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@#Leishmaniasis and toxoplasmosis are parasitic protozoal diseases that pose serious health concerns, especially for immunocompromised people. Leishmania major and Toxoplasma gondii are endemic in Saudi Arabia and are particularly common in the Qassim Region. The present work was conducted to evaluate the in vitro antileishmanial and antitoxoplasmal activity of methanolic extracts and phytochemical fractions from two plants, Euphorbia retusa and Pulicaria undulata, which are ethnobotanical agents used to treat parasitic infection. Whole E. retusa and P. undulata plants were extracted with methanol and fractionated using petroleum ether, chloroform, ethyl acetate, n-butanol, and water and then were tested in vitro against L. major promastigote and the amastigote stages of T. gondii; the cytotoxicity of the extracts was tested against Vero cell line. The methanolic extracts of E. retusa and P. undulata exhibited promising antitoxoplasmal activity against T. gondii with EC50 values 5.6 and 12.7 μg mL-1 , respectively. The chloroform fraction of P. undulata was the most potent, exhibiting an EC50 of 1.4 μg mL-1 and SI value of 12.1. It was also the most active fraction against both L. major promastigotes and amastigotes, exhibiting an EC50 of 3.9 and 3.8 μg mL-1 and SI values 4.4 and 4.5, respectively. The chloroform fraction from P. undulata is a very good candidate for the isolation of active antitoxoplasmal and antileishmanial ingredients; therefore, further phytochemical analysis for active compound isolation is highly recommended.
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Objective: Current therapy strategies of leishmaniasis have some problems such as high cost, toxicity and side effects. Plant extracts can be a source of drugs to control leishmaniasis. In this study, the effect of hydroalcoholic and chloroformic extracts of Vigna radiata, Tamarix ramosissima, and Carthamus lanatus on Leishmania major and L. tropica was studied. Methods: The plant samples were collected from west of Iran and their extracts were prepared. Anti-promastigote activity assay of all extracts was done using tetrazolium-dye assay. Results: Only high concentrations of V. radiata and C. lanatus were able to inhibit Leishmania, while both high and low concentrations of T. ramosissima had antileishmanial effect. No difference was observed between hydroalcoholic with chloroformic extract of each plant. Conclusion: Altogether, the results revealed the antileishmanial activity of T. ramosissima extracts against L. major and L. tropica, indicating its potential as an antileishmanial agent.
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The aim of the present study was to determine the main constituents of Scrophularia striata essential oil and to evaluate in vitro effect of essential oil on Leishmania tropica and Leishmania major promastigotes and axenic amastigotes. Chemical constituents of the extracted essential oil were separated by headspace solid-phase microextraction (HS-SPME) equipped with a PDMS/DVB fiber. The fiber was injected to gas chromatogram- mass spectroscopy (GC-MS) to determine their identity. Finally, after exposure of parasites to different concentrations of water soluble fraction of essential oil, viability of promastigotes and axenic amastigotes were investigated. Based on the HS-SPME results, 47 compounds representing 95.6% of the total oil, were identified in essential oil. Essential oil analysis showed that nonane (19.7%), α-terpineol (17.4%) and linalool (10.2%) were the most abundant compounds. This study indicates that water soluble fraction of S. striata essential oil has promising anti-leishmanial activity.
El objetivo del presente estudio fue determinar los principales componentes del aceite esencial de Scrophularia striata y evaluar el efecto in vitro del aceite esencial en promastigotes y amastigotes axénicos de Leishmania tropica y Leishmania major. Los componentes químicos del aceite esencial extraído se separaron mediante microextracción de fase sólida en el espacio superior (HS-SPME) equipado con una fibra PDMS/DVB. Para determinar su identidad la fibra se inyectó en un cromatógrafo de gases acoplado un espectrómetro de masas (GC-MS). Finalmente, después de la exposición de los parásitos a diferentes concentraciones de fracción soluble del aceite esencial en agua, se investigó la viabilidad de los promastigotes y los amastigotes axénicos. En base a los resultados de HS-SPME, se identificaron 47 compuestos que representan el 95.6% del aceite total en el aceite esencial. El análisis de aceites esenciales mostró que el nonano (19.7%), el α-terpineol (17.4%) y el linalol (10.2%) fueron los compuestos más abundantes. Este estudio indica que la fracción soluble en agua del aceite esencial de S. striata tiene una actividad antileishmanial prometedora.
