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Background: Ascorbate peroxidase (APX) has emerged as a promising target for chemotherapy because of its absence in humans and crucial role in the antioxidant defence of trypanosomatids. APXs, which are class I haeme-containing enzymes, reduces hydrogen peroxide using ascorbate to produce water and monodehydroascorbate, thereby preventing cell damage caused by H2O2. Methods: We aimed to create an APX-knockout L. infantum line using CRISPR/Cas9. Despite unsuccessful attempts at full knockouts, we achieved deletion of chromosomal copies post-APX episomal insertion, yielding LiΔchrAPX::LbAPX parasites. We performed phenotypic characterization to assess the impact of these genetic modifications, which included the determination of APX transcript expression levels using quantitative PCR, drug sensitivity, infectivity, and parasite survival in macrophages. Results: Quantitative polymerase chain reaction (PCR) analysis revealed a 10- to 13-fold reduction in APX transcript expression in LiΔchrAPX::LbAPX compared with wild-type (LiWT) and APX-overexpressing (Li::Cas9::LbAPX) parasites, respectively. The episomes in those knockdown parasites remained stable even after 20 drug-free passages in vitro. Li::Cas9::LbAPX parasites showed increased resistance to trivalent antimony (SbIII) and isoniazid, reduced tolerance to H2O2, and unchanged macrophage infectivity compared with LiWT. In contrast, LiΔchrAPX::LbAPX parasites were more sensitive to SbIII and isoniazid, exhibited greater susceptibility to H2O2-induced oxidative stress, and 72 h post-infection, showed fewer infected macrophages and intracellular amastigotes compared with LiWT parasites. Conclusions: Our findings hint at the indispensability of APX in L. infantum and raise the possibility of its potential as a therapeutic target for leishmaniasis. [ABSTRACT FROM AUTHOR]

Protein-energy malnutrition (PEM) is a risk factor for developing visceral leishmaniasis (VL). While nutrient deficiency can impair immunity, its mechanistic impact on protective adaptive immune responses following Leishmania infection remains unknown. To determine the potential negative impacts of malnutrition on anti-parasitic responses in chronic VL, we provided mice with a polynutrient-deficient diet (deficient protein, energy, zinc, and iron) that mimics moderate human malnutrition. The polynutrient-deficient diet resulted in growth stunting and reduced mass of visceral organs and following infection with Leishmania infantum, malnourished-mice harbored more parasites in the spleen and liver. Malnourished and infected mice also had fewer T lymphocytes, with reduced T cell production of IFN-γ required for parasite clearance and enhanced production of the immunosuppressive cytokine, IL-10. To determine if IL-10 was causative in disease progression in the malnourished mice, we treated infected mice with monoclonal antibody α-IL-10R. α-IL-10R treatment reduced the parasite number in malnourished mice, restored the number of T cells producing IFN-γ, and enhanced hepatic granuloma formation. Our results indicate that malnutrition increases VL susceptibility due to defective IFN-γ-mediated immunity attributable to increased IL-10 production. Author summary: Malnutrition contributes to the development of visceral leishmaniasis (VL) following Leishmania infection. Despite the clear association, the impact of malnutrition on the adaptive immune mechanisms required for parasite control is still unclear. We found that malnutrition disrupts the ability to control parasite replication in the spleen and liver due to defective IFN-γ-mediated immunity, reduced hepatic granuloma formation, and enhanced IL-10 production. Blocking IL-10R signaling restored the protective mechanisms to control parasite replication in the malnourished mice without interfering with the undernutrition state. Thus, we demonstrate that increased IL-10 production driven by malnutrition disrupts protective immunity against Leishmania, thereby enhancing the susceptibility of VL. Understanding the association between malnutrition and VL will provide insights into therapeutic approaches to treat this aggressive disease. [ABSTRACT FROM AUTHOR]

Dogs represent the primary reservoir for Leishmania infantum human visceral leishmaniasis (VL) through phlebotomine sand flies. Public health initiatives targeting zoonotic VL commonly focus on dogs with severe clinical disease, often in renal failure, as they have previously been considered the most infectious to sand flies. However, more recent studies suggest that dogs with mild to moderate clinical disease may be more infectious than dogs with severe disease. The mechanisms of infectiousness from the skin and how this relates to transmissibility as clinical disease progresses is largely unknown. We evaluated dermal parasitism in dogs naturally infected with L. infantum across the four LeishVet clinical stages of disease. We establish the relationship between dermal parasitism, critical, frequently observed, clinical parameters such as anemia and creatinine, and infectiousness. Using RNAscope and confocal microscopy, we found notable variation in dermal parasitism between dogs, particularly within LeishVet II. Dogs with mild disease had significantly less dermal inflammation and parasitism than dogs with moderate or severe disease. We found significant correlations between anemia, dermal parasitism, and infectiousness (p = 0.0098, r = -0.4798; p = 0.0022, r = -0.8364). In contrast, we did not observe significant correlation between creatinine, a measure of renal function, and dermal parasitism or infectiousness. Host blood cell abnormalities, including anemia, correlate with infectiousness to sand flies. As these signs of disease often appear earlier in the course of disease, this indicates that classical measures of disease severity do not necessarily correlate with infectiousness or epidemiological importance. Public health initiatives attempting to break the zoonotic cycle of L. infantum infection should therefore focus on preventing transmission from infectious, anemic dogs, and not those with the most severe disease. Author summary: Dogs infected with Leishmania can transmit parasites to humans and other dogs through sand flies. This causes human visceral leishmaniasis (VL) and canine leishmaniosis (CanL). Public health interventions removing clinically ill dogs from the population have not significantly decreased rates of human or canine disease. Additionally, studies found that dogs with mild to moderate clinical disease transmitted the most parasites to feeding sand flies. We evaluated skin sections from L. infantum-infected dogs and characterized the dermal and systemic factors associated with infectiousness to sand flies. We found that the number of parasitized immune cells positively correlated with infectiousness to sand flies. Additionally, anemia, measured by a decreasing hematocrit, significantly correlated with increased parasitized dermal immune cells and infectiousness to sand flies. In contrast, parasitized dermal immune cells did not significantly change as chronic kidney disease, measured by creatinine, progressed, even though these were the sickest animals based on the LeishVet clinical scoring system. These initial findings suggest that early host cell-parasite interactions underlying clinical parameters, more than overall clinical severity, influences infectiousness to sand flies. Public health initiatives should focus on breaking the cycle of zoonotic infection by providing insecticide intervention to dogs most infectious to sand flies. [ABSTRACT FROM AUTHOR]

Background: The Leishmania genome harbors formerly active short interspersed degenerated retroposons (SIDERs) representing the largest family of repetitive elements among trypanosomatids. Their substantial expansion in Leishmania is a strong predictor of important biological functions. In this study, we combined multilevel bioinformatic predictions with high-throughput genomic and transcriptomic analyses to gain novel insights into the diversified roles retroposons of the SIDER2 subfamily play in Leishmania genome evolution and expression. Results: We show that SIDER2 retroposons form various evolutionary divergent clusters, each harboring homologous SIDER2 sequences usually located nearby in the linear sequence of chromosomes. This intriguing genomic organization underscores the importance of SIDER2 proximity in shaping chromosome dynamics and co-regulation. Accordingly, we show that transcripts belonging to the same SIDER2 cluster can display similar levels of expression. SIDER2 retroposons are mostly transcribed as part of 3'UTRs and account for 13% of the Leishmania transcriptome. Genome-wide expression profiling studies underscore SIDER2 association generally with low mRNA expression. The remarkable link of SIDER2 retroposons with downregulation of gene expression supports their co-option as major regulators of mRNA abundance. SIDER2 sequences also add to the diversification of the Leishmania gene expression repertoire since ~ 35% of SIDER2-containing transcripts can be differentially regulated throughout the parasite development, with a few encoding key virulence factors. In addition, we provide evidence for a functional bias of SIDER2-containing transcripts with protein kinase and transmembrane transporter activities being most represented. Conclusions: Altogether, these findings provide important conceptual advances into evolutionary innovations of transcribed extinct retroposons acting as major RNA cis-regulators. [ABSTRACT FROM AUTHOR]

Dogs are the primary reservoirs of Leishmania infantum (L. infantum), but Leishmania tropica (L. tropica) infection is also possible in dogs and can transmitted to humans. The southeast of Iran experiences a high prevalence of canine leishmaniasis (CanL), and veterinarians frequently encounter symptomatic cases. Therefore, from December 2017 to November 2022, the present case series was designed to assess the prevalence of CanL in owned dogs with cutaneous manifestations resembling CanL. A total of 500 owned dogs with dermal lesions from two endemic cities, Zabol and Kerman, were enrolled. Impression smears from skin lesions and popliteal lymph nodes were prepared from all cases, whereas blood samples were gathered from amastigote-positive dogs for serological and molecular surveys. Commercial ELISA was done on sera samples, and two-step nested PCR was used on extracted DNA to amplify variable fragments of the Leishmania species' kinetoplast DNA (kDNA). Leishman bodies were microscopically detected in 7.2% (36/500) of dermal smears. Of the 360 owned dogs from Zabol, 2 have been diagnosed with L. tropica, and 10 have been diagnosed with L. infantum. Out of 140 owned dogs from Kerman, 8 were identified with L. tropica infection, and 16 were infected with L. infantum. Molecular results revealed the presence of 750 full dual-band bases related to the L. tropica species in 10/36 (27.7%) cases, which showed a considerable increase in canine cutaneous leishmaniosis compared with previous studies in southeastern Iran. The noticeable prevalence of L. tropica in owned dogs indicated that the dog's role in cutaneous leishmaniosis should be re-evaluated as a possible animal reservoir in endemic areas. [ABSTRACT FROM AUTHOR]

