TYPE Original Research PUBLISHED 01 June 2023 DOI 10.3389/fvets.2023.1183304 The local skin cellular immune OPEN ACCESS response determines the clinical outcome of sarcoptic mange in EDITED BY Francisco Javier Salguero, UK Health Security Agency (UKHSA), United Kingdom REVIEWED BY Iberian ibex (Capra pyrenaica) Stefan Hoby, Berne Animal Park, Switzerland Brianna R. Beechler, Marta Valldeperes 1,2, José Enrique Granados 1,3, Valentín Pérez 4,5, Oregon State University, United States Jorge Ramón López-Olvera 1,2*, Arián Ráez-Bravo 1,2, *CORRESPONDENCE Paulino Fandos 6, Jesús M. Pérez 1,7, Gregorio Mentaberre 1,8, Jorge Ramón López-Olvera Jordi.Lopez.Olvera@uab.cat Stefania Tampach 1,2, Ramón C. Soriguer 9 and José Espinosa 4,5 RECEIVED 09 March 2023 1 Wildlife Ecology and Health Group (WE&H), Universitat Autònoma de Barcelona (UAB), Barcelona, ACCEPTED 28 April 2023 Spain, 2 Departament de Medicina i Cirurgia Animals, Servei d’Ecopatologia de Fauna Salvatge (SEFaS), PUBLISHED 01 June 2023 Universitat Autònoma de Barcelona (UAB), Barcelona, Spain, 3 Parque Nacional y Parque Natural de CITATION Sierra Nevada, Granada, Spain, 4 Department of Animal Health-Instituto de Ganadería de Montaña (IGM), Valldeperes M, Granados JE, Pérez V, ULe-CSIC León, León, Spain, 5 Faculty of Veterinary Science, University of León, León, Spain, 6 Retired López-Olvera JR, Ráez-Bravo A, Fandos P, Researcher, Madrid, Spain, 7 Department of Animal and Plant Biology, and Ecology, Jaén University, Pérez JM, Mentaberre G, Tampach S, Campus Las Lagunillas, Jaén, Spain, 8 Departament de Ciència Animal, Universitat de Lleida (UdL), Lleida, Soriguer RC and Espinosa J (2023) The local Spain, 9 Estación Biológica de Doñana (CSIC), Sevilla, Spain skin cellular immune response determines the clinical outcome of sarcoptic mange in Iberian ibex (Capra pyrenaica). Introduction: Sarcoptic mange, caused by Sarcoptes scabiei, is a disease with Front. Vet. Sci. 10:1183304. implications for wildlife conservation and management. Its severity depends on doi: 10.3389/fvets.2023.1183304 the host’s local skin immune response, which is largely unknown in Iberian ibex COPYRIGHT (Capra pyrenaica), a mountain ungulate dramatically affected by mange. In this © 2023 Valldeperes, Granados, Pérez, López- Olvera, Ráez-Bravo, Fandos, Pérez, Mentaberre, species, the clinical outcome of sarcoptic mange varies among individuals, and Tampach, Soriguer and Espinosa. This is an the local immune response could be key to controlling the infestation. This study open-access article distributed under the terms aims to characterize the local cellular immune response and its relationship with of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction the clinical outcome. in other forums is permitted, provided the Methods: Fourteen Iberian ibexes were experimentally infested with S. scabiei original author(s) and the copyright owner(s) are credited and that the original publication in and six more served as controls. Clinical signs were monitored, and skin biopsies this journal is cited, in accordance with were collected from the withers at 26, 46, and 103 days post-infection (dpi). The accepted academic practice. No use, presence and distribution of macrophages (including M1 and M2 phenotypes), T distribution or reproduction is permitted which does not comply with these terms. lymphocytes, B lymphocytes, plasma cells, and interleukine 10 were quantitatively evaluated using immunohistochemical techniques. Results: An inflammatory infiltrate that decreased significantly from 26 to 103 dpi was observed in all the infested ibexes. The predominant inflammatory cell population in the skin of the mangy ibexes was formed by macrophages (mainly the M2 phenotype) followed by T lymphocytes, with lower numbers of B lymphocytes and plasma cells. Three clinical courses were identified: total recovery, partial recovery, and terminal stage. The inflammatory infiltrates were less pronounced in the fully recovered ibexes than in those that progressed to the terminal stage throughout the study. Discussion: The results suggest an exacerbated but effective Th1-type cellular immune response controlling mange in Iberian ibex. Furthermore, the local immune response appears to determine the variability of the clinical responses to S. scabiei infestation in this species. This first report on the progression of local skin immune cells is relevant not only for individuals but also for population management and conservation. KEYWORDS Capra pyrenaica, clinical outcome, experimental infestation, Iberian ibex, immunohistochemistry, inflammatory cell, sarcoptic mange (Sarcoptes scabiei), skin immune response Frontiers in Veterinary Science 01 frontiersin.org Valldeperes et al. 10.3389/fvets.2023.1183304 1. Introduction (Capreolus capreolus), Iberian lynxes (Lynx pardinus), wild boars (Sus scrofa), and wombats (Vombatus ursinus) (6, 19, 28–35). These studies Sarcoptic mange is an emerging parasitic transmissible disease have demonstrated not only interspecific but also intraspecific caused by the mite Sarcoptes scabiei and affects humans and animals differences in the local skin immune response to S. scabiei infestation worldwide (1–4). It can cause significant declines in wildlife (1), which can also vary between naturally and experimentally infested populations; therefore, it is relevant for wildlife conservation and animals (28). However, skin lesions and inflammatory infiltrate also management (2). In Spain, sarcoptic mange has been reported in wild depend on the stage of infestation; therefore, to precisely histologically carnivores (5, 6), lagomorphs (7, 8), and ungulates (9–11), but the characterize the local inflammatory response, a representative number most dramatic effects of the disease have been observed in mountain of histological sections taken throughout the disease course must ungulates, including Cantabrian chamois (Rupicapra pyrenaica parva) be studied. Thus, identifying inflammatory cell populations in and Iberian ibex (Capra pyrenaica), in which outbreaks have led to experimentally induced scabietic lesions during the sensitization and demographic declines of over 95% (12–16). challenge infection phases, and characterizing the timing and intensity Despite sarcoptic mange having been known for a long time and of the immunological response during the challenge phase, could help the number of studies on this disease conducted in humans, domestic elucidate their role and the mechanism for the immune response in animals, and wildlife, its clinical course and progression remain not wildlife (25, 29). yet fully understood. Sarcoptes scabiei burrows galleries in the The features of the humoral and cellular immune responses, both epidermis, feeding on host cells and lymphatic fluid (17) and inducing local in the skin and systemic in the bloodstream, are relevant for the antigenic reactions with imbalances in Th1/Th2/Th17 immune course of the disease. Consequently, they also influence sarcoptic responses (18, 19). These responses drive the pathogenesis and clinical mange