Live attenuated Leishmania major p27 gene knockout as a novel vaccine candidate: A study on safety, protective immunity and efficacy against canine leishmaniasis caused by Leishmania infantum.

Canine leishmaniasis (CanL) is an important parasitic e disease caused by Leishmania infantum and is transmitted by female phlebotomine sand flies primarily between canines and secondarily to humans. Recently, we showed that immunization with Leishmania major p27 gene knockout (Lmp27-/-) as a live attenuated vaccine was safe, induced immunogenicity, and protected against the development cutaneous and visceral leishmaniasis in mice. The p27 protein is a component of the COX protein complex which is responsible for ATP production. In this study, we analyzed the Lmp27-/- candidate vaccine potential with this regard to the safety and induction of immunogenicity and protection against CanL. Variables such a clinical manifestation, anti-Leishmania antibodies using direct agglutination test (DAT), lymphocyte proliferation, delayed-type hypersensitivity (DTH), bone marrow aspiration (BMA) and parasite burden using parasitological and molecular examinations were measured. The results demonstrated that the Lmp27-/- vaccinated group showed no clinical signs after inoculation with Lmp27-/- mutant during a 12-month follow-up, and had significantly higher T-cell responses (Lymphocyte proliferation and DTH), lower seroconversion and parasite burdens following a challenge inoculation with L. infantum after 6-mounth. In conclusion, vaccination with Lmp27-/- parasites would be safe and provide significant immunoprotectivity and efficacy against infection with wild type (WT) L. infantum.

Evaluation of curcumin and CM11 peptide alone and in combination against amastigote form of Iranian strain of L. major (MRHO/IR75/ER) in vitro.

Curcumin (diferuloylmethane) is the main phytochemical of Curcuma longa Linn, an extract of the rhizome turmeric. For thousands of years, turmeric among other natural products has been used as a dietary spice and as a medicinal plant in Asian countries. The present study reports the leishmanicidal activity of curcumin in different concentrations (10 µM, 20 µM, 40 µM). It is also showing the effect of CM11 peptide (8 µM) alone and in combination with curcumin (10 and 20 µM) as a leishmanicidal drug. The experiments were performed with the amastigote form of Leishmania major (MRHO/IR/75/ER) in vitro and the leishmanicidal activity was analyzed after 12 and 24 h of incubation by Giemsa and DAPI staining. Further investigation was done by using semi-quantitative PCR with new designed common primer pair derived from an 18S rRNA gene belonging to the L. major and mouse, which amplified the above-mentioned gene segments simultaneously with different PCR product size. Our findings showed that curcumin had leishmanicidal activity in a dose and time-dependent manner and its lowest effective dose was at concentrations of 40 µM afetr12 h and 10 µM after 24 h. The IC50 value of curcumin against amastigote forms of L. major was 21.12 µM and 11.77 µM after 12 and 24 h, respectively. Treatment of amastigote form with CM11 (8 µM) alone and in combination with curcumin (10 µM and 20 µM) showed less leishmanicidal activity. Interestingly, CM11 in combination with curcumin (10 µM and 20 µM) had even less leishmanicidal effect compared to curcumin alone in the same concentrations (10 µM and 20 µM). The semi-quantitative PCR analysis confirmed the data achieved by Giemsa and DAPI staining and showed that curcumin reduced the PCR product derived from amastigote form in concentration and time-dependent manner compared to the genome of the host cells.

Detection of Leishmania infantum Infection in Reservoir Dogs Using a Multiepitope Recombinant Protein (PQ10).

Visceral leishmaniasis is a neglected disease caused by Leishmania infantum and transmitted via female sand flies. Canine visceral leishmaniasis diagnosis should be performed as soon as possible, even on the basis of only a few or even a single clinical sign, to enhance the prediction of disease and to avoid both dog and human transmission and unnecessary euthanasia of apparently positive dogs. In the present work, we examined whether PQ10 recombinant protein could be suitable for immunological detection of Leishmania infantum infection. The coding sequence of PQ10 recombinant protein was sub-cloned in pET28 expression vector and was commercially synthesized by GENERAY Biotechnology, China. In the following process, sequencing with proper primers was done and the expression, optimization of expression and protein purification were performed. The efficacy of PQ10 for serodiagnosis was evaluated with 100 serum samples collected from dogs living in the visceral leishmaniasis endemic areas of Iran. Samples (n=20) of the dogs with other infectious disease were also be collected. The synthesized colones verified by the sequencing with proper primers. In the following process, expression, optimization of expression and protein purification performed and the purified recombinant protein confirmed by western blot. The ELISA was performed with PQ10 recombinant protein. The sensitivity of ELISA that was evaluated with sera from naturally infected dogs was 94%. The specificity value of the ELISA determined with sera from healthy dogs and from dogs with other infectious diseases was 86%. The positive predictive value (PPV) and negative predictive value (NPV) determined 87.03% and 93.47% respectively. Our findings indicated to the potential use of this recombinant protein in the diagnosis of canine visceral leishmaniasis.