Subject(s)
Oils, Volatile/pharmacology , Oils, Volatile/chemistry , Scrophularia/chemistry , Leishmania/drug effects , Terpenes/analysis , Colorimetry , Solid Phase Microextraction , Gas Chromatography-Mass SpectrometryABSTRACT
Objective To understand Leishmania infections among employees of China Petroleum First Construction Corporation returning from Uzbekistan, and take timely actions to prevent the spread of the epidemic. Methods Questionnaire survey was conducted to collect screening subjects’information. Palpation of the liver, spleen and superficial lymph nodes was performed by a physician, and the lesions on the frequently exposed skin were detected by a dermatologist. In addition, the liver and spleen sizes were measured using B-mode ultrasonography, and serum samples were collected to be subjected to an rK39-based rapid diagnostic test for detection of visceral leishmaniasis. Leishmania was detected using microscopy in the specimens sampled from the lesioned skin, and the parasites species was identified using molecular assays in parasitologically positive specimens. Results Among the 181 employees screened, enlarged cervical lymph nodes were palpable in 6 subjects, and skin lesions were found in 12 cases. B-mode ultrasonography displayed hepatosplenomegaly in 5 cases, and rK39 test were positive in 3 serum samples. Two classical lesioned skin specimens were sampled, and Leishmania was detected in one specimen. The promastigote DNA was extracted and two fragments of 120 bp and 350 bp in sizes were amplified using PCR assay with K13A/K13B and L5.8S/LITSR primers specific to Leishmania. The two amplification products were 90% and 98% homologous to the corresponding sequences of L. major (GenBank accession numbers: EU370906.1 and FN677342.1). Conclusions Six patients with cutaneous leishmaniasis were screened, including 2 uncured cases. One uncured case was diagnosed as imported cutaneous leishmaniasis caused by L. major infection.
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Objective To understand Leishmania infections among employees of China Petroleum First Construction Corporation returning from Uzbekistan, and take timely actions to prevent the spread of the epidemic. Methods Questionnaire survey was conducted to collect screening subjects’information. Palpation of the liver, spleen and superficial lymph nodes was performed by a physician, and the lesions on the frequently exposed skin were detected by a dermatologist. In addition, the liver and spleen sizes were measured using B-mode ultrasonography, and serum samples were collected to be subjected to an rK39-based rapid diagnostic test for detection of visceral leishmaniasis. Leishmania was detected using microscopy in the specimens sampled from the lesioned skin, and the parasites species was identified using molecular assays in parasitologically positive specimens. Results Among the 181 employees screened, enlarged cervical lymph nodes were palpable in 6 subjects, and skin lesions were found in 12 cases. B-mode ultrasonography displayed hepatosplenomegaly in 5 cases, and rK39 test were positive in 3 serum samples. Two classical lesioned skin specimens were sampled, and Leishmania was detected in one specimen. The promastigote DNA was extracted and two fragments of 120 bp and 350 bp in sizes were amplified using PCR assay with K13A/K13B and L5.8S/LITSR primers specific to Leishmania. The two amplification products were 90% and 98% homologous to the corresponding sequences of L. major (GenBank accession numbers: EU370906.1 and FN677342.1). Conclusions Six patients with cutaneous leishmaniasis were screened, including 2 uncured cases. One uncured case was diagnosed as imported cutaneous leishmaniasis caused by L. major infection.
