Design, synthesis and biological evaluation of antiparasitic dinitroaniline-ether phospholipid hybrids.

A series of nine novel ether phospholipid-dinitroaniline hybrids were synthesized in an effort to deliver more potent antiparasitic agents with improved safety profile compared to miltefosine. The compounds were evaluated for their in vitro antiparasitic activity against L. infantum, L.donovani, L. amazonensis, L. major and L. tropica promastigotes, L. infantum and L. donovani intracellular amastigotes, Trypanosoma brucei brucei and against different developmental stages of Trypanosoma cruzi. The nature of the oligomethylene spacer between the dinitroaniline moiety and the phosphate group, the length of the side chain substituent on the dinitroaniline and the choline or homocholine head group were found to affect both the activity and toxicity of the hybrids. The early ADMET profile of the derivatives did not reveal major liabilities. Hybrid 3, bearing an 11-carbon oligomethylene spacer, a butyl side chain and a choline head group, was the most potent analogue of the series. It exhibited a broad spectrum antiparasitic profile against the promastigotes of New and Old World Leishmania spp., against intracellular amastigotes of two L. infantum strains and L. donovani, against T. brucei and against T. cruzi Y strain epimastigotes, intracellular amastigotes and trypomastigotes. The early toxicity studies revealed that hybrid 3 showed a safe toxicological profile while its cytotoxicity concentration (CC50) against THP-1 macrophages being >100 µM. Computational analysis of binding sites and docking indicated that the interaction of hybrid 3 with trypanosomatid α-tubulin may contribute to its mechanism of action. Furthermore, compound 3 was found to interfere with the cell cycle in T. cruzi epimastigotes, while ultrastructural studies using SEM and TEM in T. cruzi showed that compound 3 affects cellular processes that result in changes in the Golgi complex, the mitochondria and the parasite's plasma membrane. The snapshot pharmacokinetic studies showed low levels of 3 after 24 h following oral administration of 100 mg/Kg, while, its homocholine congener compound 9 presented a better pharmacokinetic profile.

Synthesis and Evaluation of Marine-Inspired Compounds Result in Hybrids with Antitrypanosomal and Antileishmanial Activities.

Natural products are a very rich source for obtaining new compounds with therapeutic potential. In the search for new antiparasitic and antimicrobial agents, molecular hybrids were designed based on the structures of antimicrobial marine quinazolinones and eugenol, a natural phenolic compound. Following reports of the therapeutic potential of quinazolinones and eugenol derivatives, it was expected that the union of these pharmacophores could generate biologically relevant substances. The designed compounds were obtained by classical synthetic procedures and were characterized by routine spectrometric techniques. Nine intermediates and final products were then evaluated in vitro against Trypanosoma brucei and Leishmania infantum. Antifungal and antibacterial activity were also evaluated. Six compounds (9b, 9c, 9d, 10b, 10c, and 14) showed mild activity against T. brucei with IC50 in the range of 11.17-31.68 µM. Additionally, intermediate 9c showed anti-Leishmania activity (IC50 7.54 µM) and was six times less cytotoxic against THP-1 cells. In conclusion, novel derivatives with a simple quinazolinone scaffold showing selectivity against parasites without antibacterial and antifungal activities were disclosed, paving the way for new antitrypanosomal agents.

Characterization and Proteomic Analysis of Plasma EVs Recovered from Healthy and Diseased Dogs with Canine Leishmaniosis.

Dogs are highly valued companions and work animals that are susceptible to many life-threatening conditions such as canine leishmaniosis (CanL). Plasma-derived extracellular vesicles (EVs), exploited extensively in biomarker discovery, constitute a mostly untapped resource in veterinary sciences. Thus, the definition of proteins associated with plasma EVs recovered from healthy and diseased dogs with a relevant pathogen would be important for biomarker development. For this, we recovered, using size-exclusion chromatography (SEC), EVs from 19 healthy and 20 CanL dogs' plasma and performed proteomic analysis by LC-MS/MS to define their core proteomic composition and search for CanL-associated alterations. EVs-specific markers were identified in all preparations and also non-EVs proteins. Some EVs markers such as CD82 were specific to the healthy animals, while others, such as the Integrin beta 3 were identified in most samples. The EVs-enriched preparations allowed the identification of 529 canine proteins that were identified in both groups, while 465 and 154 were only identified in healthy or CanL samples, respectively. A GO enrichment analysis revealed few CanL-specific terms. Leishmania spp. protein identifications were also found, although with only one unique peptide. Ultimately, CanL-associated proteins of interest were identified and a core proteome was revealed that will be available for intra- and inter-species comparisons.

