Evaluation of Dried Blood Spots for Serological Surveys of Myxoma and Rabbit Hemorrhagic Disease Viruses in their Wild Reservoir

Evaluation of dried blood spots for serological surveys of myxoma and rabbit hemorrhagic disease viruses in their wild reservoir Joana Ferreira-e-Silva a,b,c , Saúl Jim´ enez-Ruiz d , Marisa Rodrigues a,b,e , Emídio Santos f , Sabrina Castro-Scholten d , Vitor Lizana g,h , Alba Martí-Marco g , Tereza Almeida a,b , Ana M. Lopes a,b,i , Joana Abrantes a,b,c , Juan B´ arcena j , Esther Blanco j , Carlos Rouco k , Ignacio García-Bocanegra d,l , Paulo C´ elio Alves a,b,c,e , Nuno Santos a,b,e,* a CIBIO, Centro de Investigaç˜ ao em Biodiversidade e Recursos Gen´ eticos, InBIO Laborat´ orio Associado, Universidade do Porto, Campus de Vair˜ ao, Vair˜ ao 4485-661, Portugal b BIOPOLIS Program in Genomics, Biodiversity and Land Planning, CIBIO, Campus de Vair˜ ao, Vair˜ ao 4485-661, Portugal c Departamento de Biologia, Faculdade de Ciˆ encias, University of Porto, R. do Campo Alegre, s/n, Porto 4169-007, Portugal d GISAZ-ENZOEM. Animal Health and Zoonoses Research Group, Competitive Research Unit on Zoonoses and Emerging Diseases, University of C´ ordoba, C´ ordoba 14014, Spain e Estaç˜ ao Biol´ ogica de M´ ertola (EBM), CIBIO, Praça Luís de Cam˜ oes, M´ ertola 7750–329, Portugal f ICNF - Instituto de Conservaç˜ ao da Natureza e das Florestas, I.P., Av. da República, 16, Lisboa 1050-191, Portugal g Servicio de An´ alisis, Investigaci´ on, Gesti´ on de Animales Silvestres (SAIGAS), Facultad de Veterinaria, Universidad Cardenal Herrera-CEU, CEU Universities, Alfara del Patriarca, Valencia 46115, Spain h Wildlife Ecology & Health Group (WE&H), Universitat Aut` onoma de Barcelona (UAB), Bellaterra PC08193, Spain i Instituto de Ciˆ encias Biom´ edicas Abel Salazar (ICBAS)/Unidade Multidisciplinar de Investigaç˜ ao Biom´ edica (UMIB), University of Porto, R. Jorge Viterbo Ferreira, 228, Porto 4050-313, Portugal j Centro de Investigaci´ on en Sanidad Animal (CISA-INIA/CSIC), Carretera Algete-El Casar de Talamanca, Km 8.1, Valdeolmos, Madrid 28130, Spain k ´ Area de Ecología, Departamento Biología Vegetal y Ecología, ´ Area de Ecología, Universidad de Sevilla, Sevilla 41012, Spain l CIBERINFEC, ISCIII-CIBER de Enfermedades Infecciosas, Instituto de Salud Carlos III, Madrid 28029, Spain ARTICLE INFO Keywords: European rabbit Oryctolagus cuniculus Epidemiology Wildlife Protein Saver cards ABSTRACT Myxoma (MYXV) and rabbit hemorrhagic disease (RHDV) viruses are pathogens of economic relevance for cuniculture and conservation concern for wild European rabbits (Oryctolagus cuniculus), recently classified as ‘Endangered’ in its native range. Large-scale serological surveys, facilitated by sample collection using dried blood spots (DBS), allow monitoring seroprevalence in the wild reservoir but require evaluating the technique for the host and pathogen of interest. This study aimed to evaluate Protein Saver 903 DBS for MYXV and RHDV (genotype GI.2) serological surveys in European rabbits. Paired serum and DBS collected from 172 rabbits harvested or found dead in the Iberian Peninsula were tested for IgG antibodies specific against MYXV and RHDV GI.2 using indirect ELISA. We found an almost perfect agreement between serum and DBS for MYXV (Cohen’s κ=0.914, CI 95 0.847 – 0.981) and a strong agreement for RHDV GI.2 (Cohen’s κ=0.808, CI 95 =0.722 – 0.893). The diagnostic sensitivity of DBS was 95.4 % (CI 95 90.3 – 97.9 %) for MYXV and 82.1 % (CI 95 73.2 – 88.5 %) for RHDV GI.2. The diagnostic specificity and positive predictive value were 100 % for both pathogens. This study supports DBS as a suitable sampling strategy for serological surveys