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Background: The current in silico study was done to determine the primary biochemical features and immunogenic epitopes of Echinococcus granulosus glutathione S-transferase protein as a potential vaccine candidate. Methods: Several web tools were employed to predict physico-chemical properties, antigenicity, allergenicity, solubility, post-translational modification (PTM) sites, subcellular localization, signal peptide, transmembrane domain, secondary and tertiary structure followed by refinement and validations. In addition, B-cell epitopes were predicted and were screened using various web servers, while MHC-binding and CTL epitopes were predicted using IEDB and NetCTL servers, respectively. Results: The protein had 219 residues with a molecular weight of 25.55 kDa and alkaline isoelectric pH (7.5). This protein was stable, thermotolerant (aliphatic index: 78.04) and hydrophilic (GRAVY: -0.440). The predicted antigenicity scores were low and the protein was non-allergenic in nature. There were no transmembrane domain and signal peptide in the sequence. Moreover, several B-cell, MHC-binding and CTL epitopes were found in the EgGST protein, which could be further used in multi-epitope vaccines. Conclusion: Further studies are needed on the development of vaccines in vivo using EgGST alone or in combination with other antigens in the future.

Abstract Background Memory impairment caused by Toxoplasma gondii infection has been documented. Berberine (BRB) is well known for its enhancing effects on memory and has shown promising results. However, the impact of BRB on T. gondii infection and schizophrenia-induced consolidation and reconsolidation memory impairment is still unclear. Here; we examined the effect of BRB on the inhibitory avoidance (IA) memory consolidation and reconsolidation impairment induced by T. gondii infection, and ketamine (Ket) as a pharmacological model of schizophrenia. Also; the brain-derived neurotrophic factor (BDNF) levels in the medial prefrontal cortex (mPFC) and hippocampus were analyzed. Methods Rats were infected with T. gondii RH strain or received Ket (30 mg/kg/day) intraperitoneally (i.p) for at least five consecutive days (as the model of schizophrenia). Then followed by oral administration with BRB (25 mg/kg/day) for five days. Finally, the IA memory retention test was examined 48 post-conditioning, and BDNF was measured. Results Results indicated IA memory impairment in T. gondii-infected animals since lower step-through latency (STL) was observed than in control animals. We found significant (P = 0.01, P = 0.001) elevations in STL and a significant decrease (P = 0.001) in total time spent in the dark area following BRB administration in infected and Ket-treated rats, indicating improvement (increased STL) in consolidation and reconsolidation memory. Moreover, BDNF levels were reduced (P = 0.01) in the hippocampus and mPFC regions of both T. gondii- infected and Ket-induced groups, which remarkably enhanced after BRB treatment. Furthermore; we found that BRB administration notably increased the mPFC BDNF levels in mPFC (P

The progression of Toxoplasma gondii (T. gondii) invasion is aided by rhoptry proteins (ROPs), which are also crucial for the parasite’s survival in host cells. In this study, in silico analysis was performed to examine the various aspects of the ROP29 protein, such as physicochemical properties, potential T- and B-cell epitopes, and other significant features. The research revealed that there were 55 possible sites for posttranslational modification in the ROP29 protein. The secondary structure of the ROP29 protein consists of a random coil, an alpha-helix, and an extended strand, which account for 49.69%, 36.81%, and 13.50%, respectively. Moreover, a number of putative T- and B-cell epitopes for ROP29 were found. The Ramachandran plot showed that 88.91% (crude model) and 97.54% (refine model) of the amino acid residues were located in the favored regions. Also, the testing of this protein’s antigenicity and allergenicity showed that it was nonallergenic and immunogenic. Our results suggested that employing in silico tools to apply structural and functional predictions to the ROP29 protein can lower the likelihood that laboratory investigations will fail. This research served as a crucial foundation for further research. More research is required in the future in suitable animal model employing ROP29 alone or in combination with other antigens.

Background: We aimed to determine species of liver fluke that predominately cause fascioliasis in sheep, goats, and cattle in the Sulaymaniyah Province, Iraq using the molecular technique of DNA sequencing and restriction fragment length polymorphism (RFLP). Methods: The samples were collected from November 2021 to May 2022. The flukes were collected from infected livers of livestock at the slaughterhouse of Sulaymaniyah Governorate, Iraq. A total of 205 flukes were collected from 56 hosts, cattle (n=22), sheep (n=28), and goats (n=6). The specific primers for FCOX1 and 28S rDNA gene amplification were used. The PCR products were subjected to restriction fragment polymorphism (RFLP) assay using Hpy188III and Dra II restriction enzymes, besides DNA sequencing. Results: The results showed the genetic polymorphisms among the flukes. Three patterns of RFLP were observed Fasciola hepatica, F. gigantica, and F. intermediate, where 28 of them displayed F. hepatica (sheep, n=14, goat, n=3 and cattle, n= 11), whereas 24 samples displayed the F. gigantica (sheep, n=12, goat, n=3 and cattle, n= 9), and only four samples belonged to F. intermediate (sheep n=3 and cattle, n=1). In addition, the result of the ribosomal DNA (28S rDNA) sequencing confirmed that the isolated flukes belonged to F. hepatica, F. gigantica and F. intermediate. Conclusion: All three main species are present in the study area and F. hepatica predominated among the animal species in this area also, our results concluded that PCR-RFLP is a rapid and reliable method for liver fluke species identification.