An MD-based systematic study on the mechanical characteristics of a novel hybrid CNT/graphene drug carrier.

This paper is aimed to assess the mechanical properties of a hybrid graphene-carbon nanotube carrier embedded with doxorubicin (DOX). Utilizing molecular dynamics simulation, the results reveal that by increasing the temperature from 309 to 313 K, the elastic modulus of the GS/CNT/DOX carrier decreases from 0.8 to 0.74 TPa. Also, it is shown that the presence of chitosan molecules enhances the mechanical characteristics of the proposed nanocarrier. Taking the chirality of the graphene sheet into account, the results indicate that by increasing the size of the graphene sheet, the failure stress is slightly increased for the armchair type. However, this value decreases as the size of the zigzag sample increases. Additionally, the influence of aspect ratio on the elastic modulus, fracture stress, and fracture strain of these systems is systematically examined. It has been shown that the failure stress may change significantly with increasing this parameter, especially for carrier systems having zigzag carbon nanostructures. Moreover considering various voids content in the CNT structure, the weakening effect of defects is systematically explored. Also, the dependence of the mechanical features of the proposed hybrid carrier on the presence of DOX molecules is studied via MD simulations. Finally, we have investigated the role of CNT physical characteristics including its size and chirality on the results. Graphical abstract.

The frequency of fungi isolated from the skin and hair of asymptomatic cats in rural area of Meshkin-shahr-Iran.

BACKGROUND: Dermatophytosis is a frequent cutaneous infection affecting the keratinized tissues of humans, pets and livestock. Animals can carry dermatophytic elements asymptomatically and are considered to play an important role in the epidemiology of the disease. As exposure to any infected lesion free animals, especially cats, may lead to the development of infection in humans. OBJECTIVES: This study was done to determine the frequency of fungal agents isolated from skin and hair of cats living in rural areas of Meshkin-shahr, Iran. ANIMALS: A total of 103 asymptomatic cats living in rural areas of the region were studied. METHODS: This cross-sectional descriptive study was performed in Medical Mycology Laboratory, School of Public Health, Tehran University of Medical Sciences from February 2015 to July 2016. A total of 103 asymptomatic cats were studied. Mycological analysis including direct examination and culture on SC, SCC and DTM of the collected samples were conducted. For molecular confirmation when needed, panfungal PCR targeting the ITS1 region of the rDNA gene cluster using primers ITS1 and ITS4 were performed. Gender and age were also recorded. RESULTS: None of the 103 cats examined were positive for fungal elements on direct examination. However, 15 (14.5%) cases showed dermatophytes growth. T. verrucosum was the most common etiologic agents of dermatophytosis. Although the gender of the cats had not significant association with dermatophytosis prevalence, age was a significant influential risk factor (P=0.019). Aspergillus spp., Alternaria spp., Rhizopus spp., Penicillium spp.and paecilomyces spp. in descending frequency were the most predominantly identified saprophytic fungi. CONCLUSION: Our findings clearly highlighted the epidemiological role of asymptomatic cats in spreading dermatophytosis to humans and other animals.

Evaluation of Th17 associated antigen in Old World Cutaneous Leishmaniasis: A comparative study in acute versus chronic human cutaneous Leishmaniasis using immunohistochemistry.