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Leishmaniasis is one of the neglected diseases that remain in need for pharmacological alternatives. In this context, N-Myristoyltransferases (NMT) arise as interesting targets to explore since they are involved in the co/post-translational processing of peptides which are responsible for host cell invasion. Studies that consider these enzymes as targets point out the potential of benzoheterocyclic compounds as inhibitors of Candida albicans's N-myristoyltransferase. Here we applied a combination of comparative binding site analysis and molecular docking studies based on a Piggyback approach in the search for new Leishmania major NMT ligands. Our results revealed that NMT enzymes from both pathogens present enough structural similarity to allow extrapolation of the knowledge available from C. albicans studies to develop new L. major NMT inhibitors. Molecular docking studies with benzoheterocyclic analogues indicate the potential of benzothiazole derivatives as L. major NMT ligands, giving rise to a completely new class of chemical compounds to be explored in the development of antileishmanial drugs.
Subject(s)
Benzofurans/pharmacology , Leishmaniasis/drug therapy , Leishmania major , Candida albicans , Enzymes/analysisABSTRACT
Aims: This study investigates the activity of tetracyclic iridoid compounds against Leishmania spp. and the mechanism(s) of action. Study Design: An experimental study. Place and Duration: Department of Parasitology, Noguchi Memorial Institute for Medical Research, between September 2017 and July 2018. Methodology: The 50 % inhibitory concentration (IC50) of compounds against Leishmania donovani and L. major promastigotes were determined after 48 hours of incubation using the Alamar blue. Cytotoxicity of compounds was determined against cell lines using MTT assay. The anti-amastigote activity of compounds was further assessed by DAPI (4′,6-diamidino-2-phenylindole) staining. The mechanism of cell death induced by compounds was determined using nexin assay. Mitosis, cytokinesis and morphometry were monitored by DAPI and Kinetoplastid Membrane Protein (KMP) staining. Cell cycle arrest induced by compounds was analyzed by FACS. Results: Molucidin and ML-F52 inhibited the growth of promastigote in L. donovani (Molucidin; IC50 = 2.94±0.60 µM, ML-F52; IC50 = 0.91±0.50 µM) and L. major (Molucidin; IC50 = 1.85± 0.20 µM, ML-F52; IC50 = 1.77± 0.20 µM). ML-F52 had a 10-fold cytotoxic effect on parasites relative to normal cell lines. Against intracellular forms, Molucidin and ML-F52 inhibited intracellular amastigote replication and infectivity. Amphotericin B, Molucidin and ML-F52, induced a dose-dependent apoptotic effect on promastigotes. Although no change in KMP-11 expression was observed, iridoids inhibited cell division and morphological changes in promastigote cultures. Molucidin and ML-F52 induced apoptotic mechanism of cell death, inhibited cytokinesis and induced phenotypic changes in promastigotes. Molucidin further induced ‘’nectomonad-like’’ forms and loss of kDNA, ML-F52 induced ‘cell-rounding’ with loss of flagellum. Molucidin also induced cell growth arrest at G2-M phase (54.5 %). A significant induction of apoptosis (P = .05) was shown by an enhanced peak in the sub-G1 confirming the apoptotic inducing properties of iridoids. Conclusion: This study shows the anti-leishmania activity of tetracyclic iridoids which could be further investigated for the development of new chemotherapy against Leishmaniasis.