Background: Due to the opportunism character of Acanthamoeba, the presence of this parasite in the thermal water of recreational baths and hospital environments can be a risk to the health of staff, patients and others. The aim of this study was to investigate the distribution of potentially pathogenic Acanthamoeba genotypes isolated from the hospital environment and the thermal water of recreational baths in Markazi Province, central Iran. Methods: Overall, 180 samples including thermal water from recreational baths in Mahallat City and dust, soil and water from different hospitals of Arak, Farahan and Komijan cities, central Iran were collected. The presence of Acanthamoeba was investigated using microscopic examination and molecular methods. The PCR and sequencing was performed based on a specific 18S fragment of ribosomal DNA. Results: Based on the microscopic survey, totally 134 positive samples were detected including 35% in thermal water samples and 44.7% in hospital samples. In molecular analysis, 53.5% of the samples were identified as Acanthamoeba and 46.7% as Protacanthamoeba bohemica. The genotypes were detected as T4 (33.3%), T2 (10%), T11 (6.7%), and T5 (3.3%). Conclusion: The T4 was the most common genotype found in hospitals sampling sites while the T2 genotype and P. bohemica were detected in thermal water sampling sites.

Objective: To evaluate the clinical and molecular features of human cystic echinococcosis in Tehran, Iran. Methods: In this cross-sectional study, all human cystic echinococcosis patients admitted to Tehran’s hospitals from 2011 to 2019 were enrolled, and demographic characteristics, clinical findings, and laboratory data were collected. Formalin-fixed, paraffin-embedded and fresh tissue samples of 175 cystic echinococcosis patients were evaluated for molecular characterization. The samples’ isolated DNA was used to amplify cytochrome c oxidase I and NADH dehydrogenase subunit I genes. Also, the morphological features of fresh samples were examined. Results: In total, 175 patients with a mean age of 45 (9-98) years were admitted to Tehran’s hospitals diagnosed with cystic echinococcosis. Moreover, the highest (26.9%) and the lowest (2.9%) prevalence of cystic echinococcosis cases were in the 16-30 and 1-15 years range, respectively. Male/female ratio was 0.96 (49.1% vs. 50.9%). The liver was affected in 92 patients (52.6%), and two or more organs were infected in 7 patients (4.0%). The cysts’ diameter varied from 1 to 25 cm, and 96.0% of the patients had a single hydatid cyst. All patients underwent radical surgery and the PAIR technique was applied for all cases. No significant difference was observed between the protoscolex hooks of pulmonary and hepatic cysts. G1-G3 had the highest percentage (99.4%) over other identified G6 genotypes (0.6%). Conclusions: The sheep-dog cycle plays an important role in transmitting the human cystic echinococcosis infection in Tehran.

Background and Aim: Acanthamoeba is an opportunistic pathogen that may cause fatal granulomatous encephalitis and ocular keratitis in humans and animals. Increasing number of contact lens wearers can lead to increased frequency of amoebic keratitis and due to lack of effective drugs treatment of this disease is difficult. This study aimed to investigate the effect of ethanolic extract of Chaerophyllum macropodum on Acanthamoeba genotype T4. Materials and Methods: In this experimental study, samples taken from the patients with keratitis were cultured on 1.5% non-nutrient agar medium. Different concentrations of the ethanolic extract (1.25, 2.5, 5, 10 mg/ml) were tested three times (24, 48, and 72 h) on trophozoites and cysts of Acanthamoeba in vitro. The number of live and dead parasites were counted by using trypan blue staining and neobar lam. Also, the percentage of cysts apoptosis was assessed by flow cytometry. Results: In the presence of 10 mg/ml ethanolic extract in the culture medium, the percentages of live trophozoites after 24, 48, and 72 h were 53.6, 15.11, and 0 percent, respectively. In the case of cysts, after 24, 48, and 72 h, 65.31, 43.31, and 0 percent of the cysts were alive, respectively. The results of flow cytometry showed that the apoptosis rate was 0.09% in the sample treated with extract after 72 h. Conclusion: Ethanolic extract of Chirophyllum macropodium can be a promising candidate for the development of anti-Acanthamoeba drugs due to its high toxic effects on the cysts. However, since the crude extract of Chaerophyllum macropodum was used in this study, further investigations are needed to find the effective fractions of the plant and determine their mechanisms of action. It can also be tested in vivo or even against other parasites

Abstract Background The species complex of Echinococcus granulosus sensu lato (s.l.) causes cystic echinococcosis distributed worldwide. There is no genotype information from hydatid cysts in the intermediate hosts in Central Iran. Therefore, in this study, we analyzed the hydatid cysts in livestock slaughtered in an abattoir in this region. Six hundred fifty-seven hydatid cysts were isolated from 97 animals, including sheep, cattle, camels, and goats slaughtered in Yazd abattoir from September 2018 to January 2020. The demographic data was collected as well as cyst location, fertility, and viability. Out of 657 samples, 164 samples were genotyped. Then, phylogenetic analysis was performed using MEGAX. Statistical analyses were done using SPSS version 16.0 by chi-square with a significant difference of less than 0.05. Results Out of 164 samples, the G1-G3 complex genotype had the most frequency in samples, with 135 cases recognized. The G6/G7 was observed in 19 isolates and G5 was reported in nine samples. One sample was detected as Taenia hydatigena. Conclusions This study showed that G1-G3 and G6/G7 genotypes were presented in all animals, but G5 was reported only in cattle, goats, and camels. It is the first molecular identification of cystic echinococcosis in Central Iran. Hence, reporting G5 in livestock in this area should be considered due to transmission to humans.

