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Background: Trypanosomatids include the genera Trypanosoma and Leishmania, which are the etiological agents of important human diseases. These pathogens present unique mechanisms of gene expression characterized by functionally unrelated genes positioned in tandem and organized into polycistronic transcription units transcribed in a large pre-mRNA by RNA Polymerase II. Since most of the genome is constitutively transcribed, gene expression is primarily controlled by post-transcriptional processes. As in other organisms, histones in trypanosomatids contain a considerable number of post-translational modifications, highly conserved across evolution, such as the acetylation and methylation of some lysines on histone H3 and H4. These modifications have been mainly studied in Trypanosoma spp. The aim of this work was to elucidate the distribution of histone H3 lysine 4 trimethylation (H3K4me3) over the chromatin landscape of Leishmania infantum, the causative agent of canine and human leishmaniasis in the Mediterranean region. To this end, we investigated by chromatin immunoprecipitation (ChIP)-sequencing either the promastigotes (the flagellated motile form) and the amastigotes (the intracellular form) in an in vitro infection model. Results: The chromatin was prepared from THP-1 cells non infected, THP-1 cells infected with L. infantum MHOM/FR/78/LEM75, and THP-1 cells non infected and mixed with L. infantum MHOM/FR/78/LEM75 promastigotes. ChIP was conducted using anti-H3K4me3 or anti-H3K27me3 antibodies and ChIP-seq was performed on an Ion S5 sequencer. We showed that histone H3K4me3 is mainly enriched at transcription start sites (67%) or internally within the polycistronic transcription units (30%), with no differences between L. infantum promastigotes and amastigotes. Moreover, the enriched regions co-localize with another hallmark of transcriptional activation (histone H3 acetylation) in L. major, a species characterized by a high degree of synteny with L. infantum. Conclusions: These findings expand our knowledge of the epigenomics of Leishmania parasites, focusing on epigenetic markers associated with transcription in L. infantum, and will contribute to elucidate the transcriptional mechanisms in these pathogens. [ABSTRACT FROM AUTHOR]

Background: This article presents the first documented case of visceral leishmaniasis in an adult patient in Slovakia, with an unusual involvement of the colon. Leishmaniasis is a tropical parasitic infection transmitted by sandflies, predominantly occurring in developing countries. Case presentation: The patient was a 57-year-old woman without comorbidities or immunocompromising conditions. The clinical presentation included non-specific symptoms such as prolonged fever, marked weakness, hepatosplenomegaly, and generalized lymphadenopathy. The diagnosis was established based on histopathological examination and bone marrow PCR, which confirmed the presence of Leishmania infantum amastigotes. After initiating treatment with amphotericin B, the patient's condition improved, but a relapse occurred shortly thereafter, presenting with colonic involvement—an extremely rare occurrence in immunocompetent patients. The diagnosis of relapse was confirmed by repeated histopathological examination and molecular analysis. Treatment was re-initiated with an extended regimen of amphotericin B and additional supportive therapy. Conclusion: This case highlights the importance of early diagnosis and targeted treatment of visceral leishmaniasis, even in non-endemic areas of the world. It emphasizes the need to consider this diagnosis in the differential diagnosis of patients with unexplained febrile conditions and hepatosplenomegaly, as the clinical presentation can often mimic various other infectious diseases or hematological malignancies, significantly complicating the diagnostic process. [ABSTRACT FROM AUTHOR]

Background: Leishmania infantum is the only prevalent Leishmania species in Europe and manifesting predominantly as cutaneous or visceral leishmaniasis, whereas new world species like Leishmania (L.) braziliensis are well known pathogens in mucocutaneous leishmaniasis. Mucosal leishmaniasis caused by L. infantum is a rare clinical condition with only few cases described in literature. In contrast to our case, mostly immunocompromised patients with no history of leishmaniasis are affected. Case presentation: We describe the case of a 77-year-old German male who developed an ulcerous lesion of the tongue. As oral cancer was suspected, the patient underwent surgery. After suspected diagnosis of Leishmania spp. in histopathology, the patient was referred to our department for further diagnostics and treatment. Relapse from a cutaneous leishmaniasis acquired in Spain is likely, as L. infantum could be identified as the causative agent. The patient recovered after treatment. Conclusions: Mucosal leishmaniasis caused by L. infantum is rare and usually mistaken for malignancy. As demonstrated, it can be preceded by cutaneous leishmaniasis of the immunocompetent. Due to possible dissemination systemic treatment should be applied. [ABSTRACT FROM AUTHOR]

Cutaneous and visceral leishmaniasis are public health problems in Africa, Asia, Europe, and America. The treatment has a high cost and toxicity. Thus, this work aims to evaluate the leishmanicidal activity of alpha-bisabolol and its three synthetic derivatives, P1, P2, and P3, on the promastigotes and amastigotes Leishmania infantum and L. amazonensis forms. Alpha-bisabolol showed the lowest IC50 with 3.43 for L. amazonensis promastigotes, while P1 was the most toxic for L. infantum with an IC50 of 9.10. The derivative P3 was better for the amastigote form, with an IC50 of 3.39 for L. amazonensis. All the compounds effectively decreased the intracellular load of amastigote and its ability to turn promastigote again. Thus, alpha-bisabolol and its three synthetic derivatives were effective in their leishmanicidal activity. Therefore, it can be an option for developing new treatments against leishmaniasis. [ABSTRACT FROM AUTHOR]

