Quantitative proteomic analysis to determine differentially expressed proteins in axenic amastigotes of Leishmania tropica and Leishmania major.

Cutaneous leishmaniasis is commonly caused by Leishmania major and Leishmania tropica. In the present study, the differential expression of proteins was identified in the amastigote-like forms of L. tropica and L. major in Iranian isolates. Initially, the samples were cultured and identified using PCR-RFLP technique. The Leishmania isolates were then grown in host-free (axenic) culture and prepared to amastigote-like forms, followed by the extraction of their proteins. To identify significant differentially expressed proteins (DEPs) of two types of Leishmania, the label-free quantitative proteomic technique was used based on sequential window acquisition of all theoretical fragment ion spectra mass spectrometry. A total of 51 up/down-DEPs (fold change >2 and p-value <.05) were identified between the axenic amastigote forms of L. major and L. tropica. Of these, 34 and 17 proteins were up-regulated in L. major and L. tropica, respectively. Several enriched GO terms were identified via biological process analyses for DEPs; furthermore, the metabolic process and translation were disclosed as top category in the up-regulated proteins of both L. major and L. tropica species. Also, the KEGG analysis revealed carbon metabolism and metabolic pathways term as the top pathways in the proteins up-regulated in L. major and L. tropica, respectively. Taken together, the numerous novel DEPs identified between the studied species could help fully understand the molecular mechanisms of pathogenesis and provide potential drug targets and vaccine candidates.

TNF-α - 308 G/A and IFN-γ + 874 A/T gene polymorphisms in Saudi patients with cutaneous leishmaniasis.

BACKGROUND: Cutaneous leishmaniasis (CL) is well linked with immunogenetic factors. This study was undertaken to test the association of TNF-α - 308 and IFN-γ + 874 gene polymorphisms with the susceptibility of Leishmania (L) species among CL patients in central region of Saudi Arabia. METHODS: This is a case-control study involved 169 Saudi subjects with different L. species and 199 healthy controls from central region of Saudi Arabia. All subjects were characterized by TNF-α - 308 G/A and IFN-γ + 874 A/T gene polymorphisms using PCR. RESULTS: Evaluation of genotyping and allelic frequency of TNF-α - 308 G/A in different L. species showed no significant association compared to controls (p > 0.05). Except, in cases of L. tropica that showed significantly higher TNF-α - 308 A versus G allele frequency (p = 0.0004). Evaluation of genotyping of IFN-γ + 874 (TT versus AA+AT recessive) and allelic frequency of IFN-γ + 874 (T versus A) showed significant higher in L. major and also in total CL cases as compared to healthy controls (p < 0.05). Furthermore, a strong association was observed between the susceptibility of L. major, L. tropica or total CL cases with synergistically combined high TNF-α 308/INF-γ 874 alleles. CONCLUSIONS: This is the first report that shows the gene polymorphisms of TNF-α - 308 G/A and IFN-γ + 874 A/T in Saudi patients with different L. species infections. Data showed that the TNF-α-308 G/A gene polymorphism is not associated with the susceptibility of CL in Saudi subjects. The only correlation was found in between A versus G allelic frequency in L. tropica. Importantly, IFN-γ + 874 A/T polymorphism was found to be associated with the susceptibility of L. major and also with total CL subjects. Moreover, data from synergistically combined high TNF-α 308/INF-γ 874 alleles strongly suggest their potential role in the susceptibility of leishmania infection.

Camel nanobodies: Promising molecular tools against leishmaniasis.