There are little information about Th17 cells and cutaneous Leishmaniasis (CL), due to an important effect of Th17 cells on immune response, it is worth to explore the role of Th17 on CL. The purpose of this study was to assess Th17 population in patients with acute vs. chronic CL lesions in comparison with skin samples collected from healthy volunteers in an endemic region of Old World CL. A total of 49 patients with clinical manifestations of chronic (n=16) and acute (n=33) CL lesions were recruited. The clinical diagnosis of CL was confirmed by direct smear or PCR. Biopsy specimens from prelesional skin of non-infectious lesions of 30 healthy individuals were used as control. Tissue sections of 3µm thickness were prepared and used for immunohistochemistry (IHC) analysis with primary antibody specific for Th17 associated antigen (CD161). For IHC, Envision+ (DakoCytomation) system was used and developed by using diaminobenzidine (DakoCytomation). The mean age of 33 patients with acute CL and the mean age of 16 patients with chronic CL were accordingly 45.24±16.43 and 33.56±15.87. In acute and chronic CL the mean (±standard deviation) and median (±interquartile range) were accordingly 2.92±2.21, 2.56±2.9 and 2.1±1.99, 1.54±2.81. In healthy controls the mean (±standard deviation) and median (±interquartile range) were 0.72±0.41 and 0.61±0.58 respectively. With pairwise comparison of acute, chronic and control groups, there were significant difference between acute and control (P value < 0.001), chronic and control (P value = 0.043). The results showed that there was an increasing cellular response of Th17 in both acute and chronic CL patients. Th17 was significantly higher in patients with acute and chronic CL lesions in comparison with healthy control group. However, there was no significant difference between acute and chronic infection concerning to Th17 cells.

Determination of the Effective Dose of Curcumin alone and in Combination with Antimicrobial Peptide CM11 on Promastigote Forms of Iranian Strain of L. major (MRHO / IR / 75 / ER).

Zoonotic cutaneous leishmaniasis t caused by Leishmania major is spread in focal areas of more than 90 countries in the tropics, subtropics, and southern Europe. In the absence of any effective vaccine, the only means to treat and control leishmaniasis is conventional medication. Glucantime is the first choice of anti-leishmanialdrug, has serious side effects like high toxicity, exorbitant cost, problems with the administration and development of resistance. Curcumin is the active component from the rhizome of herb Curcuma longa, possessing many pharmacological and biological activities with antiprotozoal and anti-proliferative effects which make it a good alternative to existing therapy. Antimicrobial peptides like CM11, a small peptide consisting of 11 amino acids, are also novel potential drugs against at least wide spectrum of microbial organisms. The aim of this study was to evaluate the effect of curcumin alone and in combination with CM11 on promastigote form of L. major (MRHO / IR / 75 / ER) for 12h and 24h in vitro. The results of Giemsa staining showed that the morphology of the flagellum and cell shape increased changed with increasing concentration of curcumin (5 &micro;M, 10 &mu;M, 20 &mu;M, 40 &mu;M and 80 &mu;M). MTT and Trypan blue results demonstrated that the promastigotes were susceptible against curcumin in dose and time dependent manner, while CM11 alone at concentration of 8 &micro;M as well as in combination with 10 and 20 &micro;M curcumin had no significant effect on promastigotes. Our results revealed that curcumin can provide a new curative candidate against cutaneous leishmaniasis.

The relation of serum prolactin levels and Toxoplasma infection in humans.

BACKGROUND: Toxoplasma gondii is an intracellular protozoan parasite distributed worldwide. Although the infection is benign in immunocompetent individuals, it is life threatening and complicated in immunocompromised patients and fetuses of pregnant women who received their first exposure to T. gondii during the pregnancy. Prolactin (PRL) is a hormone that is secreted by the pituitary gland, and it is confirmed that it plays a role in the immune system. The present study was carried out to assess the possible relation between serum PRL levels and Toxoplasma infection frequency in human. METHODS: In this cross-sectional study, 343 serum samples (240 from women and 103 from men) were collected from individuals who were referred for PRL checking in laboratories of Karaj, Iran. Blood samples were collected, and sera were separated and analyzed for the detection of anti-Toxoplasma IgG antibody by ELISA method. The levels of PRL were measured by Roche Elecsys 2010 analyzer, electrochemiluminescence technology. RESULTS: Of 343 sera, 110 samples (32%) consisting of samples from 42 men and 68 women had anti-T. gondii IgG antibody. The prevalence of T. gondii infection in women with high PRL levels was lower than that in the comparison group with normal levels of PRL and the relationship between these two parameters was statistically significant (P=0.016). In women with hyperprolactinemia, by increasing of PRL levels, the prevalence of T. gondii infection was reduced. CONCLUSION: The results of the current study confirmed the previous studies based on immunoregulatory role of PRL and indicated that high levels of PRL could be related to Toxoplasma seronegativity in women.

