Hindawi Journal of Immunology Research Volume 2020, Article ID 9602576, 8 pages https://doi.org/10.1155/2020/9602576 Research Article Toll-Like Receptors 2, 4, and 7, Interferon-Gamma, Interleukin 10, and Programmed Death Ligand 1 Transcripts in Leishmanin Skin Test-Positive Reactions of Ibizan Hound Dogs Laura Ordeix,1,2 Sara Montserrat-Sangrà,1 Pamela Martínez-Orellana,1 and Laia Solano-Gallego 1 1 Departament de Medicina i Cirurgia Animals, Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra 08193, Spain 2 Hospital Clínic Veterinari, Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra 08193, Spain Correspondence should be addressed to Laia Solano-Gallego; laia.solano@uab.cat Received 15 January 2019; Revised 14 December 2019; Accepted 16 January 2020; Published 10 March 2020 Academic Editor: Nejat K. Egilmez Copyright © 2020 Laura Ordeix et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The leishmanin skin test (LST) is an in vivo technique commonly used to evaluate the Leishmania-specific cellular immune response in dogs. However, information regarding the local immune response in LST-positive reactions is scarce. We examined the pattern of toll-like receptor 2 (TLR2), TLR4, TLR7, interleukin- (IL-) 10, interferon gamma (IFN-γ), and (program death ligand) PD-L1 gene expression in LST-positive reactions and paired normal-looking skin of nine infected Ibizan hound dogs. Healthy skin from ten seronegative dogs from a nonendemic area was analysed as a negative control. Immune gene expressions were examined by quantitative PCR (qPCR) analysis. LST-positive reactions presented significant upregulation of TLR2, TLR4, IL-10, IFN-γ, and PD-L1 and downregulation of TLR7 when compared with healthy skin of seronegative control dogs from a nonendemic area. All transcripts but TLR7 were significantly higher in LST-positive reaction than in paired normal-looking skin of Ibizan hound. The expression profile of immune genes in LST-positive reactions was similar to that previously observed in clinically lesioned skin of mildly diseased dogs with papular dermatitis due to Leishmania infantum infection. This data provide additional support for the important role of TLRs in canine leishmaniosis. 1. Introduction gamma (IFN-γ) and tumour necrosis factor alpha (TNF-α) is associated with the elimination of intracellular amastigotes Canine leishmaniosis (CanL) caused by Leishmania infan- and the control of disease progression [1, 3]. Although, much tum is a vector borne zoonotic disease endemic in many areas of the work in CanL has focused on adaptive immune around the world, including many parts of the Mediterra- response, there is a great interest in the involvement of toll- nean basin [1]. Domestic dogs are the most important mam- like receptors (TLRs) in the innate immune response against malian reservoir for human visceral leishmaniosis, and in an Leishmania infection and how their expression could modu- endemic area, the majority of the dog population becomes late adaptive immune response [4–7]. In fact, ligation of TLR infected without showing clinical evidence of disease or during the early stages of infection by pathogen-associated serum anti-Leishmania antibodies [2]. motifs provides critical costimulatory signals for the initia- The outcome of Leishmania infection in dogs is the result tion of adaptive immune responses [8]. Although numerous of a complex immune response mounted by the host against studies have demonstrated that engagement of individual the parasite [1, 3]. Many investigations on immune response TLR, mainly on dendritic cells, can influence CD4+ T cell in CanL have been focused on adaptive immune response, priming and effector differentiation by skewing the Th1- and the data on the importance of the innate immune Th2 balance, the role of different TLRs on adaptive immune responses are scarce [4]. Effective T helper 1 (Th1) cellular response in Leishmania infection remains an unresolved immunity, with the activation of macrophages by interferon issue [7]. So far, TLR2 