Background: Cutaneous leishmaniasis is a parasitic skin disease that is endemic in many parts of the world, particularly in Iran. Although the microscopic technique is a common method of diagnosis of leishmaniasis, species of leishmania cannot be differentiated by this method. So, molecular methods have special importance.Methods: In this study, kinetoplast-DNA polymerase chain reaction (kDNA-PCR) method was used to diagnose the leishmania species in Dargaz city, Iran. Direct slide smears were obtained from skin lesions of 94 patients suspected to the leishmaniasis referred to health centers of this city. Smears were stained using Gimsa method.PCR method was performed using specific kDNA primers. Data were analyzed using SPSS software.Findings: Among 94 subjects with skin ulcers suspected to cutaneous leishmaniasis, 82 (87.2%) were positive by direct microscopic smear and 85 (90.4%) by PCR in which 22 patients (26%) had Leishmania tropica and 63 (74%) had Leishmania major. Sensitivity and specificity of direct microscopic examination were calculated as 96% and 100%, respectively. There were statistically significant differences between disease occurrence and seasons (P=0.045), habitat (P=0.004), new or old building (new or old) (P=0.003), and distance to rodents' living sites (P=0.025).Conclusion: Dargaz city is a known site of zoonotic leishmaniasis in Iran; but this study shows that there are some new sites of anthroponotic leishmaniasis in this city, too. Leishmania major is the dominant species in Dargaz city but there is Leishmania tropica foci in this area, too.

Background: Leishmania is an obligate interacellular protozoa and sand fly, as a vector, transmits infectious forms of the parasite to vertebrate host. In this way it is important to find candidate antigens which could tend to prevent the disease. Methods: The gene coding mannose 1 phosphate guanyl transferase enzyme was amplified from genomic DNA isolated from the Iranian strain of L. major (MRHO/IR/75/ER) as a template. The Polymerase Chain Reaction (PCR) product was ligated into the pTZ57R plasmid and the recombinant gene was digested using restriction enzymes, BamHI and EcoRI. The fragment was ligated into the pET32a plasmid, as an expression vector. The cloned pET32a-GDP mannose was confirmed using restriction enzyme digestion method and the DNA fragment was sequenced.Findings: Electrophoresis method confirmed the PCR product is related to the enzyme mannose 1 phosphate guanyl transferase. After the ligation of the product into the pTZ57R and pET32a, and the restriction enzyme digestion by BamHI and EcoRI, the correct frame of cloned gene in vectors was confirmed.Conclusion: There was 92 percent homology between the cloned gene coding enzyme mannose 1 phosphate guanyl transferase in this study and the ones present in gene bank. It is suggested that the gene encoding mannose 1 phosphate guanyl transferase enzyme is conserved among different genera of Leishmania.

Background: Many studies have suggested the benefits of garlic oil extracts against Leishmania parasites. Recent research has proved that these derivatives (such as allicin) are cytotoxic and therapeutic effects of garlic are in fact due to an odorless compound (S-allyl-cysteine). Considering the small number of studies in this field, we evaluated the effectiveness of S-allyl-cysteine on survival, apoptosis, and proliferation of Leishmania major promastigotes in vitro.Methods: In this study Leishmania major promastigotes (MRHO/IR/75/ER) were used. The parasites were first cultured and then treated with S-allyl-cysteine at end concentrations of 2.5, 5.0, 10.0, 20.0, 40.0, and 50.0 mM. Their survival, apoptosis, and proliferation was assessed after 72 hours.Findings: None of the concentrations of S-allyl-cysteine could induce apoptosis in parasite promastigotes. Lower concentrations (5.0 and 10.0 mM) of S-allyl-cysteine had greatest effect on proliferation. On the other hand, direct observation showed that cultures comprising S-allyl-cysteine at end concentration of 20.0, 40.0, and 50.0 mM formed more rosettes.Conclusion: The antioxidant effects of S-allyl-cysteine have been previously proved. Similarly, this study showed that some concentrations of S-allyl-cysteine could affect the survival and growth of promastigotes in culture medium. Therefore, this substance seems to be a good stimulating factor for growth of parasite promastigotes in culture media.