of antibodies specific to MYXV and RHDV GI.2 in European rabbits, which generally agrees with results from other hosts and pathogens where this technique was evaluated. Abbreviations: MYXV, myxoma virus; RHDV GI.2, rabbit hemorrhagic disease virus GI.2; DBS, dried blood spots; PS, Protein Saver; IELISA, indirect enzyme-linked immunosorbent assay; NAR, normalized absorbance ratio. * Corresponding author at: CIBIO, Centro de Investigaç˜ ao em Biodiversidade e Recursos Gen´ eticos, InBIO Laborat´ orio Associado, Universidade do Porto, Campus de Vair˜ ao, Vair˜ ao 4485-661, Portugal E-mail address: [email protected] (N. Santos). Contents lists available at ScienceDirect Preventive Veterinary Medicine journal homepage: www.elsevier.com/locate/prevetmed https://doi.org/10.1016/j.prevetmed.2024.106369 Received 24 January 2024; Received in revised form 19 August 2024; Accepted 3 November 2024 Preventive Veterinary Medicine 234 (2025) 106369 Available online 6 November 2024 0167-5877/© 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ). 1. Introduction Large-scale serological surveys in wildlife are challenging to perform due to the often-secretive nature of wild animals, remote locations, and haphazard opportunities for sampling (Ryser-Degiorgis, 2013; Cardoso et al., 2022). Obtaining serum requires the refrigeration of blood samples and their prompt centrifugation, thus restricting the collection of samples to specialized personnel with access to appropriate equipment and facilities (Maceda-Veiga et al., 2015). Dried whole blood samples collected in specialized absorbent matrices (dried blood spots, herein DBS) have emerged as a viable alternative for serological surveys in humans, livestock, and wildlife (Samsonova et al., 2022). Dried blood spots can be collected by lay personnel, stored at room temperature for several weeks, and safely shipped to dedicated laboratories (Manak et al., 2018; Samsonova et al., 2022). They allow incorporating citizen science approaches in the surveillance of wildlife diseases, increasing the geographical coverage of sampling in a cost-efficient way (Peng et al., 2023). Nevertheless, they require the evaluation of the technique for the host(s) and pathogen(s) under study (Samsonova et al., 2022). Myxomatosis and rabbit hemorrhagic disease are important viral diseases of the European rabbit (Oryctolagus cuniculus) in the wild and cuniculture (Rosell et al., 2019; Delibes-Mateos et al., 2021). While these pathogens are primarily controlled through vaccination and strict biosecurity measures in cuniculture, the presence of a widespread wild reservoir continually favors their emergence (Rosell et al., 2019). European rabbits were recently classified as endangered in their native range mainly because of the demographic impact of these viral diseases (Villafuerte & Delibes-Mateos, 2019). Myxomatosis is caused by a poxvirus (myxoma virus - MYXV) transmitted by mechanical vectors or directly from infected to susceptible hosts (Kerr et al., 2015). It is characterized by tumour-like lesions (myxomas) and respiratory signs (Bertagnoli and Marchandeau, 2015). Although causing only mild symptoms in its natural host (Sylvilagus spp. lagomorphs), in European rabbits the disease can be severe (Bertagnoli and Marchandeau, 2015). Myxoma virus is currently endemic in wild European rabbits in the Iberian Peninsula (Villafuerte et al., 2017), but its case fatality rate drastically decreased since its emergence in the 1950s (Camacho-Sillero et al., 2022; Pacheco et al., 2022). Rabbit hemorrhagic disease is caused by a calicivirus (rabbit hemorrhagic disease virus - RHDV) transmitted by direct or indirect contact or mechanical vectors (Abrantes et al., 2012; Lopes et al., 2023). The disease is characterized by necrotizing hepatitis and disseminated intravascular coagulation (Neimanis et al., 2019). The currently circulating RHDV GI.2 emerged in France in 2010 (Le Gall-Recul´ e, 2013) and quickly disseminated across Europe (Aguayo-Ad´ an et al., 2022). Rabbit hemorrhagic disease virus GI.2 is endemic but not homogeneously distributed in the Iberian Peninsula, and its case fatality rate can be high in natural outbreaks (Rouco et al., 2018; Camacho-Sillero et al., 2019; Jim´ enez-Ruiz et al., 2023). Large-scale data on the seroprevalence of MYXV and RHDV GI.2 in wild European rabbit populations are invaluable to inform management of the domestic and wild compartments of their epidemiological cycles (Villafuerte et al., 2017; Ramsey et al., 2023). Despite wild European rabbits being commonly harvested, information on seroprevalence is haphazard throughout much of its range due to the difficulties in performing large-scale wildlife epidemiological surveys. This study aimed to evaluate a protocol for serological surveys of myxoma and rabbit hemorrhagic disease viruses in wild European rabbits, using DBS to facilitate large-scale and long-term monitoring. 2. Methods 2.1. Blood collection and dried blood spot elution Whole blood (1–2 ml) was collected post-mortem with a 5 ml syringe from the thoracic or abdominal cavities of wild European rabbits legally harvested for recreational purposes in Portugal (n=95) and Spain (n=70). Additional samples were collected upon standard necropsy of carcasses found in the field and frozen at −20◦C for 2–7 months before necropsy (n=7). Whole blood was collected, refrigerated until processing at the laboratory 2–24 h after blood collection, centrifuged at 1500 g for 10 min, and the serum collected and stored frozen at −20◦C until analysis. Dried blood spots were obtained from the same rabbits by placing Protein Saver (PS) 903 cards (Whatman, Maidstone, U.K.) in contact with blood in the thoracic or abdominal cavities. Whole blood collected in the PS cards was left to dry at room temperature for 2–6 weeks, protected from direct sunlight. It was then frozen at −20◦C until elution, 1–12 months later. One 5 mm diameter DBS punch, which, according to the manufacturer, should hold 11.6–12.4 µl whole blood, was extracted from each DBS and incubated with 50 µl of phosphate buffered saline (pH=7.4), after a short spin. The eluate was collected after overnight incubation at 4 ◦C followed by another short spin and kept frozen at −20◦C until analysis. All procedures were performed in compliance with relevant laws and institutional guidelines. No rabbits were killed for the purpose of this study, and no live animal experimentation was performed. 2.2. Serological assays Serum and DBS eluates were analyzed for IgG specific for myxoma and RHD GI.2 viruses by indirect enzyme-linked immune serum assays (iELISA). The in-house iELISA targeting RHDV GI.2-specific IgG was performed as described in Pacheco et al. (2022), based on B´ arcena et al. (2015). Briefly, 100 ng/well of GI.2-derived virus-like particles purified as described in Almanza et al. (2008) diluted in carbonate/bicarbonate buffer (pH=9.5) were absorbed to Nunc Maxisorp 96-well ELISA plates and incubated overnight at 4◦C. The plates were blocked with PBS–5 % skim milk solution, washed three times with PBS–0.05 % Tween 20, and the sera assayed at 1/200 dilution in PBS-5 % skim milk solution. As the concentration of IgG was expected to be higher in serum than in DBS eluates, the later were tested at higher concentrations: 1/67, 1/50, and 1/40 dilutions. We aimed to establish which DBS eluate dilution achieved