A survey of shared pathogens at the domestic-wild ruminants' interface in Donana National Park (Spain)

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ Esta es la versión aceptada del artículo publicado en: This is a accepted manuscript of a paper published in: Transboundary and Emerging Diseases (2022): May 2022 DOI: https://doi.org/10.1111/tbed.14126 Copyright: © 2021 Wiley-VCH GmbH. El acceso a la versión publicada del artículo puede requerir la suscripción de la revista. Access to the published version may require subscription. “This is the peer reviewed version of the following article: Jiménez-Ruiz S, García-Bocanegra I, Acevedo P, Espunyes J, Triguero-Ocaña R, Cano-Terriza D, Torres-Sánchez MJ, Vicente J, Risalde MÁ. A survey of shared pathogens at the domestic-wild ruminants' interface in Doñana National Park (Spain). Transbound Emerg Dis. 2022 May;69(3):1568-1576. doi: 10.1111/tbed.14126, which has been published in final form at https://doi.org/10.1111/tbed.14126. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited." 1 1 2 3 4 A survey of shared pathogens at the domestic–wild ruminants’ interface in Doñana National Park 5 (Spain) 6 7 Saúl Jiménez-Ruiz1,2 , Ignacio García-Bocanegra2 , Pelayo Acevedo1 , Johan Espunyes3,4 , 8 Roxana Triguero-Ocaña1,5 , David Cano-Terriza2, Maria J. Torres-Sánchez6 , Joaquín 9 Vicente1 , María Ángeles Risalde7,8 10 11 1Grupo Sanidad y Biotecnología (SaBio), Instituto de Investigación en Recursos Cinegéticos IREC 12 (UCLM-CSIC-JCCM), Ciudad Real, Spain 13 2Grupo de Investigación en Sanidad Animal y Zoonosis (GISAZ), Departamento de Sanidad Animal, 14 Facultad de Veterinaria, Universidad de Córdoba (UCO), Córdoba, Spain 15 3Wildlife Conservation Medicine Research Group (WildCoM), Departament de Medicina i Cirurgia, 16 Universitat Autònoma de Barcelona (UAB), Bellaterra, Spain 17 4Research and Conservation Department. Zoo de Barcelona, Barcelona, Spain 18 5VISAVET Health Surveillance Centre, Universidad Complutense de Madrid (UCM), Madrid, Spain 19 6Departamento de Microbiología, Universidad de Sevilla (USE), Sevilla, Spain 20 7Grupo de Investigación en Sanidad Animal y Zoonosis (GISAZ), Departamento de Anatomía y 21 Anatomía Patológica Comparadas y Toxicología, Facultad de 22 Veterinaria, Universidad de Córdoba (UCO), Córdoba, Spain 23 8Unidad de Enfermedades Infecciosas, Grupo de Virología Clínica y Zoonosis, Instituto Maimónides de 24 Investigación Biomédica de Córdoba (IMIBIC), Hospital Reina Sofía, Universidad de Córdoba (UCO), 25 Córdoba, Spain 26 27 *Corresponding author: joaqu[email protected]s 28 29 2 30 31 ABSTRACT 32 A cross-sectional study was carried out to evaluate shared pathogens that can be 33 transmitted by close or non-close contact at the domestic–wild ruminants’ interface. During 34 summer–autumn 2015, a total of 138 cattle and 203 wild ruminants (red deer, Cervus 35 elaphus, and fallow deer, Dama dama) were sampled in Doñana National Park (DNP, south36 western Spain), a Mediterranean ecosystem well known for the interaction network 37 occurring in the ungulate host community. Pestiviruses, bovine respiratory syncytial virus 38 (BRSV; Bovine orthopneumovirus), bovine herpesvirus 1 (BoHV-1; Bovine alphaherpesvirus 39 1) and Mycobacterium tuberculosis complex (MTC) were assessed using serological, 40 microbiological and molecular techniques. The overall seroprevalence against viruses in 41 cattle was 2.2% for pestiviruses, 11.6% for BRSV and 27.5% for BoHV-1. No virus-specific 42 antibodies were found in wildlife. MTC incidence in cattle was 15.9%, and MTC 43 seroprevalence in wild ruminants was 14.3%. The same Mycobacterium bovis spoligotypes 44 (SB1232, SB1230 and SB1610) were identified in cattle, red deer and fallow deer. The 45 serological results for the selected respiratory viruses suggest epidemiological cycles only 46 in cattle. Surveillance efforts in multi-host epidemiological scenarios are needed to better 47 drive and prioritize control strategies for shared pathogens. 48 49 K E Y WO R D S 50 bovine herpesvirus 1, bovine respiratory syncytial virus, cattle, Mycobacterium tuberculosis 51 complex, pestiviruses, wild ruminants. 52 53 54 3 55 56 INTRODUCTION 57 58 Pathogens that infect multiple hosts (multi-host pathogens) and can be transmitted between livestock 59 and/or wildlife represent a front of growing concern worldwide due to their implications on animal and 60 public health, and conservation of endangered species (Miller et al., 2013; Woolhouse et al., 2001). 