Several Ranunculaceae species are used in folk medicine to eliminate pathologies associated with oxidative stress as well as parasitic infections; however, a number of studies confirming their pharmacological properties is limited. In this study, 19 ethanolic extracts obtained from 16 Ranunculaceae species were assayed for in vitro antioxidant, antiproliferative, and antiparasitic potential. The maximum antioxidant potential in both oxygen radical absorbance capacity (ORAC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays was observed for Aconitum toxicum extract [half-maximal inhibitory concentration (IC50) 18.7 and 92.6 μg/mL]. Likewise, Anemone transsilvanica extract exerted the most promising antiproliferative activity against Caco-2 (IC50 46.9 μg/mL) and HT29 (IC50 70.2 μg/mL) cell lines in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Additionally, a dual antioxidant and cytotoxicity effect was demonstrated for Aconitum moldavicum and Caltha palustris extracts. Whilst the efficacy of extracts was modest against Trypanosoma brucei (IC50 ranging from 88.8 to 269.3 µg/mL), several extracts exhibited high potency against Leishmania infantum promastigotes (Aconitum vulparia IC50 18.8 µg/mL). We also tested them against the clinically relevant intracellular stage and found extract of A. vulparia to be the most effective (IC50 29.0 ± 1.1 µg/mL). All tested extracts showed no or low toxicity against FHs 74Int normal cell line (IC50 ranging from 152.9 to >512 µg/mL). In conclusion, we suggest the above-mentioned plant extracts as potential candidates for development of novel plant-based antioxidant and/or antiproliferative and/or antileishmanial compounds. [ABSTRACT FROM AUTHOR]

In canine leishmaniosis endemic areas, Leishmania infantum may occur in sympatry with the non-pathogenic Leishmania tarentolae, which is associated to reptiles. The potential infectivity of L. tarentolae for mammals raises questions about the interactions between the two Leishmania species, and the potential cross-immune protection in dogs. This study aimed to assess the outcome of experimental L. tarentolae infection in dogs, determining: i) the anti-L. tarentolae antibody production, ii) the duration of the immunity and cytokine expression, and iii) the possible pathogenic effect in the canine host. Twelve purpose-bred beagle dogs were randomly allocated to three groups (intravenous inoculation, G1; intradermal inoculation, G2; negative control, G3). G1 and G2 dogs were inoculated twice (day 0, day 28) with 108 promastigotes of L. tarentolae strain (RTAR/IT/21/RI-325) isolated from a Tarentola mauritanica gecko. The animals were followed until day 206. Blood, serum, conjunctival swabs and lymph node aspirate samples were collected monthly and bone marrow, liver and spleen biopsies on day 91. Hematological and biochemical parameters were assessed monthly, as well as serology (IFAT and ELISA) and molecular identification of L. tarentolae. Mononuclear cells (PBMC) were obtained to assess the cytokine expression through in vitro stimulation or (re-) infection. Data from this study demonstrated that DNA from L. tarentolae is detectable up to 3 months post-infection, with seroconversion after day 28. Moreover, the non-pathogenic nature of L. tarentolae was confirmed, with a neutral Th1/Th2 polarization, and a possible shift to Th1 phenotype after derived macrophages (re-) infection, as demonstrated by the expression of IFN-gamma. Therefore, L. tarentolae demonstrated a great potential as a surrogate pathogen and/or immune-prophylaxis/immune-therapy against Leishmania infections in dogs and humans. Author summary: The reptilian-associated Leishmania tarentolae may be infective for mammals, which raises questions about a potential cross-immune protection towards Leishmania infantum in dogs. This study aimed to assess the outcome of experimental L. tarentolae infection in the canine host. Twelve purpose-bred beagle dogs were allocated into three groups (intravenous inoculation, G1; intradermal inoculation, G2; negative control, G3). The first two groups were inoculated twice (day 0, day 28) with 108 promastigotes of L. tarentolae, and were followed until day 206. Hematological and biochemical parameters were assessed, as well as serology (IFAT and ELISA), molecular identification and cytokine expression. Results demonstrated that DNA from L. tarentolae is detectable up to 3 months post-infection, with seroconversion after day 28. Moreover, the non-pathogenic nature of L. tarentolae was confirmed, with a neutral Th1/Th2 polarization, and a possible shift to Th1 phenotype (i.e., expression of IFN-gamma). These results suggest that L. tarentolae could be a potential candidate for developing immune strategies, opening new avenues for the prevention of the canine leishmaniasis. [ABSTRACT FROM AUTHOR]

Background: Renal disease is the main cause of death in canine leishmaniosis. Detection of an active glomerular injury is important to identify early renal damage and to prevent the development of chronic kidney disease. Podocyturia can indicate renal injury, and podocyte-associated molecules such as podocin and nephrin can be used to identify podocyturia. The purpose of the study was to evaluate urinary podocin and nephrin concentrations in dogs with leishmaniosis as markers of podocyturia. Methods: A total of 35 healthy dogs and 37 dogs with leishmaniosis were enrolled in the study. Dogs with leishmaniosis were classified according to the staging of the International Renal Interest Society (IRIS). Urinary podocin and nephrin concentrations were measured in all dogs with a validated enzyme-linked immunosorbent assay test and normalized to creatinine (uPoC and uNeC, respectively). The demographic, clinical, and laboratory data from both groups were analyzed and compared. Subsequently, the laboratory results were analyzed and compared according to IRIS staging in dogs in IRIS stage I and dogs in IRIS stage II + III + IV. The Pearson's correlation test evaluated the relationship between urinary markers of podocyturia. Results: Compared with healthy dogs, lower urinary podocin [median values (IQR): 15.10 (11.75–17.87) ng/ml versus 8.63 (7.08–13.56) ng/ml; P < 0.01] and nephrin [median values (IQR): 3.2 (3.62–5.43) ng/ml versus 2.67 (2.06–3.44) ng/ml; P < 0.01] were found in infected sick dogs. No significant differences were observed in the uPoC and uNeC between the two groups. Urinary nephrin and podocin concentrations were higher in healthy dogs and in dogs in IRIS stage I (both P < 0.05) compared with dogs in IRIS stages II + III + IV. No significant differences were found for uPoC and uNeC between healthy dogs and dogs with leishmaniosis in different IRIS clinical stages. Conclusions: Dogs with leishmaniosis had a low concentration of podocin and nephrin in more advanced IRIS clinical stages, when kidney disease was more severe compared with healthy dogs and dogs in IRIS stage I with mild disease. Urinary nephrin was detectable for the first time in healthy non-infected dogs. [ABSTRACT FROM AUTHOR]

Background: Dogs may be infected with species of Leishmania parasites that are disseminated through blood circulation and invade the internal organs. In this study, we aim to detect the parasite in the blood of dogs using the PCR technique. The present work was performed from February 2022 to May 2023 in Fars Province, southern Iran, where the disease is endemic. Results: In total, 7(5.1%) out of 135 blood samples, six were identified as Leishmania tropica and one as Leishmania major. We found no trace of Leishmania infantum, which is always known for visceral infection. In addition, no sign of cutaneous lesions or a significant disease was seen in the animals infected with both species. Of 48 dogs with anemia, two were Leishmania positive. The mean value of hematological parameters in the infected dogs was within the normal range except for a significant reduction in the platelet measures (p < 0.05). Conclusions: Our data revealed that both Leishmania species, tropica and major, may manifest as viscerotropic leishmaniasis. More investigations are needed to understand the conditions under which these species choose the type of infection. Moreover, our data emphasize the role of asymptomatic dogs in carrying these parasites, a crucial factor in spreading the disease. [ABSTRACT FROM AUTHOR]

Interleukin 27 (IL‐27) is a cytokine that regulates susceptibility to Leishmania infantum infection in humans and experimental models. This cytokine has not yet been described in canine leishmaniasis (CanL). Therefore, we investigated whether IL‐27 has a regulatory role in CanL. The EBI3 and p28 subunits of IL‐27 were measured in splenic leukocytes culture supernatant from dogs with CanL and compared to control dogs. We also correlated EBI3 and p28 levels with IL‐21, anti‐L. infantum antibodies and parasite loads. We performed functional assays followed by IL‐27 blockade and measured parasite loads, production of cytokines in splenic leukocytes culture supernatant, and the expression of PD‐1, CTLA‐4, phospho‐Stat‐1/3, T‐bet, GATA3 and nitric oxide production (NO). Both IL‐27 subunits increased in the supernatant of dogs with CanL compared to control dogs. EBI3 and p28 levels showed a moderate positive correlation with IL‐21 (r = 0.67, p < 0.0001 and r = 0.45, p < 0.012, respectively), and the EBI3 subunit was positively associated with anti‐L. infantum IgG antibodies (r = 0.38, p < 0.040) and parasite load (r = 0.47, p < 0.009). IL‐27 and IL‐21 participate of immune responses in CanL. IL‐27 may be associated with the failure of immunity to control parasite replication via upregulation of the expression of PD‐1, CTLA‐4, T‐bet and NO in splenic leukocytes from dogs with CanL. These findings suggest that the pathways regulated by IL‐27 are involved in CanL pathogenesis in the host, and may be targets for new therapies. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL), caused by protozoa of the genus Leishmania, remains a significant public health concern due to its potentially lethal nature if untreated. Current chemotherapy options are limited by severe toxicity and drug resistance. Derivatives of 1,2,4-oxadiazole have emerged as promising drug candidates due to their broad biological activity. This study investigated the effects of novel 1,2,4-oxadiazole derivatives (Ox1–Ox7) on Leishmania infantum, the etiological agent of VL. In silico predictions using SwissADME suggest that these compounds have high oral absorption and good bioavailability. Among them, Ox1 showed the most promise, with higher selectivity against promastigotes and lower cytotoxicity towards L929 fibroblasts and J774.G8 macrophages. Ox1 exhibited selectivity indices of 18.7 and 61.7 against L. infantum promastigotes and amastigotes, respectively, compared to peritoneal macrophages. Ultrastructural analyses revealed severe morphological damage in both parasite forms, leading to cell death. Additionally, Ox1 decreased the mitochondrial membrane potential in promastigotes, as shown by flow cytometry. Molecular docking and dynamic simulations indicated a strong affinity of Ox1 for the L. infantum CYP51 enzyme. Overall, Ox1 is a promising and effective compound against L. infantum. [ABSTRACT FROM AUTHOR]