the best diagnostic performance using the same cut-off threshold as serum tested at 1/200 dilution. This approach has the advantage of not requiring establishing independent cut-off thresholds for each of the DBS eluate dilution evaluated. After incubation for 1 h, the conjugate goat anti-rabbit IgG / HRP (BioRad, Portugal) was added at 1/4000 dilution, incubated for 1 h, and 100 µl of the substrate 3,3 ′ ,5,5’-tetramethylbenzidine (Abcam, U.K.) was added. Reactions were stopped after 4±1 min with 65 µl of 1 M phosphoric acid, and the optical density at 450 nm (OD 450 nm ) was recorded within 15 minutes. Positive controls consisted of pooled sera from rabbits with high iELISA optical densities (Pacheco et al., 2022), and negative controls were pooled sera from unvaccinated domestic European rabbits without a history of clinical disease and kept in-house. A commercial kit (Ingezim 17.MIX.K1, Eurofins Technologies Ingenasa, Spain) targeting MYXV-specific IgG was employed according to the manufacturer’s instructions. Sera were assayed by MYXV iELISA at 1/200, and eluates at 1/67, 1/50, and 1/40 dilutions, as for RHDV GI.2. The MYXV and RHDV GI.2 assays were considered valid if the average OD 450 nm of the two replicates of the positive control was >5 times the average OD 450 nm of the two replicates of the negative control. The results of both assays were standardized as normalized absorbance ratios (NAR) (Ramanakumar et al., 2010) according to Eq. (1): NAR =average OD450 sample 2× (average OD450 negative control)(1) The dichotomous serological status (seropositive/negative) of each sample was attributed based on the cut-off thresholds previously estimated by finite mixture models for sera tested at 1/200 dilution, being NAR=2.0 for RHDV GI.2 and NAR=2.4 for MYXV. Under these J. Ferreira-e-Silva et al. Preventive Veterinary Medicine 234 (2025) 106369 2 conditions, both iELISA tests were shown to achieve 100 % sensitivity and specificity when testing wild rabbit sera at 1/200 dilution (Pacheco et al., 2022). 2.3. Dried blood spot evaluation The agreement between the dichotomous serological results, expressed as positive/negative according to the abovementioned cut-off thresholds, was assessed by Cohen’s unweighted Kappa (κ) (Cohen, 1960), estimated using the package "DescTools" (Signorell et al., 2022). The correlation between the semi-quantitative serological results (Prechl, 2021) of sera (NAR serum ) and DBS eluates (NAR eluate ) was assessed by the coefficient of determination (R 2 ) of the regression between the NAR eluate and NAR serum . The sensitivity, specificity, negative and positive predictive values of DBS eluates were estimated by comparison with serum, here assumed as the reference matrix. Statistical analyses were performed using R 4.2.1 (R Development Core Team, 2023). 3. Results The summary of the test results obtained across biological matrices and test dilutions is presented in Table 1. Cohen’s Kappa (κ) showed an almost perfect agreement between the binomial results of serum and DBS eluates for MYXV when tested at both 1/40 dilution (0.942, CI 95 0.886 – 0.998) and 1/50 dilution (0.914, CI 95 0.847 – 0.981), and a strong agreement for RHDV GI.2 when tested at 1/ 50 dilution (0.808, CI 95 0.722 – 0.893) (Table 2). The coefficient of determination (R 2 ) showed strong linear correlations between NAR serum and NAR eluate at 1/50 dilution for MYXV and RHDV GI.2 (Table 2 and Fig. 1 A and 1B). The relationship between NAR serum and NAR eluate for RHDV GI.2 at 1/50 was better described as quadratic (R 2 =0.795, Fig. 1 C). The differences in the parameters κ and R 2 between the DBS eluate’s test dilutions 1/40 and 1/50 were small (Table 2). Assuming serum as the reference matrix to detect pathogen-specific IgG, the diagnostic sensitivity of DBS eluates tested at 1/50 dilution was 82.1 % (CI 95 73.2 – 88.5 %) for RHDV GI.2 and 95.4 % (CI 95 90.3 – 97.9 %) for MYXV. The diagnostic specificity of DBS eluates tested at 1/ 50 dilution for both viruses was 100 % (Table 3). The seroprevalence of MYXV in serum tested at 1/200 was 74.3 % (CI 95 67.2 – 80.6 %) and RHDV GI.2 54.3 % (CI 95 46.6 – 62.8 %). Under these prevalences, the negative predictive value of iELISA performed on DBS eluates at 1/50 dilution was >80 % and the positive predictive value was 100 % for MYXV and RHDV GI.2. 4. Discussion Here we demonstrate the suitability of DBS eluates as alternative to sera for serological surveys of MYXV and RHDV GI.2 in wild European rabbits. Previous studies used this technique in other host(s) and pathogen(s), generally finding it an easy, affordable, and reliable sample collection method (Samsonova et al., 2022). Duncombe et al. (2013) assessed PS cards in the cattle-Brucella abortus system and found a high correlation between antibody titers obtained in paired serum and DBS eluate samples, with Pearson’s correlation being 0.975 for infected and 0.758 for non-infected animals. The duration of the storage of DBS and the location of the punch within the DBS had negligible influence on the test results (Duncombe et al., 2013). Dried blood spots in PS cards were also shown to have good sensitivity (96.1 %, CI 95 91.2–98.7 %) and specificity (98.6 %, CI 95 95.1–99.8 %) compared to sera for SARS-CoV-2 IgG in human samples (Meyers et al., 2021). Good performance of PS DBS was also shown in serological tests for animal tuberculosis in wild boar (Santos et al., 2018) and Toxoplasma gondii exposure in wild ungulates (Aston et al., 2014). Portejoie et al. (2009) assessed the use of DBS collected in a different card (Whatman 3D55) in lagomorphs to detect antibodies against European brown hare syndrome calicivirus in domestic and wild hares. The specificity of DBS was 100 % and the specificity 85 % when compared to serum but sensitivity was lower in samples with low titers. Overall, the agreement between the results from serum and DBS was good (Portejoie et al., 2009). We analyzed the agreement of the dichotomous (positive/negative) test results between paired serum and DBS eluates from European rabbits. Our strategy was to adapt the DBS eluate test dilutions to achieve a diagnostic performance as similar as possible to serum, employing the cut-off thresholds previously established by our team for the later matrix (Pacheco et al., 2022). These cut-off thresholds were established by applying finite mixture models to serological data from other populations of wild European rabbits. Finite mixture models allow to characterize the distributions of the quantitative test results of the seropositive and seronegative subgroups within datasets, without requiring samples of known serological status. These models thus estimate the probability of any given sample being positive or negative to a diagnostic test (Meyer et al., 2018). Our findings support PS DBS eluate as a viable alternative to serum for estimating seroprevalence of MYXV and RHDV GI.2 in wild rabbit populations (Tables 2 and 3), which generally agrees with research performed on other pathogens (Samsonova et al., 2022). We also investigated the correlation between the semi-quantitative iELISA standardized results (NAR) of serum and DBS eluates. The dilution factors needed to achieve broadly similar NAR with DBS eluates (1/ 50) were lower when compared to serum (1/200), suggesting a lower concentration of IgG in the former matrix with the