BackgroundToxoplasmosis is one of the most common infections in humans and animals, which is caused by an obligate intracellular opportunistic parasite known as Toxoplasma gondii (T. gondii). Some data have shown that both Rhesus (Rh)-positive and Rh-negative individuals differ in response to biological factors, including Toxoplasma infection. Therefore, this systematic review and meta-analysis was conducted to investigate the scientific evidence regarding the possible association between the Rh blood group and Toxoplasma infection and to determine the seroprevalence of T. gondii in the Rh blood group system.MethodsThe research was conducted on PubMed, ScienceDirect, ProQuest, and Google Scholar databases until January 2023. Twenty-one cross-sectional studies were included with a total of 10910 people. The data were synthesized using a random effect model with 95% confidence intervals (CIs).ResultsThe overall prevalence of T. gondii was calculated at 32.34% (CI 95%: 28.23-36.45%) and 33.35% (CI 95%: 19.73-46.96%) in Rh-positive and Rh-negative blood groups. In addition, the pooled OR for the relationship between the Rh blood group and the seroprevalence of T. gondii was 0.96 (95% CI: 0.72-1.28).ConclusionsThis meta-analysis showed a high prevalence of Toxoplasma infection in both Rh-negative and positive blood groups. This systematic review and meta-analysis revealed that no significant association was found between toxoplasmosis and Rh factor. Because of the limited number of studies in this field, more research is recommended to determine the exact relationship between toxoplasmosis and the Rh factor.

Background: Sarcocystis is a genus of coccidian protozoa that at least seven species of it can parasitize cattle. The global prevalence of Sarcocystis is close to 100% in adult cattle. The main aim of this study was to identify the infection rate of Sarcocystis spp. in heart of cattle in Tehran, Iran by microscopy and PCR-RFLP methods. Methods: Totally, 100 bovine heart samples were collected from the main slaughterhouse of Shahriar, Meysam slaughterhouse, west of Tehran in 2016. At first, heart samples were completely examined for the presence of sarcocystic macrocysts. Then, for microscopic examination, 50 g of each heart was digested in sterile condition using pepsin acid digestion method. Then, the species of the parasite were detected by PCR-RFLP technique and sequencing. Results: Overall, 97 of 100 of the heart muscle samples were infected with Sarcocystis. All the samples were detected as S. cruzi through similar patterns in PCR-RFLP. Conclusion: S. cruzi is the most common species in the heart of cattle slaughtered in Shahriar.

Background: The extract of myrtle plant contains polyphenolic compounds that show antibacterial, antiviral, and anti-parasitic properties. We aimed to investigate the therapeutic effect of aqueous and ethanolic myrtle extract against leishmaniasis caused by L. major in vivo and in vitro conditions. Methods: This study was carried out in Tarbiat Modares University, Tehran, Iran in 2018. Aqueous and ethanolic extract of myrtle plant at 6.25 to 400 mg/ml concentrations were tested on Leishmania major promastigotes, non-infected macrophages, and macrophages infected with amastigotes in vitro using counting, MTT and flow cytometry techniques. Then, BALB/c mice were treated with ethanolic, aqueous and a mixture of both extracts of myrtle plant. The treatment was carried out for four weeks. Then, the effectiveness of the herbal medicine was assessed by measuring wounds diameters, mice weights and their mortality rate on weekly basis. Results: The IC50 values of aqueous and ethanolic extracts for promastigotes were 7.86 and 11.66 μg/mL respectively. The IC50 values of the aqueous and ethanolic extracts for amastigotes were 12.5 and 47.2 μg/mL respectively. Flow cytometry indicates 62.88% and 60.16% apoptosis induced by ethanolic and aqueous extract of myrtle plant respectively. The lowest parasitic load was seen in the group treated with ethanolic extract. Conclusion: The lesion sizes for treated groups with extracts were similar to those treated with glucantime. Oral administration instead of injection is another advantage of myrtle plant over glucantime, which makes the herb easy and more practical.

Background: The genus Abbreviata (Spirurida: Physalopteridea) currently contains 47 species. Physalopteridae nematodes infect a large number of vertebrates, including mammals, birds, reptiles and amphibians. The current study is a report of the first morphological and molecular identification of A. kazakhstanica (Spirurida: Physalopteridea) in Pseudopus apodus in Iran. Methods: Eleven road-killed P. apodus, were collected from, Iran during 2016-2018. The nematodes were isolated from stomach. After morphological study, the genomic DNA of the parasites was extracted using CTAB method. The DNA was used for PCR amplification of cytochrome c oxidase subunit I (cox1). The PCR products were sequenced, the sequence data were analyzed and multiple alignments were conducted using the Clustal Omega. Results: After detailed microscopic examination, the A. kazakhstanica was identified. The cox1 sequences confirmed the species of helminth. The new sequences of A. kazakhstanica were submitted to GenBank under the accession number MK578751-2. Conclusion: Regarding the limited data on parasitological status of Iranian reptiles, more specific and comprehensive investigations are needed to identify the parasitic fauna.

