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.

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.

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.

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.

Comparison of real-time PCR and conventional PCR with two DNA targets for detection of Leishmania (Leishmania) infantum infection in human and dog blood samples.

Zoonotic visceral leishmaniasis (VL) is endemic in northwestern Iran. Real-time PCR, conventional PCR, and the direct agglutination test (DAT) were used to diagnose Leishmania infantum infection in blood samples from 100 domestic dogs and 100 humans. Based on clinical evaluation, 82 humans and 72 dogs from the endemic area were categorized as having asymptomatic infection, DAT positive with no clinical signs of VL, or symptomatic infection, DAT positive with at least one sign of VL. Eighteen human samples containing no Leishmania antibodies (DAT(-)) and 28 dog DAT(-) sera from non-endemic areas with no history of VL constituted negative controls. All 46 DAT(-) samples were also negative by Dipstick rK39. Bone marrow material was used for parasitological examinations in symptomatic VL, and peripheral blood samples were used for detection of L. infantum infection using conventional PCR and real-time PCR in non-symptomatic subjects. Two DNA targets (ITS1 kDNA) were used for conventional PCR. L. infantum antibodies in sera were detected by DAT. Parasitemia was measured by real-time PCR targeting kDNA using Taqman Assay. All 72 (100%) symptomatic (38/38) and asymptomatic (34/34) dog DAT(+)samples, 45 of 48 (93.8%) symptomatic human DAT(+) samples, and 32 of 34 (94.1%) human asymptomatic cases were identified by real-time PCR. The mean (59.19 vs 12.38 parasite equivalents/mL of blood) and median (16.15 vs 1 parasite equivalents/mL of blood) ranges of parasitemia were higher in dogs than in humans (P<0.05). The highest agreement was obtained between real-time PCR and DAT (99% in dogs and 95% in humans). Sensitivity of 100% and 93.9%, specificity of 96.4% and 100%, positive predictive values of 98.6% and 100%, and negative predictive values of 100% and 78.3% were found by real-time PCR for dog and human samples, respectively.

Genotyping of Echinococcus granulosus from domestic animals and humans from Ardabil Province, northwest Iran.

Cystic echinococcosis is endemic in Iran, particularly in Ardabil Province, where it causes health and economic problems. The genetic pattern of Echinococcus granulosus has been determined in most parts of Iran, except in this area. In the present investigation, 55 larval isolates were collected from humans (11), sheep (19), goats (4) and cattle (21). For analysis of the genetic characteristics of E. granulosus isolates, DNA sequencing of mitochondrial cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) genes was applied. Fifty isolates were successfully analysed, with 92% (46) and 8% (4) identified as G1 and G3 genotypes, respectively. The sequence analyses of the isolates displayed nine characteristic profiles in cox1 sequences and eight characteristic profiles in nad1 sequences. Based on these results, the sheep strain (G1 genotype) was the most prevalent in humans, sheep, goats and cattle. The buffalo strain (G3 genotype) was not only demonstrated in sheep (1 isolate) and cattle (1 isolate), but also for the first time in two human isolates. These findings will provide information for local control of echinococcosis.

Comparative proteomics study on meglumine antimoniate sensitive and resistant Leishmania tropica isolated from Iranian anthroponotic cutaneous leishmaniasis patients.

In order to define the protein expressional changes related to the process of meglumine antimoniate resistance in anthroponotic cutaneous leishmaniasis (CL), we performed a comparative proteomics analysis on sensitive and resistant strains of Leishmania tropica isolated from Iranian CL patients. Cell proteins were analysed with 2-dimensional electrophoresis and differentially expressed proteins were identified by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry. Image analysis of the matched maps identified 7 proteins that were either over- or down-expressed: activated protein kinase c receptor(LACK), alpha tubulin (x2), prostaglandin f2-alpha synthase, protein disulfide isomerase, vesicular transport protein and a hypothetical protein. The study shows the usefulness of proteomics in identifying proteins that may express differences between sensitive and resistant L. tropica isolates.

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.

Gene cloning, expression and serological evaluation of the 12-kDa antigen-B subunit from Echinococcus granulosus.

A 12-kDa subunit of antigen B from Echinococcus granulosus has recently been cloned, expressed and used in diagnostic ELISA to test human sera for evidence of cystic echinococcosis. The performance of the ELISA based on the recombinant antigen (rAgB) was compared with that of similar assays based on native antigen B (nAgB) or hydatid-cyst fluid. For the preparation of the rAgB, total RNA was extracted from Ec. granulosus protoscoleces so that antigen-B complementary DNA could be synthesised, amplified by PCR, and then cloned into the pQE30 expression vector. The recombinant plasmid was transformed in Escherichia coli and induced using isopropyl-beta-D-thiogalactopyrano-side. Bacterial samples were collected, lysed and then analysed by SDS-PAGE and western blotting. The recombinant protein was purified by affinity chromatography. Although the performance of the ELISA based on cyst fluid appeared identical to that of the assay based on the recombinant antigen (with a sensitivity, specificity, positive predictive value and negative predictive value of 96.0%, 97.0%, 97.2% and 95.5%, respectively), the corresponding results for the ELISA based on nAgB (98.6%, 100%, 100% and 98.5%) were slightly better. Despite this difference (which was not statistically significant), the comparative ease with which large quantities of the recombinant antigen could be produced make the antigen a potentially useful tool in the diagnosis of cystic echinococcosis.