61 Outdoor farming systems facilitate the spatio-temporal overlapping in the use of resources (e.g. food and 62 water) between domestic and wild species, which could contribute to the maintenance of shared 63 pathogens (multihost pathogens requiring at least one domestic and one wild host to be maintained) 64 (Cowie et al., 2016; Gortázar et al., 2016). In this sense, plain areas often thrive as agricultural lands, so 65 that extensive livestock production has been relegated near bush areas where wild ungulate populations 66 are continuously growing and expanding, thus favouring the maintenance of these pathogens in different 67 host communities (Gortázar et al., 2007). 68 69 The complexity of interactions established within multi-host communities and natural anthropogenic 70 factors may determinate the horizontal inter-species transmission of shared pathogens (Tompkins et al., 71 2011). Indirect transmission occurs when intermediate living (vectors) or inanimate (fomites) vehicles 72 are involved in transmitting the infection between infected and susceptible hosts (Thrusfield, 2018). 73 Among directly transmitted agents, many pathogens are mainly transmitted through physical contact 74 or very close proximity (contact with infected discharges of other animals), increasing the risk of 75 transmission in epidemiological scenarios with high population density and management practices 76 involving aggregation of animals (Gortázar et al., 2006; Sorensen et al., 2014). On the other hand, the 77 ability of other pathogens to survive under determined environmental conditions can favour their 78 transmission also through non-close interactions (Fine et al., 2011), produced between individuals 79 that share contaminated feeding or drinking resources at different times (Barasona, Latham et al., 80 2014; Kukielka et al., 2013; Sorensen et al., 2014). Nevertheless, interaction patterns are dependent on 81 the availability and distribution of resources (Triguero-Ocaña et al., 2020), the social hierarchy 82 relationships (Böhm et al., 2009) or the biosecurity measures (Barasona et al., 2013). Therefore, under83 standing infection dynamics and pathogen transmission routes is a key factor to design effective multi84 host pathogen control strategies. 85 4 In south and central regions of the Iberian Peninsula, livestock is usually raised under extensive 86 production systems, sharing habitats with wild ungulates and establishing multi-host communities that 87 favour the transmission of shared pathogens (Ministerio de Agricultura, Pesca y Alimentación, 2017). 88 89 To better understand the epidemiological implications of these communities, previous studies assessed 90 the frequency of interactions at the livestock– wildlife interface, evidencing a more relevant role in 91 the pathogen transmission of non-close contacts than close contact (Cowie et al., 2016; Kukielka et 92 al., 2013; Triguero-Ocaña et al., 2019, 2020). Interestingly, Triguero-Ocaña et al. (2020) characterized the 93 dynamic network of interactions produced between sympatric cattle, red deer (Cervus elaphus), fallow 94 deer (Dama dama) and wild boar (Sus scrofa) in Doñana National Park (DNP; south-western Spain), over 95 a period of two years, and simulated a theoretical spread of pathogens horizontally transmitted within 96 this network. Their results showed a high connection between species through non-close interactions, 97 where most of them (93.2%) involved cattle and wild ruminant species, as well as a marked seasonality 98 with maximum interaction rates in summer–autumn period. 99 100 The epidemiology of shared pathogens transmitted by nonclose contact has been frequently 101 investigated in Mediterranean ecosystems, especially in relation to Mycobacterium tuberculosis 102 complex (MTC) (Barasona, Mulero-Pázmány et al., 2014; Barroso et al., 2020; Gortázar et al., 2008, 103 2011). However, there is a gap of knowledge on pathogens with close direct transmission within these 104 domestic–wild ruminant communities. In this regard, pestiviruses, bovine respiratory syncytial virus 105 (BRSV; Bovine orthopneumovirus) and bovine herpesvirus 1 (BoHV-1; Bovine alphaherpesvirus 1) are 106 among the main pathogens involved in bovine respiratory disease (BRD) (Mosier, 2014) with the ability 107 to cross-infect several domestic and wild ruminant species (Frölich, 2000). These viruses have a limited 108 environmental survival and airborne transmission of few minutes and metres, so their main 109 transmission pathway is considered to be direct and close (Callan & Garry, 2002). In this context, the 110 hypothesis raised in this study was that multi-host pathogens transmitted by non-close contact are 111 more easily maintained within host communities in natural ecosystems than those transmitted by close 112 ones. Therefore, selected pathogens with mainly close (pestiviruses, BRSV and BoHV-1) and non-close 113 (MTC) contact direct transmission routes were evaluated at the domestic–wild ruminants’ interface in 114 DNP. 115 116 5 117 118 MATERIALS AND METHODS 119 120 Study area and design. 