Abstract Background Cystic echinococcosis (CE), is a parasitic zoonosis caused by Echinococcus granulosus (E. granulosus) larvae in liver and lungs of both humans and animals. Surgical intervention is the mainstay for CE treatment, using scolicidal agents that inactivate live protoscolices. This study evaluated the scolicidal effects of Silybum marianum ethanolic extract and its combination with albendazole in vitro for the first time. Moreover, in a literature review, we investigated the effects of a wide range of Iranian medicinal plants on protoscolices of E. granulosus. Methods S. marianum ethanolic extract was prepared and high-performance liquid chromatography (HPLC) analysis was used to establish the proportions of its component compounds in the extract. Cytotoxicity was evaluated in mouse macrophage cells (J774A.1 cell line) using MTT method. Next, the scolicidal activity of the extract alone and combined with albendazole was tested as triplicate at various concentrations incubated for 5, 10, 20, 30, and 60 min. Finally, protoscolex viability was determined using 0.1% eosin as a vital stain. PCR–RFLP and DNA sequencing techniques were used to characterize the genotype of E. granulosus. Results HPLC analysis showed that S. marianum ethanolic extract contained mostly silydianin (14.41%), isosilybin A (10.50%), and silychristin (10.46%). The greatest scolicidal effects were obtained with the combination of S. marianum with albendazole (79%), S. marianum ethanolic extract alone (77%) and albendazole (69%), at a concentration of 500 μg/ml for 60 min, respectively (P

Background: Recently, a hypothesis about the negative relationship between cancers and parasites has been proposed and investigated; some parasitic worms and their products can affect the cancer cell proliferation. Due to the potential anti-cancer effect of helminthic parasites, in the present study, the excretory–secretory protein of Toxocara canis (T. canis) parasite was used to evaluate the possible anti-cancer properties and their effect on gastrointestinal and liver cancer cell proliferation-related genes in laboratory conditions.Methods and materials: The selected synthesized peptide fraction from the T. canis excretory–secretory Troponin protein peptide (ES TPP) was exposed at 32, 64, 128, and 256 μg/ml concentrations to three gastrointestinal cancer cell lines AGS, HT-29, and Caco 2, as well as HDF cells as normal cell lines. We used the MTT assay to evaluate cellular changes and cell viability (CV). Variations in gene (Bcl-2, APAF1, ZEB1, VEGF, cyclin-D1, and caspase-3) expression were analyzed by real-time RT-PCR.Results: After 24 h of exposure to pept1ides and cell lines, a decrease in CV was observed at a concentration of 64 μg/ml and compared to the control group. Then, after 48 h, a significant decrease in the CV of Caco 2 cells was observed at a concentration of 32 μg/ml; in the other cancer cell lines, concentrations above 32 μg/ml were effective. The peptide was able to significantly alter the expression of the studied genes at a concentration of 100 μg/ml.Conclusion: Although the studied peptide at high concentrations could have a statistically significant effect on cancer cells, it is still far from the standard drug and can be optimized and promising in future studies.

BackgroundBlastocystis sp. is a common intestinal parasite, possibly responsible for diarrhea, vomiting and nausea, abdominal pain, and irritable bowel syndrome. However, many studies focused on this issue due to the uncertainty of its pathogenic potential. The extracellular vesicles (EVs) are significant mediators for cellular communication, carrying biological molecules such as proteins, lipids, and nucleic acids. Compared with other parasites, little is known about the Blastocystis EVs. Hence the present investigation was done.MethodsThe Blastocystis parasites were cultured in the DMEM medium, and a 550–585 bp fragment was amplified using PCR, and sequencing was done. A commercial kit was used for exosome extraction and dynamic light scattering (DLS), flow cytometry (CD63, CD81 markers), and electron microscopy tests to determine their morphology. The human leukemia monocytic cell line (THP-1) was exposed to Blastocystis EVs. Next, the expression of proinflammatory and anti-inflammatory cytokines, including IL-4, IL-6, IL-10, and tumor necrosis factor-alpha (TNF-α), were measured using quantitative PCR.ResultsExosomes were extracted from ST1-3 Blastocystis sp. According to the DLS assay, the size of the exosomes was in the range of 30–100 nm. Electron microscopy images and CD63 and CD81 markers also confirmed the exosome's size, structure, and morphology. According to real-time PCR results, ST1-derived exosomes caused IL-6 and TNF-α upregulation and IL-10 and IL-4 downregulation, ST2- and ST3-derived exosomes downregulated IL-10, and ST3-derived exosomes caused IL-6 upregulation. There is a statistically significant difference (P ≤ 0.05).ConclusionTo our knowledge, this is the first report of the release of exosome-like vesicles by the human parasite, Blastocystis, and the provided information demonstrates the role of this parasite, particularly ST1 on proinflammatory and anti-inflammatory cytokines and navigating the host response.

Background: Toxoplasmosis is a global public health concern with severe complications, particularly in pregnant women and immunosuppressed patients. Herein, we revealed the first genotypic evidence of Toxoplasma gondii (T. gondii) in diabetic and non-diabetic individuals referred to Imam Sajjad Hospital, Tehran, Iran, using nested-Polymerase Chain Reaction (PCR) and sequencing. Materials and Methods: We collected 98, 95, and 94 blood samples from Type-1 Diabetes Mellitus (T1DM), Type-2 Diabetes Mellitus (T2DM), and control subjects from February to August 2018, and their DNA was extracted. After amplification and visualization of dense granule antigen 6 gene (344 bp) using external and internal primer pairs, the positive samples were sent for sequencing. Results: Among 287 individuals, three T1DM patients (two IgG, one IgM) and one T2DM patient (IgG) were found positive by molecular method, which proved to be type III strain by sequencing. Conclusion: To the best of our knowledge, this is the first genotyping investigation on T. gondii in diabetic individuals. Further research should be done to better realize the association between Toxoplasma genotypes and the outcome of diabetes mellitus.