Design of a dual-promoter expression vector harboring Sag1 and Gra7 genes from Toxoplasma gondii (RH strain).

Toxoplasmosis, a parasitic disease caused by Toxoplasma gondii, has possible irreparable consequences in immunocompromised patients and fetuses. Finding an effective method of prevention, such as vaccination, is crucial because of the global distribution of the parasite and the lack of effective anti-toxoplasmosis drugs. The Sag1 and Gra7 antigens of T. gondii can induce strong humoral and cell-mediated immune responses. Therefore, to develop a novel DNA vaccine against toxoplasmosis, we prepared a eukaryotic construct expressing the Sag1 and Gra7 genes of T. gondii (RH strain). We then verified the ability of this construct to produce the corresponding Sag1 and Gra7 antigens in mammalian cells. Using specific primers, the complete coding sequences of Sag1 and Gra7 genes were amplified by polymerase chain reaction (PCR) from the genomic DNA of T. gondii. Then, both genes were subcloned into pVitro2-neo-mcs plasmid. The pVitro-Sag1-Gra7 construct was subjected to colony PCR, enzymatic digestion, and sequencing to confirm successful subcloning. Sag1 and Gra7 expression in HeLa cells was investigated. Sag1 and Gra7 were successfully subcloned in pVitro2-neo-mcs plasmid. The expression of Sag1 and Gra7 in HeLa cells was confirmed through Western blot analysis. The recombinant pVitro-Sag1-Gra7 construct that simultaneously produces Sag1 and Gra7 antigens in one mammalian cell may be used to develop a novel protective vaccine against toxoplasmosis.

Geographic distribution and spatial analysis of Leishmania infantum infection in domestic and wild animal reservoir hosts of zoonotic visceral leishmaniasis in Iran: A systematic review.

Visceral leishmaniasis (VL) is an important parasitic disease which is endemic in different parts of Iran; and domestic and wild canines are principal reservoir hosts of the disease. The objective of this study was to review the spatial distribution of canine VL (CVL) caused by Leishmania infantum in domestic and wild canines in different geographical areas of Iran. An extensive literature search was conducted in different international and national databases, including Cochrane, MEDLINE/PubMed, Scopus, Web of Science and Iran Medex to find articles with the words "visceral leishmaniasis in Iran" in their titles and "canine visceral leishmaniasis in Iran" or "feline visceral leishmaniasis in Iran" or "accidental reservoir hosts of visceral leishmaniasis in Iran" in their subtitles, irrespective of the type and duration of study. Screening of the irrelevant articles from total 36,342, yielded 61 eligible articles. More than 93% of the studies were carried out on domestic dogs (Canis familiaris, n = 57) and the remaining were on other carnivores such as wild canines including foxes (Vulpes vulpes, n = 4), jackals (C. aureus, n = 6) and wolves (C. lupus, n = 6); while studies on domestic cats (Felis catus, n = 3) as well as desert rodents (n = 2) were rare. The average rate of L. infantum infections reported among domestic dogs using direct agglutination test (DAT) in Iran was 12.5%. The highest prevalence rate (14%) was reported from the northwest regions of the country where VL is endemic. The review indicates that CVL is endemic in various parts of Iran and domestic dogs are the main and potential reservoir hosts of the disease. Other carnivores, such as domestic cats and some species of desert rodents (Cricetulus migratorius, Mesocricetus auratus and Meriones persicus) seem to be playing a role in the maintenance of transmission cycle of L. infantum in the endemic areas of the disease.

Evaluation of Toxoplasma gondii soluble, whole and excretory/secretary antigens for diagnosis of toxoplasmosis by ELISA test.