elution protocol applied. We propose testing DBS eluates at 1/50 dilution in future surveys for RHDV GI.2 and MYXV, as this dilution showed higher coefficients of determination for both pathogens and a similar agreement to the 1/40 dilution. The correlation between the NAR of DBS eluates and serum for MYXV corresponds to a linear relationship. Interestingly, the same did not apply to RHDV GI.2, where a quadratic relationship better fitted the data (Fig. 1 C). Samples with high RHDV GI.2 results in serum (NAR serum ≥9) tended to show much higher NAR eluate , up to 15.4 (Fig. 1B). The reasons behind this unexpected apparent concentration of RHDV GI.2-specific IgG in DBS eluates are unknown but could be biased by the scarce number (n=4) of samples with NAR serum and NAR eluate ≥9. Nevertheless, caution is warranted when interpreting semi-quantitative serological results of DBS eluates showing high RHDV GI.2 NAR. Table 1 Summary of the test results. Dichotomous results (positive/negative) obtained from serum tested at 1/200 and dried blood spot eluates tested at 1/67, 1/50, and 1/40 dilutions. Pathogen Test matrix Eluate 1/67 a Eluate 1/50 Eluate 1/40 Serum 1/200 Positive Negative Positive Negative Positive Negative Myxoma virus Positive 53 12 124 6 126 4 Negative 0 25 0 45 0 45 RHDV Positive 51 44 78 17 84 11 Negative 1 76 0 77 6 71 a Only 90 eluates were available to test for IgG specific for Myxoma virus at 1/67 dilution J. Ferreira-e-Silva et al. Preventive Veterinary Medicine 234 (2025) 106369 3 5. Conclusions This study evaluates the suitability of DBS eluates as an alternative tool for serological surveys of MYXV and RHDV GI.2 specific antibodies in European rabbits. Dried blood spots are an easy and relatively lowcost method of sample collection that can be used by non-skilled personnel. Furthermore, it allows to incorporate citizen science approaches in the sero-epidemiological surveillance. Combined with good diagnostic performance when tested for IgG specific for MYXV and RHDV GI.2 at 1/50 dilution, these characteristics make DBS an option for sampling at large geographical scale and in remote locations. Dried blood spots eluates can become a tool for monitoring MYXV and RGDV GI.2 in wild European rabbit populations. Such monitoring is necessary to understand the epidemiology of these pathogens in wildlife and their transmission to commercial cuniculture. CRediT authorship contribution statement Ana Margarida Lopes: Writing – review & editing, Resources, Methodology. Joana Abrantes: Writing – review & editing, Resources, Methodology. Juan B´ arcena: Writing – review & editing, Supervision, Resources, Methodology, Funding acquisition. Joana Ferreira-e-Silva: Writing – review & editing, Writing – original draft, Investigation, Formal analysis, Data curation. Esther Blanco: Writing – review & editing, Supervision, Resources, Methodology, Funding acquisition. Saúl Jim´ enez-Ruiz: Writing – review & editing, Investigation, Data curation. Carlos Rouco: Writing – review & editing, Supervision, Resources, Methodology, Funding acquisition. Marisa Rodrigues: Writing – review & editing, Resources, Investigation. Ignacio García-Bocanegra: Writing – review & editing, Supervision, Resources, Methodology, Funding acquisition. Emídio Santos: Writing – review & editing, Resources, Investigation. Paulo C´ elio Alves: Writing – review & editing, Supervision, Resources, Project administration, Funding acquisition. Sabrina Castro-Scholten: Writing – review & editing, Resources, Investigation, Data curation. Nuno Santos: Writing – review & editing, Writing – original draft, Supervision, Project administration, Methodology, Funding acquisition, Formal