Background: Iran is one of the endemic areas of Mediterranean Visceral Leishmaniasis, a disease caused by Leishmania infantum. In this work, we examined whether Proteína quimérica 10 (PQ10) recombinant protein is suitable for immunological diagnosis of human visceral leishmaniasis. Methods: The study was carried out in Tarbiat Modares University during 2016-2018. The coding sequence of PQ10 recombinant protein was sub-cloned in pET28 expression vector and was commercially synthesized by GENERAY Biotechnology, China. Sequencing with proper primers was done, the expression, optimization of expression and protein purification were performed, and the purified recombinant protein was confirmed by western blot. The efficacy of PQ10 for serodiagnosis was evaluated with 50 positive and 50 negative serum samples, which confirmed by the direct agglutination test and collected from individuals living in the visceral leishmaniasis endemic areas of Iran. ELISA was performed with the PQ10 recombinant protein. Results: The 95% CI sensitivity of ELISA that was evaluated with sera from naturally infected individuals was 84%. The 95% CI specificity value of the ELISA determined with sera from healthy individuals (50 serum samples) and from individuals with other infectious diseases was 82%. The 95% CI positive predictive value (PPV) and negative predictive value (NPV) were exterminated 82.35% and 83.67%, respectively. Conclusion: We have used a recombinant synthetic protein to improve serodiagnosis of human visceral leishmaniasis. PQ10 could be useful for diagnosis of asymptomatic cases, as well as in the early phase of infections.

Background: Fleas (Insecta: Siphonaptera) are considered as highly specialized bloodsucking on mammals such as humans, livestock, dog, cat, rabbit, squirrels, rats, and mice. The desire for blood feeding from warm-blooded animals has led to becoming an intermediate host for some tapeworms like Dipylidium. The aim of this study was to detect D. caninum larval infection in fleas of dogs living in Meshkinshahr County, northwest of Iran. Methods: Fleas were collected from 42 dogs using brushing the hair in Meshkinshahr for one year (2014-2015). After the morphological study, fleas were preserved in 90% ethanol for molecular identification. After DNA extraction, the 28S ribosomal RNA gene (~670 bp) of D. caninum was amplified using specific primers. Finally, the PCR products were sequenced. Results: Overs, 974 fleas were collected from the dogs. In the morphological study, three species Ctenocephalides canis, Ct. felis, and Pulex irritans were identified. PCR and sequence analysis results showed that 4 isolates Ct. Canis were infected with D. caninum. Also, no positive specimens were isolated from the other two species. Conclusion: Ct. canis is reported as the most important species of fleas in transferring D. caninum in that region.

Background: Studies on experimental model of cancer showed that hydatid cyst fluid (HCF) has antitumor activity. We aimed to investigate the effect of HCF and Antigen B (AgB) on 4T1 breast tumor cells in BALB/c mice. Methods: This study was carried out in the Department of Parasitology, Tarbiat Modares University, Tehran, Iran from 2019 to 2020. There were two control groups of BALB/c mice (one group were injected with aluminum sulfate and another group with PBS), and six groups, injected via the intraperitoneal route with 100, 300 and 500 μg/ml concentrations of HCF, AgB diluted in 100 μl PBS, and alum. Seven days after the last treatment, 7×105 4T1 cells were subcutaneously injected into the right flank of BALB/c mice. Results: The difference between the mean size of the tumor in the case and control groups was statistically significant (P

Background: Toxoplasma gondii is an obligate intracellular parasite that can infect humans and animals. As the choice drug have shown side effects, development a new drug with low toxicity will be necessary. Methods: BALB/c mice were infected with tachyzoiets of T. gondii. After treatment by oral and parenteral artemether (250 µg/mice) and sulfadiazine (50 µg/mice), we evaluated the rates of survival in treated and control mice. The fold change of B1 gene (target gene) expression in liver and brain of mice treated with parenteral artemether (i.p.), oral artemether (via gavage) and sulfadiazine, were detected by using the Real-Time quantitative PCR. Results: Both treatment with sulfadiazine and artemether showed significant prolongation in time to death of the infected mice compared to the control group. Median survival days for parenteral artemether, oral artemether, sulfadiazine and control group were 8, 11, 12 and 6 d respectively. Expression of B1 gene in liver and brain of mice after treatment with artemether and sulfadiazine were reduced in comparison to housekeeping gene (β-tubulin gene). The fold change (comparing to control group) for parenteral artemether, oral artemether, sulfadiazine is 0.034, 0.027 and 0.111 for liver and 0.220, 0.425 and 0.366 for brain respectively. Conclusion: Artemether is effective to control the tachyzoites of T. godii in vivo conditions and oral treatment is more effective than parenteral treatment. Due to its low cytotoxicity and its high effective action against the tachyzoietes of T. godii in susceptible animals.

