., 149 7 2012) were used as positive controls. Sterile nuclease-free water was used as a negative control. 150 The size of each PCR product was analyzed by electrophoresis in 1.5 % agarose gels stained 151 with ethidium bromide. 152 TBP species identification was based on an analysis of the nucleotide sequences of the obtained 153 amplicons. DNA purification was performed using the commercial QIAquick PCR purification 154 kit (Qiagen, Hilden, Germany). In the case of tick samples, the ectoparasites were washed twice 155 in sterile PBS solution for 5 min, shaken slowly, and then kept overnight at 4 °C. Each tick was 156 manually cut into four pieces with a sterile lancet and then suspended in 100 μl of Tris, 500 μl 157 of lysis buffer, and 20 μL of Proteinase K provided by the commercial kit. The remaining steps 158 of the purification of samples from wild ungulates and ticks were performed following the 159 manufacturer’s recommendations. Finally, DNA obtained from wild ungulates and tick samples 160 was eluted in 50 μl of Tris10 mM, pH 7.6 and 10 μL of elution buffer respectively and stored 161 at -20 °C for subsequent analysis. A volume of 5 μL of purified DNA was used in PCR 162 reactions. 163 Nucleotide sequences were assembled and edited using SeqMan (DNAStar, Lasergene, USA) 164 and MegAlign (DNAStar, Lasergene, USA) with alignments to representative sequences of 16S 165 rRNA gene for Anaplasma spp. and Borrelia spp. and 18S RNA gene for Theileria spp. and 166 Babesia divergens. Phylogenetic trees were constructed using the Neighbor-Joining method 167 (bootstrap analysis of 1000 replicates) with MEGA software version 7 (Kumar, Stecher & 168 Tamura, 2016). The trees are drawn to scale with branch lengths measured as the number of 169 substitutions per site. Only bootstrap values ≥ 70 are shown. 170 Statistical analysis 171 The prevalence of infections with A. phagocytophilum, piroplasms (Babesia/Theileria spp.) and 172 B. burgdorferi s.l. was estimated from the proportion of positives to the total number of 173 samples, with exact binomial confidence intervals of 95% (95%CI). Sample collection was 174 8 divided into three consecutive periods: 2009-2011, 2011-2013 and 2013-2015. Differences 175 between the prevalence of TBP infection and explanatory variables were analyzed using a 176 Pearson’s chi-square test or Fisher’s exact test, as appropriate. We evaluated two dependent 177 variables: prevalence of Anaplasma spp. and prevalence of Babesia/Theileria spp. Prevalence 178 of B. burgdorferi s.l. was not finally assessed because of the number of positives detected was 179 low. All the explanatory variables (Supplementary Table 1) were analyzed and those with P180 value < 0.20 were selected as potential risk factors. Cramer’s V coefficient between pairs of 181 variables was computed to assess collinearity. When collinearity (V > 0.6) occurred, only the 182 variable most clearly linked to infection was retained. The selected variables were included in 183 penalized maximum likelihood multiple logistic regression models to detect statistical 184 associations controlling for potential issues due to separation. An initial model was obtained 185 using all the selected explanatory variables and variables with a non-significant P-value were 186 removed sequentially. Model