management at both an individual and population level, as signs of sarcoptic mange, which range from mild erythema to more strategies and their associated costs must be weighed against the severe lesions, such as dermatitis, hyperkeratosis, alopecia, and risks, hazards, and impact of the disease on the population (36). The systemic signs, which sometimes eventually cause death (6, 20–23). management of sarcoptic mange in wild Caprinae, and in Iberian However, two research questions regarding the immune response to ibex in particular, is challenging and controversial, with different and S. scabiei in different wildlife species remain not yet fully clarified: (1) even contradictory measures being applied and a lack of consensus what is the effect of immunological responses on mange severity, and not only on the management options but even on the criteria for (2) what are the primary drivers in host–parasite interactions for both deciding which measure to implement (16). Understanding positive and negative clinical outcomes of mange? (24, 25). individual immune responses that affect population dynamics is not The pathogenesis of sarcoptic mange has been characterized in only relevant for individual health and welfare, but also has humans, in which the local skin immune response defines the severity population, ecological, and management implications. of the parasitosis (18, 26, 27). A Th1-mediated skin immune response Although Iberian ibex has repeatedly been reported to be capable with higher T CD4+ lymphocyte infiltrates leads to milder ordinary of recovering and surviving sarcoptic mange (37–39), previous scabies, while a response skewed toward Th2 with predominantly T studies suggest that the systemic humoral immune response is not CD8+ lymphocyte infiltrates leads to a more severe crusted scabies effective at preventing the development of advanced clinical stages of (18, 26, 27). Therefore, the detailed study of the inflammatory sarcoptic mange and eventual death. Rather, it is a non-specific response to S. scabiei in skin biopsies can reveal interspecific and/or indicator of the inflammatory process associated with the disease interindividual variability in the severity of clinical signs, as well as in (40–42). Conversely, also in Iberian ibex, the ability to cope with the development of resistance and the existence of asymptomatic sarcoptic and survive may depend on the skin local cellular immune carriers. This has led to the study of immunological and inflammatory response rather that on the both skin local and systemic humoral local skin response to sarcoptic mange both by conventional immune response (1, 3, 21, 43). histological techniques and by immunohistochemical methods in Therefore, the aim of this study is to immunohistochemically domestic animals such as goats (Capra hircus), sheep (Ovis aries), dogs characterize the cellular immune skin response of Iberian ibex to (Canis lupus familiaris), and pigs (Sus scrofa domesticus), as well as in experimental infestation with S. scabiei, describe the progression of wildlife, including chamois (Rupicapra spp.), red deer (Cervus the inflammatory infiltrate throughout the course of the disease, and elaphus), wolves (Canis lupus), red foxes (Vulpes vulpes), roe deer investigate the potential link between this response and the clinical outcome. 2. Materials and methods Abbreviations: AIC, Akaike’s information criterion; AICc, corrected Akaike’s information criterion; AICWt, Akaike’s information criterion weight; CD4, Cluster 2.1. Animals of differentiation 4; CD8, Cluster of differentiation 8; CD3, Cluster of differentiation 3; CD20, Cluster of differentiation 20; CD204, Cluster of differentiation 204; Dpi, Twenty healthy free-ranging Iberian ibexes (10 females and 10 Days post-infection/infestation; ELISA, Enzyme linked immunosorbent assay; males, aged between 1 and 11 years) were captured in the Sierra GLMM, General linear mixed model; H&E, Hematoxylin and Eosin; HRP, Horseradish Nevada Natural Space (36° 55′- 37°10′N, 2° 56′- 3° 38′W) and the peroxidase; Iba-1, ionized calcium-binding adapter molecule 1; IL, interleukin; Sierras de Cazorla, Segura y Las Villas Natural Park (37° 53′-37° 88′N, iNOS, inducible nitric oxide synthase; K, degrees of freedom; KOH, potassium 2° 53′-2°88′W), in southern Spain. The ibexes were captured and hydroxide; LMM, linear mixed model; PBS, phosphate buffered saline; Th1, type immobilized with a combination of xylazine (3.0 mg/kg) and ketamine 1T helper; Th2, type 2T helper; Th17, type 17T helper. (3.0 mg/kg) (44), using a Teleinject G.U.T 50 anesthesia ® Frontiers in Veterinary Science 02 frontiersin.org Valldeperes et al. 10.3389/fvets.2023.1183304 gas-applicator1. After firing a single dart from a distance of 10–20 m, experimental period (“totally recovered”); three ibexes had progressive the anesthesia was maintained for approximately 15 min, at which skin lesions spreading over 70% of the body surface, but with signs of point a first clinical inspection was conducted. The ibexes were recovery, such as hair growth, reduction of skin thickening, and skin inspected for clinical signs compatible with sarcoptic mange, and the smoothing (“partially recovered”); and seven ibexes had advanced presence of antibodies against S. scabiei was assessed using a validated skin lesions, including alopecia, skin desquamation, thickening and enzyme-linked immunosorbent assay (ELISA) (42). Only ibexes crusting, and pruritus, spreading over 70% of the body surface without negative for sarcoptic mange by both physical examination and signs of recovery (“terminal”) (21). serological diagnosis were retained for the study and transported to specific experimental facilities located in the Sierra de Huétor Natural Park (37° 18′-37° 30′N, 3° 28′-3° 47′W). The ibexes were divided into 2.4. Skin sampling groups of four to six individuals of mixed sexes and ages. Each group was kept in a separate pen measuring 30 m2 with access to food and Skin biopsies were collected from the withers of the infested water ad libitum. These ibex groups remained constant throughout the ibexes at 26, 46, and 103 dpi using an 8-mm diameter biopsy punch entire experimental period. ® (KRUUSE Biopsy Punch, Langeskov, Denmark), after the This study adhered to all legal requirements and guidelines related interscapular region was shaved. The control ibexes were sampled to animal welfare and experimentation in Andalusia, Spain and from the same body region using the same procedure only at 103 dpi. Europe. The handling procedures and sampling frequency were Each ibex was individually restrained in a handling crush, blindfolded, designed to minimize stress and its impact on the health of the and locally anesthetized with a combination of 10 mg of lidocaine subjects, in accordance with European (2010/63/UE) and Spanish ® hydrochloride and 0.01 mg of adrenalin (ANESVET , Ovejero Lab, (R.D 53/2013) standards. The study was approved by the Ethics on León, Spain). After the skin samples were collected, the resulting skin Animal Welfare Committee of the