Background: Malaria is one of the major health problems in endemic countries like Afghanistan. Evidence has been reported about reducing the effects of chloroquine against Plasmodium falciparum in many endemic countries. The aim of this study was to investigate the resistance mutations in pfmdr1 and pfdhfr genes of P. falciparum samples detected in blood samples of malaria patients in Laghman Province, Afghanistan. Methods: Samples were taken on DNA retention cards and 3 glass slides (thin and thick spread) from Laghman Province, Afghanistan in 2018. The pfmdr and pfdhfr mutations in 30 P. falciparum positive samples were examined using PCR-RFLP techniques. The PCR product was then sequenced to determine the mutation at the N86Y and D1246Y mutations of the pfmdr1 and N51, C59, I164, S108 and A16 points of pfdhfr genes. Results: In the pfmdr1 gene, all samples were wild-type and no mutation was detected at point 86 and D1246Y. In the pfdhfr gene sequences using CLC main workbench software no mutations were detected at codons 16, 51. However, some mutation was observed at codons 59, 108 and 164. These mutations were L164I, S108N and C59R. Conclusion: Our findings provide evidence of the possible emergence of fansidar-resistant specimens in Laghman. The data of this study provide the basis for future prospective studies in other endemic areas of Afghanistan. The absence of significant mutations in P. falciparum samples of Laghman Province may indicate that this parasite may have switched to chloroquine re-sensitization in this area.

Objective(s): Since leishmaniasis is one of the health problems in many countries, the development of preventive vaccines against it is a top priority. Peptide vaccines may be a new way to fight the Leishmania infection. In this study, a silicon method was used to predict and analyze B and T cells to produce a vaccine against cutaneous leishmaniasis.Materials and Methods: Immunodominant epitope of Leishmania were selected from four TSA, LPG3, GP63, and Lmsti1 antigens and linked together using a flexible linker (SAPGTP). The antigenic and allergenic features, 2D and 3D structures, and physicochemical features of a chimeric protein were predicted. Finally, through bioinformatics methods, the mRNA structure was predicted and was produced chemically and cloned into the pLEXY-neo2 vector.Results: Results indicated, polytope had no allergenic properties, but its antigenicity was estimated to be 0.92%. The amino acids numbers, molecular weight as well as negative and positive charge residuals were estimated 390, ~41KDa, 41, and 30, respectively. The results showed that the designed polytope has 50 post-translationally modified sites. Also, the secondary structure of the protein is composed of 25.38% alpha-helix, 12.31% extended strand, and 62.31% random coil. The results of SDS-PAGE and Western blotting revealed the recombinant protein with ̴ 41 kDa. The results of Ramachandran plot showed that 96%, 2.7%, and 1.3% of amino acid residues were located in the preferred, permitted, and outlier areas, respectively. Conclusion: It is expected that the TLGL polytope will produce a cellular immune response. Therefore, the polytope could be a good candidate for an anti-leishmanial vaccine.

Abstract Background Plasmodium vivax resistance to chloroquine (CQ) has been reported from many endemic regions in the world. Plasmodium vivax is responsible for 95% of malaria cases in Afghanistan and CQ is the first-line treatment given for vivax malaria. The pvmdr-1 and pvcrt-o (K10 insertion) genes are possible markers for CQ-resistance in P. vivax isolates. There have been no studies done on the presence or absence of molecular markers for CQ-resistance P. vivax in Afghanistan. The present work aimed to evaluate the frequency of mutations in the pvmdr-1 and K10 insertion in the pvcrt-o genes of P. vivax. Methods Plasmodium vivax isolates were collected from Laghman, Baghlan and Khost provinces. For investigation of polymorphisms of desired regions in pvmdr-1 and pvcrt-o genes, sequencing was applied on the PCR products. A new asymmetric qPCR and melting analysis assay based on unlabelled probe developed for scanning of K10 insertion in pvcrt-o gene. Results The analysis of sequencing data of the pvmdr-1 gene showed wild type Y976 and K997 and mutant M958 and L1076 in 33 isolates from three provinces. Of the 36 samples evaluated for K10 insertion in pvcrt-o, 2/18(11%), 0/10(0%) and 0/8(0%) isolates from Laghman, Baghlan and Khost province, respectively, possessed K10 insertion, confirmed by either sequencing and unlabelled probes. Conclusion Two samples with K10 insertion and 33 samples with pvmdr1 polymorphism, indicating on the possibility of CQ resistance in P. vivax populations in Afghanistan. Furthermore, unlabelled probes are simple and inexpensive alternative tools for screening of P. vivax mutations.

Background: Trichomonas vaginalis is a protozoan pathogen of the human genitourinary tract, which is the cause of trichomoniasis. The disease is one of the most important non-viral sexually transmitted infections in the world. Many studies have been performed on the molecular identification of Trichomonas in men in different parts of the world, but in Iran, such a study has been very limited. We aimed to detect T. vaginalis in urine of men with unusual sexual behavior by wet mount microscopic observation, culturing in TYM-33 and molecular method in Tehran, Iran. Methods: Totally, 47 urine samples were collected from men with high-risk sexual behaviors referred to Health Care Centers in South of Tehran, Iran during 2017-2018. After urine centrifugation and wet mount sediment preparation, the samples were cultured in TYM-33 medium and examined microscopically. Then DNA was extracted from urine sediment samples and finally the 18srRNA gene was amplified by PCR. Results: Thirty-one (65.95%) urine samples were positive for T. vaginalis by PCR. Of these, only 6 (12.76%) were detected positive by wet mount method and 25 (53.19%) by culture and successive passages. Conclusion: The high prevalence of trichomoniasis among men with high-risk sexual behavior in Tehran indicates a potent health risk condition for families. Of course, this percentage is not the real infection rate in ordinary men in Tehran. This situation could be controlled by promoting concurrent diagnosis and treatment of T. vaginalis infection in men and women, as well as strengthening religious beliefs and health education in the individuals.