selection was assessed using the Aikake’s information criterion 187 (AIC). Variables that altered the coefficients of the independent variables of interest by 30% or 188 more when removed from the model were classified as confounding factors. The model was re189 run until all remaining variables presented statistically significant values (P < 0.05) and the 190 better AIC. A receiver operating characteristic (ROC) curve was also used to evaluate the 191 accuracy of the final models. Statistical analysis was performed using Stata 14.0 software 192 (StataCorp, Texas, USA). 193 Results 194 Infection with the selected TBP was detected in 127 of 863 wild ruminants (14.7%; 95%CI: 195 12.4-17.3) but not in wild boar (0/269). A total of 79 wild ruminants were positive for 196 Anaplasma/Ehrlichia spp. (9.2%; 95%CI: 7.3-11.3), 42 for Babesia/Theileria spp. (4.9%; 197 95%CI: 3.5-6.5) and six for B. burgdorferi s.l. (0.7%; 95%CI: 0.3-1.5) (Supplementary Table 198 1). The frequencies of infections and identification with the selected pathogens are shown in 199 9 Table 1. At least one infected animal was detected in 33 (66.0%) of the 50 sampled hunting 200 estates (Figure 1, Supplementary Tables 3-6). 201 All the strains of Anaplasma/Ehrlichia spp. detected were identified as A. phagocytophilum, 202 with 99.0–100 % sequence identity with the reference strain. The percentage of sequence 203 identity was lower for other Anaplasma species: 98.0% for A. platys, 97.0% for A. centrale and 204 A. marginale, and 96.0% for A. ovis (Supplementary Figure 1). With respect to the piroplasms, 205 Babesia divergens infection was identified in 25 animals (24 red deer and one fallow deer) with 206 98.0-99.0% sequence identity with lower percentages of identity with other piroplasms (78.0% 207 with T. capreoli, 78.0% with Theileria sp. OT3, 79.0% with T. equi, and 77.0% with T. ovis). 208 Theileria capreoli was only found in two red deer (99.5% sequence identity). Theileria 209 sequences highly identical (97.1 to 99.7%) to Theileria spp. OT3 AY533145 were found in 15 210 samples from mouflons and presented lower identity with other piroplasms (91.0% with T. 211 capreoli and T. ovis, 86.0% with T. equi, and 74.0% with B. divergens) (Table 1, Supplementary 212 Figure 2). In the six B. burgdorferi s.l.-positive cervids (five red deer and one fallow deer), all 213 these samples were identified as Borrelia afzelii with sequence identity values that ranged from 214 99.4 to 100%. The sequences of A. phagocytophilum, Babesia spp., Theileria spp. and B. afzelii 215 obtained in the present study (n = 127) were deposited in the GenBank database (GenBank 216 Accession Numbers: MT614368-MT614446, MT622544-MT622568, MT622569-MT622584 217 and MW054193, and MW054157-MW054162 respectively). Their phylogenetic relationships 218 with reference strains are shown in the Supplementary Figures 2-4. Co-infections with A. 219 phagocytophilum and B. divergens, and A. phagocytophilum and Theileria spp. were detected 220 in one red deer and four mouflons, respectively. 