Simple Summary: A dog imported from Greece to Germany presented a mass in the mammary gland. Cytology of the secretion was performed, and besides the malignant epithelial cells, amastigotes of Leishmania (L.). infantum were identified. Diagnoses of mammary carcinoma and canine leishmaniasis were confirmed by histopathology, PCR testing, and serology. The detection of L. infantum amastigotes in the mammary glands indicates a possible shedding by the milk and makes trans-mammary transmission to puppies theoretically possible as another route of L. infantum transmission apart from vectorial transmission by sand flies, blood transfusion, vertical transmission, and venereal transmission. Mammary tumors are one of the most common neoplasms in female dogs, and cytology represents a non-invasive diagnostic method. The protozoal pathogen Leishmania spp. was previously demonstrated in canine mammary glands. An eight-year-old, female-spayed Doberman was imported from Crete, Greece, three years before the first presentation. The dog was presented due to a mammary tumor two years after adoption. The clinical examination revealed fever and weight loss. Smears of the mammary secretion were investigated cytologically. Multiple atypical epithelial cells with moderate to marked criteria of malignancy were detected. Furthermore, amastigotes were visible intra- and extracellularly. The diagnosis of L. infantum infection was based on a positive PCR out of the cytological smear, and a positive serology. Mammary carcinoma and granulomatous inflammation with amastigotes were confirmed by histopathology. We aimed to provide a complete report of cytological, histopathological, hematological, and biochemistry findings in a dog with L. infantum in the mammary glands with focus on trans-mammary pathogen transmission as a potential alternative way of spreading Leishmania infections. Canine leishmaniasis should be a potential differential diagnosis in dogs with lesions and/or inflammation in the mammary glands and a history of presence in areas endemic for L. infantum, especially the Mediterranean in Europe. [ABSTRACT FROM AUTHOR]

Leishmaniasis represents a severe global health problem. In the last decades, there have been significant challenges in controlling this disease due to the unavailability of licensed vaccines, the high toxicity of the available drugs, and an unrestrained surge of drug-resistant parasites, and human immunodeficiency virus (HIV)–Leishmania co-infections. Leishmania spp. preferentially invade macrophage lineage cells of vertebrates for replication after subverting cellular functions of humans and other mammals. These early events in host–parasite interactions are likely to influence the future course of the disease. Thus, there is a continuing need to discover a simple cellular model that reproduces the in vivo pathogenesis. Acanthamoeba spp. are non-mammalian phagocytic amoeba with remarkable similarity to the cellular and functional aspects of macrophages. We aimed to assess whether the similarity reported between macrophages and Acanthamoeba spp. is sufficient to reproduce the infectivity of Leishmania spp. Herein, we analyzed co-cultures of Acanthamoeba castellanii or Acanthamoeba polyphaga with Leishmania infantum, Leishmania amazonensis, Leishmania major, and Leishmania braziliensis. Light and fluorescence microscopy revealed that the flagellated promastigotes attach to the A. castellanii and/or A. polyphaga in a bipolar and or random manner, which initiates their uptake via pseudopods. Once inside the cells, the promastigotes undergo significant changes, which result in the obligatory amastigote-like intracellular form. There was a productive infection with a continuous increase in intracellular parasites. However, we frequently observed intracellular amastigotes in vacuoles, phagolysosomes, and the cytosol of Acanthamoeba spp. Our findings corroborate that Leishmania spp. infects Acanthamoeba spp. and replicates in them but does not cause their rapid degeneration or lysis. Overall, the evidence presented here confirms that Acanthamoeba spp. have all prerequisites and can help elucidate how Leishmania spp. infect mammalian cells. Future work exposing the mechanisms of these interactions should yield novel insights into how these pathogens exploit amoebae. Author summary: Leishmaniasis represents a severe global health problem, and drug resistance is a growing concern. Leishmania spp. are obligate intracellular parasites that survive within cells of the vertebrate macrophage, modulating their activation. Understanding the multilayered relationship between metabolism and function of innate immune cells during infection has great therapeutic and preventive potential. Mammalian macrophages and Acanthamoeba spp. display similarities in the molecular mechanisms involved in directional motility, recognition, binding, engulfment, and the phagolysosomal processes, and they express similar receptors. Hence, we hypothesize that Acanthamoeba spp. represent a model that can be used to evaluate macrophage–pathogen interactions from the perspective of innate immunity. However, it has not yet been described whether Leishmania spp. can survive in Acanthamoeba spp. We found that Acanthamoeba spp. support Leishmania growth. Acanthamoeba spp. contain ancient pathogen recognition mechanisms, and Leishmania spp. could manipulate amoeboid functions to favor their survival and replication based on strategies of ancestral metabolic pathways. Our robust evidence highlights that amoebae could be used as a model to understand the biology and evolution of host–Leishmania interactions. [ABSTRACT FROM AUTHOR]

Background: Leishmania infantum is endemic in Europe (and elsewhere) while L. donovani s.s., L. tropica and L. major are not but are present in neighboring countries in North Africa, the Middle East, (the Asian part of) Turkey and the Southern Caucasus. Lists of sand fly vector species in the scientific literature vary with the criteria for vector incrimination, and criteria vary because, for some, evidence is difficult to generate. With minimal criteria, about 20 sand fly species are proven or suspected vectors of L. infantum in Europe and neighboring countries, while for L. tropica and L. major, there are seven and four proven or suspected vector species, respectively, in this area. For L. donovani s.s., present in Cyprus, the Middle East and (the Asian part of) Turkey, no local vectors have been incriminated so far. The aim was to assess the degree of spatial agreement between Leishmania spp. and various vectors species and their relative contribution to the explained variation. Methods: We used multivariate regression modeling to analyze the spatial relationship between autochthonous Leishmania spp. and clinical forms in humans and animals and 14 Phlebotomus spp. in Europe and neighboring countries. Results: There was only fair agreement between parasite and vector distributions. The most parsimonious models describing the distribution of Leishmania spp. and clinical forms included three to six sand fly species and explained between 12% (L. infantum) and 37% (L. donovani) of the observed variation. Selected models included confirmed and suspected vector species as well as unexpected species. Conclusions: The relatively low agreement between Leishmania and vector distributions highlights the need to improve leishmaniasis reporting and vector surveillance in areas where no information is available, both for a better understanding of the epidemiology of infection in endemic areas and to monitor possible spread of infection into non-endemic areas. While some of the unexpected sand fly-Leishmania spp. statistical associations might be spurious, for others, the existence of sporadic or recent reports of infections warrants further vector competence studies that consider strain variation. [ABSTRACT FROM AUTHOR]

1-(3-Methylthiophen-2-yl)-N-(3,4,5-trimethoxyphenyl)methanimine (1, MTM) was synthesized from 3-methylthiophene-2-carbaldehyde and 3,4,5-trimethoxyaniline and characterized by FTIR and 1H and 13C NMR spectra. The geometric structure of the title compound was optimized, and its electronic (FMO, NLO, MEP, NBO), spectroscopic (NMR, IR, UV), and thermodynamic properties were calculated by quantum chemicial methods. The calculated and experimental geometric parameters were compared with each other. Compound 1 was tested for leishmanicidal activity against Leishmania infantum by the broth microdilution method and was found to be efficient against Leishmania infantum promastigotes in the concentration range studied. Possible interactions responsible for the antileishmanial activity were determined by molecular docking analysis against trypanothione reductase (TRe). The docking results demonstrated high inhibition constant of the title compound and supported its antileishmanial activity. [ABSTRACT FROM AUTHOR]

Leishmania infantum is the vector-borne trypanosomatid parasite causing visceral leishmaniasis in the Mediterranean basin. This neglected tropical disease is treated with a limited number of obsolete drugs that are not exempt from adverse effects and whose overuse has promoted the emergence of resistant pathogens. In the search for novel antitrypanosomatid molecules that help overcome these drawbacks, drug repurposing has emerged as a good strategy. Nitroaromatic compounds have been found in drug discovery campaigns as promising antileishmanial molecules. Fexinidazole (recently introduced for the treatment of stages 1 and 2 of African trypanosomiasis), and pretomanid, which share the nitroimidazole nitroaromatic structure, have provided antileishmanial activity in different studies. In this work, we have tested the in vitro efficacy of these two nitroimidazoles to validate our 384-well high-throughput screening (HTS) platform consisting of L. infantum parasites emitting the near-infrared fluorescent protein (iRFP) as a biomarker of cell viability. These molecules showed good efficacy in both axenic and intramacrophage amastigotes and were poorly cytotoxic in RAW 264.7 and HepG2 cultures. Fexinidazole and pretomanid induced the production of ROS in axenic amastigotes but were not able to inhibit trypanothione reductase (TryR), thus suggesting that these compounds may target thiol metabolism through a different mechanism of action. [ABSTRACT FROM AUTHOR]

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Leishmaniasis is a tropical infectious disease caused by Leishmania parasites. The disease can be spread by the bite of an infected sand fly. Currently, five chemotherapeutic drugs are available in leishmaniasis treatment. However, these drugs exhibit toxicity and serious adverse effects on infected individuals, necessitating alternative treatment strategies. One such strategy involves using combinations of existing antileishmanial drugs. In this study, we evaluated the interaction between artesunate (AS) and three antileishmanial drugs—amphotericin B (AmB), miltefosine (MF), and paromomycin (PM) against Leishmania infantum. This evaluation marks the first time such an assessment has been conducted. The Chou–Talalay combination index method was employed to analyze the drug interaction. The findings revealed that the interaction between AS and AmB ranged from antagonistic to synergistic, while the interaction between AS and MF showed moderate to strong synergism. In contrast, the interaction between AS and PM resulted in an antagonistic interaction, which differs from the combinations with AmB or MF. This study provides valuable insights for developing novel drug regimens for leishmaniasis treatment, emphasizing the potential of AS and its combination with existing antileishmanial drugs. Further research is necessary to optimize drug combinations and minimize adverse effects, leading to more effective therapeutic outcomes. [ABSTRACT FROM AUTHOR]