is one of the TLRs more frequently 2 Journal of Immunology Research associated with the pathogenesis of cutaneous lesions in Table 1: Signalment, clinicopathological, immunological, and CanL [9, 10]. parasitological data of nine Ibizan hounds. There are few and poorly standardized assays to evaluate Leishmania-specific cellular immune responses in dogs. One Parameters (reference intervals Ibizan hound dogs arithmetic and units) mean ± standard deviation of these tests is the leishmanin skin test (LST) or Montene- gro’s skin test [11–14]. The LST consists of the intradermal Sex 2 males and 7 females inoculation of Leishmania antigen and the elicitation of a Age (months) 16 (6-84)∗ delayed-type hypersensitivity (DTH) reaction in a previously Creatinine (0.5 - 1.5 mg/dL) 1 ± 0:1 infected dog [13]. Positive LST reactions in dogs are associ- Urea (21.4 - 59.9 mg/dL) 33:8 ± 7:4 ated with mild-to-moderate disease, subclinical infections, or a positive reaction in animals after successful treatment Total protein (5.4 - 7.1 g/dL) 6:3 ± 0:3 [15–17]. On the contrary, the response is low or absent in Albumin (2.6 - 3.3 g/dL) 3:5 ± 0:3 noninfected dogs or dogs with severe disease [17]. Albumin/globulin ratio (0.8-2) 1:2 ± 0:2 Ibizan hounds are considered resistant dogs to Leish- Beta globulin (0.9 - 1.6 g/dL) 1:3 ± 0:1 mania infection as they rarely manifest clinical disease and mount a significant cellular immune response to the infec- Gamma globulin (0.3 - 0.8 g/dL) 0:5 ± 0:1 tion demonstrated by a high prevalence of positive LST reac- Hematocrit (37- 55%) 49:3 ± 5:9 tions as well as a potent Leishmania-specific IFN-γ Hemoglobin (12 – 18 g/dL) 16:1 ± 1:8 production and low or null humoral response when com- pared with other breeds from the same geographical area Leishmania infantum-specific 18:3 ± 10:3 antibody levels (ELISA units) [12, 18]. The immunohistological features of LST-positive reactions in Ibizan hound dogs have been recently described Blood L. infantum-specific IFN-γ 3486 ± 5291:1 (pg/ml) [19]. LST-positive reactions 72 hours after intradermal injec- tion of Leishmania antigen were characterized by histological Blood parasite load (parasites/ml) 0:9 ± 2:2 changes indicative of a DTH reaction similar to those find- Skin parasite load (dCq) ings described for human subjects [19, 20]. A moderate-to- LST 3:8 ± 2:6a intense, perivascular-to-interstitial dermatitis with and with- Normal-looking 8:9 ± 1:9a out panniculitis was observed. Rarely, a diffuse pattern in the ELISA: enzyme-linked immunosorbent assay; dCq: delta-cycle threshold deep dermis and panniculus were also observed in a few sam- (low or negative values of dCq represented high parasite density); LST: ples. There was no evidence of granuloma formation in any leishmanin skin test. aWilcoxon signed rank test, Z = −3:0267, p = 0:002. of the dogs studied [19]. CD3+ T lymphocytes were the more ∗ Median and range. prominent cellular type in the dermal infiltrate. Further immunophenotyping of CD3+ T lymphocytes was not per- that presented papulo-crusting dermatitis on the inner aspect formed; however, marked necrosis present in all cases sug- of the pinnae suggestive of a mild form of leishmaniosis and gested the contribution of a cytotoxic T cell-mediated had no clinicopathological abnormalities [1, 21]. These dogs response in LST-positive reactions in resistant dogs as were all infected by L. infantum as shown by positive LST described for human beings [19, 20]. Moreover, TLR2 pro- reactions [12, 19]. Moreover, strong specific IFN-γ produc- tein expression was evident by immunohistochemistry in tion in stimulated cultured blood was performed as described dermal mononuclear cells [19]. Although LST is a commonly by Solano-Gallego et al. [22]. IFN-γ was not detected in all 9 used assay to evaluate the cell-mediated immune response in dogs but in 6 of them [19]. In addition, blood Leishmania dogs with leishmaniosis, to the best of the authors’ knowl- qPCR carried out as previously described [23] was negative edge, studies