University of Jaén and authorized ® wounds were topically treated with Bactrovet antiseptic sprays, by the Dirección General de Producción Agrícola y Ganadera of the composed of a mixture of micronized aluminum and silver and Consejería de Agricultura, Pesca y Medio Ambiente of the Junta de rosehip oil. Andalucía (Ref: SA/SIS/MD/ps/ October 25, 2012). The Sierra Nevada A total of 48 skin samples were obtained (Table 1). Each biopsy Natural Park staff also approved this study. was placed into 10% neutral buffered formalin for 48–72 h, then transferred to 60% ethanol and stored at 4°C until histological analysis. The biopsies were embedded in paraffin and 4-μm thick 2.2. Experimental infestation sections were either stained with hematoxylin and eosin (H&E) for skin lesion assessment (21) or used for immunohistochemical studies. After an 8-week adaption period in the facilities, 14 of the 20 ibexes were experimentally infested with S. scabiei. Two cm2 skin fragments were attached to the withers from a naturally infested free- 2.5. Immunohistochemistry ranging Iberian ibex, as described previously (21). Mite density was calculated in skin pieces adjacent to those used for the infestation Different primary antibodies raised against antigens expressed by using a stereomicroscope after overnight digestion in 5% potassium macrophages (Iba-1), including M1 (iNOS) and M2 (CD204) hydroxide (KOH) solution at 40°C (45). A thermal gradient was then subpopulations, T lymphocytes (CD3), B lymphocytes (CD20), and induced by shining a light on Petri dishes with black bottoms and plasma cells (Kappa-Lambda), as well as the cytokine interleukin 10 transparent central areas (46). The resulting estimated dose received (IL-10), were used to quantitatively assess different cell populations by each ibex was 750 ± 440 mites (mean ± standard deviation). The that play a relevant role in the local skin immune response (Table 2). remaining six ibexes served as non-infested controls. Heat-mediated antigen retrieval was performed on 4-μm-thick sections placed onto poly-L-lysine-coated slides, using the PT Link ® ® system (PT-Link, Agilent , Santa Clara, CA, USA) or proteinase K, 2.3. Clinical assessment depending on the specific antibody used (Table 2). After deparaffinization, rehydration, and drying, endogenous peroxidase The clinical signs and the extension of mange-compatible skin lesions were monitored for 103 days post-infection (dpi) and classified TABLE 1 Number of skin biopsies from the Iberian ibexes experimentally as 0 (no visible skin lesions), 1 (focal skin lesions affecting less than infested with Sarcoptes scabiei and controls used for immunohistochemical analyses. 50% of the body surface), or 2 (extended skin lesions affection more than 50% of the body surface), as described previously (14). None of Group Day post-infestation (dpi) Total the control ibexes developed any lesions or clinical signs compatible 26 46 103 with mange. Conversely, both lesions and clinical signs were observed Terminal 7 7 7 21 in all the infected ibexes, in which three different clinical courses were identified: four ibexes had mild skin lesions affecting less than 50% of Partially 3 3 3 9 the body surface that healed completely before the end of the 103-day recovered Totally recovered 4 4 4 12 Control 6 6 Total 14 14 20 48 1 https://www.telinject.de/en/products/vario-product-line/g-u-t-50/ Frontiers in Veterinary Science 03 frontiersin.org Valldeperes et al. 10.3389/fvets.2023.1183304 TABLE 2 Primary antibodies and protocols used to characterize the different cellular types analyzed in the skin of the Iberian ibexes 2.6. Evaluation of the immunostained slides experimentally infested with S. scabiei and controls. A total of 356 skin sections immunolabeled for the seven cellular Target Specificity Source Epitope Dilution markers analyzed in the 48 skin samples were evaluated, (clone) unmasking corresponding to the 20 ibexes included in the study (Table 1). Ten Macrophage randomly chosen fields were selected and photographed at a 96° 20′ buffer Iba-1 (rabbit polyclonal) Wako® Dako pH6 1:2,000 ® magnification of 400× (Nikon Eclipse Ci microscope, coupled with a MicrosCopiaDigital MDE3-6-3 digital camera) on each slide. The MØ Scavenger immunolabeled cells were counted on digital images using the Cell receptor A TransGenic Counting add-on in ImageJ (National Institutes of Health, Bethesda, (mouse Inc.® 96° 20′ buffer MD, USA). The average value for the 10 fields counted in each slide CD-204 1:400 monoclonal) (KAL- Dako pH6 was calculated for each immunolabeled cell subtype. Additionally, the (clone SRA-ES) KT022) distribution of the immunostained cells in the different skin areas M2 marker was assessed. Inducible nitric The immunostained cell count and distribution was independently oxide synthase evaluated by two of the authors (J. Espinosa and V. Pérez, the latter a 96° 20′ buffer European College of Veterinary Pathology Diplomate), and any iNOS (rabbit Novus® 1:150 Dako pH6 discordant results were reviewed using a multiheaded microscope to polyclonal) M1 marker reach a consensus. Interleukin-10 Biorbyt® 96° 20′ buffer IL-10 (mouse 1:100 monoclonal) (Orb10892) Dako pH9 2.7. Statistical analysis T lymphocyte Data normality was initially assessed for each cellular type and (rabbit 96° 20′ buffer CD3 Dako® 1:300 total cellular counts using graphical methods, skewness checks, and polyclonal) Dako pH6 Kolmogorov -Smirnov tests. As all the variables did not conform to (clone A0452) normality, non-parametric statistical methods were used. B lymphocyte Thermo® Generalized linear mixed models (GLMMs) and linear mixed CD20 (rabbit (RB9013P) No unmasking 1:150 models (LMMs) were fitted using the packages “nlme,”” lme4,” polyclonal) “mgcv,” and “MuMIn,” depending on the distribution of the variable Kappa: and using the individual identity as a random term (due to Plasma cells Dako® Kappa- Proteinase K 1:7,000 non-independency of the measures) for total inflammatory cell, total (rabbit (0191– Lambda 0.05% Lambda: macrophage, M2 macrophage, and T lymphocyte counts. The models polyclonal) 0193) 1:15,000 were selected based on the Akaike information criterion (AIC) (47). The explicative variables included in the models were dpi, the experimental group (control or infested), and clinical outcome within the infested group (recovered, partially recovered, and terminal). The was blocked by immersing the sections in 3% oxygen peroxide in effects of individual variables, such as age and sex, did not affect cell methanol solution for 30 min at room temperature in the dark. The number and were considered part of the individual identity in the sections were then incubated with the specific primary antibodies random term. When the interaction between the dpi and outcome diluted in a commercial reagent (Antibody Diluent, Agilent , Santa® was significant, different GLMMs were applied to detect specific Clara, CA, USA) (Table 2) overnight at 4°C in a humidified chamber. differences between groups. After washing, immunolabeling was performed using a