Purpose: The objectives of the present study are to determine the molecular prevalence of Leishmania spp. in the owned domestic cats in the Black Sea Region of Türkiye and analyze the associated risk factors in FeL. Methods: Conjunctival swabs (CS), blood, demographic, and clinical data were collected from 150 owned cats brought to the Veterinary Teaching Hospital during 2020–2022. Leishmania kinetoplast DNA (kDNA) from CS was screened by TaqMan Real-Time PCR (qPCR) with the genus-specific primers and a probe. Results: All qPCR positive products were also amplified and sequenced to identify Leishmania species by ITS1 primers. Molecular prevalence of L. infantum found as 12.6% (19/150) in the observed cats in the Black Sea Region of Türkiye. There was a significant difference (p < 0.05) between neutered and intact cats with regarding to L. infantum positivity. Intact cats found to be 0.368 times more prone to be L. infantum-positive (L+). Dermatological lesions were found the most common (26.3%) problems in the L + cats. The median leucocyte count was the only parameter that was found statistically (p < 0.05) lower in the L + group (6.60) than the negative group (L−) (8.96), when comparing the WBC, NEU/LYM, MONO/LYM, EOS/LYM and PLT/LYM values. Conclusion: This study presented the molecular occurrence of FeL in the Black Sea Region of Türkiye for the first time indicating that the carrier status of the cats makes them alternative reservoirs for possible zoonotic transmission of L. infantum in this zone. [ABSTRACT FROM AUTHOR]

Human zoonotic visceral leishmaniasis (ZVL) and canine leishmaniasis (CanL) constitute a major public and veterinary health concern and are both caused by the infection with the protozoan parasite Leishmania infantum. One of the main target organs in CanL is the liver. This complex organ, composed of various highly specialized cell types, has garnered significant attention from the scientific community as a crucial player in innate immune functions. In the context of CanL, liver infection by parasites and the host immune response generated strongly influence the disease outcome. Thus, taking advantage of a co-culture system involving canine hepatocytes and L. infantum-infected autologous Kupffer cells (KCs), allowing cell-to-cell interaction, the current report aims to shed light on the hepatocyte-KCs immune interaction. The co-culture of infected KCs with hepatocytes revealed a vital role of these cells in the activation of a local immune response against L. infantum parasites. Although KCs alone can be immunologically silenced by L. infantum infection, the cell-to-cell interaction with hepatocytes in co-culture can lead to local immune activation. In co-culture it was observed gene expression increased the number of innate immune receptors, specifically cell membrane TLR2 and cytoplasmatic NOD1 along with high TNF-α generation. Altogether, these results suggest that the immune response generated in co-culture could induce the recruitment of other circulating cells to contain and contribute to the resolution of the infection in the liver. This work also enhances our understanding of the liver as a vital organ in innate immunity within the context of CanL. [ABSTRACT FROM AUTHOR]

Abstract Background The Leishmania genome harbors formerly active short interspersed degenerated retroposons (SIDERs) representing the largest family of repetitive elements among trypanosomatids. Their substantial expansion in Leishmania is a strong predictor of important biological functions. In this study, we combined multilevel bioinformatic predictions with high-throughput genomic and transcriptomic analyses to gain novel insights into the diversified roles retroposons of the SIDER2 subfamily play in Leishmania genome evolution and expression. Results We show that SIDER2 retroposons form various evolutionary divergent clusters, each harboring homologous SIDER2 sequences usually located nearby in the linear sequence of chromosomes. This intriguing genomic organization underscores the importance of SIDER2 proximity in shaping chromosome dynamics and co-regulation. Accordingly, we show that transcripts belonging to the same SIDER2 cluster can display similar levels of expression. SIDER2 retroposons are mostly transcribed as part of 3'UTRs and account for 13% of the Leishmania transcriptome. Genome-wide expression profiling studies underscore SIDER2 association generally with low mRNA expression. The remarkable link of SIDER2 retroposons with downregulation of gene expression supports their co-option as major regulators of mRNA abundance. SIDER2 sequences also add to the diversification of the Leishmania gene expression repertoire since ~ 35% of SIDER2-containing transcripts can be differentially regulated throughout the parasite development, with a few encoding key virulence factors. In addition, we provide evidence for a functional bias of SIDER2-containing transcripts with protein kinase and transmembrane transporter activities being most represented. Conclusions Altogether, these findings provide important conceptual advances into evolutionary innovations of transcribed extinct retroposons acting as major RNA cis-regulators.

Abstract Background Renal disease is the main cause of death in canine leishmaniosis. Detection of an active glomerular injury is important to identify early renal damage and to prevent the development of chronic kidney disease. Podocyturia can indicate renal injury, and podocyte-associated molecules such as podocin and nephrin can be used to identify podocyturia. The purpose of the study was to evaluate urinary podocin and nephrin concentrations in dogs with leishmaniosis as markers of podocyturia. Methods A total of 35 healthy dogs and 37 dogs with leishmaniosis were enrolled in the study. Dogs with leishmaniosis were classified according to the staging of the International Renal Interest Society (IRIS). Urinary podocin and nephrin concentrations were measured in all dogs with a validated enzyme-linked immunosorbent assay test and normalized to creatinine (uPoC and uNeC, respectively). The demographic, clinical, and laboratory data from both groups were analyzed and compared. Subsequently, the laboratory results were analyzed and compared according to IRIS staging in dogs in IRIS stage I and dogs in IRIS stage II + III + IV. The Pearson’s correlation test evaluated the relationship between urinary markers of podocyturia. Results Compared with healthy dogs, lower urinary podocin [median values (IQR): 15.10 (11.75–17.87) ng/ml versus 8.63 (7.08–13.56) ng/ml; P

Background & objectives: Surveillance of canine leishmaniasis in Colombia is restricted to the appearance of visceral leishmaniasis cases in humans, and is mainly performed by serological tests. This requires blood sampling by veterinarians or technicians according to Colombian laws. The aim of this study was to evaluate the utility of conjunctival swabs in the molecular detection of Leishmania in dogs from the municipality of Ovejas, Sucre. Methods: The present study was cross-sectional and descriptive. The collection source of samples and information was primary. Blood samples and conjunctival swabs from 121 dogs were analysed by PCR-ITS1 to detect Leishmania spp. Positive samples were used to amplify a conserved region of the Leishmania infantum kinetoplast minicircle. Performance of both sample types was calculated by proportion of positive samples of each type and the degree of agreement between them was determined by Cohen’s kappa (κ) agreement index. Results: Leishmania infection was detected in 17.4% (21/121) of blood samples and in 16.5% (20/121) of conjunctival swabs. In total, 28.1% (34/121) of the canines were infected, of which 11.8% (4/34) were infected with L. infantum in the conjunctival swabs and 5.9 % (2/34) in the blood samples. The agreement between blood and conjunctiva was medium (κ = 0.207) by PCR-ITS1 amplification. Interpretation & conclusion: The use of conjunctival swab as a non-invasive sample could be used as an alternative method for surveillance of canine leishmaniasis.

Phlebotomus perniciosus is a major vector of Leishmania infantum in the Mediterranean. While the seroprevalence of leishmaniosis in Spanish dogs and cats has been studied, data on the exposure of cats to P. perniciosus bites under natural conditions without repellents is limited. Stray cats could serve as sentinels for L. infantum and P. perniciosus exposure. This study analyzed sera from 204 apparently healthy stray cats, collected from January 2021 to January 2022, for antibodies against P. perniciosus saliva and L. infantum parasites. Anti-sand fly antibodies were detected in 40.69% of cats using an ELISA with the recombinant salivary protein SP03B of P. perniciosus. Seroprevalence of L. infantum infection was 23.52% by Western blot and 27.41% by ELISA, with an overall seroprevalence of 40.69% (95% CI 34.18–47.54%). This is the first assessment of antibody response to P. perniciosus saliva and L. infantum in naturally exposed stray cats in Spain. Further research is needed to examine the salivary antigens recognized by cats and to explore the relationship between P. perniciosus exposure and L. infantum infection severity in cats.

Sand flies (Diptera: Psychodidae: Phlebotominae) are blood‐feeding insects that transmit the protozoan parasites Leishmania spp. and various arboviruses. The Balkan region, including the Republic of Kosovo, harbours a diverse sand fly fauna. Vector species of Leishmania infantum as well as phleboviruses are endemic; however, recent data are scarce. We performed a cross‐sectional study to update the current sand fly distribution in Kosovo and assess biological as well as environmental factors associated with sand fly presence. CDC light trapping was conducted at 46 locations in 2022 and 2023, specifically targeting understudied regions in Kosovo. Individual morphological species identification was supported by molecular barcoding. The occurrence data of sand flies was used to create distribution maps and perform environmental analyses, taking elevation, wind speed and climate‐related factors into account. In addition, PCR‐based blood meal analysis and pathogen screening were conducted. Overall, 303 specimens of six sand fly species were trapped, predominated by Phlebotomus neglectus (97%). Barcodes from eight of nine known endemic sand fly species were obtained. Combining our data with previous surveys, we mapped the currently known sand fly distribution based on more than 4000 specimens at 177 data points, identifying Ph. neglectus and Ph. perfiliewi as the predominant species. Environmental analyses depicted two geographical groups of sand flies in Kosovo, with notable differences between the species. In total, 223 blood meals of five sand fly species were analysed. Of seven identified host species, the predominant blood meal source was observed to be cattle, but the DNA of dogs and humans, among others, was also detected. This study assessed biological as well as ecological factors of sand fly occurrence, which should help better understand and evaluate potential hot spots of disease transmission in Kosovo. [ABSTRACT FROM AUTHOR]