Dogs are the main reservoir host of Leishmania infantum, etiological agent of zoonotic visceral leishmaniasis (ZVL). An effective vaccine against Canine Visceral Leishmaniasis (CVL) will help the control and elimination of ZVL. In this study, we evaluated in dogs the safety, immunogenicity, and efficacy of a live attenuated Leishmania major Centrin gene-deleted (LmCen−/−) as a vaccine. Two doses (106 or 107) of LmCen−/− vaccine were administered intradermally in a prime-boost regimen. Both vaccine doses induced equally high level of IgG anti-Leishmania and exhibited strong antigen-specific cellular responses with IFN-γ production by CD4 + T cells one-month post-immunization. A second cohort of dogs was vaccinated with 106LmCen−/− parasites one month prior to their transfer to a CVL endemic focus in Northern Tunisia for exposure to sand fly bites during three successive transmission seasons. Dogs were exposed to bite from naturally infected sandflies for 3–5 months per year. Our results showed that only 1/11 vaccinated dogs became PCR positive for Leishmania and developed clinical signs of CVL. In contrast, 4/11 unvaccinated dogs were tested PCR positive for Leishmania and displayed oligosymptomatic CVL, demonstrating that immunization with LmCen−/− vaccine confers long-term protection with an efficacy of 82.5% against CVL in natural transmission settings. [ABSTRACT FROM AUTHOR]

Canine leishmaniosis (CanL), caused by Leishmania infantum, is a widespread vector-borne disease. In Italy, an endemic region for CanL, overlapping transmission of L. infantum and tick-borne pathogens (TBPs) like Anaplasma phagocytophilum and Ehrlichia canis is increasingly reported. Dogs with clinical leishmaniosis often show higher co-infection rates and pronounced clinicopathological abnormalities. This study presents a systematic literature review and new findings from southern Italy, focusing on co-infections with E. canis and A. phagocytophilum in clinically healthy L. infantum seropositive and seronegative dogs. The systematic review identified two eligible studies. The first reported 34/488 (7%) dogs L. infantum seropositive, with 11.8% also seropositive for A. phagocytophilum. Among 454 seronegative dogs, 3% were seropositive for A. phagocytophilum and 2.4% for E. canis. The second study identified 154/1260 (12.2%) dogs L. infantum seropositive, with co-infection rates of 0.6% and 1.9% for A. phagocytophilum and E. canis, respectively. Among 1106 seronegative dogs, 1.3% were seropositive for A. phagocytophilum and 2.3% for E. canis. In the retrospective study from southern Italy, 90/154 (58.4%) dogs were L. infantum seropositive, with co-infection rates of 4.4% for A. phagocytophilum and 2.2% for A. phagocytophilum and E. canis. Among 64 seronegative dogs, 1.6% showed similar co-infections. This is the first systematic review in Italy, documenting low and comparable co-infection rates with A. phagocytophilum and E. canis in clinically healthy dogs, regardless of L. infantum serostatus. These findings suggest that co-infections may occur independently, offering insights into vector-borne disease dynamics in endemic areas. [ABSTRACT FROM AUTHOR]

Background: In recent years, cases of leishmaniosis have been described in animals housed in captivity in zoos in Spain [Bennett's wallaby (Macropus rufogriseus rufogriseus), orangutan (Pongo pygmaeus pygameus), and European otter (Lutra lutra)]. Some of these zoological parks are in endemic areas for both human and animal leishmaniosis, thus it should be very important to include this zoonosis in the differential diagnosis. Methods: The study was carried out in two zoological parks in Madrid, Madrid Zoo and Faunia, and analyzed seven meerkats. Serological tests [rK-39 and enzyme-linked immunosorbent assay (ELISA)] and molecular tests [nested polymerase chain reaction (PCR) and real-time PCR] were performed to detect Leishmania DNA. Additionally, an entomological study was carried out in both zoological parks, with molecular tests performed on female Phlebotomus perniciosus sand flies to determine their blood meal source and detect Leishmania DNA. Results: Two meerkats were positive for L. infantum. A 9-year-old male from the Madrid Zoo died suddenly, showing pale mucous membranes and bilateral noninflammatory alopecia and hyperpigmentation in the lateral area of the eyes. Positive results were obtained in serology, nested PCR, and real-time PCR (blood, conjunctival and oral swabs, hair, spleen, lymph node, liver, kidney, and skin), as well as numerous amastigotes in the liver and kidney tissue samples. The other meerkat, a 12-year-old male from Faunia that is still alive, presented an alopecic lesion at the base of the tail. Positive results were obtained by nested and real-time PCR from different tissues such as blood, hair, oral, and conjunctival swabs. It was treated with oral allopurinol (25 mg/kg) and miltefosine (2 mg/kg), but the molecular diagnosis remained positive after 8 months, regarding it as a mild stage of the disease. The rest of the tested meerkats were negative. The presence of P. perniciosus phlebotomine sand flies was also detected in both zoos. Although no L. infantum DNA was detected in any of sand flies analyzed, it was determined that their food sources were rabbits and humans. Conclusions: To our knowledge, this study describes, for the first time, the detection and infection by L. infantum in meerkats (Suricata suricatta). [ABSTRACT FROM AUTHOR]