121 122 DNP (37°0′N, 6°30′W; 54,252 ha) is one of the most important nature reserves in Europe in terms of 123 biodiversity, where an endemic breed of cattle (called ‘Marismeña’) and wild ungulate species, such as 124 red deer, fallow deer and wild boar, inhabit in sympatry. Livestock movements are limited by cattle-proof 125 fences in four management areas, but wildlife can easily cross them and move freely within the park. 126 Human handling on cattle is exclusively related to obligatory disease control programmes, including 127 eradication campaigns for bovine tuberculosis and brucellosis, vaccination against bluetongue virus and 128 surveillance of several OIE (World Organization for Animal Health) listed diseases (Ministerio de 129 Agricultura, Pesca y Alimentación, 2020a). Nor vaccination neither biosecurity measures are 130 implemented against the selected respiratory viruses in the study area. Likewise, wild ruminant 131 populations are only regulated from populational control, which is annually carried out by the park's 132 authorities to maintain sustainable population densities in order to preserve the biodiversity of this 133 singular ecosystem (Vicente et al., 2014). 134 135 During summer–autumn 2015, a cross-sectional study was performed to determine circulation of 136 pestiviruses, BRSV, BoHV-1 and MTC in cattle and wild ruminant populations in DNP. Sample size was 137 calculated assuming a mean prevalence of 30%, a confidence level of 95% and a desired precision of 5%. 138 This estimation was based on previous surveys on Pestivirus and MTC in cattle-deer communities in 139 Mediterranean ecosystems and within DNP, respectively (Gortázar et al., 2008; Ministerio de Agricultura, 140 Pesca y Alimentación, 2020b; Paniagua et al., 2016; Rodríguez-Prieto et al., 2016; Romero et al., 2008). 141 Sampling was homogeneously distributed across the study area, and the number of animals by species 142 was chosen to ensure a 95% probability of detecting at least one positive individual, for an assumed 143 minimum prevalence of 3%. Hence, a total of 341 domestic and wild ruminants including 138 cattle, 101 144 red deer and 102 fallow deer were randomly selected (Figure 1). Data on sex, age and location were 145 recorded for each animal. Whenever possible, cattle age was provided by veterinary services, whereas 146 wild ruminants were classified into three age groups (yearlings, sub-adults or adults) based on their 147 dentition patterns (Sáenz de Buruaga et al., 2001). 148 6 149 150 Sample collection and laboratorial analyses. 151 152 Blood samples were obtained by puncture of the medial caudal vein in cattle and from the endocranial 153 venous sinuses in wild ruminant species (Jiménez-Ruiz et al., 2016). Samples were placed into sterile 154 tubes with no anticoagulant and immediately refrigerated and centrifuged at 400 g for 10 min. Sera were 155 then stored at −20°C until analyses. Serum samples were tested by different commercial enzyme-linked 156 immunosorbent assays (ELISA) to detect specific antibodies against pestivirus (blocking ELISA for BVDV157 p80-Ab; IDEXX®, Montpellier, France), BRSV and BoHV-1 (indirect ELISAs; SVANOVIR®, Uppsala, Sweden), 158 according to manufacturers’ instructions. 159 160 Pestivirus ELISA positive sera were subsequently tested by virus neutralization tests (VNT) usingdifferent 161 pestivirus species. Isolates of Pestivirus A (Bovine Viral Diarrhoea Virus (BVDV)-1NADL strain; GenBank 162 Acc. No. M31182), Pestivirus B (BVDV-2, BVDV GBK IRTA-CRESA strain; GenBank Acc. No. MW591704) 163 and Pestivirus D (Border Disease Virus-4-BDV-pig-SP-2007 strain; GenBank Acc. No. HF567456) were used 164 for VNT since they are the main pestivirus species reported in livestock and wildlife in Spain (Elvira Partida 165 et al., 2017; Jiménez-Ruiz et al., 2020). Antibody titres were expressed as the reciprocal of the highest 166 dilution neutralizing 100 tissue culture infective doses (100 TCID50) in all cultures (Reed & Muench, 1938), 167 using the immunoperoxidase monolayer assay (IPMA) to monitor neutralization (OIE, 2019a). Sera 168 showing neutralization at dilutions ≥1:10 were considered as pestivirus positive and were only specifically 169 associated with Pestivirus A, B or D when threefold higher antibody titres between species were detected 170 (Jiménez-Ruiz et al., 2020). 