Desenvolupament de noves membranes per a la millora de la seva estabilitat : caracterització i estudi comparatiu de les diferents membranes envers al transport de metalls en solucions aquoses /

S'han preparat noves membranes híbrides pel transport de metalls amb la finalitat de millorar l'estabilitat mecànica i química, el temps de vida i la pèrdua de transportador de les membranes líquides suportades (SLM) ja existents. La inclusió del transportador a la matriu polimèrica de les membranes És una de les millores proposades respecte a la inestabilitat de les (SLM). A més, els materials híbrids presenten certs avantatges respecte als materials orgànics i inorgànics considerats de forma independent. Les noves membranes proposades estan basades en una barreja orgànica (triacetat celálulosa, CTA) i materials organosilícics (diclorodimetilsilà i tetraetoxisilà, DCDMS i TEOS, respectivament) com a suport de membranes. La preparació de les membranes s'ha optimitzat variant la quantitat de transportador àcid Di(2-etilhexilfosfòric, D2EHPA) i plastificants (2-Nitrofeniloctilèter, NPOE i/o Tris(2-butoxietil)fosfat), TBEP). Les tècniques d'Espectroscòpia Infraroja amb Reflexió Total Atenuada, la Microscòpia Electrònica de Rastreig, l'Anàlisi Termogravimètrica, la Ressonància Magnètica Nuclear de 29Si i la Difracció de Raigs-X han estat emprades per a caracteritzar les membranes híbrides i per a tenir una correlació entre les propietats estructurals amb els valors de permeabilitat dels ions metàl·lics. El transport s'ha avaluat fent servir D2EHPA i D2EHDTPA àcid Di(2¬etilhexilditofosfòric) sota les mateixes condicions experimentals. Els experiments de transport reportats tracten tant amb el transport de solucions sintètiques monocomponents com ara el transport selectiu de barreges binaries i ternàries de diferents ions metàl·lics. S'ha trobat que variant la composició de les membranes, pel que fa als diferents agents extractants emprats i a la seva proporció relativa, s'obtenen sistemes diferents que permeten separar selectivament els ions metàl·lics presents en una barreja. Així, per exemple, si s'empra una membrana amb D2EHPA es produeix el transport selectiu de zinc a la fase de recuperació, mentre que coure i cadmi resten a la fase de càrrega. D'altra banda, si es fa servir D2EHDTPA com a agent transportador, la velocitat de transport (o flux) augmenta i els perfils de selectivitat s'inverteixen respecte als dels D2EHPA. Amb una barreja d'ambdós transportadors s'observa un comportament intermedi del transport que pot ésser, en certa manera, modulat a voluntat. Aquesta tesi també descriu un estudi comparatiu entre membranes compostes activades (ACM) i les noves membranes híbrides (HM), totes dues incorporant Aliquat 336 pel transport de Pt(IV) en un medi de clorurs. Les ACM són membranes amb una estructura de dues capes: una porosa preparada per inversió de fase i una densa formada per polimerització interfacial. L'eficiència de les dues membranes front al transport de Pt(IV) ha estat investigada, i diferents paràmetres com la composició de la fase de recuperació i la selectivitat front a Pd(II) han estat avaluats. A més, estudis comparatius entre els dos sistemes s'han portat a terme amb una celála triple amb una mateixa fase de càrrega. Finalment, els resultats d'aquestes membranes també s'han comparat amb els reportats anteriorment per les membranes d'inclusió polimèrica (PIM) i les SLM pel transport de Pt(IV) sota condicions experimentals similars.New hybrid membranes for metal ion transport were synthesized with the aim of improving mechanical and chemical stabilities, mean lifetime and loss of carrier. Hybrid organic- inorganic materials present several advantages with respect to organic and inorganic materials considered independently. The new membranes proposed here are based on a mixture of organic (cellulose triacetate, CTA) and organosilicone materials (dichlorodimethylsilane and tetraethoxysilane, DCDMS and TEOS, respectively) as membrane support. Membrane preparation was optimized varying the amount of metal carrier (bis(2-ethyl hexyl phosphoric acid), D2EHPA) and plasticizer (2-Nitrophenyloctylether, NPOE and/or Tris(2butoxyethyl)phosphate), TBEP). Total Reflection Infrared Spectroscopy, Scanning Electron Microscopy, Thermogravimetric Analysis, 29Si Nuclear Magnetic Resonance and X-Ray Diffraction were used to characterize the hybrid membranes and to correlate structural properties with permeability values for metal ions. Transport behaviour was evaluated for both carriers under similar experimental conditions. The transport experiments reported here concerned transport at different cycles and selectivity towards different metal ions. Using D2EHPA the membrane provided a selective transport of zinc to the stripping compartment of the membrane cell, while copper and cadmium remained in the feed compartment. Whereas, using D2EHDTPA as carrier the transport rate increased and the selectivity profiles were inverted in relation with those of D2EHPA. With a mixture of both extracting agents it was observed an intermediate behaviour in selective transport, being possible to modulate it. This thesis also describes a comparative study of activated composite (ACM) and hybrid membranes (HM), both incorporating Aliquat 336 as a carrier, for the transport of Pt(IV) ions in chloride media. The inclusion of the carrier molecules in the polymeric network of both types of membranes is one of the approaches to overcome supported liquid membrane (SLM) instability. ACM are membranes with a bilayer structure: a porous lower polymeric support prepared by phase inversion and a dense upper layer made by interfacial polymerisation. The efficiency of both types of membrane for the transport of Pt(IV) ions has been investigated, and several parameters affecting metal transport have been evaluated, such as the composition of the stripping phase and the selectivity of the membranes in the presence of Pd(II) ions. Moreover, comparative studies for both ACM and HM systems have been made by contacting the same feed solution in a triple-compartment cell. Finally, the results of these membrane systems are compared with those previously reported using polymer inclusion membranes (PIM) and SLM for the transport of Pt(IV) under similar experimental conditions