The present study was performed to compare the soluble, whole and excretory/secretary antigens of Toxoplasma gondii (RH strain) in diagnosis of toxoplasmosis by ELISA method. Tachyzoites of T. gondii, RH strain were injected in intra-peritoneal cavity of BALB/c mice, after 4 days tachyzoites were harvested by peritoneal washing of the mice. For soluble antigen, exudates were centrifuged and sediment sonicated and then centrifuged at 4 °C, 1 h, supernatant collected and density of protein determined by Bradford method. For whole antigen after collecting, washing and centrifuging of peritoneal fluid the tachyzoites sediment was counted. In excretory/secretary antigen 1.5 × 108 tachyzoites were transferred in 1 ml tube of saline and incubated under mild agitation and after centrifuging, supernatant was collected and protein density determined by Bradford method. 176 human serum samples were evaluated for T. gondii IgG antibody with prepared antigens, and finally serum samples were evaluated by commercial ELISA kit (Trinity, USA) which was considered as gold standard method. In this study sensitivity and specificity of prepared antigens compared with commercial kit in ELISA method. Sensitivity and specificity of soluble antigen was 91.4 and 74.5 %, in whole antigen these parameters were 77.1 and 77.3 % and in excretory/secretary antigen were 28.5 and 74.5 % respectively. Soluble antigen had high levels of sensitivity and specificity in ELISA method and the results were rather resemble to commercial kit (Trinity, USA).

Comparison of the Toxoplasma gondii mice and cell culture derived antigens in ELISA assay.

Toxoplasmosis is an infectious disease caused by the coccidian parasite Toxoplasma gondii. Diagnosis is based on serological methods with detection of specific IgG and IgM antibodies. The present study was performed to compare the sensitivity and specificity of soluble antigen of T. gondii, RH strain obtained from mice and cell culture in ELISA method. Tachyzoites of T. gondii, RH strain that inoculated in mice peritoneum were collected. At the same time, tachyzoites were harvested from HeLa cell culture that infected with the parasite. Soluble antigen was prepared and ELISA method performed on 100 serum samples that were collected from different laboratories in Tehran, Iran. Commercial Trinity kit was used as gold standard. The sensitivity and specificity of T.gondii soluble antigen were higher in antigens that obtained from cell culture in comparison with mice peritoneum. T. gondii cell culture derived antigen has high sensitivity and specificity in ELISA test.

Seroprevalence of Toxoplasma gondii infection in domestic dogs in an area from northwest of Iran: a cross-sectional study using immunodominant surface antigen 1 (SAG1).

Toxoplasma gondii is an obligate intracellular protozoan parasite that infects humans and animals. T. gondii surface antigen 1 (SAG1) is an appropriate antigen with high specificity and sensitivity for the detection of T. gondii infection in humans and animal hosts. The aim of this study was to determine the seroprevalence of T. gondii infection using SAG1 antigen (P30) in ownership dogs in Meshkin-Shahr district in the northwestern Iran. The sera samples were collected from 171 domestic dogs and tested using indirect ELISA (SAG1 antigen). The data were analyzed using SPSS software version 13. From a total of 171 dogs, 82 (48 %) of them were sero-positive. No statistical significant difference was seen between T. gondii infection and gender (P = 0.995). The highest sero-prevalence of rate was observed in >5 years animals; but no statistical significant difference was seen between T. gondii infection and age (P = 0.589). Our findings indicate that Toxoplasma seropositivity rate is high in ownership dogs in northwest of Iran. This is probably due to high exposure to contaminated food, soil, or water sources with sporulated Toxoplasma oocysts.

Correlation between clinical responses with the drugsusceptibility of parasites in Iranian cutaneous leishmaniasis caused by Leishmania major.

Reviews have shown increasing number of Iranian patients with cutaneous leishmaniasis (CL) who are unresponsive to pentavalent antimonial compounds such as meglumine antimoniate (Glucantime, MA). The present investigation aims to determine the correlation between clinical responses (healing, or non-healing) with susceptibility of Leishmania parasites to glucantime. Initially, in vitro susceptibility of Leishmania parasites was carried out on 93 isolates using macrophage models. Identification of these species was also performed by molecular methods including Nested-PCR and PCR-RFLP. The f indicated that total isolated were L. major. A significant association between the clinical outcome and the in vitro effective concentration 50% (EC50) values was observed. Leishmania derived from patients with non-healing lesions had EC50 values at least 3-fold higher than parasites isolated from lesions of healing patients. By molecular methods, patterns for both sensitive and resistant samples demonstrated restriction band which is related to L. major. The obtained findings in the present study demonstrated that MA-resistant L. major field isolates are now frequent in Iran. Such studies help to find strategies for rapidly diagnosing resistance in order to improve the clinical management of CL.

Sequence variation in mitochondrial cox1 and nad1 genes of ascaridoid nematodes in cats and dogs from Iran.