Toward Chemical Validation of Leishmania infantum Ribose 5-Phosphate Isomerase as a Drug Target.

Neglected tropical diseases caused by kinetoplastid parasites (Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp.) place a significant health and economic burden on developing nations worldwide. Current therapies are largely outdated, inadequate, and face mounting drug resistance from the causative parasites. Thus, there is an urgent need for drug discovery and development. Target-led drug discovery approaches have focused on the identification of parasite enzymes catalyzing essential biochemical processes, which significantly differ from equivalent proteins found in humans, thereby providing potentially exploitable therapeutic windows. One such target is ribose 5-phosphate isomerase B (RpiB), an enzyme involved in the nonoxidative branch of the pentose phosphate pathway, which catalyzes the interconversion of d-ribose 5-phosphate and d-ribulose 5-phosphate. Although protozoan RpiB has been the focus of numerous targeted studies, compounds capable of selectively inhibiting this parasite enzyme have not been identified. Here, we present the results of a fragment library screening against Leishmania infantum RpiB (LiRpiB), performed using thermal shift analysis. Hit fragments were shown to be effective inhibitors of LiRpiB in activity assays, and several fragments were capable of selectively inhibiting parasite growth in vitro. These results support the identification of LiRpiB as a validated therapeutic target. The X-ray crystal structure of apo LiRpiB was also solved, permitting docking studies to assess how hit fragments might interact with LiRpiB to inhibit its activity. Overall, this work will guide structure-based development of LiRpiB inhibitors as antileishmanial agents.

Design, Synthesis and Antiparasitic Evaluation of Click Phospholipids.

A library of seventeen novel ether phospholipid analogues, containing 5-membered heterocyclic rings (1,2,3-triazolyl, isoxazolyl, 1,3,4-oxadiazolyl and 1,2,4-oxadiazolyl) in the lipid portion were designed and synthesized aiming to identify optimised miltefosine analogues. The compounds were evaluated for their in vitro antiparasitic activity against Leishmania infantum and Leishmania donovani intracellular amastigotes, against Trypanosoma brucei brucei and against different developmental stages of Trypanosoma cruzi. The nature of the substituents of the heterocyclic ring (tail) and the oligomethylene spacer between the head group and the heterocyclic ring was found to affect the activity and toxicity of these compounds leading to a significantly improved understanding of their structure-activity relationships. The early ADMET profile of the new derivatives did not reveal major liabilities for the potent compounds. The 1,2,3-triazole derivative 27 substituted by a decyl tail, an undecyl spacer and a choline head group exhibited broad spectrum antiparasitic activity. It possessed low micromolar activity against the intracellular amastigotes of two L. infantum strains and T. cruzi Y strain epimastigotes, intracellular amastigotes and trypomastigotes, while its cytotoxicity concentration (CC50) against THP-1 macrophages ranged between 50 and 100 µM. Altogether, our work paves the way for the development of improved ether phospholipid derivatives to control neglected tropical diseases.

The Diverse Piscidin Repertoire of the European Sea Bass (Dicentrarchus labrax): Molecular Characterization and Antimicrobial Activities.

Fish rely on their innate immune responses to cope with the challenging aquatic environment, with antimicrobial peptides (AMPs) being one of the first line of defenses. Piscidins are a group of fish specific AMPs isolated in several species. However, in the European sea bass (Dicentrarchus labrax), the piscidin family remains poorly understood. We identified six different piscidins in sea bass, performed an in-depth molecular characterization and evaluated their antimicrobial activities against several bacterial and parasitic pathogens. Sea bass piscidins present variable amino acid sequences and antimicrobial activities, and can be divided in different sub groups: group 1, formed by piscidins 1 and 4; group 2, constituted by piscidins 2 and 5, and group 3, formed by piscidins 6 and 7. Additionally, we demonstrate that piscidins 1 to 5 possess a broad effect on multiple microorganisms, including mammalian parasites, while piscidins 6 and 7 have poor antibacterial and antiparasitic activities. These results raise questions on the functions of these peptides, particularly piscidins 6 and 7. Considering their limited antimicrobial activity, these piscidins might have other functional roles, but further studies are necessary to better understand what roles might those be.

The use of Escherichia coli total antigens as a complementary approach to address seropositivity to Leishmania antigens in canine leishmaniosis.