Seroepidemiological study of visceral leishmaniasis in Booyerahmad district, south-west Islamic Republic of Iran.

Visceral leishmaniasis (VL) is endemic in parts of Islamic Republic of Iran. A cross-sectional seroprevalence study was carried out in children in Booyerahmad district in the south-west of the country. Serum samples were taken from 1628 children up to 10 years old from different areas in Booyerahmad in 2005-06. AntiLeishmania antibody was detected in 50 out of 1628 children (3.1%) by direct agglutination test (antibody titre > or = 1:3200). There was no significant difference in seropositivity between the sexes (2.8% males and 3.3% females). The highest rate of infection (5.2%) was in the age group 10 years. Further studies are needed to explore the reservoirs and vectors of the disease in this region.

Integrated visceral leishmaniasis surveillance system in primary care for children in Meshkin-Shahr district, north-western Islamic Republic of Iran.

In 2001 a visceral leishmaniasis (VL) surveillance system was set up for children aged < or = 12 years in the primary health system in Meshkin-Shahr district of Ardebil province, north-western Islamic Republic of Iran. All cases with clinical signs and symptoms of VL and positive by the direct agglutination test were referred for physical examination and treatment. The mean annual incidence of VL decreased significantly from 1.88 before (1985-2000) to 0.77 per 1000 child population after the intervention (2001-07). In a control area with no surveillance, it increased from 0.11 to 0.23 per 1000. Early detection of VL using practical serological tests and timely treatment of cases could decrease the mortality and morbidity rates of VL in endemic areas.

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.

Phlebotomus perfiliewi transcaucasicus, a vector of Leishmania infantum in northwestern Iran.

Visceral leishmaniasis is caused by Leishmania infantum, which is transmitted to humans by bites of phlebotomine sand flies and is one of the most important public health problems in Iran. To identify the vector(s), an investigation was carried out in Germi district, an important focus of the disease in Ardebil province in northwestern Iran, during July-September 2004 and 2005. Using sticky papers, CDC light traps and aspirators, 3,560 sand flies were collected and identified to species. Host bloodmeal preference and Leishmania infections in female specimens were evaluated using enzyme-linked immunosorbent assay (ELISA) for the former and microscopic examination followed by polymerase chain reaction (PCR) assay using species-specific kinetoplast minicircle primers for the latter. Nine sand fly species are present in the district, including Phlebotomus kandelakii Shchurenkova, Phlebotomus perfiliewi transcaucasicus Perfil'ev, Phlebotomus major Annandale, Phlebotomus balcanicus Theodor, Phlebotomus halepensis Theodor, Phlebotomus brevis Theodor & Meshghali, Phlebotomus papatasi Scopoli, Sergentomyia dentata Sinton, and Sergentomyia sintoni Pringle, with P. p. transcaucasicus being the most prevalent representative of the genus Phlebotomus at 45%. The anthropophilic index for P. p. transcaucasicus was 36.3%, indicating a strong preference for humans. Of 905 female P. p. transcuacasicus dissected, 10 (1.1%) were found naturally infected with promastigotes. Species-specific amplification of promastigotes eluted from Giemsa-stained slides revealed specific PCR products of L. infantum DNA. Based on its high anthropophily and natural infections with L. infantum, and the fact that it was the only species found infected with L. infantum, we conclude that P. p. transcaucasicus is the principal vector of L. infantum in northwestern Iran.

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.

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.

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.

Molecular data on vectors and reservoir hosts of zoonotic cutaneous leishmaniasis in central Iran.

Due to the increasing number of positive cases of cutaneous leishmaniasis with occurrence of new foci, a study was carried out to investigate on vectors and reservoirs of the disease in the Shahrood district, central Iran during 2005-2006. Sandflies and rodents were collected using sticky papers and Sherman live traps respectively More than 1700 sandflies were collected and identified, mainly Phlebotomus papatasi species. RAPD-PCR analysis of sandflies showed that 10% of P. papatasi and 4.2% of P. caucasicus were naturally infected with Leishmania major. Two species of rodents, potential reservoirs, Rhombomys opimus (92.5%) and Nesokia indica (7.5%) were trapped in the district. Microscopy identification from rodents confirmed that 91.9% of the Rhombomys opimus were positive to amastigotes. Species identification of isolated parasites revealed Leishmania major DNA in the infected Rhombomys using RAPD-PCR technique. This epidemiological data highlight the importance of the disease in the region and could help people involved in control programs.

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.