aimed to evaluated local immune response have or positive in five and four dogs, respectively [19]. All dogs not been previously published. The objective of the present were serologically negative except one (low positive) based study was to describe the pattern of expression of TLR2, on an in-house ELISA [19, 23]. TLR4, and TLR7, IFN-γ, IL-10, and PD-L1 in LST-positive Ten clinically healthy seronegative Beagle dogs from a reactions in Ibizan hound dogs living in a highly endemic nonendemic area (United Kingdom) were used as control area for leishmaniosis. dogs. All dogs were male and between 36 and 72 months of age. These dogs had been previously used as control subjects 2. Materials and Methods in other studies [5, 24, 25]. 2.1. Dogs. Nine Ibizan hound dogs from the Island of 2.2. Skin Biopsies. Two skin samples of 6 mm were obtained Mallorca, Spain, with a positive LST were enrolled in this from each Ibizan hound under intravenous sedation. Dogs study. These dogs were also previously described in a pub- were sedated with a combination of 0.2 ml of medetomidine lished study aimed to evaluate the histological and immuno- (Domtor®, Pfizer, Madrid, Spain), 0.2 ml of butorphanol logical changes induced by intradermal inoculation of (Torbugesic Vet 10 mg/ml, Zoetis Spain, S.L., Madrid, Spain), Leishmania antigen in resistant dogs [19]. Signalment, clini- and 0.3 ml of alphaxalone (Alfaxan 10 mg/ml®, Jurox Limited copathological, immunological, and parasitological data Microbial Developments, Malvern, UK). One skin sample from Ibizan hound dogs are summarized in Table 1. Briefly, was obtained from a LST-positive reaction at 72 hours [12] Ibizan hounds were clinically healthy, except for three dogs and another from normal-looking skin. Skin biopsies of Journal of Immunology Research 3 Table 2: Canine reference and target genes used in this study. GenBank reference Amplicon Assay ID Gene symbol Gene name GenBank mRNA sequence pairwise Cf02625049_s1 TLR2 Toll-like receptor 2 AF328930.1 NM_001005264.2 69 Cf02622203_g1 TLR4 Toll-like receptor 4 AB080363.1 NM_001002950.1 120 Cf02710573_s1 TLR7 Toll-like receptor 7 AB248956.1 NM_001048124.1 124 Cf02624265_m1 IL-10 Interleukin 10 AF328930.1 NM_001003077.1 83 Cf02623316_m1 IFN-γ Interferon gamma AF091130.1 NM_001003174.1 117 APG2FND PD-L1 Programmed dead ligand 1 NM_001291972.1 NM_001291972.1 164 Cf02643820_m1 LOC479750 Similar to CG14980-PB XM_536878.2 — 78 Succinate dehydrogenase complex; Cf02664981_m1 SDHA XM_535807.2 DQ402985.1 64 subunit A; flavoprotein LST-positive reactions were cut into two halves. One half was are listed in Table 2. Two suitable reference genes were fixed in 10% formalin for the aforementioned immunohisto- selected as previously suggested [25–27]: Succinate dehydro- logical study [19] and the other half and the whole skin genase complex; subunit A; flavoprotein (SDHA), and biopsy from normal-looking skin were submerged in similar to CG14980-PB (CG14980). PCR amplification was RNA later (RNAlater® Stabilization Solution, Ambion, Inc., performed using the QuantStudio™ 12K Flex System Real- Austin, Texas) stored at 4°C overnight and then kept at Time PCR (Thermo Fisher Scientific, Carlsbad, California, -80°C until used. USA) using TaqMan® Universal Master Mix II with UNG Moreover, skin biopsies from normal-looking skin (Applied Biosystems, Foster City, California, USA). Plates from control dogs were used as control samples for immune (96 wells/plate) were filled with 0.35 μl nuclease-free water gene expression. These samples were characterised by (Sigma, San Luis, Missouri, USA), 7.50 μl TaqMan Universal histologically normal skin and negative for Leishmania Master Mix (2×), 0.75 μl TaqMan assay 20, and 6.4 μl 1/5 quantitative PCR. cDNA. Plates were closed with an optical film (Applied Biosystems) centrifuged in order to mix the samples and 2.3. RNA Extraction. For RNA isolation, skin samples were were placed into a laboratory pipetting robot (Epmotion thawed on ice and placed in lysis solution (TRI Reagent, 5057 