Determination of dimethyl selenide and dimethyl sulphide compounds causing off-flavours in bottled mineral waters

Sales of bottled drinking water have shown a large growth during the last two decades due to the general belief that this kind of water is healthier, its flavour is better and its consumption risk is lower than that of tap water. Due to the previous points, consumers are more demanding with bottled mineral water, especially when dealing with its organoleptic properties, like taste and odour. This work studies the compounds that can generate obnoxious smells, and that consumers have described like swampy, rotten eggs, sulphurous, cooked vegetable or cabbage. Closed loop stripping analysis (CLSA) has been used as a pre-concentration method for the analysis of off-flavour compounds in water followed by identification and quantification by means of GC-MS. Several bottled water with the aforementioned smells showed the presence of volatile dimethyl selenides and dimethyl sulphides, whose concentrations ranged, respectively, from 4 to 20 ng/L and from 1 to 63 ng/L. The low odour threshold concentrations (OTCs) of both organic selenide and sulphide derivatives prove that several objectionable odours in bottled waters arise from them. Microbial loads inherent to water sources, along with some critical conditions in water processing, could contribute to the formation of these compounds. There are few studies about volatile organic compounds in bottled drinking water and, at the best of our knowledge, this is the first study reporting the presence of dimethyl selenides and dimethyl sulphides causing odour problems in bottled watersPostprint (published version

Erratum to: Leishmania infantum-specific production of IFN-γ and IL-10 in stimulated blood from dogs with clinical leishmaniosis

BACKGROUND: There is limited information available on cytokine profiles in dogs with different degrees of disease severity due to natural infection of Leishmania infantum. The aim of this study was to investigate L. infantum-specific IFN-γ and IL-10 production in blood from dogs with leishmaniosis at diagnosis and correlate these findings with disease severity, humoral immune response and blood parasitemia. METHODS: Sixty dogs were diagnosed based on physical examination, routine laboratory tests, L. infantum-specific antibody levels measured by quantitative ELISA and blood parasitemia by real-time PCR. Heparin whole blood was stimulated with L. infantum soluble antigen (LSA) and concanavalin A (ConA) and incubated for 5 days. IFN-γ and IL-10 concentrations were measured in supernatants with sandwich ELISAs. RESULTS: The majority of dogs (n = 36) were classified as LeishVet stage II (moderate disease). The rest of the dogs were classified as stage I (n = 10), III (n = 10) and IV (n = 4). Dogs classified with stage I and IIa presented significantly higher (P = 0.02) LSA IFN-γ concentrations, lower (P <0.0001) antibody levels and a tendency for lower blood parasitemia (P = 0.1) than dogs classified with stages IIb, III or IV while no differences in ConA IFN-γ or IL-10 concentrations were observed among groups. Thirty-five dogs produced significantly higher LSA IFN-γ (mean ± SD: 2320 ± 3960 pg/ml) and ConA IFN-γ (mean ± SD: 7887 ± 7273 pg/ml) when compared with 25 dogs that did not produce detectable LSA IFN-γ but produced ConA IFN-γ (mean ± SD: 4917 ± 5233 pg/ml). IFN-γ producer dogs presented lower (mean ± SD: 5750 ± 14,082 ELISA units (EU), P = 0.001) antibody levels and blood parasitemia (mean ± SD:   5 ± 10 parasites/ml, P = 0.001) when compared with IFN-γ non-producers (mean ± SD: 19,638 ± 28,596 EU and 1100 ± 5112 parasites/ml), respectively. LSA IL-10 was not detectable in 34 dogs while 49 dogs secreted ConA IL-10 (mean ± SD of 90 ± 103 pg/ml). LSA IFN-γ concentration was negatively correlated with blood parasitemia and antibody levels and positively correlated with ConA IFN-γ and LSA IL-10 concentrations. CONCLUSIONS: The results of this study demonstrate that sick dogs lacking L. infantum specific IFN-γ production in stimulated whole blood produce a strong humoral response, have a high blood parasitemia and severe clinical disease. IL-10 does not appear to be a marker of disease severity