AIM: To characterize several anti-Leishmania tropica nanobodies and to investigate their effect on Leishmania infection. METHODS: Several immunological tests were implied to characterize five different (as confirmed by sequencing) anti-L tropica nanobodies (NbLt05, NbLt06, NbLt14, NbLt24 and NbLt36) against parasite lysates or intact cells from different stages, promastigotes and amastigotes. Direct inhibitory effect of these nanobodies on parasite infection cycle on macrophages was tested in cell culture. RESULTS: All the five nanobodies (with distinguished characteristics) were more specific to L tropica than to L major, but could equally recognize the lysate and the outer surface of the intact cells from the two main stages of the parasite. Nanobodies recognized several leishmania antigens (majorly between 75 and 63 kDa), and their proteinaceous nature was confirmed. Because of its role in leishmania life cycle, gp63 was considered a potential antigen candidate for nanobodies, and bioinformatics predicted such interaction. All nanobodies have a negative effect on the infectivity of L tropica, as they decreased the number of infected macrophages and the amastigotes inside those macrophages. CONCLUSION: Such anti-leishmania nanobodies, with outstanding characteristics and important target, can be of great use in the development of promising treatment strategies against leishmaniasis.

First molecular detection and identification of Leishmania species in small wild rodents from Turkey.

Leishmaniasis is a parasitic disease infecting animals and humans. Two clinical forms (Visceral and cutaneous leishmaniasis) and four species are reported to be present in Turkey. Several studies have investigated canine and human leishmaniasis in Turkey but no study was performed to screen the infection among wild rodents, so far. The present study aims to investigate the role of small wild rodents as reservoir animals for Leishmania spp. in different regions of Turkey. Formalin-preserved tissue samples (spleen, liver, lung) of 712 rodents from 30 provinces were screened for the presence of Leishmania spp. DNA. Before DNA extraction, tissues were dried, rehydrated, and homogenated. Leishmania screening in rodent tissues and species determination was performed with a combination of real-time kDNA and ITS1 polymerase chain reaction protocols. Eight (1.12%) out of 712 animals were found to be positive for Leishmania spp. DNA and species typing revealed five L. infantum, two L. tropica and one L. major among positives. Leishmania major and L. infantum DNA were detected in Apodemus spp. from Zonguldak province located in the Western Black Sea Region, while L. tropica DNA was found in Meriones sp. and Gerbillus dasyurus from Adana and Hatay provinces located in Eastern Mediterranean Region of Turkey. The present study is first to report natural infection of L. infantum, L. major and L. tropica in small wild rodents in Turkey, suggesting their possible roles as reservoirs. Further studies are needed for planning epidemiological studies and also for developing rodent control measures in risky endemic areas to break the transmission cycle.

Molecular identification of Leishmania tropica and L. infantum isolated from cutaneous human leishmaniasis samples in central Morocco.

BACKGROUND & OBJECTIVES: Cutaneous leishmaniasis (CL) in Marrakesh-Safi region located in the central-south part of Morocco is a public health problem. This study assessed the efficiency of a microscopic examination method in establishing the diagnosis of CL and PCR for the characterization and identification of the circulating Leishmania strains in different CL foci of the study area. METHODS: A total of 297 smears obtained from cutaneous lesions of suspected patients with CL were stained with May-Grünwald Giemsa (MGG) for microscopic examination. For each positive smear, genomic DNA was extracted and PCR-analysed, targeting the small subunit ribosomal ribonucleic acid (ssu rRNA) gene to detect Leishmania DNA. Then, the internal transcribed spacer 1 (ITS1) was amplified and sequenced in order to identify the Leishmania species. The sensitivity and specificity of the conventional microscopy with ssu rRNA gene were compared by Leishmania nested PCR (LnPCR) and ITS1 gene by ITS-PCR. RESULTS: A total of 257 smears were positive in the microscopic examination, i.e. the detection rate of amastigotes by optical microscopy was 86.53% (257/297). The LnPCR was found to have a specificity and a sensitivity of 100%, each. Interestingly, the sequencing results showed that 99.61% (256/257) of the isolates had Leishmania tropica and 0.39% (1/257) had L. infantum infection. INTERPRETATION & CONCLUSION: Though, classical microscopic examination is useful and economical, it is not sensitive enough, especially in endemic regions where several Leishmania species coexist. In such situations, PCR constitutes a complementary method for the identification of the causal species. The results indicate that both the L. tropica (dominant) and L. infantum are the causative agents of CL in the Marrakesh-Safi region. The rate of CL infection is high in Imintanout, and Chichaoua provinces. Hence, early diagnosis and prompt treatment of CL patients is necessary to prevent its extension to neighboring localities.