Objective(s): Toxoplasma gondii is an obligate intracellular protozoan parasite that causes toxoplasmosis in humans and animals. Micronemes (MICs) are effective candidates for DNA vaccine. Materials and Methods: In this study, we evaluated the immune response of BALB/c mice against MIC3 gene of Toxoplasma gondii and interleukin 12 (IL-12) as DNA vaccine. The MIC3 gene was cloned into the PTZ57R/T vector before sub-cloning in pcDNA3. Recombinant pc-MIC3 was transformed into Escherichia coli (TOP10 strain). The pc-MIC3 plasmid was then transfected into Chinese Hamster Ovary (CHO) cells, and the expression of the MIC3 gene was evaluated by SDS-PAGE and Western blotting. Sixty female BALB/c mice were divided into 6 groups. Each group received 3 intramuscular immunizations on days 0, 21st and 42nd using one of the following stimulants: phosphate-buffered saline, pcDNA3, pCAGGS-IL12, pc-MIC3 (100 µg), pc-MIC3 (50 µg), or combined pCAGGS-IL12 (50 µg) and pc-MIC3 (50 µg). The enzyme-linked immunosorbent assays was applied to evaluate interferon gamma (IFN-γ) and IL-4 cytokines excretion of lymphocytes stimulated with tachyzoites lysate antigen, as well as the total levels of immunoglobulin G (IgG), IgG2a and IgG1 in immunized mice sera.Results: Our results showed that mice challenged with pc-MIC3 (100 µg) had the highest longevity and quantity of immunoglobulin. Moreover, the highest expression level of IFN-γ was found in mice injected with combined pcMIC3 and pCAGGS-IL12 (P

Background: Toxoplasmosis can cause miscarriage or complications in the fetus. Diagnosis and treatment of this disease by anti-parasitic drugs especially in early pregnancy can help to prevent fetal infection and its complications. This study aimed to determine T. gondii infection in pregnant women, evaluate risk factors in the transmission of the disease and congenital toxoplasmosis. Methods: Overall, 360 sera of pregnant women from 5 cities in the Hormozgan Province in southern Iran with different climate were evaluated from 2015-2016 for T. gondii infection by using ELISA method and positive cases of IgM and IgG were tested again using Avidity IgG ELISA. All cases were evaluated according to climate, acute and chronic of toxoplasmosis, number of pregnancy and abortion, epidemiological factors and food habits. Results: Among 360 specimens T. gondii IgG + IgM antibodies were found positive in 0. 8% subjects and also 27% of samples had IgG seropositivity. A significant relationship was observed between age, sampling place, consumption of raw and half cooked meat, history of contact with cats, abortion history, number of children, and parity with IgG positive. In Avidity IgG ELISA test, 13 people with low avidity, 3 people with borderline avidity were reported. Conclusion: 72. 2% of the population had no antibody against the disease that this could be a warning to the people and requires education of preventive and prenatal care and routine screening of women at childbearing age.

Objective(s): Because leishmaniasis is related to the impaired functioning of T-cells, the use of an immunomodulator can increase the efficacy of antileishmanial therapy in visceral leishmaniasis. In this study, we used shark cartilage extract with artemisinin and glucantime against visceral leishmaniasis in BALB/c mice, and evaluated the synergistic therapeutic effect. Materials and Methods: The culturing method and quantitative real-time PCR by using the kDNA gene was used to detect parasite loads in the spleen and liver. INF-γ and IL-4 cytokine levels and survival rates were assayed.Results: The drug therapy with target drugs reduced parasite burden in the spleen and liver significantly. Although parasite burden was lower in the artemisinin treated group than in the glucantime treated group (P

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Background: Leishmania is a mandatory intracellular pathogen and causing neglected disease. Hence, protection against leishmaniasis by a development vaccine is an important subject. This study aimed to design a poly-epitope vaccine for cutaneous leishmaniasis. Methods: The present study was conducted in the Parasitology Department of Tarbiat Modares University, Tehran, Iran during 2017-2019. Several bioinformatics methods at online servers were used for prediction of different aspects of poly-epitope, including, physico-chemical attributes, allergenicity, antigenicity, secondary and tertiary structures, B-cell, T-cell and MHC (I, II) potential epitopes of LACK, LEIF, GP63 and SMT antigens of L. major. Results: After designing the construct (GLSL), the outputs of PTM sites demonstrated that the poly-epitope had 57 potential sites for phosphorylation. Furthermore, the secondary of GLSL structure includes 59.42%, 20.94% and 19.63% for random coil, extended strand and alpha-helix, respectively. The GLSL is an immunogenic protein with an acceptable antigenicity (0.8410) and non-allergen. Afterward, 20 potential epitopes of LACK, LEIF, GP63 and SMT antigens were linked by a flexible linker (SAPGTP), then was synthesized, and sub-cloned in pLEXY– neo2. The results were confirmed the expression of 38.7 kDa poly-epitope in secretory and cytosolic sites, separately. Conclusion: A good expression in the L. tarentulae and confirmation of the GLSL poly-epitope could be a basis for developing a vaccine candidate against leishmaniasis that should be confirmed via experimental tests in BALB/c mice.