Caracterización de los mecanismos de captación de hierro de Pasteurella multocida

Consultable des del TDXTítol obtingut de la portada digitalitzadaLos objetivos de la presente tesis doctoral han sido la caracterización de los mecanismos de captación de hierro de Pasteurella multocida, con el fin de contribuir al diseño de una vacuna contra este patógeno basada en la sobreexpresión de proteínas de membrana externa, receptoras de hemoglobina y/o hemina, o en la expresión constitutiva de las proteínas de membrana reguladas por hierro (IROMPs). Se ha identificado el gen fur de P. multocida y se ha estudiado el perfil electroforético de las proteínas de membrana externa de dicho patógeno, descubriéndose un control de la expresión de una proteína mayoritaria de membrana (OmpH) por parte de la proteína Fur. Se ha construido un mutante fur y se ha demostrado que la mutación de este gen no presenta ningún efecto sobre la virulencia de la cepa. Así mismo, se ha caracterizado la región cromosómica de P. multocida que contiene los genes exbB, exbD y tonB, detectándose la existencia de promotores internos en dicha región y por lo tanto la expresión independiente de cada uno de los tres genes del sistema. Se ha estudiado la regulación de la expresión de todos ellos, demostrándose que pertenecen al regulón Fur, y se han construido mutantes en los genes exbB, exbD y tonB. Se ha calculado la dosis letal 50 (DL50) de cada una de las cepas construidas, determinándose que cada uno de estos genes es imprescindible para el desarrollo normal de una infección por P. multocida, ya que en todos los casos se obtuvo un incremento de la DL50 superior a los tres órdenes de magnitud con respecto la cepa salvaje. Por otro lado, se ha analizado el genoma de P. multocida Pm70 y se han estudiado 9 proteínas que, por similitud con los receptores descritos en el banco de datos, podrían actuar como receptores de hemoglobina y/o hemina. Así se ha podido determinar que algunas de ellas son capaces de unir hemoglobina y hemina (PM0040, PM0236, PM0300, PM0741, PM1081 y PM1428), mientras que otras tan sólo interaccionan con la hemoglobina (PM0576) o la hemina (PM1282), y que otra (PM1078) no reconoce ninguna de estas fuentes de hierro. De entre todas estas proteínas se ha elegido la PM0300, que se ha denominado HgbA, para realizar un estudio más detallado de este tipo de receptores de membrana. Así, se ha demostrado que el gen hgbA forma una unidad transcripcional con los genes PM0298 y PM0299. Estos genes presentan similitud con hugX y hugZ de Plesiomonas shigelloides respectivamente, y están implicados en procesos de detoxificación de hierro. Se ha demostrado que, en P. multocida, las proteínas PM0298 y PM0299 son esenciales para la viabilidad de la cepa. Por otro lado, se ha determinado que el gen hgbA se expresa en las primeras dos horas de la infección y que, in vitro, su expresión se induce en ausencia de hierro. Además, se ha construido un mutante deficiente para esta proteína y se ha comprobado que no presenta disminuida ni su capacidad de unir hemoglobina in vitro ni su virulencia. Por otro lado, se ha comprobado la distribución prácticamente universal de este gen en cepas de P. multocida de distintos serotipos y orígenes animales, hecho que propone a esta proteína como candidata para el desarrollo de un método de identificación rápido y específico de este microorganismo. Por último, se ha analizado la capacidad inmunogénica y protectora de los receptores de hemina y/o hemoglobina. Así, se ha demostrado que por lo menos tres de estas proteínas (PM0236, PM0741 y HgbA) son inmunogénicas. Sin embargo, la inoculación en ratones de las proteínas HgbA y PM0741, ya sea de forma individual o conjunta, no induce protección ante un enfrentamiento homólogo.In the present work, the Pasteurella multocida iron uptake mechanisms have been characterised. These results could be use to the obtention of one vaccine against this pathogen. The P. multocida fur gene has been identified and it has been demonstrated that the porine OmpH is under the control of the Fur protein. On the other hand, a P. multocida Fur mutant was constructed and its LD50 was calculated demonstrating that a mutation in this gene has no effect in the virulence of the strain. Moreover, the region which contains P. multocida exbB, exbD and tonB genes has been characterised. The results obtained demonstrate that these three genes do not constitute a single transcriptional unit, but all these genes possess its own promoter which is under the Fur-Fe(II) regulation. It has been obtained a ExbB, ExbD and TonB mutants and these mutants present a decrease in their virulence to increase their LD50 by more than three orders of magnitude when were inoculated via intraperitoneal. These data demonstrate that exbB, exbD and tonB genes are essential to P. multocida infectious process. Furthermore, the P. multocida Pm70 genome was analysed and nine putative haemin and haemoglobin-binding proteins have been identified by similarity with the same molecules of other bacterial pathogens. Quantitative binding assays have demonstrated that PM0040, PM0236, PM0300, PM0741, PM1081 and PM1428 bind both haemin and haemoglobin, whereas PM0576 and PM1282 only bind either haemoglobin or haemin, respectively. PM1078, despite presents similarity with a Yersinia enterocolitica haemin receptor, does not recognize none of these iron sources. PM0300 was named hgbA and it has been demonstrate that this gene and the PM0298 and PM0299 ORFs, which present similarity with hugX and hugZ of Plesiomonas shigelloides, respectively, constitute a single transcriptional unit. PM0298 and PM0299 are essential to the viability of P. multocida cells. The hgbA gene is expressed in vivo within the two first hours post-inoculation, and in vitro is repressed by iron. Moreover, a HgbA mutant binds haemoglobin to the same extent as the wild-type strain and, in agreement with this, virulence of the P. multocida hgbA cells was no affected. On the other hand, the hgbA gene is present in almost all the strains of P. multocida analysed, independently of their serotype or their animal source. This result propose hgbA as a putative candidate to develop a fast and specific method to identify this pathogen. Finally, the immumogenicity and the ability to induce protection of all these haemin and haemoglobin receptors were analysed. The results obtained have demonstrated that, despite PM0236, PM0741 and HgbA are immunogens, inoculation of mice with any single one of these binding proteins alone is not protective against P. multocida infection