[Cutaneous leishmaniasis of the vermilion border of the upper lip extending to the oral mucosa]. / Leishmaniose cutanée du vermillon avec atteinte muqueuse de la lèvre supérieure.

BACKGROUND: Cutaneous leishmaniasis is endemic in Morocco. Mucosal involvement is rare. We report a case in Morocco of cutaneous leishmaniasis of the vermilion border of the upper lip extending to the oral mucosa due to Leishmania tropica. PATIENTS AND METHODS: A 15-year-old girl was seen with 2 ulcerated lesions, present for 4 months, situated on the left cheek and vermilion border and extending to the oral mucosa. The diagnosis of leishmaniasis was confirmed by direct examination revealing high numbers of Leishmania amastigotes. Culture of the offending organism in NNN medium and isoenzymatic characterization resulted in identification of L. tropica. Treatment with meglumine antimoniate (Glucantime) was ineffective. The outcome was good after treatment with fluconazole. CONCLUSION: In Morocco, cutaneous leishmaniasis with mucosal involvement is rare, and usually develops as a complication of cutaneous leishmaniasis via direct extension.

Prevalence of Leishmania species among patients with cutaneous leishmaniasis in Qassim province of Saudi Arabia.

BACKGROUND: Leishmaniasis is a parasitic infection endemic in more than ninety countries of the world. The cutaneous leishmaniasis (CL) is a most common form of leishmaniasis and it remains to be a major public health issue in Saudi Arabia. This study was undertaken to investigate the Leishmania species responsible for CL infection in different provinces of Qassim, Saudi Arabia. METHODS: Skin biopsies were obtained from CL patients and DNA was extracted using the Magna pure system. Leishmania species were identified by highly specific/sensitive quantitative and qualitative PCR. RESULTS: Out of total 206 CL biopsies, 49.5% biopsies were found to be positive for Leishmania major (L. major), 28.6% biopsies were positive for Leishmania tropica (L. tropica), 3.9% were found to be positive for Leishmania infantum/donovani (L. infantum/donovani). Not only have these, all tested CL biopsies showed negative test for Leishmania mexicana (L. mexicana) and Leishmania viannia (L. viannia). CONCLUSIONS: This is the first comprehensive study that shows the majority of CL in Qassim was caused by L. major and L. tropica. To the best of our knowledge, this is the very first report that shows the occurrence of L. infantum/donovani in Saudi Arabia. This requires higher alert to the Ministry of Health of Saudi Arabia to take proactive actions in preventing the onset of L. major, L. tropica, L. infantum and L. donovani infections.

Epithelial homelessness: an atypical form of anoikis triggered by Leishmania interaction with epithelial cells.

Aim: Leishmaniasis is characterized by a spectrum of diseases with two main clinical forms, cutaneous and visceral, caused by Leishmania tropica and Leishmania donovani, respectively. Studying Leishmania's interaction with the epithelial barrier at the initial site of a bite is crucial to understanding the establishment of the disease. Materials & methods: To discern parasite-host epithelial interaction, we developed in vitro cellular models involving co-cultures of Leishmania and MDCK epithelial cells. Results: Both L. donovani-MDCK and L. tropica-MDCK co-culture models demonstrated a phenomenon known as atypical anoikis apoptosis, typically identified by distinctive 'flipping in' of cell membranes and disordered cytoskeletal frameworks. Conclusion: This study bridges the gap in the fundamental understanding of the intricate latticework involving vector-Leishmania-host and may inform drug development strategies.