analysis, Data curation, Conceptualization. Vitor Lizana: Writing – review & editing, Resources, Investigation, Data curation. Alba Martí-Marco: Writing – review & editing, Resources, Investigation. Tereza Almeida: Writing – review & editing, Resources, Methodology. Declaration of Competing Interest None. Table 2 Agreement between the serological results of the matrices serum and dried blood spot eluate. Confidence intervals 95 % are shown in brackets. Matrix Rabbit hemorrhagic disease virus GI.2 Myxoma virus Serum Test dilution Dried blood spot eluate Test dilution Cohen’s Kappa (κ) Coefficient of determination (R 2 ) Cohen’s Kappa (κ) Coefficient of determination (R 2 ) 1/200 1/67 0.503 (0.393–0.613) 0.610 (0.522 – 0.699) 0.711 (0.565 – 0.856) 0.755 (0.670 – 0.840) 1/50 0.808 (0.722 – 0.893) 0.761 (0.700 – 0.822) 0.914 (0.847 – 0.981) 0.931 (0.912–0.950) 1/40 0.801 (0.712 – 0.891) 0.716 (0.646 – 0.786) 0.942 (0.886 – 0.998) 0.926 (0.905 – 0.947) Fig. 1. Relationship between the semi-quantitative serological results in paired samples of serum and dried blood spot eluates. Normalized absorbance ratios of paired serum and dried blood spot eluates tested by indirect ELISA at 1/50 dilution: A) myxoma virus; B) rabbit hemorrhagic disease virus GI.2 (linear); C) rabbit hemorrhagic disease virus GI.2 (quadratic). Non-concordant binomial results as white dots. The cut-off thresholds for positivity as dashed grey lines. Table 3 Diagnostic performance of dried blood spot eluates tested at 1/50 dilution. Diagnostic sensitivity and specificity were estimated assuming serum as the reference matrix. Pathogen Diagnostic performance (%) (95 % confidence interval) Sensitivity Specificity Positive predictive value Negative predictive value Rabbit hemorrhagic disease virus GI.2 82.1 (73.2 – 88.5) 100 (95.4 – 100) 100 (95.3 – 100) 82.5 (75.4 – 87.9) Myxoma virus 95.4 (90.3 – 97.9) 100 (92.1 – 100) 100 (97.0 – 100) 88.2 (77.4 – 94.3) J. Ferreira-e-Silva et al. Preventive Veterinary Medicine 234 (2025) 106369 4 Acknowledgements This work was supported by Fundaç˜ ao para a Ciˆ encia e Tecnologia (FCT) [grant SFRH/BPD/116596/2016 to N.S., CEECIND/01388/2017 to A.M.L. and CEECIND/00078/2017 to J.A.] and co-funded by the project NORTE-01–0246-FEDER-000063, supported by Norte Portugal Regional Operational Programme (NORTE2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This study was partially funded by project LAGMED (www.lagmed.eu), supported by FCT (PRIMA/0003/2018), the Spanish Ministry of Science, Innovation and University (PRIMA S2–11PCI2019–103732 to E.B.) and PRIMA programme, an Art. 185 initiative supported and funded under Horizon 2020, the European Union’s Framework Programme for Research and Innovation. This work benefited from research grants funded by the Spanish Ministry of Science and Innovation (projects Iber-Lagohealth; PID2023–151954NB100 and LagoHealth; PID2019–111080RB-C21). It was also partially funded by the Sub-modality 2.4. "UCOLIDERA" of the "Enrique Aguilar Benítez de Lugo" Research Plan of the University of Cordoba and CIBER -Consorcio Centro de Investigaci´ on Biom´ edica en Red- (CB 2021), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovaci´ on and European Union – NextGenerationEU. S.J.R. is supported by a ‘Juan de la Cierva’ contract (JDC2022–048850-I) funded by the MCIN/AEI/ 10.13039/501100011033 and by the European Union “NextGenerationEU”/PRTR. S.C.S. is supported by an FPU grant from the Spanish Ministry of Universities (FPU19/06026). This work was supported by Life Iberconejo (LIFE20-GIE ES000731). References Abrantes, J., van Der Loo, W., le Pendu, J., Esteves, P.J., 2012. 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