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ABSTRACT Background Leishmania major is a causative agent of zoonotic cutaneous leishmaniasis in the center of Iran, Abarkouh district. Molecular characterization and precise incrimination of Leishmania species was carried out to perform controlling measurements and to design treatment programs for zoonotic cutaneous leishmaniasis. Methods All smears isolated from ulcers of suspected patients were examined under a light microscope and graded for amastigotes frequency. Extraction of DNA, PCR, RFLP and sequencing of ITS-rDNA genotype were done to increase the efficacy of Leishmania parasites identification at their species-specific level and to detect any Leishmania infections within. Results Humans were found to be infected with L. major with high infection frequency and also Leishmania tropica was identified with low occurrence for the first time as non-native species using molecular analyses. The rates of infections was considerable with microscopic observation (n= 65, 73%) out of 89 smears prepared from suspected patients. Molecular analyses showed that the density of L. major was significantly higher (n= 48, 53.93%) than L. tropica (n= 4, 4.49%) (Mann-Whitney U test: p< 0.05) and two samples (2.25%) remained ambiguous after several sequencing. L. major did not have diversity with two common haplotypes but L. tropica were found to exhibit high diversity with three novel haplotypes. Conclusion L. major was considered the causative agent of leishmaniasis in the region, but the identification of a non-native L. tropica revealed the importance of further isolation of Leishmania parasites following molecular analyses and confirmation, and also revealed the importance of further isolation of Leishmania parasites from patients of the field areas who do not have easily access to health care centers for specialized treatment strategies.
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Humans , Animals , Male , Female , Leishmania tropica/genetics , Leishmaniasis, Cutaneous/parasitology , Leishmania major/genetics , Rural Population , Haplotypes , Polymorphism, Restriction Fragment Length , Leishmania tropica/isolation & purification , Leishmania tropica/ultrastructure , Polymerase Chain Reaction , DNA, Protozoan/isolation & purification , DNA, Protozoan/genetics , Leishmaniasis, Cutaneous/pathology , Leishmaniasis, Cutaneous/epidemiology , Leishmania major/isolation & purification , Endemic Diseases , IranABSTRACT
Cutaneous leishmaniasis (CL) can be seen in 2 forms, zoonotic and anthroponotic, in Iran. In this study, epidemiological aspects of CL were studied during an 8-year period (2009–2016) in city of Kashan, central Iran. The demographic and epidemiological data, including age, sex, occupation, number and site of the lesions, treatment regimen, past history of CL, and season of all patients were gathered from the health centers. Descriptive statistics were used to describe features of the study data. Total 2,676 people with CL were identified. The highest annual incidence was estimated to be 182 per 100,000 population in 2009 and the least was in 2016 (47 per 100,000 population). The highest frequency affected age groups were observed in 20–29 year-old patients (20.9%). More than 51% of the patients were under 30 years old. The maximum frequency of the disease, 1,134 (43.3%), was seen in autumn. The most common location of lesions was hands (61.4%). Most of the patients (81.6%) were treated by systemic glucantime regimen. In the city of Kashan, the incidence rate of the CL disease is significantly higher than many other regions of Iran. To reduce the risk of disease, control of reservoir hosts and vectors of disease, and education of individual protection are strongly recommended.
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Humans , Education , Epidemiologic Studies , Epidemiology , Hand , Incidence , Iran , Leishmania major , Leishmania tropica , Leishmaniasis, Cutaneous , Occupations , Seasons , UrbanizationABSTRACT
BACKGROUND: Cutaneous leishmaniasis is a tropical infection of public health importance. Numerous treatment approaches are in practice with variable degree of success however its management has no universal consensus or practice guidelines to follow. OBJECTIVE: Analyze the management of cutaneous leishmaniasis retrospectively at a central hospital of Jazan Province, Kingdom of Saudi Arabia to identify the current treatment pattern and compare the outcomes. METHODS: This cross-sectional study was conducted based on the hospital records of patients who attended the dermatology clinic for cutaneous leishmaniasis during the year 2012 to 2015. RESULTS: Forty three patients were included in the study. There was a male preponderance (65.1%) among the patients and 60.5% of them were of pediatric age group. Monotherapy was the initial choice for 58.1% of the patients. Intralesional sodium stibogluconate (SS-IL) was the most preferred treatment for initial therapy, as monotherapy and as part of combination therapy. A complete response was achieved in 22 patients (51.2%) with initial therapy. Among the different treatment groups, SS-IL+itraconazole showed significantly higher complete response rate compared to other treatments offered as initial therapy (p<0.01). Initial SS-IL monotherapy provided complete response in 41.2% patients receiving it, while itraconazole monotherapy provided complete response in 75% and 90.9% of the patients receiving initial itraconazole+SS-IL combination therapy with achieved complete response. CONCLUSION: The findings and observations suggest that initial combination therapy with SS-IL+itraconazole significantly improved the complete response rates and thus reduced the need for additional or prolonged therapies.