221 Multiple regression models showed that frequency of infection with A. phagocytophilum and 222 piroplasms was associated only with species of infected hosts. Anaplasma phagocytophilum 223 infection was significantly lower in red deer, roe deer and wild boar than in mouflon, although 224 16 Surveillance programs should also be implemented to characterize the risk of TBD transmission 375 in this country. This may also be important in countries in the Mediterranean Basin that present 376 similar ecological variations. In addition to this, further research is warranted to assess the 377 prevalence of other TBP not included in the present study in wild ungulates in southern Spain. 378 Acknowledgements 379 This work has benefited from the financial aid of research grants funded by Spanish Ministry 380 of Economy and Competitiveness (AGL2013-49159-C2-2-R). We also want to thank the 381 collaboration of all involved hunting states and game reserves and the dedicated assistance of 382 their game wardens, as well as to many colleagues and fellow students who participated in the 383 field sampling. 384 Conflict of interest 385 The authors declare that they have no competing interests. The authors certify that they have 386 no affiliation with or financial involvement in any organization or entity with a direct financial 387 interest in the subject matter or materials discussed in the manuscript. 388 Ethical approval 389 No animals were killed specifically for this study. 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Piroplasmosis in wildlife: Babesia and Theileria affecting 642 free-ranging ungulates and carnivores in the Italian Alps. Parasites and Vectors, 7, 1– 643 7. https://doi.org/10.1186/1756-3305-7-70 644 Zeman, P., & Pecha, M. (2008). Segregation of genetic variants of Anaplasma phagocytophilum 645 circulating among wild ruminants within a Bohemian forest (Czech Republic). 646 International Journal of Medical Microbiology, 298, 203–210. 647 https://doi.org/10.1016/j.ijmm.2008.03.003 648 649 650 651 25 Table legends 652 Table 1. Prevalence of selected tick-borne pathogens in wild ungulates (n = 1,132) in southern 653 Spain. 654 Table 2. Results of the penalized regression models for the association with the presence of 655 DNA of Anaplasma spp. (Anaplasma phagocytophillum) and piroplasms (Babesia/Theileria 656 spp.) in wild ungulates. 657 Table 3. Distribution of TBP in ticks (n = 89) removed from TBP-positive wild ruminants (n = 658 31) sampled in southern Spain. The number of sampled animals within each sampling point is 659 shown in brackets. 660 Figure legend: 661 Figure 1. Spatial distribution of tick-borne pathogens in wild ungulate species in southern 662 Spain. The number of sampled animals in each location is shown in brackets. 663 664 32 688 Supplementary Table 2. Primers used in PCR for detection and identification of 689 Anaplasma/Ehrlichia spp., Borrelia burgdorferi sensu lato and Babesia/Theileria spp. 690 691 692 †For this PCR protocol, we used a concentration of primer of 25 μM instead of the 10 693 μM used by these authors. 694 References 695 Adaszek, Ł., Winiarczyk, S., Łukaszewska, J., (2009). A first case of ehrlichiosis in a horse in 696 Poland. Deutsche Tierärztliche Wochenschrift. 116, 330-334 697 Adaszek, Ł., García-Bocanegra, I., Arenas-Montes, A., Carbonero, A., Arenas, A., & 698 Winiarczyk, S. (2012). Identification of piroplasms isolated from asymptomatic equine 699 species from southern Spain. Berliner und Münchener Tierärztliche Wochenschrift, 125, 700 509-512. 