Fenómenos de la diglosia árabe: de la Edad Media a la Edad Contemporánea

Artículo introductorio a la sección monográfica

Separation and Recycling of Concentrated Heavy Metal Wastewater by Tube Membrane Distillation Integrated with Crystallization

Tube membrane distillation (MD) integrated with a crystallization method is used in this study for the concurrent productions of pure water and salt crystals from concentrated single and mixed system solutions. The effects of concentrated Zn 2+ and Ni 2+ on performance in terms of membrane flux, permeate conductivity, crystal recovery rates, and crystal grades are investigated. Preferred crystallization and co-crystallization determinations were performed for mixed solutions. The results revealed that membrane fluxes remained at 2.61 kg·m −2 ·h −1 and showed a sharp decline until the saturation increased to 1.38. Water yield conductivity was below 10 μs·cm −1. High concentrated zinc and nickel did not have a particular effect on the rejection of the membrane process. For the mixed solutions, membrane flux showed a sharp decrease due to the high saturation, while the conductivity of permeate remained below 10 μs·cm −1 during the whole process. Co-crystallization has been proven to be a better method due to the existence of the SO 2− common-ion effect. Membrane fouling studies have suggested that the membrane has excellent resistance to fouling from highly concentrated solutions. The MD integrated with crystallization proves to be a promising technology for treating highly concentrated heavy metal solutions

Histological and parasitological distinctive findings in clinically-lesioned and normal-looking skin of dogs with different clinical stages of leishmaniosis