Canine leishmaniosis (CanL) is a major veterinary concern and a public health issue. Serological data are essential for disease management. Several antigens used in serological assays have specificity related problems preventing relevant seropositivity values establishment. Herein we report significant seropositivity level disparity in a study cohort with 384 dogs from eight countries, for antigens traditionally used in CanL - soluble promastigote Leishmania antigens (SPLA) and K39 recombinant protein (rK39): 43·8 and 2·9% for SPLA and rK39, respectively. To better understand the reasons for this disparity, CanL-associated serological response was characterized using, for complement serological evaluation, a ubiquitous antigen - soluble Escherichia coli antigens (SECAs). Using cohorts of CanL dogs and dogs without clinical evidences of CanL from non-endemic regions of Portugal, the serological response of CanL animals followed specific trend of seropositivity rK39 > SPLA > SECA absent in non-diseased animals. Using receiver operating characteristic curve analysis, these characteristic trends were converted in ratios, SPLA/SECA, rK39/SECA and rK39/SPLA, that presented high predictive for discriminating the CanL cohort that was potentiated when applied in a scoring system involving positivity to four out of five predictors (rK39, SPLA, SPLA/SECA, rK39/SECA and rK39/SPLA). In fact, this approach discriminated CanL with similar sensitivity/specificity as reference antigens, diminishing seropositivity in European cohort to 1·8%. Ultimately, non-related antigens like SECA and seropositivity ratios between antigens enable different perspectives into serological data focusing on the search of characteristic serological signatures and not simple absolute serology values contributing to comprehensive serological status characterization.

Chroman-4-One Derivatives Targeting Pteridine Reductase 1 and Showing Anti-Parasitic Activity.

Flavonoids have previously been identified as antiparasitic agents and pteridine reductase 1 (PTR1) inhibitors. Herein, we focus our attention on the chroman-4-one scaffold. Three chroman-4-one analogues (1-3) of previously published chromen-4-one derivatives were synthesized and biologically evaluated against parasitic enzymes (Trypanosoma brucei PTR1-TbPTR1 and Leishmania major-LmPTR1) and parasites (Trypanosoma brucei and Leishmania infantum). A crystal structure of TbPTR1 in complex with compound 1 and the first crystal structures of LmPTR1-flavanone complexes (compounds 1 and 3) were solved. The inhibitory activity of the chroman-4-one and chromen-4-one derivatives was explained by comparison of observed and predicted binding modes of the compounds. Compound 1 showed activity both against the targeted enzymes and the parasites with a selectivity index greater than 7 and a low toxicity. Our results provide a basis for further scaffold optimization and structure-based drug design aimed at the identification of potent anti-trypanosomatidic compounds targeting multiple PTR1 variants.

The impact of distinct culture media in Leishmania infantum biology and infectivity.

An ideal culture medium for Leishmania promastigotes should retain the basic characteristics of promastigotes found in sandflies (morphology and infectivity). Furthermore, the media should not create a bias in experimental settings, thus enabling the proper extrapolation of results. To assess this we studied several established media for promastigote growth. We analysed morphology, viability, cell cycle progression, metacyclic profile, capacity to differentiate into axenic amastigotes and infectivity. Furthermore, using a rational approach from the evaluated media we developed a simple serum-free medium (cRPMI). We report that parasites growing in different media present different biological characteristics and distinct in vitro and in vivo infectivities. The developed medium, cRPMI, proved to be a less expensive substitute for traditional serum-supplemented media for the in vitro maintenance of promastigotes. In fact, cRPMI is ideal for the maintenance of parasites in the laboratory, diminishing the expected loss of virulence over time typical of the parasite cultivation. Ultimately this report is a clear warning that the normalization of culture media should be a real concern in the field as media-specific phenomena are sufficient to induce biological bias with consequences in infectivity and general parasite biology.

Exploring hydrophilic 2,2-di(indol-3-yl)ethanamine derivatives against Leishmania infantum.

Herein we report the design and the synthesis of a library of new and more hydrophilic bisindole analogues based on our previously identified antileishmanial compound URB1483 that failed the preliminary in vivo test. The novel bisindoles were phenotypically screened for efficacy against Leishmania infantum promastigotes and simultaneously for toxicity on human macrophage-like THP-1 cells. Among the less toxic compounds, eight bisindoles showed IC50 below 10 µM. The most selective compound 1h (selectivity index = 10.1, comparable to miltefosine) and the most potent compound 2c (IC50 = 2.7 µM) were tested for their efficacy on L. infantum intracellular amastigotes. The compounds also demonstrated their efficacy in the in vitro infection model, showing IC50 of 11.1 and 6.8 µM for 1h and 2c, respectively. Moreover, 1h showed a better toxicity profile than the commercial drug miltefosine. For all these reasons, 1h could be a possible new starting point for hydrophilic antileishmanial agents with low cytotoxicity on human macrophage-like cells.