Leishmaniasis is a neglected tropical disease caused by a protozoan of the genus Leishmania, which has visceral and cutaneous forms. The symptoms of leishmaniasis include high fever and weakness, and the cutaneous infection also causes lesions under the skin. The drugs used to treat leishmaniasis have become less effective due to the resistance mechanisms of the protozoa. In addition, the current compounds have low selectivity for the pathogen, leading to various side effects, which results in lower adherence to treatment. Various strategies were developed to solve this problem. The bioconjugation between natural compounds with antimicrobial activity and cell-penetrating peptides could alleviate the resistance and toxicity of current treatments. This work aims to conjugate the cell penetration peptide TAT to the guanidine GVL1. The GVL1-TAT bioconjugate exhibited leishmanicidal activity against Leishmania amazonensis and Leishmania infantum with a high selectivity index. In addition, the bioconjugate was more active against the intracellular enzyme CPP than the individual compounds. This target is very important for the viability and virulence of the parasite within the host cell. Docking studies confirmed the higher interaction of the conjugate with CPP and suggested that other proteins, such as trypanothione reductase, could be targeted. Thus, the data indicated that guanidines conjugated with cell-penetrating peptides could be a good approach for developing antileishmanial molecules. [ABSTRACT FROM AUTHOR]

In Brazil, Visceral Leishmaniases is caused by Leishmania infantum, and domestic dogs are the main reservoirs in its urban transmission cycle. As an alternative to euthanizing dogs, miltefosine has been used to treat canine visceral leishmaniasis since 2016. In this study, we have assessed the efficacy of miltefosine for treating canine visceral leishmaniasis in a new endemic area through follow-up of naturally infected dogs was evaluated. The clinical, parasitological, and histopathological characteristics of 21 dogs naturally infected with L. infantum were assessed at three time points: on the day before initiating miltefosine treatment (T0), immediately after treatment completion (T1), and 6 months after treatment completion (T2). Three dogs were treated exclusively with miltefosine, while eighteen received combination therapy with miltefosine with other treatments such as allopurinol, domperidone and immunotherapy. Skin biopsies were obtained from the abdomen to assess inflammatory responses and to quantify parasite loads using qPCR. The parasites were isolated using aspirates acquired from popliteal lymph nodes. Molecular and parasitological analyses confirmed the presence of L. infantum in all dogs, validating the effectiveness of skin and lymph node samples for diagnosis. The clinical conditions of the infected animals were improved and the skin parasite load decreased after treatment, even when distinct combination therapies were performed. The histopathological assessment revealed a miltefosine-induced reduction in the inflammatory response and a decrease in amastigotes number. Furthermore, a positive correlation was established between the reduction in parasite load and the enhancement of clinical scoring, as well as a reduction in the skin inflammatory response. Our findings suggest that miltefosine-based combination therapies reduce skin parasite load and improve clinical outcomes, while the dogs treated with miltefosine alone showed increased parasitic load and worsened clinical staging at T2. Considering this data belonging to a recent transmission area, treatment strategy suggests effective in controlling canine visceral leishmaniasis. [ABSTRACT FROM AUTHOR]

Background: We standardized two recombinase polymerase amplification (RPA) assays coupled with lateral flow (LF) strips for the detection of Leishmania braziliensis and Leishmania infantum kinetoplast DNA (kDNA). Methods: The RPA-LF assays were tested at different temperatures and reaction times, using DNA from cultured L. braziliensis and L. infantum. The L. infantum RPA-LF was also tested using clinical samples (bone marrow and skin) from infected and uninfected dogs. Results: The detection limits (analytical sensitivity) of the assays were 0.04 pg/μl and 0.04 ng/μl for L. braziliensis and L. infantum kDNA, respectively. Using clinical samples, the L. infantum RPA-LF successfully detected the parasite kDNA in bone marrow (21/30; 70.0%) and skin samples (23/30, 76.6%) from naturally infected dogs. We found an almost perfect agreement (kappa = 0.807) between RPA-LF for L. infantum and our reference quantitative real-time polymerase chain reaction (qPCR), considering clinical samples with a quantification cycle (Cq) < 30, whereas the agreement with samples with a Cq > 30 (lower parasite loads) was moderate (kappa = 0.440). Conclusions: The RPA-LF assays developed here may be promising diagnostic tools for point-of-care diagnosis of L. infantum and L. braziliensis infection in dogs, particularly in remote rural areas lacking laboratory infrastructure. [ABSTRACT FROM AUTHOR]