ready-to-use B lymphocyte and plasma cell counts were too low to perform any ® ® kit EnVision System (Agilent , Santa Clara, CA, USA) for the statistical models. Therefore, Mann–Whitney U tests were carried out appropriate monoclonal or polyclonal antibodies, and slides were to compare these two variables between the experimental groups incubated for 40 min at room temperature. After two washes in (control vs. infested). Differences in B lymphocyte and plasma cell phosphate-buffered saline (PBS), antibody localization was counts among dpi (26, 46, and 103 dpi) within the infested clinical ® determined using 3,3-diaminobenzidine (Agilent , Santa Clara, CA, outcome groups (healthy, totally recovered, partially recovered, and USA) as a chromogenic substrate for horseradish peroxidase (HRP) terminal) were assessed using non-parametric Friedman’s tests for of the secondary antibody. The reaction was stopped with tap water repeated measures, whereas the differences among the three different after 3–4 min or, for plasma cells, using the commercial ImmPact ® clinical outcome groups for each dpi were assessed through Kruskall– ® Vector kit (Red Substrate Kit) (Vector Lab, Newark, CA, USA). Wallis tests and pairwise comparisons using a Wilcoxon rank sum test Finally, the slides were counterstained with Harris’s hematoxylin. with Bonferroni correction (48). Finally, Spearman’s rank correlation Appropriate species- and isotype-matched immunoglobulins were test was applied to establish possible correlations among the different used as controls, including sections with an isotype control for the cell types analyzed. primary antibody and those for which the primary antibody was All the statistical procedures were performed with R 4.1.2 software omitted. The same examined sections were used to determine the using the functions “ggdensity,” “stats_overlay_normal_density,” and optimal dilution and incubation pH. “skewness,” and the packages “nortest,” “plotrix,” “coin,” “lme4,” and Frontiers in Veterinary Science 04 frontiersin.org Valldeperes et al. 10.3389/fvets.2023.1183304 “AICcmodavg” (R Development Core Team 2022). Statistical dpi with the partially recovered outcome approached significance significance was set at p < 0.05. (Table 4). Total cell counts consistently decreased throughout the study in all the infested groups (Figure 1; Supplementary Table S2). Despite 3. Results this decrease, the total cell counts of the infested ibexes at 103 dpi were still higher than those of the control ibexes at 103 dpi (p = 1.4e-08) 3.1. Differences between control and (Figure 2; Table 3). However, this difference from the controls was infested ibexes only significant for the partially recovered and terminal groups, but not for the totally recovered ibexes (Supplementary Table S3). Throughout the experimental infestation, five cell types At 26 dpi, the total cell counts were higher in the terminal ibexes (macrophages; M2 macrophages; T lymphocytes; B lymphocytes; than in the partially recovered ones. At 46 dpi, the total cell counts of and plasma cells) were detected in the skin of the infested (26, 46, the terminal group were higher than those of the totally and partially and 103 dpi) and control ibexes (103 dpi) (Figure 1; Table 3). The recovered groups. At 103 dpi, the totally recovered ibexes had identification of M1 macrophages through iNOS marker and IL-10- significantly lower total cell counts than the partially recovered and immunomarked cells was anecdotal. The positively immunolabeled terminal groups (Figure 3; Table 3; Supplementary Table S4). cells were identified by their morphology and deep brown (macrophages and lymphocytes) or red (plasma cells) granular 3.2.2. Total macrophage counts stain. When analyzing the cell counts of all the dpi altogether, all Total macrophage counts were also explained by the interaction the cell types were significantly (p < 0.001) more abundant in the between dpi and outcome, according to the most parsimonious model skin of the infested ibex than in the control ones. When analyzing (AICc = 4390.2, K = 10, AICcWt = 1.0) (Supplementary Table S5). only the samples from 103 dpi, the difference between the control The effects of all the sampling time points, all the outcomes, and and the infested ibexes was significant only for total macrophage the interaction of 103 dpi with all the terminal outcomes were and M2 macrophage counts (Table 3). significant on the total macrophage counts (Table 5). In the infested ibexes, total macrophages (Iba-1) and M2 In all the infected groups, the total macrophage counts consistently (CD204) macrophages were observed in the superficial and decreased for each time point (Supplementary Table S6; Figures 1, 4). intermediate dermis as well as in the epidermis, but not in the control Despite this decrease, the total macrophage counts of the infested ibexes. T lymphocytes (CD3+ immunolabeled) were observed in all ibexes at 103 dpi were still higher than those of the control ibexes the layers of the dermis, forming multifocal to confluent aggregates (p = 5.0e-09) (Figure 2; Table 3). However, this difference from the interspersed with macrophages. Additionally, intraepidermal foci of controls was only significant for the partially recovered and terminal lymphocytic exocytosis were observed, mainly in samples from 26 groups, but not for the totally recovered ibexes dpi. Finally, antibody-producer cells (B lymphocytes and plasma (Supplementary Table S7). cells) were less abundant in the immune cellular skin infiltrates At 26 dpi, the total macrophage counts of the terminal ibexes were identified in all the groups and from all the time points (Figure 1; higher than both those of the totally and partially recovered groups. Table 3). Both cell types were distributed mainly at the intermediate At 46 and 103 dpi, the total macrophage counts of the terminal ibexes and deep dermis, but while B lymphocytes were diffusely distributed, were only significantly higher than those of the totally recovered plasma cells were seen mostly as perivascular infiltrates, mainly in the group (Supplementary Table S8; Figure 4). deep dermis (Figure 1). Macrophages, both overall and especially M2, were the 3.2.3. M2 macrophage counts predominant cell type in the inflammatory infiltrate in the skin of the As for total cell counts and total macrophage counts, the M2 infested ibexes (Figure 2; Table 3). Consequently, total (Iba-1) and M2 macrophage counts were again explained by the interaction between (CD204) macrophage counts significantly correlated (R2 = 0.827, dpi and clinical outcome, according to the most parsimonious model p < 0.001). T lymphocytes were the second most abundant cell (AICc = 3931.3, K = 10, AICcWt = 1.0) (Supplementary Table S9). population. Finally, B lymphocytes and plasma cell numbers were less The effects of 103 dpi, the interaction of 46 dpi with the terminal abundant and significantly correlated (R2 = 0.707; p < 0.001). outcome, and the interactions of 103 dpi with the partially recovered and the terminal group outcomes were significant in terms