Small parasites called Leishmania are passed to humans through the bites of sandflies. These parasites cause three deadly forms of disease: one that affects the organs, one that causes skin lesions and one that affects organ linings. This study looked at how Leishmania parasites behave when they enter through the skin. We found that when the parasites were in contact with cells, the cells changed their shape and lost contact with neighboring cells. This led to a type of cell death known as anoikis, a Greek term meaning 'homelessness'.

Thymoquinone Effect on Leishmania tropica/infantum and Leishmania-Infected Macrophages.

INTRODUCTION: Leishmania is a parasitic protozoan that tries to enter and amplify within macrophages. Macrophage cells are also immune defense cells that phagocyte many microbes like bacteria, fungi, as well as parasites like Leishmania spp. However, they are unable to kill this parasite that resides in the phagosomes of contaminated macrophages and multiplies in these macrophages, leading to the destruction of contaminated macrophages and the emerging of Leishmania wounds. A large number of current therapies for Leishmania cure have adverse effects, or parasites have developed resistance to some of these therapies, so a better therapy for the cure of Leishmania is required. Thymoquinone is one of the Nigella Sativa ingredients with numerous biological effects, such as antioxidant as well as antimicrobial effects on a variety of microbes, namely fungi, bacteria, as well as parasites like Leishmania spp. The impacts of Thymoquinone on Leishmania tropica and Leishmania infantum, as well as Leishmania-infected macrophages, were examined in this study. METHODS: The impact of various Thymoquinone dosages on L. tropica and L. infantum promastigotes and amastigotes was examined in vitro. Flow cytometry, as well as MTT, was also applied to examine the cytotoxic activity of Thymoquinone on promastigotes of L. tropica and L. infantum, as well as the incidence of apoptosis. The amastigote assay is also utilized to calculate the % of contaminated macrophages as well as the number of the present parasites in each macrophage. RESULTS: The percentage of macrophages contaminated with L. tropica and L. infantum amastigotes after medicating with 20 µM of Thymoquinone was 23% and 19%, respectively. Also, after medicating with 10 µM of Thymoquinone, these percentages were 32% and 31%, respectively. Flow cytometry indicated that Thymoquinone caused 33.9% and 31.4% apoptosis in L. tropica and L. infantum, respectively. As determined by the promastigote assay, the inhibitory concentration (IC50) of Thymoquinone for L. tropica and L. infantum was 9.49 µM and 12.66 µM, respectively. The results of the promastigote and amastigote assay show that with an increase in Thymoquinone doses, its ability to kill Leishmania parasites increases, too. CONCLUSION: According to the results of the study, Thymoquinone has a potentially lethal impact on L. tropica and L. infantum promastigotes as well as amastigotes (within leishmania contaminated macrophages).

Design, synthesis, in vitro - In vivo biological evaluation of novel thiazolopyrimidine compounds as antileishmanial agent with PTR1 inhibition.

The leishmaniasis are a group of vector-borne diseases caused by a protozoan parasite from the genus Leishmania. In this study, a series of thiazolopyrimidine derivatives were designed and synthesized as novel antileishmanial agents with LmPTR1 inhibitory activity. The final compounds were evaluated for their in vitro antipromastigote activity, LmPTR1 and hDHFR enzyme inhibitory activities, and cytotoxicity on RAW264.7 and L929 cell lines. Based on the bioactivity results, three compounds, namely L24f, L24h and L25c, were selected for evaluation of their in vivo efficacy on CL and VL models in BALB/c mice. Among them, two promising compounds, L24h and L25c, showed in vitro antipromastigote activity against L. tropica with the IC50 values of 0.04 µg/ml and 6.68 µg/ml; against L. infantum with the IC50 values of 0.042 µg/ml and 6.77 µg/ml, respectively. Moreover, the title compounds were found to have low in vitro cytotoxicity on L929 and RAW264.7 cell lines with the IC50 14.08 µg/ml and 21.03 µg/ml, and IC50 15.02 µg/ml and 8.75 µg/ml, respectively. LmPTR1 enzyme inhibitory activity of these compounds was determined as 257.40 µg/ml and 59.12 µg/ml and their selectivity index (SI) over hDHFR was reported as 42.62 and 7.02, respectively. In vivo studies presented that L24h and L25c have a significant antileishmanial activity against footpad lesion development of CL and at weight measurement of VL group in comparison to the reference compound, Glucantime®. Also, docking studies were carried out with selected compounds and other potential Leishmania targets to detect the putative targets of the title compounds. Taken together, all these findings provide an important novel lead structure for the antileishmanial drug development.