The European Bison (Bison bonasus) is the largest mammal in Europe and is classified as an endangered species. Leishmaniosis is a vector-borne disease caused by the protozoan Leishmania infantum. In general, this infection has been associated with dogs, cats, and humans. However, epidemiologic studies and reports confirm that the parasite is able to infect many other mammalian species. Recent evidence has demonstrated that ruminants in endemic areas are exposed to L. infantum infection. Moreover, climate change has allowed the northward spread of vector species, causing the expansion of L. infantum infection in regions traditionally classified as nonendemic in Europe. The aim of this study was to determine the presence or absence of anti–L. infantum antibodies in serum samples from 343 European bison in Poland, collected from 2017 to 2023. For this purpose, the presence of anti-Leishmania antibodies was analyzed using an in-house multispecies ELISA. Anti-Leishmania antibodies were detected in four animals, an overall seroprevalence of 1.17%. The results provide scientific evidence of serologic exposure to the parasite in Poland, a country previously considered nonendemic for L. infantum infection. [ABSTRACT FROM AUTHOR]

Dogs act as definitive hosts for several diseases caused by protozoa, some of which are zoonotic. Due to their close contact with humans and other animals, they play a crucial role in the transmission of these diseases. Although infection with Neospora caninum or Leishmania infantum is not a determining factor for another, co-infection with these protozoa can aggravate clinical signs and increase the mortality rate. Though, there are reports of success in the treatment of neosporosis, the prognosis is generally considered unfavorable, especially in young dogs. The objective of this study is to report a case of infection by both protozoa in a dog treated at a university veterinary hospital, highlighting the clinical remission of nervous signs of neosporosis after treatment with the combination of sulfadoxine and clindamycin, followed by reduction of clinical signs of canine leishmaniasis (CanL) with treatment with miltefosine and allopurinol. Despite the worse prognosis for co-infection with N. caninum and L. infantum, the dog presented remission of neurological signs and a reduction in parasite load and clinicopathological signs associated with CanL.

Abstract Background COVID-19 is a pandemic involving coinfection with other opportunistic microorganisms, including parasites such as Leishmania infantum. The present study aimed to determine the frequency of L. infantum infection and its role in disease and mortality among symptomatic COVID-19 patients in comparison with the non-COVID-19 control group in the endemic area of visceral leishmaniasis (VL) in Iran. Methods A total of 250 blood samples from hospitalized COVID-19 patients and 231 blood samples from non-COVID-19 patients as a control group were collected in Mashkin Shahr city, Iran from October 2020 to May 2021, and anti-Leishmania infantum antibodies were detected via direct agglutination test (DAT). For confirmation of non-COVID-19 samples as the control group, anti-COVID-19 IgG antibodies were measured via indirect ELISA. Additionally, demographic data, clinical data, and disease outcomes were recorded. Results Overall, 46 (18.4%) COVID-19 patients and 18 (7.8%) non-COVID-19 patients had anti-Leishmania infantum antibodies at titers of 1:200 or higher according to the DAT (p value $$\:

International pet travel and commercial operations have increased animal disease importation risks, including for Leishmania infantum, a deadly parasite of humans and domestic dogs. Collaborating as an interdisciplinary working group, we developed an operational tool for veterinary and public health practitioners to assess and manage L. infantum risk in dogs imported to the United States. Overall risk varies by dog, human, and geographic factors but could be high without proper controls. We determined dog risk management strategies should include application of sand fly insecticides and repellents, sterilization, and treatment. US public health authorities can use a One Health approach to manage L. infantum importation risks via infected dogs.

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.

Objective: To determine the overall and pooled prevalence of Leishmania (L.) infantum in sandfly vectors in Iran. Methods: The present research conducted a systematic review and meta-analysis and searched regional databases such as PubMed, Scopus, Web of Science (WoS), Embase, PAHO Iris, LILACS, WHO Iris, and local databases named: SID, Magiran, Civilica, and also grey literatures. The current research included studies that were conducted in Iran and examined L. infantum in different sandfly vectors. The studies’ quality assessment/risk of bias assessment was evaluated by the Joanna Briggs Institute Critical Appraisal Checklist for prevalence data studies, and the data were analyzed by Stata 14 software. In addition, we examined 22 primary studies to estimate the overall prevalence of L. infantum among various vectors of visceral leishmaniasis. Results: According to the meta-analysis, the pooled prevalence of Phlebotomus (Ph.) tobbi, Ph. alexandri, Ph. kandelaki, Ph. perfiliewi, Ph. major, Ph. keshishiani were 5.34%, 4.36%, 2.23%, 1.79%, 4.37% and 1.18%. Ph. tobbi has the highest infection rate (25.00%) of L. infantum among the sandfly vectors. Conclusions: Visceral leishmaniasis is widespread in Fars, Ardebil, and East-Azerbaijan provinces, which are the most important endemic regions in Iran.