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Humans , Male , Antimony Sodium Gluconate , Consensus , Cross-Sectional Studies , Dermatology , Hospital Records , Injections, Intralesional , Itraconazole , Leishmania major , Leishmania tropica , Leishmaniasis, Cutaneous , Observational Study , Public Health , Retrospective Studies , Saudi ArabiaABSTRACT
Objectives: To detect Leishmania species in human patients, animal reservoirs and Phlebotomus sandflies in Waziristan, Pakistan. Methods: Tissue smears and aspirates from 448 cutaneous leishmaniasis (CL) suspected patients were analyzed. To sort out role of the reservoir hosts, skin scrapings, spleen and liver samples from 104 rodents were collected. Furthermore, buffy coat samples were obtained from 60 domestic animals. Sandflies were also trapped. All human, animals and sandfly samples were tested by microscopy, kinetoplastic PCR and internal transcribed spacer 1 (ITS1) PCR followed by restriction fragment length polymorphism for detection of Leishmania species. Results: An overall prevalence of 3.83% and 5.21% through microscopy and ITS1 PCR respectively was found. However, the statistically non-significant correlation was found between area, gender, and number of lesions. The presence of rodents, sandflies, domestic animals and internally displaced people increased the risk of CL. Using ITS1-PCR-RFLP, Leishmania tropica (L. tropica) was confirmed in 106 samples while 25 of the isolates were diagnosed as Leishmania major (L. major). Similarly, 3/104 rodents were positive for L. major and 14 pools of DNA samples containing Phlebotomus sergenti sandflies were positive for L. tropica. None of samples from domestic animals were positive for leishmaniasis. Conclusions: In the present study, L. tropica and L. major are found to be the main causative agents of CL in study area. Movement of internally displaced people from CL endemic areas presents a risk for nearby CL free areas. To the best of our knowledge, we report for the first time L. major infection in rodents (Rattus rattus) and L. tropica in Phlebotomus sergenti sandflies trapped in Waziristan, Pakistan.
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Objective: To appraise the activity of voriconazole against Leishmania major (L. major) in vitro and its effectiveness on wound regeneration in cutaneous leishmaniasis in BALB/c mice. Methods: The IC
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Objectives:To detect Leishmania species in human patients, animal reservoirs and Phlebotomus sandflies in Waziristan, Pakistan.Methods:Tissue smears and aspirates from 448 cutaneous leishmaniasis (CL) suspected patients were analyzed. To sort out role of the reservoir hosts, skin scrapings, spleen and liver samples from 104 rodents were collected. Furthermore, buffy coat samples were obtained from 60 domestic animals. Sandflies were also trapped. All human, animals and sandfly samples were tested by microscopy, kinetoplastic PCR and internal transcribed spacer 1 (ITS1) PCR followed by restriction fragment length polymorphism for detection of Leishmania species.Results:An overall prevalence of 3.83% and 5.21% through microscopy and ITS1 PCR respectively was found. However, the statistically non-significant correlation was found between area, gender, and number of lesions. The presence of rodents, sandflies, domestic animals and internally displaced people increased the risk of CL. Using ITS1-PCR-RFLP, Leishmania tropica (L. tropica) was confirmed in 106 samples while 25 of the isolates were diagnosed as Leishmania major (L. major). Similarly, 3/104 rodents were positive for L. major and 14 pools of DNA samples containing Phlebotomus sergenti sandflies were positive for L. tropica. None of samples from domestic animals were positive for leishmaniasis.Conclusions:In the present study, L. tropica and L. major are found to be the main causative agents of CL in study area. Movement of internally displaced people from CL endemic areas presents a risk for nearby CL free areas. To the best of our knowledge, we report for the first time L. major infection in rodents (Rattus rattus) and L. tropica in Phlebotomus sergenti sandflies trapped in Waziristan, Pakistan.