Leishmania and HIV co-infection: first naturally Leishmania strain presenting decreased susceptibility to miltefosine, recovered from a patient in Portugal.

BACKGROUND: In Europe, up to 70% of visceral leishmaniasis (VL) cases occurring in adults living with HIV. People living with HIV with VL co-infection often display persistent parasitemia, requiring chronic intermittent anti-Leishmania therapies. Consequently, frequent VL relapses and higher mortality rates are common in these individuals. As such, it is of paramount importance to understand the reasons for parasite persistence to improve infection management. METHODS: To outline possible causes for treatment failure in the context of HIV-VL, we followed a person living with HIV-VL co-infection for nine years in a 12-month period. We characterized: HIV-related clinicopathological alterations (CD4+ T counts and viremia) and Leishmania-specific seroreactivity, parasitemia, quantification of pro-inflammatory cytokines upon stimulation and studied a Leishmania clinical isolate recovered during this period. RESULTS: The subject presented controlled viremia and low CD4+ counts. The subject remained PCR positive for Leishmania and also seropositive. The cellular response to parasite antigens was erratic. The isolate was identified as the first Leishmania infantum case with evidence of decreased miltefosine susceptibility in Portugal. CONCLUSION: Treatment failure is a multifactorial process driven by host and parasite determinants. Still, the real-time determination of drug susceptibility profiles in clinical isolates is an unexplored resource in the monitoring of VL.

Identification of Innovative Folate Inhibitors Leveraging the Amino Dihydrotriazine Motif from Cycloguanil for Their Potential as Anti-Trypanosoma brucei Agents.

Folate enzymes, namely, dihydrofolate reductase (DHFR) and pteridine reductase (PTR1) are acknowledged targets for the development of antiparasitic agents against Trypanosomiasis and Leishmaniasis. Based on the amino dihydrotriazine motif of the drug Cycloguanil (Cyc), a known inhibitor of both folate enzymes, we have identified two novel series of inhibitors, the 2-amino triazino benzimidazoles (1) and 2-guanidino benzimidazoles (2), as their open ring analogues. Enzymatic screening was carried out against PTR1, DHFR, and thymidylate synthase (TS). The crystal structures of TbDHFR and TbPTR1 in complex with selected compounds experienced in both cases a substrate-like binding mode and allowed the rationalization of the main chemical features supporting the inhibitor ability to target folate enzymes. Biological evaluation of both series was performed against T. brucei and L. infantum and the toxicity against THP-1 human macrophages. Notably, the 5,6-dimethyl-2-guanidinobenzimidazole 2g resulted to be the most potent (Ki = 9 nM) and highly selective TbDHFR inhibitor, 6000-fold over TbPTR1 and 394-fold over hDHFR. The 5,6-dimethyl tricyclic analogue 1g, despite showing a lower potency and selectivity profile than 2g, shared a comparable antiparasitic activity against T. brucei in the low micromolar domain. The dichloro-substituted 2-guanidino benzimidazoles 2c and 2d revealed their potent and broad-spectrum antitrypanosomatid activity affecting the growth of T. brucei and L. infantum parasites. Therefore, both chemotypes could represent promising templates that could be valorized for further drug development.

Discovery of 1,3,4-Oxadiazole Derivatives as Broad-Spectrum Antiparasitic Agents.

Vector-borne parasitic diseases (VBPDs) pose a significant threat to public health on a global scale. Collectively, Human African Trypanosomiasis (HAT), Leishmaniasis, and Malaria threaten millions of people, particularly in developing countries. Climate change might alter the transmission and spread of VBPDs, leading to a global burden of these diseases. Thus, novel agents are urgently needed to expand therapeutic options and limit the spread of drug-resistant parasites. Herein, we report the development of broad-spectrum antiparasitic agents by screening a known library of antileishmanial and antimalarial compounds toward Trypanosoma brucei (T. brucei) and identifying a 1,3,4-oxadiazole derivative (19) as anti-T. brucei hit with predicted blood-brain barrier permeability. Subsequently, extensive structure-activity-relationship studies around the lipophilic tail of 19 led to a potent antitrypanosomal and antimalarial compound (27), with moderate potency also toward Leishmania infantum (L. infantum) and Leishmania tropica. In addition, we discovered a pan-active antiparasitic molecule (24), showing low-micromolar IC50s toward T. brucei and Leishmania spp. promastigotes and amastigotes, and nanomolar IC50 against Plasmodium falciparum, together with high selectivity for the parasites over mammalian cells (THP-1). Early ADME-toxicity assays were used to assess the safety profile of the compounds. Overall, we characterized 24 and 27, bearing the 1,3,4-oxadiazole privileged scaffold, as broad-spectrum low-toxicity agents for the treatment of VBPDs. An alkyne-substituted chemical probe (30) was synthesized and will be utilized in proteomics experiments aimed at deconvoluting the mechanism of action in the T. brucei parasite.