Tape-disc-loop-mediated isothermal amplification (TD-LAMP) method as noninvasive approach for diagnosis of cutaneous leishmaniasis caused by L. tropica.

Cutaneous leishmaniasis (CL) is a parasitic disease caused by the bite of infectious female sand flies with high socioeconomic burdens. There is currently no non-invasive, point-of-care, diagnostic method with high sensitivity and specificity available for CL. We herein report the development of a non-invasive tape disc (TD) sampling method combined with a loop-mediated isothermal amplification (LAMP) assay using primer sets targeting kinetoplast DNA (kDNA) of Leishmania tropica (L. tropica) with a colorimetric readout for species-specific diagnosis of CL. We tested our Tape-Disc (TD)-LAMP method on a panel of skin samples collected by TD from 35 confirmed L. tropica patients, 35 healthy individuals and 35 patients with non-L. tropica infections. The detection limit of the TD-LAMP assay was determined as 1 fg (fg), and the assay sensitivity and specificity of 97 % and 100 % for L. tropica infection, respectively. This non-invasive, sensitive and rapid diagnostic method warrants further exploration of its use for differential diagnosis of CL in disease endemic settings.

Clinical and immunological spectra of human cutaneous leishmaniasis in North Africa and French Guiana.

Cutaneous leishmaniasis (CL) caused by infection with the parasite Leishmania exhibits a large spectrum of clinical manifestations ranging from single healing to severe chronic lesions with the manifestation of resistance or not to treatment. Depending on the specie and multiple environmental parameters, the evolution of lesions is determined by a complex interaction between parasite factors and the early immune responses triggered, including innate and adaptive mechanisms. Moreover, lesion resolution requires parasite control as well as modulation of the pathologic local inflammation responses and the initiation of wound healing responses. Here, we have summarized recent advances in understanding the in situ immune response to cutaneous leishmaniasis: i) in North Africa caused by Leishmania (L.) major, L. tropica, and L. infantum, which caused in most cases localized autoresolutives forms, and ii) in French Guiana resulting from L. guyanensis and L. braziliensis, two of the most prevalent strains that may induce potentially mucosal forms of the disease. This review will allow a better understanding of local immune parameters, including cellular and cytokines release in the lesion, that controls infection and/or protect against the pathogenesis in new world compared to old world CL.

Efficacy of malachite green mediated photodynamic therapy on treatment of Cutaneous Leishmaniasis: In vitro study.

BACKGROUND: Leishmaniasis is a common zoonotic disease that is transmitted by phlebotomus and causes several clinical conditions, from self healing lesion to deadly internal organ involvement. Photodynamic therapy (PDT) is a treatment method that leads to the generation of cytotoxic species and consequently to cell death and tissue destruction by visible light in the presence of a photosensitizer and oxygen. The aim of this study was to investigate effect of malachite green (MG)-mediated PDT in Leishmania tropica (L. tropica) promastigotes. MATERIAL AND METHODS: Parasites were incubated with 0.19, 0.39, 1.56, 3.25 and 6.25 µM of MG for one hour and subjected to 46.4 J/cm2 light irradiation. Trypan blue assay was used to evaluate the viability of the cells and mitochondirial activity alteration was determined by MTT. Morphological changes were analyzed by Giemsa staining and Scanning electron microscopy (SEM) analyses. Flow cytometry was used to quantify the fluorescence emitted by cell volume, JC-1, Cell Cycle and Annexin V/PI staining reagents. RESULTS: Malachite green mediated photodynamic therapy at 1.56 and 3.125 µM decreased the viability of the L. tropica promastigotes and induced changes in the mitochondrial membrane potential. L.tropica promastigotes was bloked in G0/G1 phase. The morphology of the parasite was affected at the 1.56 and 3.125 µM MG+PDT, resulting in rounded cells with loss of flagellum and irregular shape. CONCLUSIONS: This study demonstrated that antileishmanial effects through mitochondrial dysfunction, cell cycle arrest, and apoptosis-like cell death to parasites. This work showed PDT with MG effectedparasites. Therefore, MG-mediated PDT may provide a promising approach for L. tropica promastigotes.