 Regarding the high prevalence of Cutaneous Leishmaniasis the diagnosis, cure of it, also the problems and expense of culture of parasite, are four important characters. The effect of different bloods group of human, Cow, Sheep and goat on growth of promastigote of Leishmania tropica, L. major were examined and compare with the blood of rabbit. This study was performed during 1998-99 in Kashan.This study was performed on promastigote L.tropica, L.major with an analytical studies which was confirmed by World Health Organization. The examination was repeated 10 times. The time of compatibility with culture medium, maximum and minimum growth based on decrease of active parasite comparing with the basic number on the time of culture were determined and judged statistically.The time of compatibility with culture medium was equal in all cases and it was about 1 to 3 days. Musing blood of human, sheep and goat growth of parasite was reached at maximum in shorter time comparing with musing blood of cow. The minimum growth in the blood of rabbit and cow was more than blood of human, goat and sheep. The growth of 2 kinds of parasites in the blood of rabbit was more than blood of human, goat and sheep (P<0.05), but it was similar to the amount of growth of these parasites in blood of rabbit and cow.According to the importance of time of growth of promastigote, it is possible to replace the blood of man with the blood of rabbit.    

Leishmaniasis is a complex disease, with a wide spectrum of clinical manifestation affects over 12 million people globally. Cutaneous leishmaniasis is still an important public health problem in many parts of the world, specially the Middle East. In spite of various forms of local treatment none is generally suitable for all forms of the disease in all. In this research, therapeutic effects of paromomyeine sulfate in combination with gentamicine sulfate films on cutaneous leshmaniasis in Balb/c mice have been studied. According to data and experiments available in Iran and some endemic countries about treatment of the disease by paromonycin, a new physical form of the drug was designed (drug film) and parasiticidal effects were evaluated in mice model. The base of the films, ethyl cellulose and HPMC (Hydroxy propyl Methyl Cellulose) contained paromomycin 15% and gentamicine 0.5%. In this regard female Balb/c mice were infected with 2×106 L.major promastigotes (MRHO/1R/75/ER). The mice were divided in three groups, of ten mice each. The placebo and test groups were received prepared placebo and drug films respectively. The remaining group, control received no treatment. The results indicated that utilization of this physical form of the drug in a mentioned period of time caused significant cure effects in the test group, so that 80% of Balb/c mice were cured at the end of the treatment period and there was no sign of cure in placebo and control groups. In general and because of good characters of the drug films and related results in animals, it is possible to use the new form of the drug in a human trial.

Leishmaniasis is a protozoan zoonosis that causes problems in public health. The use of pentavalent antimony compounds as chemical drugs in the treatment of cutaneous leishmaniasis are associated with various limitations and side effects. Today, the plant extracts and their derivations are the promising sources in herbal medicine. The purpose of this study was to evaluate the effectiveness of oil extract Pistacia atlantica from Uramanat area in Kurdistan province, Iran, on promastigotes of standard strain Leishmania major in vitro. In this study, 10, 15, 25, and 50% concentrations of oily extract were added to the plate containing promastigotes of Leishmania major. The effectiveness of different concentrations of extract was determined by MTT (3-[4, 5-(dimethylthiazole-2-yl]-2, 5-diphenyltetrezolium bromide) colorimetric method and counting of living promastigotes. The results of present study showed, different concentrations of oil extract has an inhibitory effect on promastigotes of Leishmania and the extract 50% has the most effect. The results showed a significant different between in the number of promastigotes of Leishmania major in different concentrations of extract and exposure times (24, 48, and 72 hours) (P≤0. 05). The results show that extract of pistacia atlantica has an appropriate effect on Leishmania major in vitro. Therefore, it is suggested to be evaluated the effect of the extract Pistacia atlantica on leishmaniasis wounds.