The study was conducted to determine the sequence variation in two mitochondrial genes, namely cytochrome c oxidase 1 (pcox1) and NADH dehydrogenase 1 (pnad1) within and among isolates of Toxocara cati, Toxocara canis and Toxascaris leonina. Genomic DNA was extracted from 32 isolates of T. cati, 9 isolates of T. canis and 19 isolates of T. leonina collected from cats and dogs in different geographical areas of Iran. Mitochondrial genes were amplified by polymerase chain reaction (PCR) and sequenced. Sequence data were aligned using the BioEdit software and compared with published sequences in GenBank. Phylogenetic analysis was performed using Bayesian inference and maximum likelihood methods. Based on pairwise comparison, intra-species genetic diversity within Iranian isolates of T. cati, T. canis and T. leonina amounted to 0-2.3%, 0-1.3% and 0-1.0% for pcox1 and 0-2.0%, 0-1.7% and 0-2.6% for pnad1, respectively. Inter-species sequence variation among the three ascaridoid nematodes was significantly higher, being 9.5-16.6% for pcox1 and 11.9-26.7% for pnad1. Sequence and phylogenetic analysis of the pcox1 and pnad1 genes indicated that there is significant genetic diversity within and among isolates of T. cati, T. canis and T. leonina from different areas of Iran, and these genes can be used for studying genetic variation of ascaridoid nematodes.

Emergence of a new focus of visceral leishmaniasis due to Leishmania infantum in Golestan Province, north-eastern of Iran.

Over the last decade, a few cases of visceral leishmaniasis (VL) have been reported in some districts of the province of Golestan, in north-eastern Iran. The aim of the present study was to investigate the prevalence of Leishmania infantum infection among humans and domestic dogs by using direct agglutination test (DAT) and PCR assays in the eastern zone of the province. Between 2011 and 2012, blood samples were randomly collected from 450 humans and 50 domestic dogs, in the eastern zone of Golestan Province including 7 villages from Marave-tappeh district where new cases of human VL had been recorded there. Each of these samples was tested for anti-Leishmania antibodies, in DAT, and for L. infantum kinetoplast DNA on whole blood, in PCR-based assays. A total of 450 human samples, 6 (1.33 %) were found seropositive and 13 (2.8 %) was found PCR-positive. Of the 50 dog samples, 16 (32 %) were found seropositive and 15 (30 %) were PCR-positive. All PCR-positive dogs were found seropositive except one as well as 6 (46.2 %) PCR-positive humans were also found seropositive. Moreover, the species of L. infantum was detected in all PCR-positive samples. The high prevalence of VL in the study areas offer it has emerged as an endemic focus in the province. Further investigations on the vectors, reservoirs and human population are recommended.

Interleukin 4 (IL-4) gene promoter polymorphisms in Rhombomys opimus, the main reservoir of zoonotic cutaneous leishmaniasis.

Great gerbils (Rhombomys opimus) are the most common gerbils in center to northeast of Iran as well as central Asia and serve as reservoirs for the zoonotic agents, including Leishmania major, the principal etiologic agent of zoonotic cutaneous leishmaniasis (ZCL). The outcome of L. major infection in gerbils is not uniform. Among several immune-related factors including cytokine genes, the polymorphism in interleukin 4 (IL-4) promoter gene showed a great impact on outcome and pathological symptoms of L. major infection at least in mouse model. In this study gerbils' IL-4 promoter gene polymorphism is assessed. Specific primers were designed to develop a PCR-based assay to amplify IL-4 promoter gene to possibly define IL-4 promoter gene polymorphism in great gerbil populations with a range of Leishmania infection and symptoms collected from different foci of the central, north and northeast regions of Iran. The results showed that the designed primers amplify 689bp of the promoter gene. Sequence analysis of the promoter gene revealed five polymorphic sites assembly six haplotypes among the gerbil populations. Further studies are needed to assess whether or not the five polymorphisms cause different outcome phenotypes following infection with L. major in great gerbils. The data might be used to characterize the immune responses of R. opimus against L. major infection.

Gene regulation of pteridine reductase 1 in leishmania promastigotes and amastigotes using a full-length antisense construct.