of the M2 macrophage counts, while the effect of the terminal outcome 3.2. Comparison among clinical outcomes approached significance (Table 6). in the infested ibexes The M2 macrophage counts decreased consistently in all the study periods in the terminal group, while such a decrease was only 3.2.1. Total cell counts significant for 103 dpi in the totally and partially recovered ibexes According to the most parsimonious model, total inflammatory (Supplementary Table S10; Figures 1, 5). Despite these decreases, the cell count trend was explained by the interaction between dpi and M2 macrophage counts of the infested ibexes altogether and for each outcome (corrected AICc [AICc] = 5072.0; degrees of freedom K = 10; clinical outcome group were still higher than those of the control AIC weight [AICcWt] = 1.0) (Supplementary Table S1). ibexes at 103 dpi (p = 2e-14) (Figure 2; Table 3; The variables significantly influencing total inflammatory cell Supplementary Table S11). count were all the time points; the terminal outcome; the interaction Statistically significant differences in the M2 macrophage counts of all the time points with the terminal outcome; and the interaction among the clinical outcomes were only found at 26 dpi, when the of 103 dpi with the partially recovered outcome. The interaction of 46 values of the terminal ibexes were significantly higher than those of Frontiers in Veterinary Science 05 frontiersin.org Valldeperes et al. 10.3389/fvets.2023.1183304 FIGURE 1 Photomicrographs of sections of mangy skin of Iberian ibexes experimentally infested with Sarcoptes scabiei at 26, 46, and 103days post-infestation (dpi), showing immunolabeling of total macrophages (Iba-1 marker), the M2 macrophage subtype (CD204), T lymphocyte (CD3), B lymphocytes (CD20), and plasma cells (Kappa-Lambda). The positively immunostained macrophages (total and the M2 phenotype) and T and B lymphocytes appear brown, whereas plasma cells are red. Magnification: 200×. the partially recovered group, approaching significance with the The effects of all the dpi and the partially recovered outcome were totally recovered ibexes (Supplementary Table S12; Figure 5). significant on T lymphocyte counts, as were the interactions between the clinical outcomes and the sampling dpi (Table 7). 3.2.4. T lymphocyte counts The T lymphocyte counts decreased consistently in each sampling The T-lymphocyte count trend was also explained by the interval in all the experimentally infested groups, except for the interaction between clinical outcome and dpi according to the most partially recovered ibexes between 46 dpi and 103 dpi parsimonious model (AICc = 4587.3, K = 10, AICcWt = 1.0) (Supplementary Table S14; Figures 1, 6). The T lymphocyte counts of (Supplementary Table S13). the infested ibexes altogether were not significantly different at any Frontiers in Veterinary Science 06 frontiersin.org Valldeperes et al. 10.3389/fvets.2023.1183304 TABLE 3 Total cell counts for the control Iberian ibexes at 103days post- infestation (dpi) and the infested Iberian ibexes altogether at 26, 46, and 3.2.5. B lymphocyte and plasma cell counts 103 dpi. The differences in B lymphocyte counts among clinical outcomes were only significant at 26 dpi, when they were higher in the terminal Number of cells per field at 400× than in the partially recovered ibexes (p = 0.023). The decrease in (mean±standard error) B lymphocyte counts was consistent in the totally recovered (26 dpi to Control Infested Infested Infested 46 dpi p = 0.0499; 46 dpi to 103 dpi p = 0.0056) and terminal (26 dpi to (103 dpi) (26 dpi) (46 dpi) (103 dpi) 46 dpi p = 0.00272; 46 dpi to 103 dpi p = 6.2e-06) groups, while they Macrophages decreased more progressively in the partially recovered ibexes 7.29 ± 1.79* 45.97 ± 3.73a 26.09 ± 2.02b 13.98 ± 2.08c* (Iba-1) (statistically significant differences were only found between 26 dpi M2 and 103 dpi, p = 0.016) (Figure 7). macrophages 1.83 ± 0.42* 31.76 ± 2.73a 25.57 ± 2.17b 11.50 ± 2.24c* Plasma cell counts were higher in the terminal group than in both (CD204) the totally (p = 0.0450) and partially (p = 0.0350) recovered groups at T lymphocyte 26 dpi. While plasma cell counts remained stable in the totally (CD3) 11.03 ± 0.70 33.01 ± 3.04a 19.11 ± 2.19b 15.36 ± 1.48b recovered group (p = 0.174), they decreased consistently throughout the study period in the other two groups (partially recovered: 26 dpi B lymphocyte 0.11 ± 0.06 2.66 ± 0.69a 1.24 ± 0.21ab 0.33 ± 0.09b to 46 dpi, p = 0.0098; 46 dpi to 103 dpi, p = 0.0346; terminal: 26 dpi to (CD20) 46 dpi, p = 0.00056; 46 dpi to 103 dpi, p = 0.00036). Nevertheless, the Plasma cells plasma cell counts of the terminal group were still higher than those (Kappa- 0.19 ± 0.10 2.84 ± 0.56a 2.95 ± 1.55a 0.26 ± 0.22b of the partially recovered group at 46 dpi (p = 0.043) and the totally Lambda) recovered group at 103 dpi (p = 0.026) (Figure 8). Total cells 20.44 ± 2.63* 116.24 ± 8.08a 74.96 ± 3.64b 41.44 ± 4.67c* a, b, and c means with different superscripts are significantly different from each other among time points within the infested Iberian ibexes; *statistically significant differences 4. Discussion between the control and the infested Iberian ibexes at 103 dpi. The capacity of Iberian ibexes to recover from and survive sarcoptic mange has been repeatedly reported (37–39). The underlying pathophysiological mechanisms of this recovery seem to rely on the local skin cellular immune response, which is driven by differences in genomic expression, rather than on any humoral immune response, either local or systemic (1, 3, 41–43, 49, 50). This longitudinal study describes for the first time the progression of the local skin immune cell response in Iberian ibexes experimentally infested with S. scabiei. Moreover, this is also the first report of such an analysis in any wild ungulate. More importantly, this study also provides the first evidence linking the clinical outcome of sarcoptic mange with the local skin immune cell response, specifically identifying the immune cell types involved and their relationship with the clinical outcome and eventual mortality or survival of the Iberian ibexes affected by this disease. FIGURE 2 Cell counts (mean±standard error) of positively inmunolabeled total Compared with the control ibexes, sarcoptic mange caused a macrophages (Iba-1 marker), M2 macrophages (CD204), T severe inflammatory response in the Iberian ibex epidermis and lymphocytes (CD3), B lymphocytes (CD20), and plasma cells (Kappa- dermis, which can be attributed to the mechanical, excoriating, and Lambda) in the skin of the non-infested ibexes and the mangy skin of the infested ibexes altogether at 26, 46, and 103days post-infestation allergenic action of the mites (51). This inflammatory infiltrate (dpi). The total cell counts are shown as “TOTAL.” a, b, and c means corresponded to the lesions previously reported in these same with different superscripts are significantly different from each other individuals, including severe hyperkeratosis, crusts, irregular among time points within