701 Altay, K., Aydin, M.F., Dumanli, N., & Aktas, M. (2008). Molecular detection of Theileria and 702 Babesia infections in cattle. Veterinary Parasitology, 158, 295–301. 703 https://doi.org/10.1016/j.vetpar.2008.09.025. 704 705 Pathogen Primers Target gene Amplicon size Reference Anaplasma/Ehrlichia spp. EHR 521: (5′-TGT AGG CGG TTC GGT AAG TTA AAG-3′) EHR 747: (5′-GCA CTC ATC GTT TAC AGC GTG-3′) 16S 247 bp Adaszek, Winiarczyk & Lukaszewska, (2009); Hodzic et al., (1998) Borrelia burgdorferi s. l. M1: (5′-ACG ATG CAC ACT TGG TGT TAA-3′) M2: (5′-TCC GAC TTA TCA CCG GCA GTC A-3′) 16S 357 bp Lee et al., (2019)† Babesia/Theileria spp. RLB R2: (5’-CTA AGA ATT TCA CCT CTG ACAGT-3’) RLB F2 (5’- GAC ACA GGG AGG TAG TGA CAAG-3’) hypervariable V4 region of the 18S 390-430 bp Adaszek et al., (2012); Altay, Aydin, Dumanli & Aktas, (2008) 33 Hodzic, E., Fish, D., Maretzki, C.M., De Silva, A.M., Feng, S., & Barthold, S.W. (1998). 706 Acquisition and transmission of the agent of human granulocytic ehrlichiosis by Ixodes 707 scapularis ticks. Clinical Journal of Microbiology, 36, 3574–3578. 708 Lee S.H., Healy J.E., & Lambert J.S. (2019). Single Core Genome Sequencing for Detection of 709 both Borrelia burgdorferi Sensu Lato and Relapsing Fever Borrelia Species. 710 International Journal of Environmental Research and Public Health. 16, 1779 711 712 713 34 Supplementary Table 3. Distribution of wild ungulates positive to Anaplasma 714 phagocytophilum infection in southern Spain. 715 ID Species Hunting estate Province Sampling hunting season Sequence groups† GenBank Accession number 1061 Fallow deer 1 Cadiz 2009-2010 A MT614368 1062 Fallow deer 1 Cadiz 2009-2010 A MT614369 1074 Fallow deer 1 Cadiz 2010-2011 A MT614370 1063 Mouflon 1 Cadiz 2009-2010 B MT614371 1064 Mouflon 1 Cadiz 2009-2010 B MT614372 1075 Mouflon 1 Cadiz 2010-2011 B MT614373 1076 Mouflon 1 Cadiz 2010-2011 B MT614374 1077 Fallow deer 2 Cadiz 2010-2011 I MT614427 1065 Roe deer 3 Cadiz 2009-2010 C MT614407 1070 Red deer 4 Cordoba 2009-2010 C MT614381 1071 Red deer 4 Cordoba 2009-2010 C MT614382 1082 Red deer 5 Cordoba 2011-2012 C MT614383 1083 Red deer 5 Cordoba 2011-2012 C MT614384 1069 Red deer 6 Cordoba 2009-2010 D MT614408 1092 Red deer 9 Cordoba 2011-2012 F MT614412 1093 Red deer 9 Cordoba 2011-2012 F MT614413 1121 Red deer 10 Cordoba 2014-2015 C MT614385 1120 Red deer 10 Cordoba 2014-2015 C MT614406 1085 Red deer 11 Cordoba 2011-2012 C MT614386 1119 Red deer 12 Cordoba 2014-2015 C MT614387 1098 Mouflon 14 Cordoba 2012-2013 B MT614375 1099 Mouflon 14 Cordoba 2012-2013 B MT614376 1100 Mouflon 14 Cordoba 2012-2013 B MT614377 1088 Red deer 14 Cordoba 2011-2012 C MT614388 1089 Red deer 14 Cordoba 2011-2012 C MT614389 1090 Red deer 14 Cordoba 2011-2012 C MT614390 1091 Red deer 14 Cordoba 2011-2012 C MT614391 1094 Red deer 15 Cordoba 2012-2013 C MT614392 1125 Red deer 15 Cordoba 2014-2015 C MT614393 1073 Red deer 16 Cordoba 2010-2011 C MT614394 1123 Red deer 16 Cordoba 2014-2015 C MT614395 1124 Red deer 16 Cordoba 2014-2015 C MT614396 1095 Mouflon 17 Cordoba 2012-2013 B MT614378 1096 Mouflon 17 Cordoba 2012-2013 B MT614379 1097 Mouflon 17 Cordoba 2012-2013 B MT614380 1108 Red deer 17 Cordoba 2013-2014 G MT614414 1116 Mouflon 17 Cordoba 2013-2014 G MT614415 1110 Mouflon 17 Cordoba 2013-2014 G MT614416 1111 Mouflon 17 