Antimony resistance mechanism in genetically different clinical isolates of Indian Kala-azar patients.

The central theme of this enterprise is to find common features, if any, displayed by genetically different antimony (Sb)-resistant viscerotropic Leishmania parasites to impart Sb resistance. In a limited number of clinical isolates (n = 3), we studied the breadth of variation in the following dimensions: (a) intracellular thiol content, (b) cell surface expression of glycan having N-acetyl-D-galactosaminyl residue as the terminal sugar, and (c) gene expression of thiol-synthesizing enzymes (CBS, MST, gamma-GCS, ODC, and TR), antimony-reducing enzymes (TDR and ACR2), and antimonial transporter genes (AQP1, MRPA, and PRP1). One of the isolates, T5, that was genotypically characterized as Leishmania tropica, caused Indian Kala-azar and was phenotypically Sb resistant (T5-LT-SSG-R), while the other two were Leishmania donovani, out of which one isolate, AG83, is antimony sensitive (AG83-LD-SSG-S) and the other isolate, T8, is Sb resistant (T8-LD-SSG-R). Our study showed that the Sb-resistant parasites, regardless of their genotype, showed significantly higher intracellular thiol compared with Sb-sensitive AG83-LD-SSG-S. Seemingly, T5-LT-SSG-R showed about 1.9-fold higher thiol content compared with T8-LD-SSG-R which essentially mirrored cell surface N-acetyl-D-galactosaminyl expression. Except TR, the expression of the remaining thiol-synthesizing genes was significantly higher in T8-LD-SSG-R and T5-LT-SSG-R than the sensitive one, and between the Sb-resistant parasites, the latter showed a significantly higher expression. Furthermore, the genes for Sb-reducing enzymes increased significantly in resistant parasites regardless of genotype compared with the sensitive one, and between two resistant parasites, there was hardly any difference in expression. Out of three antimony transporters, AQP1 was decreased with the concurrent increase in MRPA and PRP1 in resistant isolates when compared with the sensitive counterpart. Interestingly, no difference in expression of the above-mentioned transporters was noted between two Sb-resistant isolates. The enduring image that resonated from our study is that the genetically diverse Sb-resistant parasites showed enhanced thiol-synthesizing and antimony transporter gene expression than the sensitive counterpart to confer a resistant phenotype.

Geographical distribution and molecular epidemiology of cutaneous leishmaniasis in Fars Province, southern Iran.

Cutaneous leishmaniasis (CL) is one of the important zoonotic diseases in Iran, particularly in Fars Province, southern Iran. The purpose of this descriptive cross-sectional study was to investigate the molecular identification and geographical distribution of anthroponotic and zoonotic CL in southern Iran, during 2018-2019. Overall, 161 patients with CL referred to the Leishmaniasis Diagnostic Laboratory, Valfajr Health Center, Shiraz, Iran, were included in this study. The smears were prepared from the lesion borders of patients and stained with Giemsa and diagnosed microscopically. For molecular identification, the genomic DNA of each sample was extracted, and PCR method was used. The geographical distribution map was prepared using ArcMap software. Finally, the possible correlation between the frequencies of CL in various subgroups was statistically analyzed by Chi-square test, using SPSS software. Of 161 positive samples confirmed by both microscopy and PCR, 126 (78.3%) and 35 (21.7%) samples were shown to be L. major and L. tropica, respectively. Also, 87 (54%) patients were male, and 74 (46%) were female. The study showed that anthroponotic cutaneous leishmaniasis (ACL) was detected only in Shiraz city, while zoonotic cutaneous leishmaniasis (ZCL) was observed both in Shiraz and most counties of Fars Province. Furthermore, the most CL infections occurred in district 8 among the different districts of Shiraz municipality, which requires serious attention.