the infested ibexes; *statistically significant differences between the control and the infested ibexes at 103 dpi. epidermal hyperplasia with rete ridges, spongiotic areas with apoptotic keratinocytes, and lymphocyte exocytosis foci in the epidermis. Additionally, these lesions were related to the abundance of mites, their detritus, and galleries in the histological sections (21, 38). Such time point from those of the control ibexes at 103 dpi (Figure 2); parallelism between skin lesions and inflammatory skin infiltrate has however, the terminal group had higher T lymphocyte counts than the also been reported in other wildlife species, such as chamois and control ibexes at 103 dpi (Supplementary Table S15; Figure 6). wombats (25, 28). At 26 dpi, the partially recovered ibexes had lower T lymphocyte The absence of mites and the decrease of immune cells observed counts than the totally recovered and terminal groups. At 46 dpi, no at 103 dpi is consistent with the previously reported tissue restoration statistically significant differences in T lymphocyte counts were (21) and corresponds with the decline in mite numbers (38). This detected among the three clinical outcomes. At 103 dpi, the recovered reduction in mite numbers could be attributed to the exacerbated local ibexes had significantly lower values than the terminal group, inflammatory response observed at 26 dpi. Although this response approaching significance versus the partially recovered ibexes causes severe tissue damage, it could control and eliminate the mite or (Supplementary Table S16; Figure 6). promote mite displacement toward other dermal areas where the Frontiers in Veterinary Science 07 frontiersin.org Valldeperes et al. 10.3389/fvets.2023.1183304 TABLE 4 Summary of the most parsimonious model explaining the TABLE 5 Summary of the most parsimonious model explaining the evolution of total inflammatory cells in the mangy skin of Iberian ibexes evolution of total macrophage counts by days post-infection (dpi) and by days post-infection (dpi) and outcome (recovered, partially recovered, Iberian ibex outcome (recovered, partially recovered, and terminal). and terminal). Fixed Estimate SE z-value p-value Fixed Estimate SE z-value p-value effects effects dpi46 −0.53 0.046 −11.68 <2.2e-16 dpi46 −0.58 0.030 −19.45 <2.2e-16 dpi103 −1.42 0.063 −22.45 <2.2e-16 dpi103 −1.44 0.041 −35.13 <2.2e-16 Partially 0.24 0.112 2.17 0.030 Partially recovered −0.16 0.115 −1.36 0.175 recovered Terminal 0.58 0.092 6.34 2.3e-10 Terminal 0.22 0.094 2.36 0.018 dpi46* −0.34 0.067 −0.58 0.565 dpi46* partially partially 0.09 0.047 1.85 0.064 recovered recovered dpi103* 0.28 0.086 3.28 0.001 dpi103* partially partially 0.63 0.058 10.85 <2.2e-16 recovered recovered dpi46* −0.05 0.053 −0.95 0.343 dpi46* terminal −0.08 0.036 2.18 0.030 terminal dpi103* 0.25 0.071 3.64 2.7e-04 dpi103* terminal 0.48 0.047 10.25 <2.2e-16 terminal SE, standard error. SE, standard error. FIGURE 3 FIGURE 4 Cell counts (mean±standard error) of the all the inmunostained cell Cell counts (mean±standard error) of positively immunolabeled types grouped for the clinical outcome groups at 26, 46, and 103 dpi. Iba-1 cells (macrophages) for the clinical outcome groups at 26, 46, a and b means with different superscripts are significantly different and 103days post-infestation (dpi). a and b means with different from each other among the clinical outcome groups at the same superscripts are significantly different from each other among the time point; x, y, and z means with different superscripts are clinical outcome groups at the same time point; x, y, and z means significantly different from each other among time points within the with different superscripts are significantly different from each other same clinical outcome group. among time points within the same clinical outcome group. inflammatory response is still reduced (18, 19). Alternatively, the affected ibexes compared with the control group could be due to reduction of the inflammatory infiltrate throughout the dpi may not exocytosis foci of macrophages migrating from the dermis to have caused the decrease in mite numbers, but rather be the phagocytise the mite or increased numbers of Langerhans cells, as consequence of mites displacing toward unaffected skin areas richer described in chamois and related to antigen presentation to T in nutritional factors (38). lymphocytes (25, 32, 33, 53). This skin inflammatory response observed in the infested Iberian In humans, the more severe crusted scabies is associated with fewer ibex was characterized by a predominance of macrophages (mainly macrophages infiltrating the skin and an unbalanced Th1/Th17 the M2 subtype), followed by T lymphocytes, with a lower involvement immune response, whereas in ordinary scabies, M2 macrophages are of antibody-producing cells (B lymphocytes and plasma cells) more abundant in the inflammatory infiltrate, indicating a balanced (Figures 2–8). This type of inflammatory response is similar to that Th1/Th2 immune response (26). Both a suppressive effect mediated by reported in other mountain ungulate and wildlife species (25, 32, 33, a macrophage migration inhibitory factor expressed by S. scabiei and/ 52). The higher abundance of intra-epithelial macrophages in the or an induction of mixed M1/M2 macrophage polarization against the Frontiers in Veterinary Science 08 frontiersin.org Valldeperes et al. 10.3389/fvets.2023.1183304 TABLE 6 Summary of the most parsimonious model explaining the evolution of macrophage phenotype M2 counts by days post-infection interpreted as a delayed type IV hypersensitivity reaction (5, 25, 33, (dpi) and Iberian ibex outcome (recovered, partially recovered, and 62). This matches the dermis histopathology previously reported in terminal). these same Iberian ibexes, consistent with type I and type IV hypersensitivity responses (21). Overall, the consistent decrease of Fixed Estimate SE z-value p-value effects total and M2 macrophage counts in all the clinical outcome groups in the infested Iberian ibexes, combined with the consistently higher dpi46 −0.02 0.042 −0.41 0.686 values in the terminal group (Figures 4, 5), indicate a successfully dpi103 −1.42 0.068 −20.92 <2.2e-16 balanced Th1/Th2 immune response that resolves the inflammatory Partially condition after the infestation and eliminates or displaces the mite to −0.14 0.171 −0.80 0.423 recovered other areas of the body (18, 19). Such an immune response was more Terminal 0.26 0.140 1.87 0.062 intense and, therefore, required a longer and more sustained response in the terminal ibexes than in either the fully or partially recovered dpi46* ones (Figures 4, 5). The same explanation of a higher inflammatory partially 0.02 0.067 0.30 0.767 response in the severely affected ibexes resolving later and more slowly recovered than the milder increase in the totally recovered ibexes could account dpi103* for the similar trend observed in T and B lymphocytes and plasma partially 0.56 0.096 5.88 4.2e-09 cells (Figures 6–8). To fully distinguish the local skin immune recovered responses to sarcoptic mange determining the clinical outcome in the dpi46* host, macrophage subpopulations and cytokines should be assessed −0.38 0.057 −7.21 5.6e-13 terminal and further characterized, not only in Iberian ibex but also in other dpi103* species, to detect potential common inflammatory recovery patterns. 