Cordoba 2013-2014 G MT614417 1112 Mouflon 17 Cordoba 2013-2014 G MT614418 1113 Mouflon 17 Cordoba 2013-2014 C MT614419 35 1114 Mouflon 17 Cordoba 2013-2014 G MT614420 1115 Mouflon 17 Cordoba 2013-2014 G MT614421 1117 Mouflon 17 Cordoba 2013-2014 G MT614422 1118 Mouflon 17 Cordoba 2013-2014 G MT614423 1109 Mouflon 17 Cordoba 2013-2014 H MT614424 1128 Red deer 17 Cordoba 2014-2015 H MT614425 1129 Red deer 17 Cordoba 2014-2015 H MT614426 1068 Red deer 18 Cordoba 2009-2010 D MT614442 1078 Red deer 19 Cordoba 2011-2012 C MT614397 1079 Red deer 19 Cordoba 2011-2012 C MT614398 1130 Red deer 20 Cordoba 2014-2015 C MT614399 1066 Red deer 22 Cordoba 2009-2010 I MT614429 1067 Red deer 22 Cordoba 2009-2010 I MT614430 1126 Red deer 23 Cordoba 2014-2015 C MT614401 1084 Red deer 24 Cordoba 2011-2012 C MT614402 1127 Red deer 25 Cordoba 2014-2015 I MT614431 1086 Red deer 26 Cordoba 2011-2012 C MT614403 1087 Red deer 26 Cordoba 2011-2012 C MT614404 1106 Red deer 26 Cordoba 2013-2014 I MT614432 1107 Red deer 26 Cordoba 2013-2014 I MT614433 1056 Red deer 27 Cordoba 2009-2010 C MT614405 1101 Red deer 27 Cordoba 2013-2014 E MT614409 1102 Fallow deer 27 Cordoba 2013-2014 I MT614434 1103 Fallow deer 27 Cordoba 2013-2014 I MT614435 1104 Fallow deer 27 Cordoba 2013-2014 I MT614436 1105 Fallow deer 27 Cordoba 2013-2014 I MT614437 1057 Fallow deer 27 Cordoba 2009-2010 J MT614438 1058 Fallow deer 27 Cordoba 2009-2010 J MT614439 1059 Fallow deer 27 Cordoba 2009-2010 J MT614440 1060 Fallow deer 27 Cordoba 2009-2010 J MT614441 1122 Fallow deer 27 Cordoba 2014-2015 E MT614444 1131 Fallow deer 27 Cordoba 2014-2015 E MT614445 1132 Fallow deer 27 Cordoba 2014-2015 E MT614446 1080 Red deer 28 Huelva 2011-2012 F MT614410 1081 Red deer 28 Huelva 2011-2012 F MT614411 1072 Red deer 29 Jaen 2009-2010 D MT614443 1054 Red deer 31 Sevilla 2009-2010 C MT614400 1055 Red deer 31 Sevilla 2009-2010 I MT614428 †Sequences with 100% nucleotide identity were gathered in the same group (A-J). 716 717 718 36 Supplementary Table 4. Distribution of wild ungulates positive to Babesia divergens infection 719 in southern Spain 720 ID Species Hunting estate Province Sampling hunting season Sequence groups† GenBank Accession number 233 Red deer 1 Cadiz 2009-2010 A MT622550 234 Red deer 1 Cadiz 2009-2010 A MT622551 302 Red deer 1 Cadiz 2010-2011 C MT622545 238 Red deer 6 Cordoba 2009-2010 A MT622553 788 Red deer 7 Cordoba 2014-2015 A MT622566 236 Red deer 8 Cordoba 2009-2010 C MT622544 1119 Red deer 12 Cordoba 2014-2015 C MT622548 686 Red deer 13 Cordoba 2012-2013 C MT622547 586 Red deer 16 Cordoba 2011-2012 B MT622555 756 Red deer 17 Cordoba 2013-2014 B MT622564 306 Red deer 18 Cordoba 2010-2011 B MT622554 593 Red deer 21 Cordoba 2011-2012 A MT622560 759 Red deer 21 Cordoba 2013-2014 A MT622565 237 Red deer 22 Cordoba 2009-2010 A MT622552 687 Red deer 25 Cordoba 2012-2013 A MT622561 688 Red deer 25 Cordoba 2012-2013 A MT622562 944 Red deer 25 Cordoba 2014-2015 A MT622568 590 Red deer 26 Cordoba 2011-2012 B MT622558 591 Red deer 26 Cordoba 2011-2012 B MT622559 755 Red deer 26 Cordoba 2013-2014 B MT622563 588 Red deer 26 Cordoba 2011-2012 B MT622556 818 Red deer 27 Cordoba 2014-2015 A MT622567 1028 Fallow deer 27 Cordoba 2014-2015 B MT622549 587 Red deer 28 Huelva 2011-2012 C MT622546 589 Red deer 30 Seville 2011-2012 A MT622557 †Sequences with 100% nucleotide identity were gathered in the same group (A-J). 