171 172 Serum samples of wild ruminant species were tested to detect specific antibodies against MTC using an 173 indirect in-house ELISA (P22-ELISA). As antigen, this ELISA included an immunopurified subcomplex 174 obtained from bovine purified protein derivative (bPPD), called P22 (Infantes-Lorenzo et al., 2017). Protein 175 G-horseradish peroxidase (HRP) was used as conjugate (Sigma-Aldrich Quimica SA®, Madrid, Spain), 176 following the protocol described by Thomas et al. (2019). The P22-ELISA results were expressed as an 177 ELISA percentage (E%), calculated by the formula as follows: sample E%= (mean sample OD/2 × mean of 178 negative control OD) x 100. As an anti-P22-positive control, a serum positive to P22-ELISA (OD > 1) 179 7 180 181 originated from red deer previously confirmed as Mycobacterium bovis (M. bovis) culture positive was 182 used; while the negative control used was a negative serum to P22-ELISA (OD ≤ 0.2) obtained from TB-free 183 red deer previously confirmed as M. bovis culture negative. Positive and negative controls were tested in 184 quadruplicate on every plate. 185 186 Tissue samples from the wild ruminants that showed compatible tuberculosis-like lesions (TBLL) were 187 collected as described by Gortázar et al., (2008) and placed into sterile containers for freezing at −20°C until 188 analyses. In order to confirm MTC infection, a representative number of TBLL were subjected to specific 189 MTC culture in the biosecurity level 3 laboratory of Virgen del Rocio Hospital (Seville, Spain) using standard 190 procedures (OIE, 2019b). Identification of MTC from suspected colonies was performed by a DNA probe 191 system (GenotypeHMTBC, Hain Lifescience GmbH, Germany), and spoligotypes were then identified using 192 the standardized membrane with 43 spacers (Kamerbeek et al., 1997). 193 Tuberculosis in cattle was evaluated inside of the bovine tuberculosis eradication programme using the 194 single intradermal tuberculin test (SITT) by government veterinarians. The SITT was performed according 195 to European and Spanish legislation (EU Council Directive 64/432/CEE and R.D. 2611/1996), which is based 196 on the delayed hypersensitivity reaction to the intradermal inoculation of bPPD and measurement of the 197 skin thickness after 72 hr. The positive animals were slaughtered (Directives 64/432/EC and 78/52/EC). 198 Tissue samples were obtained during routine slaughterhouse inspections by government veterinarians and 199 then submitted to the official laboratory for MTC culture and molecular analyses. The incidence of MTC 200 (total population is tested annually) was estimated taking into account the official results of SITT, while both 201 official MTC culture and spoligotyping results served as further confirmation of MTC infection in the animal 202 reactor to bPPD. 203 204 Statistical analyses. 205 206 The seroprevalence/incidence of each pathogen was calculated from the proportion of positives to the total 207 number of animals examined (in a 1-year period in case of incidence), with exact binomial confidence in208 tervals of 95% (95% CI) (Thrusfield, 2018). Differences in the infection rates between pathogens, host 209 species and individual factors (sex and age), as well as coinfections were analysed by the chi-square or 210 Fisher's tests. Missing values were omitted, and values with p < .05 were considered statistically significant. 211 8 Statistical analyses were performed using SPSS v. 25 software (IBM Corp., Armonk, NY, USA). 212 213 15 376 377 REFERENCE S 378 Arnaiz, I., Diéguez, J., Erais, C., Sanjuán, M. L., & Yus, E. (2012). Programas de control de IBR y BVD en 379 España. Producción Animal, 27, 22–36. 380 Barasona, J. A., Latham, M. C., Acevedo, P., Armenteros, J. A., Latham, 381 A. D. M., Gortazar, C., Carro, F., Soriguer, R. C., & Vicente, J. (2014a). Spatiotemporal interactions 382 between wild boar and cattle: Implications for cross-species disease transmission. Veterinary 383 Research, 45, 122. https://doi.org/10.1186/s13567-014-0122-7 384 Barasona, J. A., Mulero-Pázmány, M., Acevedo, P., Negro, J. J., Torres, 385 M. J., Gortázar, C., & Vicente, J. (2014b). Unmanned aircraft systems for studying spatial abundance of 386 ungulates: Relevance to spatial epidemiology. PLoS One, 9, e115608. https://doi.org/10.1371/journ 387 al.pone.0115608 388 Barasona, J. A., VerCauteren, K. C., Saklou, N., Gortazar, C., & Vicente, 389 J. (2013). Effectiveness of cattle operated bump gates and exclusion fences in preventing ungulate 390 multi-host sanitary interaction. Preventive Veterinary Medicine, 111, 42–50. https://doi.org/10.1016/j. 391 prevetmed.2013.03.009 392 Barasona, J. A., Vicente, J., Díez-Delgado, I., Aznar, J., Gortázar, C., & Torres, M. J. (2017). Environmental 393 presence of Mycobacterium tuberculosis complex in aggregation points at the wildlife/livestock inter394 face. Transboundary and Emerging Diseases, 64, 1148–1158. https:// doi.org/10.1111/tbed.12480 395 Barroso, P., Barasona, J. A., Acevedo, P., Palencia, P., Carro, F., Negro, J. J., Torres, M. J., Gortázar, C., 396 Soriguer, R. C., & Vicente, J. (2020). Longterm determinants of tuberculosis in the ungulate host 397 community of Doñana National Park. Pathogens, 