Normal-looking skin of dogs with leishmaniosis frequently shows microscopic lesions along with the presence of Leishmania amastigotes. However, histological lesions with or without detection of amastigotes might not occur in less severe clinical cases. In addition, comparative studies between paired clinically-lesioned and normal-looking skin samples from dogs with different disease severity are lacking. The objective of this study was to compare histological and parasitological findings by Leishmania immunohistochemistry (IHC) and quantitative PCR (qPCR) on paired clinically-lesioned and normal-looking skin biopsies from 25 dogs with different clinical stages of leishmaniosis, 11 with stage I-mild disease (papular dermatitis) and 14 with stage II-III (ulcerative or exfoliative dermatitis). The study demonstrated microscopic lesions in 14 out of 25 (56%) samples from normal-looking skin biopsies. In those samples, perivascular to interstitial dermatitis composed by macrophages with lymphocytes and plasma cells was observed mainly in the superficial and mid-dermis. The intensity of the dermatitis was mild to moderate and always less prominent than in the clinically-lesioned skin. In normal-looking skin samples, the presence of parasites was detected by histology, IHC and qPCR in 5/25 (20%), 8/25 (32%) and 18/25 (72%), respectively. Leishmania was encountered in 11/25 (44%), 23/25 (92%) and 25/25 (100%) of clinically-lesioned skin samples by histology, IHC and qPCR, respectively. Normal-looking skin from dogs with stage I-mild disease was less frequently inflamed (P = 0.0172). Furthermore, Leishmania was more easily demonstrated by histology (P = 0.0464), IHC (P = 0.0421) or qPCR (P = 0.0068) in normal-looking skin of dogs with stage II-III-moderate to severe disease. In addition, in the latter group, there was a significantly higher parasite load studied by means of qPCR than in dogs with less severe disease (P = 0.043). Clinically-lesioned skin from dogs with stage I disease was more frequently characterised by the nodular to diffuse pattern and granuloma formation (P = 0.0166) and by a lower parasite load studied by means of qPCR (P = 0.043) compared with more diseased dogs. Normal-looking skin from dogs with stage I is less likely to present histological lesions as well as harbour the parasite when compared with dogs with moderate to severe leishmaniosis

A bedside swallowing screen for the identification of post-extubation dysphagia on the intensive care unit – validation of the Gugging Swallowing Screen (GUSS)—ICU

Purpose Screening for dysphagia at the intensive care unit (ICU) soon after extubation can prevent aspiration, pneumonia, lower mortality, and shorten re‑feeding interval. This study aimed to modify the Gugging Swallowing Screen (GUSS), which was developed for acute stroke patients, and to validate it for extubated patients in the ICU. Methods In this prospective study, forty‑five patients who had been intubated for at least 24 h were recruited consecutively at the earliest 24 h after extubation. The modified GUSS‑ICU was performed twice by two speech and language therapists independently. Concurrently, gold standard the flexible endoscopic evaluation of swallowing (FEES) was performed by an otorhinolaryngologist. Measurements were conducted within a three‑hour period; all testers were blinded to each other’s results. Results According to FEES, 36 of 45 (80%) participants were diagnosed with dysphagia; 13 of those were severe, 12 moderate, and 11 mild. Compared to FEES, the GUSS‑ICU predicted dysphagia well (area under the curve for the initial rater pair: 0.923, 95% CI 0.832–1.000 and 0.923, 95% CI 0.836 ‑1.000 for the second rater pair). The sensitivity was 91.7% (95% CI 77.5–98.3%) and 94.4% (95% CI 81.3–99.3%); the specificity was 88.9% (51.8–99.7%) and 66.7% (29.9–92.5%); the positive predictive values were 97.1% (83.8–99.5%) and 91.9% (81.7–96.6%), and the negative predictive values were 72.7% (46.8–89%) and 75% (41.9–92.6%) for the first and second rater pairs, respectively. Dysphagia severity classification according to FEES and GUSS‑ICU correlated strongly (Spearman’s rho: 0.61 for rater 1 and 0.60 for rater 2, p < 0.001). Agreement by all testers was good (Krippendorffs Alpha: 0.73). The interrater reliability showed good agreement (Cohen`s Kappa: 0.84, p < 0.001). Conclusion The GUSS‑ICU is a simple, reliable, and valid multi‑consistency bedside swallowing screen to identify post‑extubation dysphagia at the ICU

Leishmania infantum-specific IFN-γ production in stimulated blood from dogs with clinical leishmaniosis at diagnosis and during treatment