0.53 0.078 6.87 6.6e-12 terminal Higher counts of T lymphocytes have been described in response SE, standard error. to sarcoptic mange in chamois, wild boars, goats, sheep, and pigs (19, 30, 32, 34, 35), whereas in foxes, wolves, red deer, and roe deer, T lymphocytes were less abundant in the inflammatory infiltrate (5, 33, 62, 63). Finally, the scarce infiltrate of B lymphocytes and plasma cells matches previous reports in mangy chamois and domestic goats and sheep (30, 34, 62). Although antibodies against S. scabiei have been broadly used for surveys of sarcoptic mange in wildlife, their role in providing protection against the disease is unclear. In Iberian ibex they increase with clinical severity and, generally, are an indicator of the contact and intensity of the infestation rather than protection (1, 3, 43, 63, 64), corresponding to the low involvement of antibody- producing cells in the skin found in this study. Despite the common decreasing trend in all the cellular types observed in all three clinical outcome groups (totally recovered, partially recovered, and terminal), the differences among these three groups indicate a correlation between the local skin cellular immune FIGURE 5 response and the clinical outcome. The skin immune response to Cell counts (mean±standard error) of positively immunolabeled CD204 cells (M2 phenotype macrophages) for the clinical outcome sarcoptic mange varies among species and individuals, although it groups at 26, 46, and 103days post-infestation (dpi). a and b means usually corresponds to a type I or type IV hypersensitivity response. with different superscripts are significantly different from each other In the type IV hypersensitivity response, the memory of T lymphocytes among the clinical outcome groups at the same time point; x, y, and z means with different superscripts are significantly different from seems to play a key role in limiting infection at the initial phase (18, each other among time points within the same clinical outcome 24, 25). Moreover, the type of T cells involved (CD4+ or CD8+) and group. the proportion of macrophages in the local skin immune cellular response is a differential trait between ordinary and crusted scabies in humans (26, 27). Similarly, in domestic goat and sheep, the T scabies mite have been suggested to explain the lower macrophage lymphocyte subpopulations shift to CD4+ over CD8+ lymphocytes in counts and shift to the M2 phenotype in scabietic humans (54–56). the local skin immune response to sarcoptic mange (30, 34). Thus, the Although differential staining of macrophage populations (iNOS higher initial (26 dpi) T lymphocyte count of the totally recovered targeting M1 and CD204 targeting M2) has been attempted previously ibexes (Figure 6), leading to lower total cell, macrophage (both total for other parasitic diseases (57–61), to the authors’ knowledge this is and the M2 phenotype), T lymphocyte, and plasma cell counts at the the first study analyzing macrophage differential expression in a end of the study period (103 dpi) than in the terminal ibexes parasitosis caused by an arthropod. This allowed the macrophages (Figures 3–6, 8), seems to confirm that an initial effective local skin infiltrating the skin of the experimentally infested ibexes to T-cell immune response is crucial for controlling the spread of be identified primarily as M2, which suppress local inflammatory sarcoptic mange in Iberian ibex. Further characterization of this T-cell response (54). Similarly, in wild carnivores, macrophages have been immune response, including the cell types, cytokines, and gene associated, along with neutrophils, with the milder alopecic form, expression involved, should help us to fully understand the Frontiers in Veterinary Science 09 frontiersin.org Valldeperes et al. 10.3389/fvets.2023.1183304 TABLE 7 Summary of the most parsimonious model explaining the evolution of T lymphocyte counts by days post infection (dpi) and Iberian ibex outcome (recovered, partially recovered, and terminal). Fixed Estimate SE z-value p-value effects dpi46 −0.87 0.046 −19.15 <2.2e-16 dpi103 −1.37 0.055 −24.92 <2.2e-16 Partially −0.66 0.171 −3.89 1.0e-04 recovered Terminal −0.19 0.138 −1.44 0.151 dpi46* 0.63 0.076 8.36 <2.2e-16 partially recovered FIGURE 7 Cell counts (mean±standard error) of positively immunolabeled dpi103* 1.10 0.082 13.43 <2.2e-16 CD20 cells (B lymphocytes) for the clinical outcome groups at 26, partially 46, and 103days post-infestation (dpi). a and b means with different recovered superscripts are significantly different from each other among the clinical outcome groups at the same time point; x, y, and z means dpi46* 0.50 0.056 8.90 <2.2e-16 with different superscripts are significantly different from each other terminal among time points within the same clinical outcome group. dpi103* 0.77 0.065 11.86 <2.2e-16 terminal FIGURE 8 FIGURE 6 Cell counts (mean±standard error) of positively immunolabeled Cell counts (mean±standard error) of positively immunolabeled CD3 Kappa-Lambda cells (plasma cells) for the clinical outcome groups at cells (T lymphocytes) for the clinical outcome groups at 26, 46, and 26, 46, and 103days post-infestation (dpi). a and b means with 103days post-infestation (dpi). a and b, means with different different superscripts are significantly different from each other superscripts are significantly different from each other among the among the clinical outcome groups at the same time point; x, y, and clinical outcome groups at the same time point; x, y, and z, means z means with different superscripts are significantly different from with different superscripts are significantly different from each other each other among time points within the same clinical outcome among time points within the same clinical outcome group. group. pathophysiological and immune mechanisms responsible for the The M2 macrophage phenotype predominantly induced by control or extension of sarcoptic mange in Iberian ibex (21, 24–26, 49, S. scabiei has immunoregulatory effects and a reparative action, which 50, 54). could contain the mite, limit the inflammatory infiltrate, and aid in To summarize, this study reports for the first time the progression restoring tissue integrity. However, the decrease in inflammatory of the local skin immune cell response over time, not only to S. scabiei infiltrate could instead be a consequence of mite displacement to infestation in Iberian ibex but to any ectoparasite in any wild ungulate unaffected skin areas, rather than a direct effect of the M2 species. The skin immune cell infiltrate elicited by mange in Iberian macrophages. The higher initial T lymphocyte counts in the ibexes ibex, predominantly formed by macrophages and T lymphocytes, that had a