721 722 723 37 Supplementary Table 5. Distribution of wild ungulates positive to Theileria spp. infection in 724 southern Spain 725 ID Species Hunting estate Province Sampling hunting season Sequence groups† GenBank Accession number 235 Mouflon 1 Cadiz 2009-2010 A MT622570 303 Mouflon 1 Cadiz 2010-2011 A MT622571 304 Mouflon 2 Cadiz 2010-2011 B MT622572 305 Mouflon 2 Cadiz 2010-2011 B MT622573 685 Red deer 13 Cordoba 2012-2013 C MT622569 ‡ 681 Red deer 17 Cordoba 2012-2013 D MW054193 ‡ 1116 Mouflon 17 Cordoba 2013-2014 E MT622582 1115 Mouflon 17 Cordoba 2013-2014 E MT622581 1117 Mouflon 17 Cordoba 2013-2014 E MT622583 1118 Mouflon 17 Cordoba 2013-2014 E MT622584 682 Mouflon 17 Cordoba 2012-2013 E MT622575 683 Mouflon 17 Cordoba 2012-2013 E MT622576 684 Mouflon 17 Cordoba 2012-2013 E MT622577 757 Mouflon 17 Cordoba 2013-2014 E MT622578 758 Mouflon 17 Cordoba 2013-2014 E MT622579 1012 Mouflon 20 Cordoba 2014-2015 E MT622580 592 Mouflon 26 Cordoba 2011-2012 E MT622574 †Sequences with 100% nucleotide identity were gathered in the same group (A-J). ‡T. capreoli 726 strains. 727 728 729 730 38 Supplementary Table 6. Distribution of wild ungulates positive to Borrelia afzelii infection in 731 southern Spain 732 ID Species Hunting estate Province Sampling hunting season Sequence groups† GenBank Accession number 275 Fallow deer 1 Cadiz 2010-2011 A MW054157 868 Red deer 16 Cordoba 2014-2015 B MW054159 1003 Red deer 20 Cordoba 2014-2015 A MW054162 934 Red deer 25 Cordoba 2014-2015 A MW054160 803 Red deer 27 Cordoba 2014-2015 B MW054158 955 Red deer 28 Jaen 2014-2015 B MW054161 †Sequences with 100% nucleotide identity were gathered in the same group (A-J). 733 734 39 Supplementary Figure 1. Phylogenetic tree of Anaplasma spp. (based on 245 nt of 106 735 sequences) strains obtained in the present study and representative sequences available in 736 GenBank. Sequences obtained of Anaplasma spp. in the present study (n = 79) are showed in 737 bold. Strains obtained from the same host animal species that showed 100% homology 738 were grouped (the number of sequences within each group is shown between brackets). Each 739 strain or group of strains are identified by Anaplasma species/GenBank accession 740 number(s)/host/sampling year(s)/country, whenever possible. 741 742 40 Supplementary Figure 2. Phylogenetic trees of Babesia divergens (A) (n = 25) 743 and Theileria spp. (B) strains (n = 17) obtained in the present study and representative 744 sequences deposited in GenBank. Sequences obtained in the present study are in bold. The 745 analysis involved 408 nt of 39 sequences and 428 nt of 33 sequences of Babesia divergens 746 and Theileria spp., respectively. Each strain is identified by piroplasm 747 species/GenBank accession number/host/sampling year/country, whenever possible. 748 749 750 41 Supplementary Figure 3. Phylogenetic tree of B. afzelii strains (n = 6) obtained in the present 751 study and representative sequences deposited in GenBank. Sequences obtained in the present 752 study are in bold. The analysis involved 357 nt of 31 sequences of Borrelia spp. Each strain is 753 identified by species/GenBank accession number/host/sampling year/country, whenever 754 possible. 755 756 757