9, 445. https://doi.org/10.3390/ pathogens9060445 398 Boelaert, F., Speybroeck, N., de Kruif, A., Aerts, M., Burzykowski, T., Molenberghs, G., & Berkvens, D. L. 399 (2005). Risk factors for bovine herpesvirus-1 seropositivity. Preventive Veterinary Medicine, 69, 285– 400 295. https://doi.org/10.1016/j.prevetmed.2005.02.010 401 Böhm, M., Hutchings, M. R., & White, P. C. (2009). Contact networks in a wildlife-livestock host 402 community: Identifying high-risk individuals in the transmission of bovine TB among badgers and 403 cattle. PLoS One, 4, e5016. https://doi.org/10.1371/journal.pone.0005016 404 Callan, R. J., & Garry, F. B. (2002). Biosecurity and bovine respiratory disease. Veterinary Clinics of North 405 America: Food Animal Practice, 18, 57–77. https://doi.org/10.1016/S0749-0720(02)00004-X 406 Cano-Terriza, D., Risalde, M. A., Jiménez-Ruiz, S., Vicente, J., Isla, J., Paniagua, J., Moreno, I., Gortázar, C., 407 Infantes-Lorenzo, J. A., & García-Bocanegra, I. (2018). Management of hunting waste as control 408 measure for tuberculosis in wild ungulates in south-central Spain. Transboundary and Emerging 409 Diseases, 65, 1190–1196. https:// doi.org/10.1111/tbed.12857 410 Cardoso-Toset, F., Gómez-Laguna, J., Amarilla, S. P., Vela, A. I., Carrasco, L., Fernández-Garayzábal, J. F., 411 Astorga, R. J., & Luque, I. (2015). Multi-etiological nature of tuberculosis-like lesions in condemned pigs 412 at the slaughterhouse. PLoS One, 10, e0139130. https://doi. org/10.1371/journal.pone.0139130 413 Corbett, C. S., De Buck, J., & Barkema, H. W. (2018). Effects of freezing on ability to detect Mycobacterium 414 avium subsp. paratuberculosis from bovine tissues following culture. Journal of Veterinary Diagnostic 415 Investigation, 30, 743–746. https://doi.org/10.1177/1040638718790781 416 Cowie, C. E., Hutchings, M. R., Barasona, J. A., Gortázar, C., Vicente, J., & White, P. C. (2016). Interactions 417 between four species in a complex wildlife: Livestock disease community: Implications for 418 Mycobacterium bovis maintenance and transmission. European Journal of Wildlife Research, 62, 51– 419 64. https://doi.org/10.1007/s1034 4-015-0973-x 420 16 Diéguez, F. J., Cerviño, M., & Yus, E. (2017). Bovine viral diarrhea virus (BVDV) genetic diversity in Spain: 421 A review. Spanish Journal of Agricultural Research, 15, e05R01. https://doi.org/10.5424/ sjar/2017152422 10619 423 Elvira Partida, L., Fernández, M., Gutiérrez, J., Esnal, A., Benavides, J., Pérez, V., de la Torre, A., Álvarez, 424 M., & Esperón, F. (2017). Detection of bovine viral diarrhoea virus 2 as the cause of abortion outbreaks 425 on commercial sheep flocks. Transboundary and Emerging Diseases, 64, 19–26. 426 https://doi.org/10.1111/tbed.12599 427 European Union (2015). Animal Health-Regulatory Committee Presentations. Evolution of the National 428 Eradication Programme on Bovine tb 2014 in Spain. Retrieved from ec.europa.eu/food/sites/ 429 food/files/animals/docs/reg-com_ahw_20150506_pres_bovinetuberculosis_spain.pdf (Accessed 7th 430 November 2020). 431 Fernández-Aguilar, X., López-Olvera, J. R., Marco, I., Rosell, R., ColomCadena, A., Soto-Heras, S., Lavín, 432 S., & Cabezón, O. (2016). Pestivirus in alpine wild ruminants and sympatric livestock from the 433 Cantabrian Mountains, Spain. Veterinary Record, 128, 586–591. https://doi. org/10.1136/vr.103577 434 Fine, A. E., Bolin, C. A., Gardiner, J. C., & Kaneene, J. B. (2011). A study of the persistence of Mycobacterium 435 bovis in the environment under natural weather conditions in Michigan, USA. Veterinary Medicine 436 International, 2011, https://doi.org/10.4061/2011/765430 437 Frölich, K. (2000). Viral diseases of northern ungulates. Rangifer, 20, 438 83–97. https://doi.org/10.7557/2.20.2-3.1505 439 Frölich, K., Thiede, S., Kozikowski, T., & Jakob, W. (2002). A review of mutual transmission of important 440 infectious diseases between livestock and wildlife in Europe. Annals of the New York Academy of 441 Sciences, 969, 4–13. https://doi.org/10.1111/j.1749-6632.2002. tb04343.x 442 García-Bocanegra, I., de Val, B. P., Arenas-Montes, A., Paniagua, J., Boadella, M., Gortázar, C., & Arenas, 443 A. (2012). Seroprevalence and risk factors associated to Mycobacterium bovis in wild artiodactyl 444 species from southern Spain, 2006–2010. PLoS One, 7, e34908. 