Introduction: Leishmaniasis stands out as a public health problem in the state of Minas Gerais, Brazil, especially in the Midwest region. However, the entomological aspects in several municipalities remain unknown. Therefore, this study aimed to investigate the sand fly fauna in Bambuí, encompassing ecological dynamics and molecular detection of Leishmania. Methods: Monthly collections were conducted using CDC light traps from September 2018 to August 2020 across 16 selected points with urban and rural characteristics, chosen based on the coverage area of the Municipal Health Department and the occurrence of canine and human visceral leishmaniasis (VL) cases. Ecological indices of the sand fly population (Chao1, Shannon, Simpson and Pielou) were assessed, and sand fly abundance was correlated to climatic variables (humidity, temperature and rainfall). Results: A total of 8838 specimens representing 17 species within nine genera were collected (estimated species richness by Chao 1 estimator = 17; SE ± 1.8). Predominantly, Lutzomyia longipalpis, Nyssomyia whitmani and Evandromyia cortelezzii constituted approximately 98% of all captured sand flies. While species richness and diversity displayed variations throughout the study, a positive correlation emerged between temperature (p < 0.0001; r = 0.7767), monthly rainfall (p < 0.0001; r = 0.7810) and sand fly abundance. Molecular analysis revealed Leishmania DNA in 2.05% of female sand flies, with the presence of Leishmania infantum in Lu. longipalpis and both Le. infantum and Leishmania braziliensis in Ev. cortelezzii. Conclusions: The entomological data, coupled with the occurrence of autochthonous cases of canine visceral leishmaniasis, offer valuable insights for evidence‐based strategies to prevent leishmaniasis in Bambuí. [ABSTRACT FROM AUTHOR]

Abstract Background Renal disease in canine leishmaniosis is of great importance owing to increased risk of mortality. In human visceral leishmaniosis, monocyte chemoattractant protein-1 (MCP-1) has been used as a marker of renal damage and inflammation. The purpose of this study was first to determine the serum MCP-1 and urinary MCP-1-to-creatinine ratio (uMCP-1/Cr) in healthy dogs and dogs with leishmaniosis at diagnosis, and second to determine whether these markers can differentiate disease severity at diagnosis. Methods In total, 19 healthy seronegative dogs and 38 dogs with leishmaniosis were included in the study. Dogs with leishmaniosis were classified as LeishVet clinical staging and as International Renal Interest Society (IRIS) staging. Serum and urinary MCP-1 concentrations were measured with an enzyme-linked immunosorbent assay. A receiver operating characteristic (ROC) curve determined disease severity at diagnosis between two LeishVet groups (Stage II versus stage III and IV). Results Dogs in Leishvet stages IIb, III, and IV had a median serum MCP-1 and uMCP-1/Cr concentration higher than healthy dogs (P

Abstract Background Cats are now recognized as competent hosts for Leishmania infantum and a blood source for sand fly vectors. Although canine leishmaniosis (CanL) is endemic in Mediterranean Basin countries, large-scale epidemiological studies are lacking for feline leishmaniosis (FeL). This study aimed to assess the prevalence of L. infantum infections, associated risk factors, clinical signs, and clinicopathological abnormalities in domestic cat populations from six Mediterranean Basin countries. Methods From 2019 to 2022, blood and serum samples of cats (n = 2067) living in Italy (n = 300), Greece (n = 297), Portugal (n = 295), France (n = 231), Israel (n = 313), and Spain (n = 631) were collected along with animal data (i.e., age, sex, breed, housing conditions, and geographical origin), clinical signs, and laboratory blood test parameters. Cats were grouped according to their age as kittens (up to 1 year), young (older than 1 and younger than 7 years), mature (between 7 and 10 years), and senior (older than 10 years). Serum samples were tested for L. infantum by immunofluorescence antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA), and blood samples of seropositive cats were tested for L. infantum kinetoplast deoxyribonucleic acid (kDNA). Viral infection by feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) was molecularly addressed in all cats enrolled. Statistical analysis was performed to evaluate the association between the risk of L. infantum infection and independent variables, and among co-infection of L. infantum with FIV and/or FeLV, clinical signs, and clinicopathological abnormalities. Results Overall, 17.3% (358/2067) of cats scored positive for L. infantum by serological tests. Specifically, 24.7% were from Portugal, 23.2% from Greece, 16.6% from Israel, 15% from Spain, 13.3% from France, and 12.6% from Italy. Leishmania infantum DNA was detected in 15 seropositive animals. Housing condition and FIV infection proved to be risk factors for FeL. Leishmania seropositivity was significantly associated with weight loss, lymphadenomegaly, gingivostomatitis, and oral ulcers, as well as with reduced albumin and albumin/globulin ratio, increased total globulins and total proteins, leukocytosis, and thrombocytosis. Conclusions This study provides, for the first time, a large-scale epidemiological survey on FeL and its clinical presentation, revealing that L. infantum circulates among domestic cats, especially shelter/free-roaming and FIV-infected animals, living in CanL endemic countries of the Mediterranean Basin. Graphical Abstract

Abstract Background Abiotic factors play a significant role in the evolution of Leishmania infantum infection due to its vectorial nature. This study aims to assess the evolution in the detection of new L. infantum infection cases in Valdeorras (Ourense, Northwestern Spain) over a 20-year period and how different climatic variables and preventive measures may have affected it. Methods Indirect immunofluorescence antibody tests (IFAT) were performed on serum samples collected from dogs attending the ‘Servicios Veterinarios de Sil’ veterinary clinic (Valdeorras, Northwestern Spain) between May 2003 and April 2023 to detect L. infantum exposure. The percentage of new cases of L. infantum infection was calculated from May of one year to April of the following year. Climatic conditions in the region, global sales of ectoparasiticides and the number of vaccines against L. infantum delivered in the veterinary clinic from 2003 to 2022 were recorded. Statistical analyses were conducted to determine the associations between these factors and the percentage of new cases of L. infantum infection. Results A total of 2909 dogs were assessed, and 3785 IFAT tests were performed between May 2003 and April 2023. The mean percentage of new seropositive cases over the 20-year period studied was 21.65 ± 10.8%, with a decline from the beginning to the end of the period studied. The percentage was significantly higher between May 2003 and April 2008 compared with the other periods (May 2008 to April 2013, May 2013 to April 2018 and May 2018 to April 2023). There was a positive correlation between the percentage of new cases of L. infantum infection and the maximum relative humidity in winter. Conversely, there was a negative correlation between the percentage of new cases and sales of ectoparasiticides and vaccination against L. infantum. Conclusions This study is one of the longest evaluations of the evolution of L. infantum infection in a fixed location and its association with external factors including climatic conditions and preventive measures. The results confirm that Valdeorras is a high-risk area for L. infantum infection. The use of ectoparasiticides and vaccines against L. infantum has been shown to play a significant role in preventing L. infantum infection, highlighting the crucial role of veterinarians in the fight against this disease. Graphical Abstract

Background and Aim: In urban environments, dogs serve as the primary reservoir for visceral leishmaniasis (VL). Rapidly diagnosing canine VL through tests enables early treatment and a favorable prognosis. This study aimed to assess the diagnostic performance of the SensPERT® Leishmania test kit (Dechra®), Alere® Leishmaniasis Ac test kit, and the rapid test dual path platform (TR-DPP®) Bio-Manguinhos in detecting VL. Materials and Methods: 30 serum samples from reactive VL dogs and 30 serum samples from healthy dogs were employed for assessing the sensitivity and specificity variation between SensPERT® Leishmania test kit, Alere® Leishmaniasis Ac test kit, and rapid test dual platform – TR-DPP®. Results: The SensPERT® Leishmania test outperformed Alere® and TR-DPP® in terms of sensitivity, specificity, positive and negative predictive values and demonstrated near-perfect concordance with Alere® and substantial concurrence with TR-DPP®. Conclusion: The SensPERT® Leishmania rapid test proved to be a promising test in the detection of VL in dogs.

Abstract Background The sand fly Nyssomyia neivai is one of the most abundant species in Southern Brazil. It is frequently found in areas that are foci of visceral leishmaniasis in the state of Santa Catarina, caused by Leishmania infantum. In this region, the main vector of L. infantum, Lutzomyia longipalpis, has not been detected. In the absence of L. longipalpis, this study aimed to identify the sand fly fauna and diagnose any potential Leishmania spp. infection in sand flies and in dogs in a region of Southern Brazil that experienced a recent canine visceral leishmaniasis outbreak. Methods This report includes a survey of the sand fly fauna at the Zoonosis Control Center of the Municipality of Tubarão (Santa Catarina, Brazil). Molecular tests were conducted to investigate Leishmania spp. natural infection in sand flies using polymerase chain reaction (PCR). In positive females, in addition to morphological identification, molecular analysis through DNA barcoding was performed to determine the sand fly species. Additionally, the dogs were tested for the presence of Leishmania spp. using a non-invasive technique for the collection of biological material, to be assessed by PCR. Results A total of 3419 sand flies, belonging to five genera, were collected. Nyssomyia neivai was the most abundant species (85.8%), followed by Migonemyia migonei (13.3%), Pintomyia fischeri (0.8%), Evandromyia edwardsi (

Leishmaniasis is a chronic global arthropod‐borne zoonotic disease produced by several species of Leishmania with cutaneous, mucocutaneous, and visceral clinical manifestations. In Spain, only Leishmania infantum has been reported so far, although other species of Leishmania, such as L. tropica and L. major, are present in surrounding countries. The aim of this work is to analyze the occurrence of Leishmania spp. infection in European wildcats (Felis silvestris) as sentinels, including their genotypic characterization. Necropsies of 18 road‐killed wildcats were conducted. Samples of ear skin and spleen were taken for DNA isolation and PCR of the highly sensitive SSU-rDNA target. Subsequent PCR tests were performed using more specific targets for the determination of Leishmania species: hsp70 and ITS1. Positive samples were sequenced, and phylogenetic trees were constructed. Seven wildcats were found positive for Leishmania spp. Based on the hsp70 and ITS1 sequences, an animal was found to be infected only with L. tropica in ear skin samples, while two cats were found to be infected with L. infantum in both the ear skin and the spleen. In one animal, a clear sequence of L. infantum ITS1 and a sequence of L. tropica hsp70 were obtained from the ear skin. Since hsp70 and ITS1 sequencing was not possible in three cats, the species of Leishmania infecting them was not determined. This is the first report of autochthonous infection with L. tropica in the Iberian Peninsula. Health care professionals, including physicians, dermatologists, and veterinarians, must be aware of this for a correct diagnosis, treatment, and management of possible coinfections. [ABSTRACT FROM AUTHOR]