Canine leishmaniasis (CanL) is a zoonotic disease caused by infection with Leishmania infantum and transmitted by female phlebotomine sandflies. It is prevalent in the Mediterranean basin, including the Maghreb region which comprises of Algeria, Tunisia, and Morocco. The environmental conditions in this area provide a suitable habitat for the proliferation of the phlebotomine sandfly, making it an endemic region for CanL. In this context, we carried out a systematic review and meta-analysis on the prevalence of canine leishmaniasis in these three Maghreb countries, identifying the risk factors for L. infantum infection. Data were collected from 27 papers published between 1973 and 2022 selected from five databases: PubMed, Google Scholar, ResearchGate, ScienceDirect and Scopus. Our selection criteria included both descriptive and analytical studies on canine leishmaniasis in Maghreb, as well as epidemiological studies that reported the prevalence of leishmaniasis infection. The meta-analysis was performed using Review Manager (RevMan) software version 5.4.1. based on predefined inclusion criteria. A total of 11,736 dogs were included in the analysis, among which 2481 were tested Leishmania positive cases. Over the years, the highest prevalence (68.32%) was recorded in Tiaret (Algeria) and the lowest (4.3%) in Tunisia. The examination of the risk factors revealed a heightened incidence of the disease in dogs of shepherd breeds, living in rural environments, displaying no apparent symptoms, and falling within the age range of 2 to 4 years. The findings presented in this study contribute a significant value to the exploration of leishmaniasis in this region, particularly by shedding light on the associated risk factors. Ultimately, substantial endeavors are warranted to enhance prevention, treatment, and overall control of the disease.

Abstract 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. Graphical Abstract

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

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.

Background: COVID-19 is a pandemic involving coinfection with other opportunistic microorganisms, including parasites such as Leishmania infantum. The present study aimed to determine the frequency of L. infantum infection and its role in disease and mortality among symptomatic COVID-19 patients in comparison with the non-COVID-19 control group in the endemic area of visceral leishmaniasis (VL) in Iran. Methods: A total of 250 blood samples from hospitalized COVID-19 patients and 231 blood samples from non-COVID-19 patients as a control group were collected in Mashkin Shahr city, Iran from October 2020 to May 2021, and anti-Leishmania infantum antibodies were detected via direct agglutination test (DAT). For confirmation of non-COVID-19 samples as the control group, anti-COVID-19 IgG antibodies were measured via indirect ELISA. Additionally, demographic data, clinical data, and disease outcomes were recorded. Results: Overall, 46 (18.4%) COVID-19 patients and 18 (7.8%) non-COVID-19 patients had anti-Leishmania infantum antibodies at titers of 1:200 or higher according to the DAT (p value 0.05). In the COVID-19 group, there was no statistically significant association between coinfection with L. infantum and sex, residency, treatment outcome, or symptoms. Conclusion: The results of this study revealed that the overall seroprevalence of L. infantum infection among patients with COVID-19 was 2.66 times greater than that in the control group, which was statistically significant. It seems that L. infantum infection can be considered a risk factor for COVID-19, particularly in VL endemic areas. Clinical trial number: Not applicable. [ABSTRACT FROM AUTHOR]

Background: Visceral leishmaniosis (VL) is the most severe form of human leishmaniosis, with an estimated 95% case fatality if left untreated. Dogs act as peridomestic reservoir hosts for the protozoan parasite Leishmania infantum, a causative agent for human leishmaniosis, endemic throughout the Mediterranean basin. To assure consistent and accurate surveillance of canine infection and prevent transmission to people, consistent diagnosis of canine L. infantum infection across this region is essential for protecting both human and animal health. Our goal was to compare the accuracy, sensitivity and specificity of enzyme-linked immunosorbent assays (ELISA) and immunofluorescence antibody tests (IFAT), performed at seven academic veterinary diagnostic centres across southern Europe and Israel. Methods: We performed a known sample "ring" trial to compare L. infantum quantitative serological tests. Two hundred seventy-two (n = 272) canine serum samples of known serological status were chosen from these sites, representative of the region. In-house or commercial ELISA and IFAT were performed according to each laboratory's specifications. Latent Class Analysis (LCA) was used to determine sensitivity and specificity of each test. True and false positives were calculated to determine the probability of identifying samples. Results: Sensitivity and specificity for ELISA ranged from 95 to 99% and 92% to 97%, respectively, with moderate variability from one site. Sensitivity and specificity for IFAT ranged from 89 to 99% and 83% to 94%, respectively, with increased variability compared to ELISA. Overall test agreement was 78% with a pair-wise agreement between 65 and 89%. Conclusions: All sites demonstrated substantial comparative diagnostic accuracy, with good agreement based on known seropositive and seronegative samples. Studies and interventional trials that use these tests will remain valid because of high diagnostic agreement between sites. [ABSTRACT FROM AUTHOR]