There is limited data regarding Leishmania infantum specific T cell mediated immunity in naturally infected sick dogs at the time of diagnosis and during anti-Leishmania treatment. Our aim was to investigate the kinetics of L. infantum specific IFN-γ production in dogs with leishmaniosis at the time of diagnosis and during treatment and to correlate it with specific L. infantum antibodies, blood parasitemia and clinicopathological findings. Thirty-four dogs were diagnosed with leishmaniosis based on physical examination, routine laboratory tests and L. infantum-specific antibody levels by quantitative ELISA. Heparinized whole blood was stimulated with L. infantum soluble antigen (LSA) and concanavalin A (ConA) and incubated for 5 days. IFN-γ concentration was evaluated in supernatants of stimulated blood using a commercial sandwich ELISA. Leishmania real-time PCR was also performed for assessing blood parasitemia. Dogs were treated with meglumine antimoniate and allopurinol. Sixteen dogs were classified as IFN-γ non-producers after LSA stimulation (mean ± SD: 0 ± 0 pg/mL) and 18 dogs as IFN-γ producers (mean ± SD: 2885.3 ± 4436.1 pg/mL) at the time of diagnosis (P < 0.0001). IFN-γ non-producers were classified in a more severe clinical staging than IFN-γ producers that presented a mild to moderate clinical staging (P = 0.03). In the IFN-γ non-producer group, production of IFN-γ after LSA stimulation was significantly increased during treatment especially at day 365 (P = 0.018) together with clinical improvement when compared with day 0. In contrast, IFN-γ producers maintained their IFN-γ production after LSA stimulation and no statistically significant changes were found during treatment follow-up. At diagnosis, IFN-γ non-producers showed a significantly higher blood parasitemia versus IFN-γ -producers (P = 0.005). IFN-γ non-producers drastically reduced blood parasitemia to minimum values at day 365 when compared with day 0 (P = 0.017). No significant differences were found at day 365 in blood parasitemia of IFN-γ producers compared to pre-treatment. At diagnosis, L. infantum specific antibodies were higher in IFN-γ non-producers than IFN-γ producers (P = 0.014). A marked reduction of antibody levels was found at day 365 when compared with day 0 in IFN-γ non-producers (P = 0.005) and producers (P = 0.001). These results demonstrate that IFN-γ concentration increases with long-term anti-Leishmania treatment together with clinical improvement in dogs that do not produce IFN-γ at diagnosis. Together with clinical recovery, reduction in blood parasitemia and L. infantum specific antibodies, tracking IFN-γ concentration could constitute an important prognostic tool for immune monitoring in CanL

Toll-like receptors 2, 4 and 7, interferon-gamma and interleukin 10, and programmed death ligand 1 transcripts in skin from dogs of different clinical stages of leishmaniosis

Background: Canine leishmaniosis (CanL) caused by Leishmania infantum can have several dermatological manifestations. The type of immune response elicited against the parasite appears to be at the basis for such clinical variability. Much of the work in CanL has focused on adaptive immune response and there are scarce data on the importance of the innate immune responses. Moreover, few studies have evaluated the immunological response in the cutaneous lesions in dogs naturally infected with L. infantum and with different degrees of disease severity, and no study has compared clinically-lesioned with normal-looking skin. Methods: We determined and compared the transcription of toll like receptors (TLRs) 2, 4 and 7, interferon gamma (IFN-γ), interleukin (IL) 10 and programmed cell death protein ligand (PD-L) 1 by real-time PCR in paired clinically-lesioned and normal-looking skin from 25 diseased dogs (mild disease-stage I (n = 11) and moderate to severe disease-stages II and III (n = 14) as well as in normal-looking skin from healthy dogs (n = 10) from a non-endemic area. We also assessed the association between the transcripts in clinically-lesioned and normal-looking skin of dogs with leishmaniosis with clinicopathological, immunological and parasitological findings. Results: Clinically-lesioned skin from mildly affected dogs was characterized by a significant upregulation of TLR2 (P < 0.0001) and IL-10 (P = 0.021) and downregulation of TLR7 (P = 0.004) when compared with more severely affected dogs. Normal-looking skin of mildly affected dogs was characterized by a significant lower expression of TLR7 (P = 0.003), IFN-γ(P < 0.0001) and PD-L1 (P = 0.001) when compared with more severely affected dogs. TLR2, TLR4, IL-10 and IFN-γupregulation in clinically-lesioned skin was correlated with lower disease severity while TLR7 upregulation was correlated with markers of disease severity. Upregulation of TLR7, IL-10, IFN-γand PD-L1 in normal-looking skin was correlated with disease severity. Conclusions: This study demonstrated different expression profiles of immune genes in clinically-lesioned and normal-looking skin among mildly and more severely affected dogs. These immunological conditions might favor the maintenance and replication of the parasite in the skin of more severely affected dogs