Background: Renal disease in canine leishmaniosis is of great importance owing to increased risk of mortality. In human visceral leishmaniosis, monocyte chemoattractant protein-1 (MCP-1) has been used as a marker of renal damage and inflammation. The purpose of this study was first to determine the serum MCP-1 and urinary MCP-1-to-creatinine ratio (uMCP-1/Cr) in healthy dogs and dogs with leishmaniosis at diagnosis, and second to determine whether these markers can differentiate disease severity at diagnosis. Methods: In total, 19 healthy seronegative dogs and 38 dogs with leishmaniosis were included in the study. Dogs with leishmaniosis were classified as LeishVet clinical staging and as International Renal Interest Society (IRIS) staging. Serum and urinary MCP-1 concentrations were measured with an enzyme-linked immunosorbent assay. A receiver operating characteristic (ROC) curve determined disease severity at diagnosis between two LeishVet groups (Stage II versus stage III and IV). Results: Dogs in Leishvet stages IIb, III, and IV had a median serum MCP-1 and uMCP-1/Cr concentration higher than healthy dogs (P < 0.0001). No statistical differences were found in serum MCP-1 and uMCP-1/Cr between dogs in LeishVet stage IIa and healthy dogs. The dogs in LeishVet stage IV had significantly higher serum MCP-1 and uMCP-1/Cr compared with the dogs in LeishVet stage IIa (P < 0.0001). Serum MCP-1 and uMCP-1 were significantly higher in dogs in IRIS stage I and II + III + IV compared with healthy dogs. Dogs stage II + III + IV of IRIS had a significantly higher serum MCP-1 compared with dogs in IRIS stage I (P < 0.0001). The area under the ROC curve for serum MCP-1 was 0.78 [95% confidence interval (CI) 0.64–0.93] and for uMCP-1/Cr it was 0.86 (95% CI, 0.74–0.99). The optimal cutoff value for serum MCP-1 and uMCP-1/Cr was 336.85 pg/ml (sensitivity of 79% and specificity of 68%) and 6.89 × 10−7 (sensitivity of 84% and specificity of 79%), respectively. Conclusions: Serum MCP-1 and uMCP-1/Cr are increased in dogs with leishmaniosis compared with healthy dogs, suggesting the presence of inflammation and renal injury. Serum MCP-1 and uMCP-1/Cr were more elevated in the advanced stages of the disease compared with the moderate stages and, therefore, can be markers of the severity of the disease process. [ABSTRACT FROM AUTHOR]

Background: Coronavirus disease 2019 originated in China and swiftly spread worldwide, posing a significant threat to public health. Caused by SARS-CoV-2, it manifests as a flu-like illness that can escalate to Acute Respiratory Distress Syndrome, potentially resulting in fatalities. In countries where HIV/Leishmania infantum is endemic, the occurrence of concurrent SARS-CoV-2/HIV/Leishmania infantum infections is a reality, prompting inquiries into appropriate clinical management. Case presentation: We present the case of a 48-year-old woman who was hospitalized for 36 days across three different hospitals in the state of Pernambuco, Brazil. She was diagnosed with SARS-CoV-2/HIV/L. infantum coinfection. The patient exhibited severe COVID-19 symptoms, including fever, productive cough, and dyspnea. Throughout her hospitalization, she experienced oxygen saturation levels of ≤ 93%, along with fluctuations in blood pressure, respiratory rate, and heart rate. Her blood tests revealed lymphopenia, leukopenia, and neutropenia, while laboratory results indicated abnormal levels of d-dimer, AST, ALT, lactate dehydrogenase, ferritin, and C-reactive protein. A computed tomography scan revealed 75% involvement of the lung parenchyma with patchy ground-glass opacities. Conclusion: Against all odds, the patient was discharged. The leukopenia associated with HIV/L. infantum may have played a decisive role. Further studies are necessary to better understand diagnostic strategies and clinical management measures for HIV/L. infantum coinfected patients who are susceptible to SARS-CoV-2 infection. [ABSTRACT FROM AUTHOR]

Background: Leishmaniosis caused by Leishmania infantum, L. major and L. tropica is endemic in Morocco. Growing evidence of both human and canine Leishmania infections in urban centres has been reported. Since many forms of the disease are zoonotic, veterinarians play an important role in leishmaniosis control by intervening at the parasite host level. This study aimed to bring together One Health principles to connect canine and feline leishmaniosis epidemiology within urban centres of Morocco (Rabat and Fez) and assess the level of awareness of Moroccan veterinarians about facing this threat. Methods: A molecular survey was conducted for Leishmania DNA detection in canine (n = 155) and feline (n = 32) whole-blood samples. Three conventional polymerase chain reaction (PCR) protocols were implemented. The first PCR aimed at identifying infected animals by targeting Leishmania spp. kinetoplast minicircle DNA (kDNA). The second and third PCR targeted the Leishmania internal transcribed spacer region (ITS-1) and the Leishmania small subunit ribosomal RNA (SSUrRNA) gene, respectively, aiming at identification of the infecting species after Sanger sequencing-positive amplicons. Total immunoglobulin G (IgG) against Leishmania spp. was evaluated in 125 dogs by enzyme-linked immunosorbent assays (ELISA) using an in-house protocol, including three Leishmania-specific antigens (SPLA, rKDDR and LicTXNPx). Sera from 25 cats were screened for total IgG to Leishmania spp. by an indirect immunofluorescence antibody test (IFAT). An online questionnaire was presented to Moroccan veterinarians addressing their knowledge and practices towards animal leishmaniosis. Results: Overall, 19.4% of the dogs tested positive for Leishmania kDNA and ITS-1 and sequencing revealed infection with L. infantum among PCR-positive dogs. These animals presented a wide range of ELISA seropositivity results (16.7%, 34.9% and 51.6%) according to the tested antigens (rKDDR, SPLA and LicTXNPx, respectively). Use of kDNA-PCR revealed 12.5% cats positive to Leishmania spp. otherwise found to be seronegative by IFAT. Conclusions: A considerable prevalence of infection was identified in dogs from urban centres of Morocco. Additionally, this is the first report of feline infection with Leishmania spp. in this country and in urban settings. Moroccan veterinarians are aware that animal leishmaniosis is endemic in Morocco, representing a public health threat, and are knowledgeable about canine leishmaniosis diagnosis and treatment. [ABSTRACT FROM AUTHOR]

Background: Cats are now recognized as competent hosts for Leishmania infantum and a blood source for sand fly vectors. Although canine leishmaniosis (CanL) is endemic in Mediterranean Basin countries, large-scale epidemiological studies are lacking for feline leishmaniosis (FeL). This study aimed to assess the prevalence of L. infantum infections, associated risk factors, clinical signs, and clinicopathological abnormalities in domestic cat populations from six Mediterranean Basin countries. Methods: From 2019 to 2022, blood and serum samples of cats (n = 2067) living in Italy (n = 300), Greece (n = 297), Portugal (n = 295), France (n = 231), Israel (n = 313), and Spain (n = 631) were collected along with animal data (i.e., age, sex, breed, housing conditions, and geographical origin), clinical signs, and laboratory blood test parameters. Cats were grouped according to their age as kittens (up to 1 year), young (older than 1 and younger than 7 years), mature (between 7 and 10 years), and senior (older than 10 years). Serum samples were tested for L. infantum by immunofluorescence antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA), and blood samples of seropositive cats were tested for L. infantum kinetoplast deoxyribonucleic acid (kDNA). Viral infection by feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) was molecularly addressed in all cats enrolled. Statistical analysis was performed to evaluate the association between the risk of L. infantum infection and independent variables, and among co-infection of L. infantum with FIV and/or FeLV, clinical signs, and clinicopathological abnormalities. Results: Overall, 17.3% (358/2067) of cats scored positive for L. infantum by serological tests. Specifically, 24.7% were from Portugal, 23.2% from Greece, 16.6% from Israel, 15% from Spain, 13.3% from France, and 12.6% from Italy. Leishmania infantum DNA was detected in 15 seropositive animals. Housing condition and FIV infection proved to be risk factors for FeL. Leishmania seropositivity was significantly associated with weight loss, lymphadenomegaly, gingivostomatitis, and oral ulcers, as well as with reduced albumin and albumin/globulin ratio, increased total globulins and total proteins, leukocytosis, and thrombocytosis. Conclusions: This study provides, for the first time, a large-scale epidemiological survey on FeL and its clinical presentation, revealing that L. infantum circulates among domestic cats, especially shelter/free-roaming and FIV-infected animals, living in CanL endemic countries of the Mediterranean Basin. [ABSTRACT FROM AUTHOR]

Entomological investigations were conducted for the first time in urban forest remnants of Porto Velho, state of Rondônia, Brazil, to explore the transmission dynamics of Leishmania. Sand fly collections were carried out at ten sites, encompassing both canopy and ground strata, from October to December 2021. A total of 1,671 sand flies were collected, representing 42 species within 12 genera. Nyssomyia Antunesi (n = 384) and Psychodopygus davisi (n = 111) were the most abundant species. Molecular analyses targeting the V7V8 region (18S gene) unveiled the presence of sequences 100% identical to Leishmania infantum in females of Bichromomyia flaviscutellata (1), Nyssomyia Antunesi complex (6), Nyssomyia umbratilis (1), Nyssomyia sp. (1), Psychodopygus ayrozai (1), Ps. davisi (3), Psychodopygus paraensis (1), and Sciopemyia sordellii (1). Sequences 100% similar to Trypanosoma minasense were found in two samples of the Nyssomyia Antunesi complex, and two samples of Sc. sordellii presented 100% identity to a Trypanosoma sp. strain, previously identified in this same sand fly in Rondônia. Sequencing of Cytb fragment suggested Homo sapiens, Dasypus novemcinctus and Tamandua tetradactyla as the blood source for distinct sand flies. The identification of sequences similar to L. infantum in sand flies collected in urban forest fragments is noteworthy, correlating with the recent local and regional occurrence of autochthonous cases of human visceral leishmaniasis. However, further studies are imperative to ascertain the presence of hosts/reservoirs and evaluate the risk of L. infantum transmission to humans. [ABSTRACT FROM AUTHOR]