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Objective:To appraise the activity of voriconazole against Leishmania major (L. major) in vitro and its effectiveness on wound regeneration in cutaneous leishmaniasis in BALB/c mice.Methods:The ICResults:The ICConclusions:Our results demonstrate that voriconazole can be an option in treating the cutaneous leishmaniasis by L. major.
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BACKGROUND Leishmania major is an Old World species causing cutaneous leishmaniasis and is transmitted by Phlebotomus papatasi and Phlebotomus duboscqi. In Brazil, two isolates from patients who never left the country were characterised as L. major-like (BH49 and BH121). Using molecular techniques, these isolates were indistinguishable from the L. major reference strain (FV1). OBJECTIVES We evaluated the lipophosphoglycans (LPGs) of the strains and their behaviour in Old and New World sand fly vectors. METHODS LPGs were purified, and repeat units were qualitatively evaluated by immunoblotting. Experimental in vivo infection with L. major-like strains was performed in Lutzomyia longipalpis (New World, permissive vector) and Ph. papatasi (Old World, restrictive or specific vector). FINDINGS The LPGs of both strains were devoid of arabinosylated side chains, whereas the LPG of strain BH49 was more galactosylated than that of strain BH121. All strains with different levels of galactosylation in their LPGs were able to infect both vectors, exhibiting colonisation of the stomodeal valve and metacyclogenesis. The BH121 strain (less galactosylated) exhibited lower infection intensity compared to BH49 and FV1 in both vectors. MAIN CONCLUSIONS Intraspecific variation in the LPG of L. major-like strains occur, and the different galactosylation levels affected interactions with the invertebrate host.
Subject(s)
Humans , Leishmania major , Lysosome-Associated Membrane Glycoproteins , Psychodidae , Host-Parasite InteractionsABSTRACT
Objective:To investigate the leishmanicidal effects of two antioxidants,caffeic acid and quercetin on Leishmania major (L.major) promasdgotes in vitro,and their immunomodulatory effects on infected phagocytes derived from susceptible BALB/c mice.Methods:Caffeic acid and quercetin-induced cell death was examined by Pi-Hoechst double staining of L.major promastigotes and MTT assay,in the presence or absence of protease inhibitors in vitro.Caffeic acid or quercetin were administered subcutaneously to BALB/c mice infected with L.major promastigotes through a dorsal air pouch.Nitric oxide and superoxide anion production by phagocytes infiltrating the air pouch and the expression of inducible nitric oxide synthase (iNOS),tumor necrosis factor alpha (TNF-α) and nuclear factor kappa B in the air pouch membrane were therefore evaluated using appropriate methods.Results:Caffeic acid and quercetin displayed a dose-dependent cytotoxic effect against L.major promastigotes,and induced cell death via caspases-independent pathways.In vivo,L.major promastigotes inoculation into air pouch cavity of BALB/c mice leads to a sequential influx of neutrophils (hours),followed by macrophages (days).Results showed that L.major delayed apoptosis of infected neutrophils and macrophages by the cleavage of the nuclear factor kappa B p65RelA subunit,and persisted by inhibiting TNF-α and iNOS expression and reactive oxygen species generation.Caffeic acid or quercetin restored reactive oxygen species production and TNF-α-induced iNOS activity,and abrogate apoptosis delay of infected phagocytes.Conclusions:The leishmanicidal effect of caffeic acid and quercetin on promastigotes and amastigotes,as well as reactivation of infected phagocytes apoptosis,suggested a potential therapeutic role against cutaneous leishmaniasis.