The discovery of aryl-2-nitroethyl triamino pyrimidines as anti-Trypanosoma brucei agents.

Pteridine reductase 1 (PTR1) is a catalytic protein belonging to the folate metabolic pathway in Trypanosmatidic parasites. PTR1 is a known target for the medicinal chemistry development of antiparasitic agents against Trypanosomiasis and Leishmaniasis. In previous studies, new nitro derivatives were elaborated as PTR1 inhibitors. The compounds showing a diamino-pyrimidine core structure were previously developed but they showed limited efficacy. Therefore, a new class of phenyl-, heteroaryl- and benzyloxy-nitro derivatives based on the 2-nitroethyl-2,4,6-triaminopyrimidine scaffold were designed and tested. The compounds were assayed for their ability to inhibit T. brucei and L. major PTR1 enzymes and for their antiparasitic activity towards T. brucei and L. infantum parasites. To understand the structure-activity relationships of the compounds against TbPTR1, the X-ray crystallographic structure of the 2,4,6-triaminopyrimidine (TAP) was obtained and molecular modelling studies were performed. As a next step, only the most effective compounds against T. brucei were then tested against the amastigote cellular stage of T. cruzi, searching for a broad-spectrum antiprotozoal agent. An early ADME-Tox profile evaluation was performed. The early toxicity profile of this class of compounds was investigated by measuring their inhibition of hERG and five cytochrome P450 isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4), cytotoxicity towards A549 cells and mitochondrial toxicity. Pharmacokinetic studies (SNAP-PK) were performed on selected compounds using hydroxypropyl-ß-cyclodextrins (50 % w/v) to preliminarily study their plasma concentration when administered per os at a dose of 20 mg/kg. Compound 1p, showed the best pharmacodynamic and pharmacokinetic properties, can be considered a good candidate for further bioavailability and efficacy studies.

Advances in Leishmania Research: From Basic Parasite Biology to Disease Control.

The genus Leishmania (Trypanosomatida: Trypanosomatidae) currently comprises just over 50 species, of which about 20 cause several syndromes in humans, collectively known as leishmaniasis or "leishmaniases" [...].

High-Throughput Phenotypic Screening and Machine Learning Methods Enabled the Selection of Broad-Spectrum Low-Toxicity Antitrypanosomatidic Agents.

Broad-spectrum anti-infective chemotherapy agents with activity against Trypanosomes, Leishmania, and Mycobacterium tuberculosis species were identified from a high-throughput phenotypic screening program of the 456 compounds belonging to the Ty-Box, an in-house industry database. Compound characterization using machine learning approaches enabled the identification and synthesis of 44 compounds with broad-spectrum antiparasitic activity and minimal toxicity against Trypanosoma brucei, Leishmania Infantum, and Trypanosoma cruzi. In vitro studies confirmed the predictive models identified in compound 40 which emerged as a new lead, featured by an innovative N-(5-pyrimidinyl)benzenesulfonamide scaffold and promising low micromolar activity against two parasites and low toxicity. Given the volume and complexity of data generated by the diverse high-throughput screening assays performed on the compounds of the Ty-Box library, the chemoinformatic and machine learning tools enabled the selection of compounds eligible for further evaluation of their biological and toxicological activities and aided in the decision-making process toward the design and optimization of the identified lead.

Guidelines for the purification and characterization of extracellular vesicles of parasites.