Leishmaniasis is an infectious disease caused by protozoa of the genus Leishmania, which is endemic in certain areas of Europe, such as southern Spain. The disease manifests in various clinical phenotypes, including visceral, cutaneous, mucosal, or asymptomatic leishmaniasis. This diversity in clinical outcomes may be influenced by the host immune response, with human leukocyte antigen (HLA) molecules playing a crucial role in determining susceptibility and progression of the infection. This study explores the association between specific HLA variants and Leishmania infantum infection. We recruited four cohorts: a control group, asymptomatic individuals, patients with symptomatic disease, and cohabitants of infected individuals. HLA typing was performed for all participants, followed by an association analysis with infection status and disease progression. Our findings indicate that the HLA-B*38 and HLA-C*03 alleles are associated with protection against L. infantum infection. These results contribute to a better understanding of the disease's progression, offer potential for new therapeutic approaches such as vaccines, and expand the existing knowledge in the literature. [ABSTRACT FROM AUTHOR]

Objective: We contribute to the understanding of the transmission dynamics of Leishmania infantum suggesting the involvement of rabbits as wild reservoirs. Results: The prevalence of infection was 86.0% (270/314 wild rabbits) ranging from 18.2% to 100% in natural geographical regions. The estimated average parasite load was 324.8 [CI 95% 95.3–554.3] parasites per mg of ear lobe ranging from 0 to 91,597 parasites/mg per tissue section. Conclusions: A positive correlation was found between skin parasite load in wild rabbits and human incidence with evidence of the presence of the same L. infantum genotypes in rabbits and humans, providing new epidemiological and biological basis for the consideration of wild rabbits as a relevant L. infantum wild reservoir. Molecular parasite surveillance reflects the great genotypic variability of the parasite population in wild rabbits. Most of these genotypes have also been found to infect humans, dogs and sandflies in the region. Our findings also highlight that direct genotyping of the parasite in host tissues should be used for molecular surveillance of the parasite instead of cultured isolates. [ABSTRACT FROM AUTHOR]

Feline leishmaniosis is infrequent worldwide, and cats have been suggested as secondary reservoirs for the parasite. However, specific diagnostic techniques for feline samples are scarce. In this study, we standardized an in-house indirect enzyme-linked immunosorbent assay (ELISA) using crude Leishmania infantum antigen to detect antibodies in feline samples from an endemic canine visceral leishmaniosis (CVL) area in the western border of Brazil. The results were compared with those of an indirect immunofluorescence assay (IFA). We tested semi-domiciled felines residing in Uruguaiana and Barra do Quaraí, Rio Grande do Sul. Among the 41 samples, 25 (61%) were positive using ELISA and 24 (58%) were positive using IFA (1:40). Our findings demonstrated a high seropositivity of feline samples from the endemic CVL area in the western border of Brazil, and we proposed the use of an in-house ELISA with crude antigen for population screening. This is the first serological survey on felines in a region where CVL is well established. [ABSTRACT FROM AUTHOR]

Canine leishmaniosis is a vector-borne disease caused by Leishmania infantum, and clinical manifestations of infection range from absent or severe to fatal and result from immune-mediated mechanisms. In dogs, the most common clinical signs of leishmaniosis include skin lesions and lymphadenomegaly. However, the presence of other nontypical signs has been described, and diagnosing these cases can be challenging. The aim of the present short communication was to describe the impact of the formation of circulating immunocomplexes due to L. infantum in a dog with leishmaniosis affected by a massive venous thrombus of the caudal vena cava and external iliac veins. On admission, the dog presented bilateral cutaneous vasculopathy of the thigh and renal disease due to L. infantum infection. Two weeks after starting anti-Leishmania treatment based on meglumine antimoniate and allopurinol administration, the animal developed acute claudication of the hind limbs with the presence of a thrombus in the caudal vena cava and the external iliac veins and a high level of circulating immunocomplexes detected by enzyme-linked immunosorbent assay. Exacerbation of the humoral immune response, along with deposition of circulating immune complexes in the tissues and the concurrent presence of kidney and liver damage, might have contributed to an imbalance in haemostasis in this patient. Future studies should evaluate and analyse the pathological mechanisms contributing to thrombosis in dogs with leishmaniosis. [ABSTRACT FROM AUTHOR]

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Leishmania infantum is considered the predominant Leishmania species responsible for visceral leishmaniasis (VL) in Greece but limited molecular-typing-based studies have been performed so far. We retrospectively analyzed data and serum samples collected from 3661 individuals suspected for VL in a sixteen-year period, from 2005 to 2020, to study the seasonality and demographic characteristics of VL cases and to define the L. infantum genotypes circulating in the country. Serum samples were tested with immunofluorescence assay and/or molecular assay. qPCR Leishmania-positive samples were subjected to genotypic analysis based on polymorphisms in 12 microsatellite regions of the internal transcribed spacers (ITSs) 1 and 2. We diagnosed 219 definite (6%, sample with a positive molecular assay and/or antibody titer ≥ 1:400) and 230 probable (6.3%, sample with antibody titer between 1:100 and 1:200) VL cases. Data analysis revealed that amongst VL-definite cases, the age group (≥65) constitutes the most affected factor, since 36.9% of the VL cases belonged to this age group. Amongst the VL definite cases, the most frequently reported symptoms were fever (83%), splenomegaly (49%), and hepatomegaly (40%), but this was not the case for immunocompromised patients that developed non-typical symptoms of leishmaniasis. Although no statistically significant differences in the overall seasonality of VL cases were observed, February and June showed a significantly higher proportion of VL cases compared to August and December. Genotyping of ITS1 and ITS2 regions revealed that all VL cases belong to ITS type A of L. infantum. Our study provides epidemiological information on VL and demonstrates for the first time, providing genotypic data, the circulation of ITS type A L. infantum in Greece. [ABSTRACT FROM AUTHOR]

Leishmaniosis is a vector-borne disease caused by protozoan parasites of the genus Leishmania, which are zoonotic and have an important impact on animal and public health globally. Between 2009 and 2023, blood samples from domestic dogs with clinical suspicion of leishmaniosis were received from 286 veterinary medical centres throughout mainland Portugal. An enzyme-linked immunosorbent assay (ELISA) was utilised to detect antibodies against Leishmania infantum antigens. Additionally, a complete blood count and tests for total proteins, urea, creatinine and alanine aminotransferase, as well as protein electrophoresis, were also performed. No significant relationship between sex and breed was observed. The age distribution was bimodal, with the highest prevalence of disease occurring at 2–5 years of age and a secondary peak occurring at 6 years or over (p < 0.001). No statistical correlation was observed between creatinine and urea across the ELISA serological groups. In contrast, both the gamma globulin levels (r = 0.45; p < 0.001) and the albumin/globulin ratio (r = −0.36; p < 0.001) exhibited moderate correlations with the ELISA. These findings support recent seroprevalence studies in dogs, with some geographical areas in Northern Portugal exhibiting the highest values, which may be the result of geographical shifts in parasite circulation due to climate change. [ABSTRACT FROM AUTHOR]

In the Americas, L. infantum (syn. chagasi) is the main cause of human visceral leishmaniasis. The role of neutrophils as part of the innate response to Leishmania spp. infection is dubious and varies according to the species causing the infection. Global expression of coding RNAs, microRNAs and long non-coding RNAs changes as part of the immune response against pathogens. Changes in mRNA and non-coding RNA expression resulting from infection by Leishmania spp. are widely studied in macrophages, but scarce in neutrophils, the first cell to encounter the trypanosomatid, especially following infection by L. infantum. Herein, we aimed to understand the expression patterns of coding and non-coding transcripts during acute in vitro infection of human neutrophils by L. infantum. We isolated neutrophils from whole blood of healthy male donors (n = 5) and split into groups: 1) infected with L. infantum (MOI = 5:1), and 2) uninfected controls. After 3 hours of exposure of infected group to promastigotes of L. infantum, followed by 17 hours of incubation, total RNA was extracted and total RNA-Seq and miRNA microarray were performed. A total of 212 genes were differentially expressed in neutrophils following RNA-Seq analysis (log2(FC)±0.58, FDR≤0.05). In vitro infection with L. infantum upregulated the expression of 197 and reduced the expression of 92 miRNAs in human neutrophils (FC±2, FDR≤0.01). Lastly, 5 downregulated genes were classified as lncRNA, and of the 10 upregulated genes, there was only 1 lncRNA. Further bioinformatic analysis indicated that changes in the transcriptome and microtranscriptome of neutrophils, following in vitro infection with L. infantum, may impair phagocytosis, apoptosis and decrease nitric oxide production. Our work sheds light on several mechanisms used by L. infantum to control neutrophil-mediated immune response and identifies several targets for future functional studies, aiming at the development of preventive or curative treatments for this prevalent zoonosis. Author summary: Visceral leishmaniasis is a neglected tropical disease that causes fever, weight loss, anemia and swelling of liver and spleen. About 2500 cases are reported annually in the Americas, with a high mortality rate. Understanding how the immune system of people with visceral leishmaniasis responds to this parasite is essential for the development of preventive and curative methods. In order to understand how gene expression is modulated during visceral leishmaniasis, we infected in vitro cultured human neutrophils, the first immune cells to be recruited in this infection, with Leishmania infantum, the protozoan that causes visceral leishmaniasis in the Americas. Next, we measured the expression of coding RNAs, responsible for the production of proteins required for an effective immune response, and of non-coding RNAs, able to control these coding RNAs, thus helping or hindering host response to infection. Analysis of coding and non-coding RNAs points to an attempt by the parasite to modulate the transcriptome of host cells, influencing the host's response to infection. Our work identifies several targets for future functional studies, aiming at the development of preventive or curative treatments for this prevalent zoonosis. [ABSTRACT FROM AUTHOR]