445 https://doi.org/10.1371/journal.pone.0034908 446 Gay, E., & Barnouin, J. (2009). A nation-wide epidemiological study of acute bovine respiratory disease in 447 France. Preventive Veterinary Medicine, 89, 265–271. https://doi.org/10.1016/j.preve 448 tmed.2009.02.013 449 Giovannini, A., Cancellotti, F. M., Turilli, C., & Randi, E. (1988). Serological investigations for some bacterial 450 and viral pathogens in fallow deer (Cervus dama) and wild boar (Sus scrofa) of the San Rossore 451 Preserve, Tuscany, Italy. Journal of Wildlife Diseases, 24, 127–132. https://doi. org/10.7589/0090452 3558-24.1.127 453 González-García, M. A., Maldonado, J. L., Gómez-Pacheco, J. M., ArenasCasas, A., Carbonero-Martínez, 454 A., Borges-Rodríguez, C., García Bocanegra, I., & Perea-Remujo, J. A. (2009). Seroprevalence and risk 455 factors associated with bovine herpesvirus type 1 (BHV1) infection in non-vaccinated cattle herds in 456 Andalusia (South of Spain). SpanishJournal of Agricultural Research, 7, 550–554. 457 https://doi.org/10.5424/ sjar/2009073-439 458 Gortázar, C., Acevedo, P., Ruiz-Fons, F., & Vicente, J. (2006). Disease risks and overabundance of game 459 species. European Journal of Wildlife Research, 52, 81–87. https://doi.org/10.1007/s10344-005-0022460 2 461 Gortázar, C., Ferroglio, E., Höfle, U., Frölich, K., & Vicente, J. (2007). Diseases shared between wildlife and 462 livestock: A European perspective. European Journal of Wildlife Research, 53, 241–256. https:// 463 doi.org/10.1007/s10344-007-0098-y 464 Gortazar, C., Torres, M. J., Acevedo, P., Aznar, J., Negro, J. J., de la Fuente, J., & Vicente, J. (2011). Fine465 tuning the space, time, and host distribution of mycobacteria in wildlife. BMC Microbiology, 11, 27. 466 https://doi. org/10.1186/1471-2180-11-27 467 17 Gortázar, C., Torres, M. J., Vicente, J., Acevedo, P., Reglero, M., de la Fuente, J., Negro, J. J., & Aznar468 Martín, J. (2008). Bovine tuberculosis in Doñana Biosphere Reserve: The role of wild ungulates as 469 disease reservoirs in the last Iberian lynx strongholds. PLoS One, 3, e2776. 470 https://doi.org/10.1371/journal.pone.0002776 471 Gortázar, C., Ruiz-Fons, J. F., & Höfle, U. (2016). Infections shared with wildlife: An updated 472 perspective. European Journal of Wildlife Research, 62, 511–525. https://doi.org/10.1007/s10344473 016-1033-x Graham, D. A., Gallagher, C., Carden, R. F., Lozano, J. M., Moriarty, J., & O’Neill, R. 474 (2017). A survey of free-ranging deer in Ireland for serological evidence of exposure to bovine 475 viral diarrhoea virus, bovine herpes virus-1,bluetongue virus and Schmallenberg virus. 476 Irish Veterinary Journal, 70, 13. https://doi.org/10.1186/s1362 0-017-0091-z 477 Hägglund, S., & Valarcher, J. F. (2016). 48 Bovine Respiratory Syncytial Virus. In L. Dongyou (Ed.), 478 Molecular detection of animal viral pathogens (1st ed., p. 413). : CRC Press Taylor and Francis Group. 479 Hardstaff, J. L., Marion, G., Hutchings, M. R., & White, P. C. (2014). Evaluating the tuberculosis hazard 480 posed to cattle from wildlife across Europe. Research in Veterinary Science, 97, 86–93. https://doi. 481 org/10.1016/j.rvsc.2013.12.002 482 Hoppe, I. B. A. L., de Medeiros, A. S. R., Arns, C. W., & Samara, S. I. (2018). Bovine respiratory syncytial 483 virus seroprevalence and risk factors in non-vaccinated dairy cattle herds in Brazil. BMC Veterinary 484 Research, 14, 208. https://doi.org/10.1186/s12917-018-1535-8 485 Houe, H. (1995). Epidemiology of bovine viral diarrhea virus. Veterinary Clinics of North America: Food 486 Animal Practice, 11, 521–547. https:// doi.org/10.1016/S0749-0720(15)30465-5 487 Infantes-Lorenzo, J. A., Moreno, I., Risalde, M. D. L. Á., Roy, Á., Villar, M., Romero, B., Ibarrola, N., de 488 la Fuente, J., Puentes, E., de Juan, L., Gortázar, C., Bezos, J., Domínguez, L., & Domínguez, M. (2017). 489 Proteomic characterisation of bovine and avian purified protein derivatives and identification of 490 specific antigens for serodiagnosis of bovine tuberculosis. Clinical Proteomics, 14, 36. https://doi. 491 org/10.1186/s12014-017-9171-z 492 Jiménez-Ruiz, S., Arenas-Montes, A., Cano-Terriza, D., Paniagua, J., Pujols, J., Miró, F., Fernández-Aguilar, 493 X., González, M. Á., Franco, J. J., & García-Bocanegra, I. (2016). Blood extraction method by endo494 cranial venous sinuses puncture in hunted wild ruminants. European Journal of Wildlife Research, 62, 495 775–780. https://doi.org/10.1007/ s10344-016-1056-3 496 Jiménez-Ruiz, S., Vicente, J., García-Bocanegra, I., Cabezón, Ó., Arnal, M. C., Balseiro, A., & Acevedo, P. 497 (2020). Distribution of Pestivirus exposure in wild ruminants in Spain. Transboundary and Emerging 498 Diseases. https://doi.org/10.1111/tbed.13827 499 Kahrs, R. F. (1977). Infectious bovine rhinotracheitis: A review and update. Journal of the American 500 Veterinary Medical Association, 171, 1055–1064. 501 Kamerbeek, J., Schouls, L., Kolk, A., van Agterveld, M., van Soolingen, D., Kuijper, S., Bunschoten, A., 502 Molhuizen, H., Shaw, R., Goyal, M., & van Embden, J. (1997). Simultaneous detection and strain 503 differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. Journal of Clinical 504 Microbiology, 35, 907–914. https://doi.org/10.1128/ JCM.35.4.907-914.1997 505 Kukielka, E., Barasona, J. A., Cowie, C. E., Drewe, J. A., Gortazar, C., Cotarelo, I., & Vicente, J. (2013). 