A nanoemulsion-based nanogel of Citrus limon essential oil with leishmanicidal activity against Leishmania tropica and Leishmania major.

Cutaneous leishmaniasis is one of the diseases that severely affects human skin. Nanogels are the well-known formulation for topical drug delivery due to easy usage, high loading capacity, and physical and chemical stabilities. In this study, the toxicity effect of three essential oils, including Mentha piperita, Anethum graveolens, and Citrus limon (CLEO), was evaluated against Leishmania major and Leishmania tropica. Ingredients of CLEO as the most potent essential oil were identified using GC-MS analysis. The five major components were limonene (61.83%), sabinene (16.99%), trans-limonene oxide (3.08%), cis-limonene oxide (2.27%), and 1,2-cyclohexane diol, 1-methyl-4-(1-methylethenyl) (1.50%). The nanogel of CLEO (CLNgel) was prepared by the addition of carbomer 940 (1% w/v) to the prepared nanoemulsion with a droplet size of 146 ± 12 nm. The viscosity of CLNgel was fitted with a regression of non-Newtonian materials, Carreau-Yasuda. Interestingly, CLNgel at a concentration of 80 µg/mL reduced the viability of both species to 0%. Therefore, the prepared prototype can/could/would be used as an excellent nanoformulation for in vivo studies.

Identification of differential protein expression and putative drug target in metacyclic stage of Leishmania major and Leishmania tropica: A quantitative proteomics and computational view.

Cutaneous leishmaniasis (CL) is an infectious disease that commonly caused by Leishmania (L.) major and L.tropica. Recently there has been a growing interest in proteomics analysis on Leishmania for drug target discovery. Therefore, we aimed to distinguish proteins which might be characteristic for each of the species from those shared by both to the detection of drug targets, which may become helpful for designing new drugs for CL. To identify differences in protein profiles of L. major and L. tropica, we conducted a Sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) analysis. Totally 67 differentially expressed proteins (DEPs) (fold change> 2 and p < 0.05) were identified between species. Of these, 42 and 25 proteins were up-regulated in L. major and L. tropica, respectively. Several enriched GO terms were identified via biological process of up-regulated proteins. Furthermore, the small molecule metabolic process and translation were detected as significant biological processes for up-regulated proteins in L. major, while translation was identified for L. tropica. Also, KEGG analysis has revealed glycolysis/gluconeogenesis and translation as the top pathways in the proteins up-regulated in L. major and L. tropica, respectively. Finally glycosomal malate dehydrogenase was identified as putative drug target using network and homology analyses. The DEPs between the species are essential in host-pathogen interactions and parasite survival in the macrophage. Furthermore, L. major and L. tropica possibly uses different pathogenicity mechanisms that leads to anthroponotic or zoonotic CL. Our results may help in the drug discovery and chemotherapeutic interventions.

Phylogenetic analysis of kinetoplast DNA: kDNA of Leishmania tropica in Thi-Qar province, Iraq.