Liquid-handlingrobot, Eppendorf, Hamburg, Germany) RiboPure™ kit, Ambion, Inc., Austin, Texas) and homoge- to generate a 384-well plate. The 384-well plates were nized with a rotor-stator homogenizer (T 10 basic ULTRA- then transferred into a real-time PCR device. The PCR TURRAX 230 V IKA 3420000) using standard procedures. components and PCR cycler conditions were identical for Total RNA was then isolated using the RiboPure™ kit the all target and reference genes. The denaturation program (Ambion, Inc., Austin, Texas) under strict RNase-free (95°C, 10 min), amplification, and quantification program conditions according to the manufacturer’s protocol. In were repeated 40 times (95°C for 15 s, 60°C for 10 s, and order to remove contaminating DNA, a DNase digestion step 72°C for 60 s) with a single fluorescence measurement. The was included using TURBO DNA-free™ DNase treatment baseline and threshold were automatically defined for the and removal reagents (Ambion, Inc., Austin, Texas) follow- program in each run. Each sample was performed in tripli- ing the manufacturer’s instructions. RNA concentration cate for all the target and reference genes, and a calibrator was determined by a Nanodrop device (Thermo Fisher sample (one sample from control group) was employed as Scientific Inc.). Samples had a final concentration of RNA control in each plate. All target genes per each dog were of 34.7–251.6 ng/μl. RNA integrity and quality were assessed run on the same day and in the same plate. Data were by using an Agilent 2100 Bioanalyzer (Agilent Technologies, processed while applying the relative quantification method Santa Clara, USA). The majority of samples included in comparable to the delta-delta-cycle threshold value (ddCt)- this study had an RNA integrity number value greater method. For normalization of target gene expression, the than 7. The recovered RNA was stored at −80°C until arithmetic mean of the two reference genes was taken for cDNA synthesis. the calculation of a reference gene index [6]. Quantitative PCR data analyses were done by the CloudSuite software 2.4. cDNA Synthesis. cDNA was generated from 4 μl of (Life Technologies™, Thermo Fisher Scientific). RNA using the SuperScript™ VILO™ cDNA Synthesis kit (Invitrogen, Thermo Fisher Scientific) according to the man- 2.5.2. Skin Parasite Load. DNA was purified from the inter- ufacturer’s instructions. cDNA was aliquoted and stored phase and organic phase generated from the RNA purifica- at −20°C until used for qPCR. tion process by means of QIAamp DNA Mini Kit (Qiagen, 2.5. Quantitative PCR Manchester, UK) following the manufacturer’s instructions with slight modifications. Briefly, 20 μl of proteinase K solu- 2.5.1. Immune and Reference Genes. The primers used in this tion and 200 μl of tissue sample were used in all samples. The study (Thermo Fisher Scientific, Carlsbad, California, USA) other steps were performed as per manufacturer’s protocol. A 4 Journal of Immunology Research fragment of skin from a control dog was used as a control for skin of Ibizan hounds compared with healthy skin of sero- DNA contamination during DNA extraction. qPCR was per- negative dogs from a nonendemic area. formed with a relative quantification described elsewhere [21]. Briefly, PCR mix reaction was prepared with 4 μl of 4. Discussion DNA, 10 μl of master mix (TaqMan® Fast Advanced Master Mix, Thermo Fisher Scientific Inc.), 1 μl of Leishmania To the best of the authors’ knowledge, no studies have evalu- primers and probes (Custom TaqMan® Gene Expression ated immune genes’ transcripts in LST-positive reactions of Assay, ThermoFisher Scientific Inc., Waltham, USA) or 1 μl dogs resistant to leishmaniosis. Moreover, similar studies of another type of assay primers and probes [Eukaryotic have not been performed in humans or experimental animal 18S rRNA Endogenous Control (VICTM/MGB Probe, models of leishmaniosis. Remarkably, we compared gene Primer Limited, ThermoFisher Scientific Inc., Waltham, transcription in LST-positive reactions with paired