Background: Leishmaniasis, caused by Leishmania protozoan parasites transmitted by Phlebotomine sand flies, is a significant public health concern in the Mediterranean basin. Effective monitoring of Leishmania-infected sand flies requires standardized tools for comparing their distribution and infection prevalence. Consistent quantitative real-time PCR (qPCR) parameters and efficient DNA extraction protocols are crucial for reliable results over time and across regions. However, the absence of standardized technical recommendations for Leishmania DNA detection hinders effective surveillance. This study aimed to compare different DNA extraction protocols and conduct a qPCR-based External Quality Assessment (EQA) through a multicenter study involving nine reference laboratories, with a focus on optimizing Leishmania DNA detection in sand fly. Methodology/Principal findings: EQA samples consisted of Leishmania infantum and L. major species, at concentrations ranging from 101 to 104 parasites/mL. All but one center detected all concentrations, demonstrating strong diagnostic proficiency. The ability to detect low concentrations highlighted the robustness of the qPCR assay used, though variations in Cq values indicated differences in sensitivity related to technical capabilities or DNA extraction kit performance. A comparative analysis of seven DNA extraction methods identified the EZ1 DSP Virus Kit and QIAamp DNA mini-kit as the most efficient, supporting their use in standardized protocols. The study also assessed the effects of lyophilization and shipment conditions, showing no significant compromise in Leishmania detection despite slight variations in Cq values. Experimentally infected sand flies were included to simulate field conditions, and all centers successfully detected positive samples with varying Cq values, probably reflecting differences in infection load. Conclusion and significance: This study emphasizes the importance of standardized DNA extraction protocols and continuous quality assurance for accurate Leishmania DNA detection. The results highlight the superior performance of certain extraction kits and the need for ongoing technical training, essential for reliable leishmaniasis surveillance, particularly in field settings with low infection densities. Author summary: Leishmaniasis is a disease caused by Leishmania parasites, transmitted by sand flies, and poses a major health risk in the Mediterranean region. Monitoring the spread of infected sand flies is crucial for controlling the disease. This study focused on improving the methods used to detect Leishmania in sand flies by comparing different DNA extraction techniques and assessing the accuracy of these methods across nine reference laboratories. All centers, except one, efficiently detected all Leishmania concentrations, demonstrating proficiency in diagnostic protocols. Moreover, we found that two specific DNA extraction kits, the EZ1 DSP Virus Kit and QIAamp DNA mini-kit, were the most effective for Leishmania detection. We also tested how sample preparation and shipping conditions affected the results, ensuring that our methods would work in real-world settings. Even under these conditions, the detection methods proved reliable. This work helps to standardize the detection of Leishmania, making surveillance more accurate and consistent. Continuous training and calibration are essential to ensure uniform diagnostic performance across laboratories, enhancing epidemiological surveillance and disease control strategies and enabling appropriate treatment. [ABSTRACT FROM AUTHOR]

Background: In Europe, canine leishmaniasis is commonly caused by Leishmania infantum. Allopurinol is the main drug for long-term management of the disease, and clinical relapses of L. infantum infection treated with this drug are described. Resistance to allopurinol has been demonstrated in-vitro, but there is only little knowledge on in vivo resistance in dogs. Findings: A two-year-old female spayed Akita Inu that was adopted from a breeding facility near Nice in France was initially diagnosed with primary immune-mediated hemolytic anemia. Immunosuppressive treatment was initiated, and the dog was referred for a second opinion to the Clinique Veterinaire Alliance in France. PCR testing for L. infantum was performed out of EDTA blood and IFA as well as ELISA testing out of serum. Resistance to allopurinol was associated with chromosome and gene copy number (CN) variations including a decrease in the S-adenosylmethionine synthetase (METK) gene CN. Results: The dog showed pale mucous membranes, fever (39.1 °C), and a relapse of the anemia. The diagnosis of leishmaniasis was based on the cytological finding of Leishmania amastigotes (bone marrow, spleen, liver), positive PCR testing, and positive IFAT serology. The dog was treated with allopurinol over a period of 1316 days and additionally received two cycles of Glucantime® (meglumine antimoniate), before samples were submitted to the LABOKLIN laboratory to test for resistance against allopurinol. The laboratory work-up revealed mild thrombocytopenia, mild hyperproteinemia with hyperglobulinemia, a marked elevation of the c-reactive protein, and decreased iron concentration. Serum protein electrophoresis showed a polyclonal peak in the gamma globulins. Serology was positive in both ELISA (21.5 LE) and IFAT (1:1024). Quantitative PCR testing of blood was positive with low numbers of Leishmania (10/ml blood) at the timepoint of suspicion for resistance. The urinary protein-to-creatinine ratio was markedly elevated (2.5) and xanthine crystalluria was detected. A CN level of below 3 is considered suspicious for resistance, as revealed in the described Akita Inu dog. Conclusions: Relapse of L. infantum infection after applying allopurinol for 1316 days due to resistance was suspected clinically. Positive PCR testing, consistent hematological and biochemistry abnormalities, and reduction in the METK gene CN backed up the clinical suspicion of resistance. Dogs infected with allopurinol resistant strains of L. infantum may represent a great risk for infection of naïve dogs, cats, and humans. [ABSTRACT FROM AUTHOR]