milder clinical outcome and completely healed suggest that indicates a locally intense cellular inflammatory response. The this early T lymphocyte immune response plays a key role in evolution over time of mite presence and the inflammatory response controlling the spread and severity of sarcoptic mange in this species, must be considered when assessing mange lesions through skin limiting the inflammatory infiltrate and leading to a faster resolution biopsies, as false negative results could occur due to mite absence and of the disease. Moreover, even if this first antigen-presenting response the complete restoration of tissue integrity as soon as 3 months after fails, differences in the local inflammatory response and consequently the establishment of the mite (21, 49). in the clinical outcome occur, with some individuals recovering and Frontiers in Veterinary Science 10 frontiersin.org Valldeperes et al. 10.3389/fvets.2023.1183304 healing and others progressing to severe stages of the disease that and curated the samples, performed the immunohistopathological ultimately lead to death. analyses, drafted the original manuscript, and elaborated the final Therefore, the development of severe sarcoptic mange and version of the manuscript. All authors contributed to the article and eventual death requires a sequence of different conditions. Firstly, the approved the submitted version. ibex must come into contact with an effective infective dose of S. scabiei mites to become infested. Secondly, the local immune skin response, which involves antigen presentation by T lymphocytes and Funding is described in the totally recovered ibexes in this study, must fail to control the spread of the disease. Thirdly, the inflammatory response This project was funded by the Consejería de Medio Ambiente de must be intense and not resolve or heal spontaneously, unlike the la Junta de Andalucía (project 173/2009/M/00;03/15/M/00; findings in the partially recovered ibexes in this study. Finally, the 861_11_M_00 and 2016/00014/M) and the Spanish Ministerio de uncontrolled infestation must spread and become severe enough to Economía y Competitividad (projects CGL2012-40043-C02-01, exhaust the pathophysiological resources of the host. CGL2012-40043-C02-02, and CGL2016-80543-P). The authors’ Further investigation is necessary to gain a comprehensive research activities are partially supported by the Plan Andaluz de understanding of the mechanisms underlying resistance to sarcoptic Investigación (RNM-118 group). MV is supported by a FI-GENCAT mange in Iberian ibex, as reported in this study. Immunological, Fellowship (2020_FI_B2_00049, which is cofinanced by the Agència immunohistochemical, genetic, and genomic research would de Gestió d’Ajuts Universitaris i de Recerca and the European Social be particularly informative in this regard. The present findings Fund). GM is a Serra Húnter Fellow. contribute to the growing body of evidence refuting the previously held belief that S. scabiei infestation in this species inevitably leads to mortality. Therefore, these findings have significant implications not Acknowledgments only for individual disease outcomes but also for the management of the Iberian ibex populations affected by sarcoptic mange. The authors would like to thank the Consejería de Medio Ambiente de la Junta de Andalucía and in particular the Sierra Nevada Natural Space for their logistical support and the Sierra de Huétor Data availability statement Natural Park for the transfer of their experimental facilities. We also give special thanks to the park wardens and fieldworkers in the SNNS The original contributions presented in the study are included in and above all to Apolo Sánchez, José López, Isidro Puga, Elías the article/Supplementary material, further inquiries can be directed Martínez, Francisco Felipe, and Antonio Rodríguez for their to the corresponding author. professional and personal involvement in the study. The authors are also grateful to Manolo Herrera for the maintenance of the ibex and the facilities during the experimental phase, Emmanuel Serrano for Ethics statement advice, and the Service of Veterinary Pathology of the UAB for the histological preparation of samples. The technical and human support The animal study was reviewed and approved by the Ethics on provided by Marta Silva is gratefully acknowledged. Animal Welfare Committee of the University of Jaén and authorized by the Dirección General de Producción Agrícola y Ganadera of the Consejería de Agricultura, Pesca y Medio Ambiente of the Junta de Conflict of interest Andalucía (Ref: SA/SIS/MD/ps/ October 25, 2012). The authors declare that the research was conducted in the absence of any commercial or financial relationships that could Author contributions be construed as a potential conflict of interest. MV analyzed the data, drafted the original manuscript, and elaborated the final version of the manuscript. JG designed the Publisher’s note experiment, obtained funding, performed the experimental infestation, obtained and curated the samples, and critically revised All claims expressed in this article are solely those of the authors the manuscript. VP performed the immunohistopathological analyses, and do not necessarily represent those of their affiliated organizations, and critically revised the manuscript. JL-O designed the experiment, or those of the publisher, the editors and the reviewers. Any product obtained funding, performed the experimental infestation, obtained that may be evaluated in this article, or claim that may be made by its and curated the samples, drafted the original manuscript, and manufacturer, is not guaranteed or endorsed by the publisher. elaborated the final version of the manuscript. AR-B performed the experimental infestation, obtained and curated the samples, and critically revised the manuscript. PF, JP, and GM designed the Supplementary material experiment, obtained funding, and critically revised the manuscript. ST participated in data analysis, and critically revised the manuscript. The Supplementary material for this article can be found online RS designed the experiment, obtained funding, and critically revised at: https://www.frontiersin.org/articles/10.3389/fvets.2023.1183304/ the manuscript. JE performed the experimental infestation, obtained full#supplementary-material Frontiers in Veterinary Science 11 frontiersin.org Valldeperes et al. 10.3389/fvets.2023.1183304 References 1. Bornstein S, Mörner T, Samuel WM. Sarcoptes scabiei and sarcoptic mange In: WM 25. Turchetto S, Obber F, Rossi L, Amelio SD, Cavallero S, Poli A, et al. Sarcoptic Samuel, MJ Pybus and AA Kocan, editors. Parasitic diseases of wild mammals. Ames, IA: mange in wild caprinae of the Alps: could pathology help in filling the gaps in Iowa Sate University Press (2001). 107–19. knowledge? Front Vet Sci. (2020) 7:193. doi: 10.3389/fvets.2020.00193 2. Pence DB, Ueckermann E. Sarcoptic mange in wildlife. Rev Sci Tech. (2002) 26. Bhat SA, Mounsey KE, Liu X, Walton SF. 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