normal- USA)] and 5 μl of H2O. The parasite load was measured with looking skin of the same dogs and healthy skin of seronega- the calculation of the delta Cq (dCq = the difference of tive control dogs from a nonendemic area. expression between mean values of duplicate determination LST-positive reactions were characterized by upregula- of Leishmania Cq and 18S rRNA Cq). Therefore, low or tion of all immune genes studied except for TLR7, when negative values of dCq represented higher parasite load than compared with normal-looking skin of Ibizan hounds and elevated dCq. skin of seronegative control dogs from a nonendemic area. That is not an unexpected finding, given that a strong 2.6. Statistical Analysis. Statistical analysis was performed inflammatory reaction in LST was observed in these dogs using the blorr, generalhoslem, and Deducer packages of the based on the immunohistological study previously described R software i386 version 3.4.2 (R Development Core Team) elsewhere [19]. Therefore, the upregulation of immune genes for Windows software. Quantitative data were expressed in LST-positive skin reactions was the result of a severe as the geometric mean and 95% confidence interval or inflammation. Moreover, parasite DNA in LST-positive reac- arithmetic mean ± standard deviation. The nonparametric tions was higher than in paired normal-looking skin of Ibizan Wilcoxon signed-rank test and Mann–Whitney U test hound dogs and it was assumed that LST-positive reactions were used to compare related and independent variables, presented more prominent microscopic inflammatory respectively. Differences were considered significant with a lesions than paired normal-looking skin as described for 5% significance level (p < 0:05). normal-looking skin of mildly affected dogs [19, 21]. TLRs are the first to recognize Leishmania through various 2.7. Ethics Approval and Consent to Participate. Dog owners Leishmania-associated molecular reaction patterns leading were informed about the study objectives and the procedure. to chemokine and cytokine expression and recruitment of An informed consent was obtained from all owners for inflammatory cells to the site of infection as observed in the participation in the study. The procedure regarding healthy present study [7]. In addition, these TLRs act as a connecting Beagle dogs followed ethical guidelines in accordance with link between innate and adaptive immune responses [7]. European Union regulations and was approved by the local Interestingly, immune gene expression profiles observed Ethical Review Committee from the Royal Veterinary in LST-positive reactions was comparable with that observed College (University of London, UK). in papular lesions of dogs with mild disease [26]. A signifi- cant upregulation of TLR2, TLR4, IL-10, IFN-γ, and PD-L1 3. Results was found in clinically lesioned skin of dogs with mild disease when compared with healthy skin from the same group of The expression of the immune genes studied in LST-positive control dogs studied herein [24]. In that study, TLR2, reactions and paired normal-looking skin from Ibizan hound TLR4, IL-10, and IFN-γ upregulation in clinically lesioned and healthy skin from seronegative control dogs from a skin was correlated with lower disease severity [24]. Instead, nonendemic area is shown in Figure 1. Concisely, TLR2 downregulation of TLR2 and TLR4 and other TLRs such as (p = 0:002), TLR4 (p = 0:04), IL-10 (p < 0:0001), IFN-γ TLR3 were noted in the skin and other organs in a susceptible (p < 0:0001), and PD-L1 (p < 0:0001) transcripts were signif- experimental model of canine L. infantum infection [5]. icantly higher in LST-positive reaction skin samples than in Therefore, the immune profile detected in LST-positive reac- healthy skin from control dogs from a nonendemic area. tions in Ibizan hound dogs agree with a competent immune Relative quantification of TLR7 was significantly lower in response in these dogs. The protective role of TLR2 in several LST-positive skin than in healthy skin