INTRODUCTION: Cutaneous leishmaniasis (CL) is one of wobbling endemic disease in Iraq, that cause intracellular obligate protistan parasite returned to the genusLeishmania. This study is aimed to identify epidemiology of CL, detect the prevalence of Leishmania tropica and find the phylogenetic relationship. METHODOLOGY: The current study was conducted in the main hospitals of Thi-Qar province-south of Iraq for period from November 2018 to October 2019. Nested-PCR was used to amplify kinetoplast minicircle fragments DNA. RESULTS: It was recorded 247 clinical cases with CL, the infections of males were higher than females, while infection rate appeared gradual reduction with age progress. Furthermore, the most CL infections were as single lesions and occurred in December. The infections of upper limbs were high when compared with other body regions. The molecular diagnosis showed L. tropica was more frequently. DNA sequences of kDNA gene of L. tropica showed confirmative genetic detection of local isolates using NCBI-Blast data and phylogenetic tree analysis after comparison with global recorded isolates. The local L. tropica isolates showed genetically closed related to NCBI-Blast L. tropica with accession number AB678350.1. Generally, the analysis of kDNA nitrogen bases sequences showed that all of samples were consistent with those recorded at the NCBI. CONCLUSION: The kDNA minicircle sequences analysis results showed mismatching of the local isolates decrease whenever approached from the Iranian border. In addition, genetic heterogeneity diagnosis is important for detection of therapy, control and epidemiological studies.

Investigation of antileshmanial activities of Cuminum cyminum based green silver nanoparticles on L. tropica promastigotes and amastigotes in vitro.

Leishmaniasis is one of the most important parasitic diseases, which is caused by Leishmania species. Nowadays; although pentavalent antimonials are used as the basic treatment option for Cutaneous Leishmaniasis, high cost, toxicity and resistance of the parasites to the medication over time are some important limitations causing several problems in the treatment. In recent years, the progress in the field of green nanotechnology provides the development of green nanoparticle-based treatment methods for Cutaneous Leishmaniasis. The importance of green nanoparticles has gradually increased due to their special reductive, stabilizing, antioxidant and non-toxic properties. Although there are many studies based on green nanoparticles against Leishmania parasites, we have not found any research about antileishmanial activities of biosynthesized silver nanoparticles (Bio-AgNPs) using Cuminum cyminum L (Cumin) seed extract. Therefore for the first time in this study in vitro antileishmanial effects of Bio-AgNPs prepared from Cumin seed extract were examined on L. tropica promastigote and amastigote forms and their efficacies were compared with chemically synthetized AgNPs. During the experiments, antileishmanial effects of synthetized nanoparticles were determined on both promastigote and amastigote forms of Leishmania parasites by detecting different parameters such as proliferation, infection index and produced nitric oxide (NO) amounts from macrophages. According to the results, it was shown that Bio-AgNPs and AgNPs excessively inhibited L. tropica promastigotes and amastigotes by significantly decreasing proliferation rates of promastigotes and metabolic activities of amastigotes, as well. Moreover, infection index rates of macrophages revealed remarkable anti-amastigote performances of Bio-AgNPs. Besides, Bio-AgNPs stimulated macrophages to release NO to kill Leishmania parasites. Consequently, for the first time, Bio-AgNPs were found to be more effective on both forms of Leishmania parasites than AgNPs. Obtained results indicated high antileishmanial potential of green nanoparticles on L. tropica parasites, causative agents of Cutaneous Leishmaniasis. Thus, obtained results demonstrated that green nanoparticles can lead to the development of new, safer, stable and more effective antileishmanial formulations against Cutaneous Leishmaniasis.

Apigenin effect against Leishmania tropica amastigotes in vitro.

Cutaneous Leishmaniasis is a current public health problem in Syria. It causes different skin lesions that vary in their severity from spontaneously heal lesions to permanent deformity ones. However, the used treatments have many disadvantages as their high toxicity and many side effects. Flavonoids including Apigenin reported to have many anti parasitic properties. As well as their preference as potential therapeutic alternatives in the treatment of Leishmaniasis due to its low side effects and toxicity. This study aims to evaluate the efficacy of Apigenin against L.tropica amastigotes in vitro using Leishmania-Macrophage Interaction Assay. Our study demonstrated the possibility of efficacy of Apigenin against L.tropica amastigotes since Apigenin reduced the infection index at IC50 60.44 µM and this requires subsequent studies in humans and using Apigenin as a candidate for the chemotherapeutic treatment against Leishmaniasis.