BACKGROUND: Pteridine metabolic pathway is unusual features of Leishmania, which is necessary for the growth of parasite. Leishmania has evolved a complex and versatile pteridine salvage network which has the ability of scavenging a wide area of the conjugated and unconjugated pteridines especially folate and biopterin. In this study, we focus on the inhibition of ptr1 gene expression. METHODS: L. major ptr1 gene was cloned into pcDNA3 and digested using KpnI and BamHI. The gene was subcloned so that antisense will transcribe and called pcDNA-rPTR. Leishmania major was cultured and late logarithmic-phase promastigotes were harvested. The promastigotes were divided into two groups. One group was transfected with 50 µg of pcDNA-rPTR, whereas the other group was transfected with pcDNA3. Transfected cells were cultured and plated onto semi-solid media. Mouse pritonean macrophages were transfected using pcDNA-rPTR-tansfected promastigotes. Western blotting was performed on mouse transfected pritonean macrophages and extracts from transfected promastigotes of L. major using a L. major ptr1 antibody raised in rabbits. RESULTS: The PTR1 protein was not expressed in pcDNA-rPTR- tansfected promastigotes and mouse macrophage transfected with pcDNA-rPTR- tansfected promastigotes. CONCLUSION: This approach might be used to study the pteridine salvage pathway in Leishmania or to assess the possibility of using gene expression inhibition in the treatment of leishmaniasis.

Brain Tissue Cysts in Infected Mice with RH-Strain of Toxoplasma gondii and Evaluation of BAG1 and SAG1 Genes Expression.

BACKGROUND: Toxoplasma gondii is an obligate intracellular parasite that infects humans at high prevalence rates. The virulent RH strain of T. gondii is generally considered to have lost its cyst forming capacity. This study performed to obtain tissue cysts in mice infected with tachyzoites of RH strain treated with sulfadiazine (SDZ). It provides the opportunity to analyze the conversion of tachyzoite to bradyzoite stage of the RH strain, followed by stage-specific gene-expression analyzing. METHODS: Two groups of Swiss-Webster and BALB/c mice were infected subcutaneously with 10(4) tachyzoites of T. gondii, RH strain and given SDZ (300 mg/l) with NaHCO3 (5 g(-1)) in drinking water from day1 to day 14 post infection (p.i). The infected mice were sacrificed on day 50 post infection. Their brains were removed and the numbers of tissue cysts were microscopically counted. Total RNA was extracted from brains and cDNA synthesis was carried out. Finally, RT-PCR (Reverse transcription PCR) was used to detect the expression of bradyzoite (BAG1) and tachyzoite (SAG1) specific genes during tachyzoite / bradyzoite stage conversion. RESULTS: Sixty five percent of all infected mice were survived. Cysts were detectable in mice brain (45%) on day 50 p.i. Also RT-PCR of the brain samples was positive for SAG1 and BAG1. CONCLUSION: It seems that conversion of tachyzoites to bradyzoites in brain of mice undergoing SDZ was not completed until 50 days after inoculation.

Genotyping of Hydatid Cyst Isolated from Human and Domestic Animals in Ilam Province, Western Iran Using PCR-RFLP.

BACKGROUND: Hydatidosis or cystic hydatid disease is one of the most important diseases in human and animals. Identification of strains is important for improvement of control and prevention of disease. The aim of this study was to determine the strains isolated from human and domestic animals in Ilam Province, Iran, using PCR-RFLP method. METHODS: Respectively, 30 and 4 animal and human hydatid cysts were collected from different slaughterhouses and hospitals of the province. Protoscolices were separated and their DNA genome was extracted by extraction kit. rDNA-ITS1 of each isolated samples was duplicated by BD1(Forward) and 4s (Reverse) Primers. PCR products were studied by electrophoresis and then were digested using TaqI, HpaII, RsaI and AluI restriction enzymes. RFLP products were studied using electrophoresis on 1% agar gel. RESULT: A fragment of 1000bp was produced from amplification of rDNA-ITS1 of protoscolices using PCR method. After digestion of PCR product by AluI enzyme, 200bp and 800bp, by RsaI, 655bp and 345bp and by HpaII 700bp and 300bp sizes were obtained. TaqI enzyme had no change in fragment size and it remained 1000bp. Considering the method, Ilam strains was specified as E. granulosus sensu stricto (G1-G3). CONCLUSIONS: Although sheep strain (G1) is dominated in human and different animal in Iran and the world, but more efforts should be done to clarify the true genotype of Ilam strains specified as E. granulosus sensu stricto (G1-G3).