from seronegative Leishmania species such as L. infantum is described else- control dogs from a nonendemic area (p = 0:002). where [7] and is in agreement with the present findings as All the transcripts of the immune genes studied, but well as the TLR4-related anti-L. infantum effects [28]. TLR7, were significantly higher in LST-positive reactions Noteworthy, relative quantification of TLR7 was signifi- from Ibizan hound dogs than in paired normal-looking skin. cantly downregulated in LST-positive reactions from resis- Only TLR4 (p = 0:03), IL-10 (p < 0:0001), and PD-L1 tant Ibizan hounds when compared with healthy skin of (p < 0:0001) transcripts were significantly higher in normal- seronegative control dogs from a nonendemic area. That looking skin from Ibizan hound dogs than in healthy skin was an interesting finding and in agreement with a previous from seronegative control dogs from nonendemic area. study that associated TLR7 upregulation with moderate-to- TLR7 (p = 0:0006) was downregulated in normal-looking severe disease [25]. In that previous study, TLR7 gene Journal of Immunology Research 5 p = 0.002 p = 0.04 25 p = 0.004 400 p = 0.004 p = 0.03 20 TLR4 transcripts (Rq) TLR2 transcripts (Rq) 300 15 200 10 100 5 0 0 LST NL H LST NL H Ibizan hound Ibizan hound Control dogs Control dogs (a) (b) p = 0.002 p < 0.0001 40 p = 0.0006 20 p = 0.02 p < 0.0001 IL-10 transcripts (Rq) 15 TLR7 transcripts (Rq) 30 20 10 10 5 0 0 LST NL H LST NL H Ibizan hound Ibizan hound Control dogs Control dogs (c) (d) p < 0.0001 p < 0.0001 800 40 p = 0.01 p < 0.004 p < 0.0001 PD-L1 transcripts (Rq) IFN-𝛾 transcripts (Rq) 600 30 400 20 200 10 0 0 LST NL H LST NL H Ibizan hound Ibizan hound Control dogs Control dogs (e) (f) Figure 1: Relative quantification of the immune genes studied. (a) TLR2 transcripts. (b) TLR4 transcripts. (c) TLR7 transcripts. (d) IL-10 transcripts. (e) IFN-γ transcripts. (f) PD-L1 transcripts. Circles and squares represent individual data of each dog. Horizontal and vertical lines represent geometric mean and 95% confidence interval, respectively. Solid lines with p values: Mann–Whitney U test; dashed lines with p values: Wilcoxon signed rank test. Abbreviations: LST: leishmanin skin test; NL: normal-looking skin from Ibizan hounds; H: healthy skin from control dogs from nonendemic area; Rq: normalized relative quantification. expression was significantly lower in both clinically lesioned over, TLR7 overexpression was associated with altered clini- and normal-looking skin of dogs mildly affected and with copathological parameters suggestive of disease severity [25]. papular dermatitis than that in more severely diseased dogs In agreement with these findings, the pathogenic role of this or healthy skin from seronegative control dogs [25]. More- receptor in visceral leishmaniosis due to L. donovani in mice 6 Journal of Immunology Research has been recently suggested [29, 30]. In this rodent model that changes in immune genes’ transcripts in normal- of Leishmania infection, an innate activation of B cells looking skin of infected Ibizan hounds were specifically through endosomal TLRs, such as TLR7, induced cytokine related to Leishmania infection. (IFN type I and IL-10), and endosomal TLR expression, followed by disease exacerbation and hypergammaglobuli- 5. Conclusions nemia [29]. Moreover, local tissue damage mediated by persistent inflammation lead to suppression of protective The present study describes the pattern of immune gene T cell responses during chronic visceral leishmaniosis due expression in the skin of LST-positive reactions and paired to L. donovani in mice via TLR7 signalling [30]. Therefore, normal-looking skin of Ibizan hound dogs infected by lack of TLR7 overexpression in LST-positive reactions points L. infantum, for the first time. LST-positive reactions pre- out a protective immune response in resistant Ibizan hounds. sented significant upregulation of TLR2, TLR4, IL-10, Normal-looking skin of infected Ibizan hound dogs IFN-γ, and PD-L1 and downregulation