Parasites are responsible for the most neglected tropical diseases, affecting over a billion people worldwide (WHO, 2015) and accounting for billions of cases a year and responsible for several millions of deaths. Research on extracellular vesicles (EVs) has increased in recent years and demonstrated that EVs shed by pathogenic parasites interact with host cells playing an important role in the parasite's survival, such as facilitation of infection, immunomodulation, parasite adaptation to the host environment and the transfer of drug resistance factors. Thus, EVs released by parasites mediate parasite-parasite and parasite-host intercellular communication. In addition, they are being explored as biomarkers of asymptomatic infections and disease prognosis after drug treatment. However, most current protocols used for the isolation, size determination, quantification and characterization of molecular cargo of EVs lack greater rigor, standardization, and adequate quality controls to certify the enrichment or purity of the ensuing bioproducts. We are now initiating major guidelines based on the evolution of collective knowledge in recent years. The main points covered in this position paper are methods for the isolation and molecular characterization of EVs obtained from parasite-infected cell cultures, experimental animals, and patients. The guideline also includes a discussion of suggested protocols and functional assays in host cells.

Pyrimido[5,4-d]pyrimidine-Based Compounds as a Novel Class of Antitrypanosomal and Antileishmanial Agents.

Sleeping sickness and leishmaniasis are neglected tropical diseases that threaten millions of people. The currently available therapies present several limitations, including high toxicity, lack of efficacy, and emerging drug resistance, prompting a search for novel therapeutic agents. In this work, we designed, synthesized, and in vitro evaluated the activity of new pyrimido[5,4-d]pyrimidines against Trypanosoma brucei and Leishmania infantum (promastigote and amastigote forms). The cytotoxicity of the compounds against the THP1 cell line was also assessed. Most tested compounds presented low micromolar activity against T. brucei with IC50 values in the range between 0.9 and 13.4 µM, and one compound also showed activity against L. infantum (IC50 = 3.13 µM). Several molecules presented a selectivity index higher than 10. The most active compound against booth parasites is derivative 4c, with IC50 = 0.94 µM (SI > 107) against T. brucei and IC50 = 3.13 µM (SI > 32) against L. infantum. This data enabled the identification of a new promising molecular scaffold for developing a novel class of antitrypanosomal and antileishmanial agents.

Leishmania Vesicle-Depleted Exoproteome: What, Why, and How?

Leishmaniasis, a vector-borne parasitic protozoan disease, is among the most important neglected tropical diseases. In the absence of vaccines, disease management is challenging. The available chemotherapy is suboptimal, and there are growing concerns about the emergence of drug resistance. Thus, a better understanding of parasite biology is essential to generate new strategies for disease control. In this context, in vitro parasite exoproteome characterization enabled the identification of proteins involved in parasite survival, pathogenesis, and other biologically relevant processes. After 2005, with the availability of genomic information, these studies became increasingly feasible and revealed the true complexity of the parasite exoproteome. After the discovery of Leishmania extracellular vesicles (EVs), most exoproteome studies shifted to the characterization of EVs. The non-EV portion of the exoproteome, named the vesicle-depleted exoproteome (VDE), has been mostly ignored even if it accounts for a significant portion of the total exoproteome proteins. Herein, we summarize the importance of total exoproteome studies followed by a special emphasis on the available information and the biological relevance of the VDE. Finally, we report on how VDE can be studied and disclose how it might contribute to providing biologically relevant targets for diagnosis, drug, and vaccine development.

Serological and Molecular Survey of Leishmania infantum in a Population of Iberian Lynxes (Lynx pardinus).

Leishmania infantum, the sand fly-transmitted protozoan parasite responsible for leishmaniasis in humans, dogs, and cats, is endemic in the Iberian Peninsula. However, the impact of L. infantum infection on the conservation of the endangered Iberian lynx (Lynx pardinus) is unknown. Herein, we describe for the first time the occurrence of L. infantum infection among a population of reintroduced and wild-born L. pardinus living in the Portuguese Guadiana Valley Park. The presence of infection was addressed by molecular detection of Leishmania kinetoplast DNA (kDNA) in 35 lynxes, with further confirmation of L. infantum species performed by an internally transcribed spacer (ITS)-1 sequencing. Eight blood samples were positive for kDNA, and ITS-1 sequencing confirmed the presence of L. infantum in two of those samples. Exposure to Leishmania was screened in a group of 36 lynxes using an immunofluorescence antibody test (IFAT) and a multi-antigen enzyme-linked immunosorbent assay (ELISA), using SPLA, rK39, and CPX as Leishmania-specific antigens. Four animals presented a positive IFAT at a dilution of 1:40. Eight samples were considered seropositive to all ELISA Leishmania-specific antigens. Agreement between PCR, IFAT, and all ELISA antigens was found for 1 in 27 samples. These results highlight the susceptibility of autochthonous L. pardinus to L. infantum infection. Further investigation is required to assess the impact of L. infantum infection on this wild species conservation.