ABSTRACT Aims Methods and Results Conclusions Leishmaniasis is an endemic disease in several regions of Brazil, a tropical country that presents specific environmental conditions that contribute to the development of phlebotomine vectors. This study aimed to detect Leishmania species in naturally infected bats from 17 municipalities in the São Paulo state.Spleen and liver samples from 203 bats were analysed by real‐time PCR and confirmed by conventional PCR followed by gene sequencing. Leishmania DNA was amplified by real‐time PCR in 6.4% of the bats and by conventional PCR followed by sequencing in 3.4% of the bats. Positive samples were characterised and included in GenBank. Leishmania species were confirmed in M. molossus, M. nigricans and E. glaucinus bats. Leishmania (Leishmania) amazonensis and L. infantum (syn. L. chagasi) were identified. This is the first detection of Leishmania spp. in bats in the studied areas. All the positive bats came from urban areas. Insectivorous bats were statistically more positive. There was similarity between our sequences and those of a human isolate and a phlebotomine from the region.This result points to bats as important possible reservoir of Leishmania in Brazil and guides the country's health authorities towards epidemiological surveillance, control and prevention actions in endemic areas. [ABSTRACT FROM AUTHOR]

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]

Simple Summary: Canine leishmaniosis (CanL) is a zoonotic disease caused by a parasite that can be transmitted by a mosquito-like insect called a sandfly. In dogs, the disease can range from mild to very severe, depending on the state of the dog's immune system. For this reason, it is important to detect the infection early. Whole blood assays (WBAs) are easy tests that allow for the rapid detection of immunity in response to a pathogen. As a result, the WBA is an important diagnostic tool for immune monitoring in CanL. In dogs, however, few tests are available to monitor their immune response to pathogens. This study aims to fill this gap by adapting a human-derived WBA technique for canine use. We compare a novel, faster, and cost-effective WBA in tubes (WBA-T) with a standardized, longer version (WBA-S) in dogs at various stages of Leishmania infantum infection. The results obtained showed that WBA-T performed similarly to WBA-S. Therefore, by implementing the WBA-T technique, results can be obtained more quickly and simply, which favors the identification of infected animals at an earlier stage, permitting early control of the infection and thus contributing to minimizing this infectious disease. Canine leishmaniosis (CanL) is a zoonotic disease caused by Leishmania infantum, where increased interferon-gamma (IFN-γ) levels are associated with controlling the infection and mild to moderate disease. Therefore, monitoring IFN-γ concentrations is essential for monitoring the immune responses in CanL. This study compared a faster, cost-effective IFN-γ release whole blood assay in tubes (WBA-T) with a standardized version (WBA-S) in 41 dogs at different states of L. infantum infection. WBA-T was performed at 24, 48, and 72 h of incubation with three conditions: blood, blood with L. infantum-soluble antigen (LSA), and blood with mitogen ConA. WBA-S was performed in plates, with blood diluted and incubated for five days using the same conditions. Supernatants (WBA-S) or plasma (WBA-T) were harvested for IFN-γ measurement by ELISA. No significant differences were observed in terms of IFN-γ concentration between WBA-T and WBA-S under LSA conditions. However, the 48 h incubation period during WBA-T showed the highest median of IFN-γ concentration compared to other incubation periods and WBA-S. The IFN-γ concentrations under ConA stimulation in WBA-S were significantly higher than in WBA-T at all incubation times studied. In conclusion, WBA-T stimulated with LSA at 48 h incubation time was shown to be the most appropriate for assessing IFN-γ production. [ABSTRACT FROM AUTHOR]

Species of Leishmania and Trypanosoma genera are the causative agents of relevant parasitic diseases. Survival inside their hosts requires the existence of a potent antioxidant enzymatic machinery. Four iron superoxide dismutases have been described in trypanosomatids (FeSODA, FeSODB1, FeSODB2, and FeSODC) that hold a potential as therapeutic targets. Nonetheless, very few studies have been developed that make use of the purified enzymes. Moreover, FeSODC remains uncharacterised in Leishmania. In this work, for the first time, we describe the purification and enzymatic activity of recombinant versions of the four Leishmania FeSOD isoforms and establish an improved strategy for developing inhibitors. We propose a novel parameter [(V*cyt. c − Vcyt. c)/Vcyt. c] which, in contrast to that used in the classical cytochrome c reduction assay, correlates linearly with enzyme concentration. As a proof of concept, we determine the IC50 values of two ruthenium carbosilane metallodendrimers against these isoforms. [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]