of TLR7 when showed some changes in immune genes’ transcripts when compared with normal skin of control healthy dogs from compared with healthy skin of seronegative control dogs. a nonendemic area. Immune gene expression profiles in Noteworthy, TLR2 and IFN-γ expressions in normal- LST-positive reactions were similar to those previously looking skin of infected Ibizan hound dogs were similar to observed in clinically lesioned skin of mildly diseased dogs healthy skin from seronegative control dogs from a nonen- with papular dermatitis due to L. infantum infection. This demic area. TLR2 overexpression is associated with an data provides additional support for the important role of inflammatory process [5] and probably denotes lack of or less TLRs in canine leishmaniosis. prominent microscopic inflammatory lesions in normal- looking skin of infected Ibizan hound dogs as suggested for Data Availability normal-looking skin of mild diseased dogs [21]. The absence of differences in a proinflammatory cytokine such as IFN-γ The datasets used and/or analysed during the current study transcription in normal-looking skin of resistant Ibizan available from the corresponding author on reasonable hounds described herein is in agreement with this. request. The Montenegro skin test reaction in human beings has been suggested as a surrogate of the early response to Disclosure Leishmania infection, based on similarities in cellular immunophenotyping between acute lesions in cutaneous Part of the information published herein has been presented leishmaniosis and Montenegro skin testing of the same at the 27th Conference of the World Association for the patient [20]. Papular dermatitis is a mild cutaneous man- Advancement of Veterinary Parasitology. ifestation of L. infantum infection classically diagnosed in young dogs, generally under one year of age, which has Conflicts of Interest been clinically and experimentally associated with sand fly bite sites [31, 32]. It is likely that this is a clinical entity The authors declare that there is no conflict of interest observed after the first contact with L. infantum inoculated regarding the publication of this paper. by sand flies in an immunocompetent dog. Therefore, similar- ities in immune gene expression among LST-positive reac- Acknowledgments tions in resistant dogs and papular dermatitis due to Leishmania infection [24] may reinforce the idea that papular This study was supported by a Spanish ministry grant, dermatitis is an early clinical manifestation of this infection. Ministerio de Economía y Competitividad and Fondos Some limitations in the present study should be FEDER (EU) (AGL2012-32498 and AGL2015-68477), and highlighted. Firstly, the number of Ibizan hounds evaluated the European Society Veterinary Dermatology (ESVD) was low. However, this study was performed under field research grant 2012. The authors are grateful to Joan Llull conditions with privately owned hunting dogs, limiting sam- for his help in collecting skin biopsies from Ibizan hounds ple size. Moreover, it would also be of interest to evaluate and Dr. Shazia Hosein for providing the skin samples from LST-positive reactions from resistant dogs of other breeds. the control group (Royal Veterinary College, University of In addition, DTH skin reactions caused by etiologies different London). We also thank Lorena Serrano and Dra. Laura Altet than L. infantum were not evaluated in Leishmania-negative from Vetgenomics and Dr. Anna Mercader from Servei de Ibizan hounds in order to demonstrate that changes if Genètica from Veterinary Faculty (UAB) for their help and immune gene transcripts were specific of Leishmania-associ- advices on molecular work. We are also grateful to Betlem ated skin inflammation or not. Unfortunately, histological Cabrera from the Departament de Genètica Animal del Cen- examination of normal-looking skin of Ibizan hounds could tre de Recerca Agrigenòmica for her advices on the technical not be performed. 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