Cullin-1-RING ubiquitin ligases (CRL1) or SCF1 (SKP1-CUL1-RBX1) E3 ubiquitin ligases are the largest and most extensively investigated class of E3 ligases in mammals that regulate fundamental processes, such as the cell cycle and proliferation. These enzymes are multiprotein complexes comprising SKP1, CUL1, RBX1, and an F-box protein that acts as a specificity factor by interacting with SKP1 through its F-box domain and recruiting substrates via other domains. E3 ligases are important players in the ubiquitination process, recognizing and transferring ubiquitin to substrates destined for degradation by proteasomes or processing by deubiquitinating enzymes. The ubiquitin-proteasome system (UPS) is the main regulator of intracellular proteolysis in eukaryotes and is required for parasites to alternate hosts in their life cycles, resulting in successful parasitism. Leishmania UPS is poorly investigated, and CRL1 in L. infantum, the causative agent of visceral leishmaniasis in Latin America, is yet to be described. Here, we show that the L. infantum genes LINF_110018100 (SKP1-like protein), LINF_240029100 (cullin-like protein-like protein), and LINF_210005300 (ring-box protein 1 –putative) form a LinfCRL1 complex structurally similar to the H. sapiens CRL1. Mass spectrometry analysis of the LinfSkp1 and LinfCul1 interactomes revealed proteins involved in several intracellular processes, including six F-box proteins known as F-box-like proteins (Flp) (data are available via ProteomeXchange with identifier PXD051961). The interaction of LinfFlp 1–6 with LinfSkp1 was confirmed, and using in vitro ubiquitination assays, we demonstrated the function of the LinfCRL1(Flp1) complex to transfer ubiquitin. We also found that LinfSKP1 and LinfRBX1 knockouts resulted in nonviable L. infantum lineages, whereas LinfCUL1 was involved in parasite growth and rosette formation. Finally, our results suggest that LinfCul1 regulates the S phase progression and possibly the transition between the late S to G2 phase in L. infantum. Thus, a new class of E3 ubiquitin ligases has been described in L. infantum with functions related to various parasitic processes that may serve as prospective targets for leishmaniasis treatment. Author summary: In eukaryotes, intracellular proteolysis is mostly performed by the ubiquitin-proteasome system (UPS), which regulates numerous cellular functions by ubiquitinating regulatory proteins that may be degraded by the proteasome. Substrate recruitment and ubiquitination are mediated by E3 ubiquitin ligases. CRL1 (Cullin-1-RING ligases) are the largest and most researched family of E3 proteins in eukaryotes, comprising SKP1, Cullin1, RBX1, and F-box proteins. The life cycle and infection of Leishmania depends on the UPS, and the role of CRL1 in these parasites has not been studied. Here, we identified CRL1 proteins in L. infantum and demonstrated the assembly of LinfCRL1, which shares a three-dimensional structure with the H. sapiens complex, suggesting a functional relationship. A set of LinfSkp1 and LinfCul1 protein partners were found to be associated with several cellular processes, indicating their pleiotropic effects. Six F-box proteins were found interacting with LinfSkp1 and were designated F-box-like proteins (Flp). Flp1 assembly in an active LinfCRL1(Flp1) complex capable of ubiquitin transfer. Our study demonstrated the critical role of LinfSKP1 and LinfRBX1 in the parasite, whereas knockout of LinfCUL1 resulted in impaired proliferation and duplication, along with the stimulation of rosette formation. In addition, LinfCul1 regulates L. infantum cell cycle progression. Overall, we characterized a novel E3 ubiquitin ligase class that could potentially be targeted by antileishmanial drugs. [ABSTRACT FROM AUTHOR]

[Display omitted] • Risk factors shaping host-parasite interactions impact the clinical management of canine leishmaniosis. • Renal and ocular findings are major risk factors for LeishVet stages III and IV. • Diagnosis of comorbidities is key to managing dogs with leishmaniosis, although independent of their clinical outcome. • A complex polygenic component underlies the development of severe clinical forms of leishmaniosis in certain purebred dogs. • Protective immunity against Leishmania parasites is related to genetic factors involved in the ER stress and UPR pathways. Canine leishmaniosis (CanL), caused by Leishmania infantum , is a complex disease of growing importance in Europe. Clinical manifestations result from the down-modulation of the host immune response through multiple host-parasite interactions. Although several factors might influence CanL progression, this is the first known study evaluating risk factors for its different clinical stages in a large referral hospital population (n = 35.669) from an endemic area, over a 20 year period. Genome-wide scans for selection signatures were also conducted to explore the genomic component of clinical susceptibility to L. infantum infection. The prevalence of CanL was 3.2% (16.7% stage I; 43.6% stage II; 32.1% stage III; 7.6% stage IV). Dog breed (crossbreed), bodyweight (<10 kg), living conditions (indoors), regular deworming treatment, and being vaccinated against Leishmania significantly decreased the transmission risk and the risk for developing severe clinical forms. Conversely, the detection of comorbidities was associated with advanced clinical forms, particularly chronic kidney disease, neoplasia, cryptorchidism, infectious tracheobronchitis and urate urolithiasis, although those did not impact the clinical outcome. Significant associations between an increased risk of severe clinical stages and findings in the anamnesis (renal or skin-related manifestations) and physical examination (ocular findings) were also detected, highlighting their diagnostic value in referred cases of CanL. Sixteen breeds were found to be significantly more susceptible to developing severe stages of leishmaniosis (e.g. Great Dane, Rottweiler, English Springer Spaniel, Boxer, American Staffordshire Terrier, Golden Retriever), while 20 breeds displayed a clinical resistantance phenotype and, thus, are more likely to mount an efficient immune response against L. infantum (e.g. Pointer, Samoyed, Spanish Mastiff, Spanish Greyhound, English Setter, Siberian Husky). Genomic analyses of these breeds retrieved 12 regions under selection, 63 candidate genes and pinpointed multiple biological pathways such as the IRE1 branch of the unfolded protein response, which could play a critical role in clinical susceptibility to L. infantum infection. [ABSTRACT FROM AUTHOR]

A series of 61 thiazolidine-2,4-diones bearing a styryl group at position 5 was synthesized in 2–5 steps and their structure was proved by elemental and spectral analyses. The compounds obtained were evaluated in vitro against the promastigote stage of the kinetoplastid parasite Leishmania infantum and the human HepG2 cell line, to determine selectivity indices and to compare their activities with those of antileishmanial reference drugs. The study of structure–activity relationships indicated the potential of some derivatives bearing a nitro group on the phenyl ring, especially when located at the meta position. Thus, among the tested series, compound 14c appeared as a hit compound with good antileishmanial activity (EC50 = 7 µM) and low cytotoxicity against both the hepatic HepG2 and macrophage THP-1 human cell lines (CC50 = 101 and 121 µM, respectively), leading to good selectivity indices (respectively, 14 and 17), in comparison with the reference antileishmanial drug compound miltefosine (EC50 = 3.3 µM, CC50 = 85 and 30 µM, SI = 26 and 9). Regarding its mechanism of action, among several possibilities, it was demonstrated that compound 14c is a prodrug bioactivated, predominantly by L. donovani nitroreductase 1, likely leading to the formation of cytotoxic metabolites that form covalent adducts in the parasite. Finally, compound 14c is lipophilic (measured CHI LogD7.7 = 2.85) but remains soluble in water (measured PBS solubility at pH7.4 = 16 µM), highlighting the antileishmanial potential of the nitrostyrylthiazolidine-2,4-dione scaffold. [ABSTRACT FROM AUTHOR]

Simple Summary: In Europe, the protozoan parasite Leishmania infantum is transmitted by sandflies. A recent study found that sheep had antibodies against this parasite, triggering questions about the role of these animals in the life cycle. Therefore, blood samples from sheep and goats from two southern German states, Baden-Wuerttemberg (BW) and Bavaria (BAV), were tested for antibodies using a method called ELISA. Details, such as the species, sex and age, were recorded to assess any association between seropositivity and these animal characteristics. A total of seven sheep flocks from BW and seven from BAV were included, comprising 274 sheep and 10 goats in BW, and 277 sheep and 78 goats in BAV. In BW, four sheep from three flocks had antibodies, and in BAV, the same number of sheep tested positive but from four different flocks. Overall, 1.45% of sheep had antibodies against L. infantum, while all goats tested negative. No significant links were found between the presence of antibodies and the factors examined. Our study shows that sheep in areas not typically associated with Leishmania can still be exposed to it. Further research is needed to determine if sheep could help us identify new areas where sandflies live and where the disease could spread. In Europe, Leishmania infantum is the most prevalent Leishmania species, and this protozoan is transmitted by phlebotomine sandflies. A recent publication has shown that sheep harbor L. infantum antibodies. This raises questions about the epidemiological role of small ruminants. Therefore, sera from small ruminants located in two southern German federal states, Baden-Wuerttemberg (BW) and Bavaria (BAV), were analyzed with an ELISA to determine the presence of L. infantum antibodies. The species, sex and age (gimmer vs. ewe) were recorded, and a univariate analysis was conducted to determine possible associations. In total, seven sheep flocks (274 sheep/10 goats) from BW and seven sheep flocks (277 sheep/78 goats) from BAV were examined. In BW, four sheep from three flocks tested positive for L. infantum antibodies. In BAV, the same number of positive sheep were detected but in four flocks. The total seropositivity rate in sheep was 1.45%. All goats tested negative. No significant association (p > 0.05) was detected between Leishmania seropositivity and the variables evaluated. Our study reveals the exposure of sheep to L. infantum in a non-endemic area. Further investigation is needed to determine whether sheep can be used as sentinels to identify new phlebotomine habitats and Leishmania risk areas. [ABSTRACT FROM AUTHOR]