506 Spatial and temporal interactions between livestock and wildlife in South Central Spain assessed by 507 camera traps. Preventive Veterinary Medicine, 112, 213–221. https:// 508 doi.org/10.1016/j.prevetmed.2013.08.008 509 Maas, M., Michel, A. L., & Rutten, V. P. (2013). Facts and dilemmas in diagnosis of tuberculosis in wildlife. 510 Comparative Immunology, Microbiology and Infectious Diseases, 36, 269–285. https://doi. 511 org/10.1016/j.cimid.2012.10.010 512 Mainar-Jaime, R. C., Berzal-Herranz, B., Arias, P., & Rojo-Vázquez, F. 513 A. (2001). Epidemiological pattern and risk factors associated with bovine viral-diarrhoea virus (BVDV) 514 18 infection in a non-vaccinated dairy-cattle population from the Asturias region of Spain. Preventive 515 Veterinary Medicine, 52, 63–73. https://doi.org/10.1016/S0167 516 -5877(01)00239-2 517 Menzies, F. D., Abernethy, D. A., Stringer, L. A., Honhold, N., & Gordon, A. W. (2012). A matched 518 cohort study investigating the risk of Mycobacterium bovis infection in the progeny of infected 519 cows. Veterinary Journal, 194, 299–302. https://doi.org/10.1016/j. tvjl.2012.05.005 520 Miller, R. S., Farnsworth, M. L., & Malmberg, J. L. (2013). Diseases at the livestock-wildlife interface: 521 Status, challenges, and opportunities in the United States. Preventive Veterinary Medicine, 110, 522 119–132. https://doi.org/10.1016/j.prevetmed.2012.11.021 523 Ministerio de Agricultura, Pesca y Alimentación (2017). Plan de Actuación sobre Tuberculosis en Especies 524 silvestres. Retrieved from www.mapa. gob.es/es/ganaderia/temas/sanidad-animal-higiene-ganadera/ 525 patubes2017_3_tcm30-378321.pdf (Accessed 8th October 2020). 526 Ministerio de Agricultura, Pesca y Alimentación (2018). Informe Resultados Programa de Vigilancia 527 Rinotraqueítis Infecciosa Bovina (IBR). Retrieved from www.mapa.gob.es/es/ganaderia/temas/sanid 528 ad-animal-higiene-ganadera/informeresultadosprogramavigilancia ibr2018_tcm30-519724.pdf 529 (Accessed 16th October 2020). 530 Ministerio de Agricultura, Pesca y Alimentación (2020). Enfermedades de los animales. Retrieved from 531 www.mapa.gob.es/es/ganaderia/temas/ sanidad-animal-higiene-ganadera/sanidad532 animal/enfermedades/ (Accessed 19th October 2020). 533 Ministerio de Agricultura, Pesca y Alimentación (2020). Programa nacional de erradicación de 534 tuberculosis bovina presentado por España para el año 2020. Retrieved from 535 www.mapa.gob.es/es/ganaderia/temas/ sanidad-animal-higiene-ganadera/pnetb_2020final_tcm30536 523317. pdf (Accessed 19th October 2020). 537 Mollema, L., Rijsewijk, F. A. M., Nodelijk, G., & De Jong, M. C. M. (2005). Quantification of the 538 transmission of bovine herpesvirus 1 among red deer (Cervus elaphus) under experimental 539 conditions. Veterinary Microbiology, 111, 25–34. https://doi.org/10.1016/j. vetmic.2005.09.007 540 Mosier, D. (2014). Review of BRD pathogenesis: The old and the new. Animal Health Research Reviews, 541 15, 166. https://doi.org/10.1017/ S1466252314000176 542 OIE. World Organisation for Animal Health (2019a). Manual of Diagnostic Tests and Vaccines for 543 Terrestrial Animals 2019. Chapter 3.7.1. Border disease. 2018. Retrieved from 544 www.OIE.int/fileadmin/Home/eng/ Health_standards/tahm/3.07.01_BORDER_DIS.pdf (Accessed 7th 545 September 2020). 546 OIE. World Organisation for Animal Health (2019b). Manual of Diagnostic Tests and Vaccines for 547 Terrestrial Animals 2019. Chapter 3.4.6. Bovine tuberculosis. 2018. 548 www.OIE.int/fileadmin/Home/eng/Health_ standards/tahm/3.04.06_BOVINE_TB.pdf (Accessed 7th 549 September 2020). 550 Paniagua, J., García-Bocanegra, I., Arenas-Montes, A., Berriatua, E., Espunyes, J., Carbonero, A., & 551 Cabezón, O. (2016). Absence of circulation of Pestivirus between wild and domestic ruminants 552 insouthern Spain. Veterinary Record, 178, 215. https://doi.org/10.1136/vr.103490 553 Peón, N., Peralta, E., San Miguel, J. M., & Suárez-Inclán, A. (2018). Prevalencia de la ERB en la producción 554 de vacuno de leche de España. Retrieved from vacapinta.com/media/files/fichero/prevalencia_erb_ 555 vaca_pinta3_castelan.pdf (Accessed 24th October 2020). 556 Phillips, C. J. C., Foster, C. R. W., Morris, P. A., & Teverson, R. (2003). The transmission of 557 Mycobacterium bovis infection to cattle. Research in Veterinary Science, 74, 1–15. 558 https://doi.org/10.1016/S0034 559 -5288(02)00145-5 560 Posado, R., Bartolomé, D., San Miguel, J. M., & García, J. J. (2013). Rinotraqueitis infecciosa bovina y virus 561 19 respiratorio sincitial bovino en ganado de Lidia en Salamanca. Archivos De Zootecnia, 62, 181–190. 