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Objective To evaluate the effect of doxorubicin and its pegylated liposomal formulation (Doxil, Caelyx) on in vitro susceptibility of promastigote and amastigote stages of Leishmania major. Methods Throughout in vitro assays the IC
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OBJECTIVE@#To evaluate the effect of doxorubicin and its pegylated liposomal formulation (Doxil, Caelyx) on in vitro susceptibility of promastigote and amastigote stages of Leishmania major.@*METHODS@#Throughout in vitro assays the IC was calculated in the promastigotes and amastigotes forms in J774 macrophage cell line. Also as cytotoxicity in J774 cell line macrophages.@*RESULTS@#Doxorubicin and Doxil showed the same activity against promastigote form with IC values of 10.49 μg/mL and 9.63 μg/mL, respectively. Similarly, the amastigote stage was susceptible at concentration of at least 1 μg/mL when compared to positive control (P < 0.0001). Also, cytotoxicity assay against macrophage revealed no toxicity on the host cells at IC concentrations.@*CONCLUSIONS@#Our findings demonstrated the efficacy of both doxorubicin and its pegylated liposomal formulation on L. major at low concentrations. Further researches are needed for evaluating the safety of drugs in animal model particularly as topical formulation.
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Objective To investigate the leishmanicidal effects of two antioxidants, caffeic acid and quercetin on Leishmania major (L. major) promastigotes in vitro, and their immuno-modulatory effects on infected phagocytes derived from susceptible BALB/c mice. Methods Caffeic acid and quercetin-induced cell death was examined by Pi-Hoechst double staining of L. major promastigotes and MTT assay, in the presence or absence of protease inhibitors in vitro. Caffeic acid or quercetin were administered subcutaneously to BALB/c mice infected with L. major promastigotes through a dorsal air pouch. Nitric oxide and superoxide anion production by phagocytes infiltrating the air pouch and the expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α) and nuclear factor kappa B in the air pouch membrane were therefore evaluated using appropriate methods. Results Caffeic acid and quercetin displayed a dose-dependent cytotoxic effect against L. major promastigotes, and induced cell death via caspases-independent pathways. In vivo, L. major promastigotes inoculation into air pouch cavity of BALB/c mice leads to a sequential influx of neutrophils (hours), followed by macrophages (days). Results showed that L. major delayed apoptosis of infected neutrophils and macrophages by the cleavage of the nuclear factor kappa B p65
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Despite the broad distribution of leishmaniasis among Iranians and animals across the country, little is known about the genetic characteristics of the causative agents. Applying both HSP70 PCR-RFLP and sequence analyses, this study aimed to evaluate the genetic diversity and phylogenetic relationships among Leishmania spp. isolated from Iranian endemic foci and available reference strains. A total of 36 Leishmania isolates from almost all districts across the country were genetically analyzed for the HSP70 gene using both PCR-RFLP and sequence analysis. The original HSP70 gene sequences were aligned along with homologous Leishmania sequences retrieved from NCBI, and subjected to the phylogenetic analysis. Basic parameters of genetic diversity were also estimated. The HSP70 PCR-RFLP presented 3 different electrophoretic patterns, with no further intraspecific variation, corresponding to 3 Leishmania species available in the country, L. tropica, L. major, and L. infantum. Phylogenetic analyses presented 5 major clades, corresponding to 5 species complexes. Iranian lineages, including L. major, L. tropica, and L. infantum, were distributed among 3 complexes L. major, L. tropica, and L. donovani. However, within the L. major and L. donovani species complexes, the HSP70 phylogeny was not able to distinguish clearly between the L. major and L. turanica isolates, and between the L. infantum, L. donovani, and L. chagasi isolates, respectively. Our results indicated that both HSP70 PCR-RFLP and sequence analyses are medically applicable tools for identification of Leishmania species in Iranian patients. However, the reduced genetic diversity of the target gene makes it inevitable that its phylogeny only resolves the major groups, namely, the species complexes.