Although Switzerland is currently not endemic for canine leishmaniosis (CanL), imported cases of this emerging zoonosis are regularly detected. Also, phlebotomine sand flies, vectors of the causative agent Leishmania infantum, are present in the southern part of the country, in Canton Ticino, and endemic foci of CanL have been recently described in neighboring Italian municipalities. In 2022 and 2023, we evaluated the distribution of phlebotomine sand flies and the presence of antibodies against L. infantum in domestic dogs and cats in Ticino and Mesolcina (Canton of Grisons). An entomological survey was also carried out in the northwest of Switzerland (cantons Basel-Stadt and Basel-Landschaft) close to an area in Germany where potential vectors are present. No sand flies were caught there. In Ticino, 15 out of 20 sites surveyed (75%) were positive for phlebotomine sand flies. Phlebotomus perniciosus, a potential vector of L. infantum, was the most abundant species, with site densities ranging from 0.1 to 5.3. The parasite was not detected in females. Leishmania infantum seroprevalences of 3.0% and 1.6% were observed in 101 and 126 dog and cat sera, respectively. Although, at this time, the risk of endemic CanL is extremely low, integrated surveillance and prevention measures should be considered. [ABSTRACT FROM AUTHOR]

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]

Leishmania, a protozoan parasite, is responsible for signifi- cant morbidity and mortality worldwide, manifesting as cutaneous, mucocutaneous, and visceral leishmaniasis. These diseases pose a substantial burden, especially in impoverished regions with limited access to effective medical treatments. Current therapies are toxic, have low efficacy, and face growing resistance. Understanding the metabolic pathways of Leishmania, particularly those differing from its host, can unveil potential therapeutic targets. In this study, we investigated the acetyl-CoA synthetase (ACS) enzyme from Leishmania infantum (LiAcs1), which, unlike many organisms, also exhibits acetoacetyl-CoA synthetase (KBC) activity. This dual functionality is unique among acyl-CoA/NRPS/luciferase superfamily enzymes and crucial for the parasite's reliance on leucine catabolism, energy production, and sterol biosynthesis. Our biochemical characterization of LiAcs1 revealed its ability to utilize both acetate and acetoacetate substrates. Additionally, LiAcs1 displayed a distinct CoA substrate inhibition pattern, partially alleviated by acetoacetate. Structural analysis provided insights into the substrate binding flexibility of LiAcs1, highlighting a more promiscuous substrate pocket compared to other ACS- or KBC-specific enzymes. Substrate mimetics elucidated its ability to accommodate both small and large AMP-ester derivatives, contributing to its dual ACS/KBC functionality. These findings not only advance our understanding of Leishmania metabolism but also present LiAcs1 as a promising drug target. The dual functionality of LiAcs1 underscores the potential for developing selective inhibitors that could disrupt critical metabolic pathways across Leishmania spp. as it appears this enzyme is highly conserved across this genus. This paves the way for developing novel effective treatments against this devastating disease. [ABSTRACT FROM AUTHOR]

Simple Summary: Canine leishmaniosis can cause inflammation and renal disease that is considered the main cause of death in dogs with leishmaniosis. In the past, various biomarkers have been investigated to assess the severity of the disease and the response to anti-Leishmania treatment; however, the need for new markers remains. In the study, 30 dogs diagnosed with leishmaniosis were divided into different groups based on the degree of the disease and evaluated at diagnosis and after anti-Leishmania treatment. Parasite load in the bone marrow, blood, and urine, previously investigated, as well as new inflammatory and renal biomarkers were evaluated before and post-treatment. Treated dogs showed a significant decrease in the parasite load in the various tissues evaluated and a significant variation of various inflammatory and renal biomarkers. Among various biomarkers, a new one was identified and could be useful to monitor treatment response and to classify disease severity at the time of diagnosis. Various inflammatory and renal biomarkers have already been assessed for monitoring the response to anti-leishmanial therapy in canine leishmaniosis. This study assessed the parasite load, various inflammatory and renal biomarkers pre- and post-treatment, and any association between the studied variables and the degree of disease severity at diagnosis. This is a prospective cohort study of 30 client-owned dogs with leishmaniosis, classified according to LeishVet's guidelines as stage I (n = 2), stage IIa (n = 7), stage IIb (n = 6), stage III (n = 8), and stage IV (n = 7). In addition to Leishmania real-time PCR in the bone marrow, blood and urine, previously studied biomarkers, and several inflammatory and renal markers never investigated in canine leishmaniosis, such as fibrinogen, antithrombin, urinary fractional excretion of sodium, and urinary amylase-to-creatinine ratio were measured pre- and post-treatment (meglumine antimoniate or miltefosine + allopurinol). A positive Leishmania real-time PCR in the blood at diagnosis predicted a positive Leishmania real-time PCR in the bone marrow post-treatment (p = 0.003). Following treatment, antithrombin and urinary amylase-to-creatinine ratio were significantly changed (p < 0.001, respectively). Urinary amylase-to-creatinine ratio, total iron-binding capacity, and antithrombin were the variables most strongly associated with disease severity (p < 0.005, respectively). Urinary amylase-to-creatinine ratio can be a useful marker to monitor treatment response and to classify the degree of disease severity. [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]