562 https://doi.org/10.4321/S0004-05922013000200003 563 Reed, L. J., & Muench, H. (1938). A simple method of estimating fifty percent endpoints. American Journal 564 of Epidemiology, 27, 493–497. https://doi.org/10.1093/oxfordjournals.aje.a118408 565 Rodríguez-Prieto, V., Kukielka, D., Rivera-Arroyo, B., Martínez-López, B., de las Heras, A. I., Sánchez566 Vizcaíno, J. M., & Vicente, J. (2016). Evidence of shared bovine viral diarrhea infections between red 567 deer and extensively raised cattle in south-central Spain. BMC Veterinary Research, 12, 11. 568 https://doi.org/10.1186/s12917-015-0630-3 569 Rola, J., Larska, M., Socha, W., Rola, J. G., Materniak, M., Urban-Chmiel, R., Thiry, E., & Żmudziński, J. F. 570 (2017). Seroprevalence of bovine herpesvirus 1 related alphaherpesvirus infections in free-living and 571 captive cervids in Poland. Veterinary Microbiology, 204, 77–83. https:// 572 doi.org/10.1016/j.vetmic.2017.04.006 573 Romero, B., Aranaz, A., Sandoval, Á., Álvarez, J., de Juan, L., Bezos, J., Sánchez, C., Galka, M., Fernández, 574 P., Mateos, A., & Domínguez, L. (2008). Persistence and molecular evolution of Mycobacterium bovis 575 population from cattle and wildlife in Doñana National Park revealed by genotype variation. Veterinary 576 Microbiology, 132, 87–95. https:// doi.org/10.1016/j.vetmic.2008.04.032 577 Ruiz-Fons, F., Sánchez-Matamoros, A., Gortázar, C., & SánchezVizcaíno, J. M. (2014). The role of 578 wildlife in bluetongue virus maintenance in Europe: Lessons learned after the natural infection in 579 Spain. Virus Research, 182, 50–58. https://doi.org/10.1016/j.virus res.2013.12.031 580 Sáenz de Buruaga, M., A. Lucio-Calero, & F. J. Purroy-Iraizoz (Eds.) (2001).Reconocimiento de sexo y edad 581 en especies cinegéticas. , 1st ed. Edilesa. 582 Sorensen, A., van Beest, F. M., & Brook, R. K. (2014). Impacts of wildlife baiting and supplemental feeding 583 on infectious disease transmission risk: A synthesis of knowledge. Preventive Veterinary Medicine, 584 113, 356–363. https://doi.org/10.1016/j.prevetmed.2013.11.010 585 Thomas, J., Infantes-Lorenzo, J. A., Moreno, I., Romero, B., Garrido, J. M., Juste, R., Domínguez, M., 586 Domínguez, L., Gortazar, C., & Risalde, 587 M. A. (2019). A new test to detect antibodies against Mycobacterium tuberculosis complex in red deer 588 serum. Veterinary Journal, 244, 98–103. https://doi.org/10.1016/j.tvjl.2018.12.021 589 Thrusfield, M (Ed.) (2018). Veterinary epidemiology, 4th ed. Wiley Blackwell. 590 Tompkins, D. M., Dunn, A. M., Smith, M. J., & Telfer, S. (2011). Wildlife diseases: From individuals to 591 ecosystems. Journal of Animal Ecology, 80, 19–38. https://doi.org/10.1111/j.1365592 2656.2010.01742.x 593 Triguero-Ocaña, R., Barasona, J. A., Carro, F., Soriguer, R. C., Vicente, J., & Acevedo, P. (2019). Spatio594 temporal trends in the frequency of interspecific interactions between domestic and wild ungulates 595 from Mediterranean Spain. PLoS One, 14, e0211216. https://doi. org/10.1371/journal.pone.0211216 596 Triguero-Ocaña, R., Martínez-López, B., Vicente, J., Barasona, J. A., Martínez-Guijosa, J., & Acevedo, P. 597 (2020). Dynamic network of interactions in the wildlife-livestock interface in mediterranean spain: 598 An epidemiological point of view. Pathogens, 9, 120. https://doi. org/10.3390/pathogens9020120 599 Vicente, J., Soriguer, R., Gortázar, C., Carro, F., Acevedo, P., Barasona, J. A., & Negro, J. J. (2014). Bases 600 técnicas para una extracción sostenible de ungulados del Parque Nacional de Doñana. Unpublished 601 report. 602 Woolhouse, M. E., Taylor, L. H., & Haydon, D. T. (2001). Population biology of multihost pathogens. 603 Science, 292, 1109–1112. https://doi. 604 20 FIGURE 1 Sampling distribution and results for selected respiratory viral agents and Mycobacterium tuberculosis complex in Doñana National Park. Pie charts indicate the number of positive (red) and negative (green) cattle per management area. 21 TABLE 1 Tests results for selected respiratory viral agents (Pestivirus, bovine respiratory syncityal virus [BRSV] and bovine herpesvirus 1 [BoHV-1]) and Mycobacterium tuberculosis complex (MTC) by domestic and wild ruminant species in Doñana National Park Number of positives/total number of individuals (95% confidence interval) PESTIVIRUS ELISA VNT BRSV ELISA BoHV-1 ELISA Cattle 3/138 2.2% (0.0–4.6) 3/3a 16/138 11.6% (6.3–16.9) 38/138 27.5% (20.1–35.0) Red deer 0/101 0.0% (0.0–3.7) – 0/101 0.0% (0.0–3.7) 0/101 0.0% (0.0–3.7) Fallow deer 0/102 0.0% (0.0–3.6) – 0/102 0.0% (0.0–3.6) 0/102 0.0% (0.0–3.6) MTC SITT P22-ELISA Culture Typing Cattle 22/138 15.9% (9.8–22.0) – 6/12 (M. bovis) SB1230 (1/6) SB1232 (3/6) SB1610 (2/6) Red deer – 16/101 15.8% (8.7–23.0) 12/18 (M. bovis) SB1230 (4/12) SB1232 (6/12) SB1610 (2/12) Fallow deer – 13/102 12.7% (6.3–19.2) 10/12 (M. bovis) SB1230 (5/10) SB1232 (3/10) SB1610 (2/10) aPestivirus A reactivity confirmed in one serum sample.