Background and purpose: Leishmania is flagellated protozoa and causative agent of leishmaniosis. It is one of the main public health problems in developing countries. Cantharidin is terpenoid component that exists in Meloidae and Oedemeridae beetles. Cantharid in is vesicant and could induce apoptosis in cancerous cells. This study was performed to determine the role of cantharid in in inducing apoptosis in the Leishmania promastigotes and macrophages infected with parasite.Materials and methods: In this experimental study the effect of cantharid in was evaluated at concentration range of 0.5-50 mg/mL on the L. major promastigotes and concentration range of 5, 20 and 50 mg/mL on macrophages infected with L. major after 24, 48 and 72 hrs by flow cytometry assay.Results: Results showed that cantharid in with concentration levels of 50 mg/mL and 0.5 mg/mL has 68.50% cytotoxicity (as 63.23% apoptosis, 5.27% late apoptosis and 0% necrosis) and 14.29% cytotoxicity (as 13.12% apoptosis, 1.12% late apoptosis and 0.05% necrosis) in promastigotes after 72hrs, respectively. Cytotoxicity 0.5 mg/mL and 50 mg/mL cantharid in in infected macrophages after 48hrs was 61.81% (as 43.42% apoptosis, 1.27% late apoptosis and 17.11% necrosis) and 44.44% (as 31.05% apoptosis, 10.08% necrosis and 3.31% late apoptosis), respectively. Cytotoxicity of 50 mg/mL and 5 mg/mL cantharidin in non-infected macrophages after 48hrs was also 49.34% (as 21.35% apoptosis, 4.23% late apoptosis and 23.76% necrosis) and 43.79% (as 34.90% apoptosis, 7.27% necrosis and 1.61% late apoptosis), respectively.Conclusion: This research indicates that cantharid in at different concentration and times could induce apoptosis in L. major promastigotes and macrophages infected with amastigotes.

Objective: Leishmania major P4 gene is normally expressed during amastigote form of the parasite and can be good candidate for producing an effective vaccine. In this study we cloned this gene in suitable vector (pQE-30) for further vaccine preparation studies.Materials and Methods: Leishmania promastigotes were grown in N.N.N. medium and culture in RPMI 1640 cell culture medium. Total genomic DNA was extracted by centrifugation of promastigotes. The pellet was suspended in lysis buffer and followed by boiling method. PCR was carried out using P4 gene specific primers. PCR product was detected by agaros gel electrophoresis and cloned into Bluescript plasmid via T/A cloning method. Reaction was transformed into XL1- Blue competent cell and recombinant plasmid screened using agar plate contained X-gal and IPTG. The product was extracted, digested by restriction enzyme and electrophoresed on agarose gel.Results: Plasmid was extracted and cloned gene was released by restriction enzyme and subcloned into pQE-30 expression vector.Conclusion: This construct is ready for protein expression in in-vitro.

Background: Cutaneous leishmaniasis is an endemic disease in many countries, including Iran. Pentavalent antimony compounds used to treat this disease is associated with adverse effects for patients. The aim of this study was to evaluate the effectiveness of the alcoholic extract of Rumex on experimental lesion of Leishmania major in BALB/c mice.Methods: In this study, thirty mice were divided into 6 groups A to F, 5 mice in each group, and then inoculated subcutaneously at the base of the tail with 100 micro liter liquid phase culture containing promastigotes (1´106) of active Leishmania major promastigotes standard strain (MRHO/IR/75/ER). Cutaneous lesions were appeared approximately after 3 weeks. Three different concentrations of Rumex seeds alcoholic extract (0.3, 0.5 and 0.9) were used as an injection to Group D-F. Three other groups were considered as control; group A received no treatment, groups B and C were received ethanol alone and glucantime, respectively. All injections were performed 2 times a day for 15 days and every week the lesion diameter was measured in all groups. The number of parasites in the lesions stained smears were examined under a microscope. Information were recorded and analyzed using one-away ANOVA and post-hoc Tukey’s test.Results: The mean diameter of lesions decreased in concentration of 9mg/ml after 5 week and complete healing was observed in this group. Also the parasitic load was decreased significantly in comparison with glucantime injected group (p£0.05).Conclusions: The results suggest that the effective concentration of the herbs mentioned in this article (9mg/ml) could be used as an appropriate alternative medicine on human cutaneous leishmaniasis.