Toxoplasma gondii is one of the most common meat-born zoonoses that infect all warm-blooded animals and humans. Sheep (Ovis aries) is one of the main reservoirs of T. gondii worldwide, and the infections induce various sequels, such as abortion and stillbirth. The present study aimed to identify the effects of humidity on the prevalence of T. gondii in sheep in high- and low-humidity regions. Heart samples from 200 slaughtered sheep (140 samples from a high-humidity region and 60 samples from a low-humidity region) were collected from Hormozgan Province (south of Iran). The samples were tested by nested PCR targeting the RE gene. Genotyping was performed by the PCR-RFLP method using the SAG3 and GRA6 genes. Some isolates were sequenced and recorded in the GenBank. T. gondii DNA was detected in 10.71 percent of the samples from the highly humid region, whereas no positive samples were detected in the low-humidity region. Genotyping revealed that all isolates belonged to the T. gondii type III genotype. Our study indicated that humidity is an important factor for the prevalence of T. gondii in sheep. Additionally, our study also showed the dominance of type III strain of T. gondii in sheep in the south of Iran.

Background: The immunomodulatory role of many parasites is well-documented. The current study designed to assess the immunoregulatory effects of the somatic extract (SE) of Toxocara canis on murine model of airway inflammations. Methods: The experiment was performed in department of parasitology of Tarbiat Modares University, Tehran, Iran from November 2018 to May 2019. Totally 30 female BALB/c mice divided into one control group and two experimental groups (10 mice in each group). The ovalbumin (OVA) group was sensitized with OVA in alum, while the SE group was administered with SE and OVA in alum intraperitoneally. The control group was injected with PBS in alum. Then, SE and OVA groups were intranasally challenged with OVA for three consecutive days and the control group encountered with PBS at the same time. One day after the last challenge, real-time PCR and histopathology survey were conducted on isolated lung tissues. Results: The gene expression of IL-25, IL-33, TNF-α and TLR-4 in SE group was significantly lower than OVA group (P

Background: In this study, mutations in the tripanothion reductase of Leishmania tropica isolated from Iran was investigated using sequencing and simulation of the enzyme by the molecular dynamic method. Methods: Fifteen susceptible and 15 clinical resistant L. tropica specimens were collected from skin lesions from different regions of Iran in 2017. After DNA extraction, trypanothione reductase (TRYR or TPR), gene fragment was amplified using PCR and sequencing methods. In the case of structural mutations, the components were simulated by molecular dynamics using the GROMACS software. Results: Some structural mutations were observed in 9 amino acids surrounding the active site of the TRYR gene of L. tropica with three-dimensional trypanothione reductase alteration. Conclusion: Change in the active site of TRYR of L. tropica, could probably contribute to the development of resistant L. tropica to glucantime. Because of the likely occurrence of mutations in glucantime as well as the ease of development of L. tropica resistant populations, more samples are needed to demonstrate the relationship between mutations in this enzyme and clinical resistance to glucantime. On the other hand, it is recommended that enzymatic studies be performed to confirm the role of mutation in the function and expression of trypanothione reductase in glucantime resistant and susceptible populations.

Vaginal infections are one of the major reasons women visit a gynecologist. Increased re­sistance to conventional antibiotics is one of the main factors mitigating the development of new antimicrobial agents, especially those of natural origin. In traditional Persian medicine, Trachyspermum ammi has been claimed to clear vagina from excessive discharge. Therefore, in this study, the antimicrobial activity of Ajwain essential oil was evaluated against some vaginal pathogens. The essential oil of ajwain was picked up and the minimum inhibitory and bactericidal concentrations (MIC and MBC) were revealed. The most frequently detected microorganisms involved in genital infections including Candida spp., Gardnerella vagina­lis, Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae and Lactobacillus acidophilus were considered. Evaluation of the essential oil of Trichomonas vaginalis was done by calculation of percent of growth inhibition. The essential oil showed a remarkable activity against the studied bacteria and fungi with MIC at a range of 0.0315 - 0.5 mg/ml and MBC at a range of 0.125 - 4 mg /ml. The highest inhibition and bactericidal activity was ob­served in S. agalactiae and G. vaginalis. 100% inhibition of T. vaginalis growth was shown at a concentration of 2000 μg/ml after 48 h by essential oil. The antimicrobial activity of the essential oil was more than that of thymol. Supposedly essential oil of Trachyspermum ammi fruit could inhibit vaginal pathogens growth .Further preclinical and clinical studies are re­quired to confirm the efficacy of this natural agent in vaginitis.

Background: Malaria is threatening more than half of Afghanistan population. Asymptomatic malaria is notable problem against malaria controlling strategies. In this study we evaluated the asymptomatic malaria status in Nangarhar Province, Afghanistan in 2017. Methods: Overall, 296 finger blood samples were taken on DNA Banking Cards and microscopic slides from asymptomatic individuals in Jalalabad city. We used a novel post real time PCR high resolution melting analysis beside microscopy and semi-nested multiplex PCR to evaluate status of asymptomatic malaria in this city. Results: The prevalence of asymptomatic malaria in Jalalabad city was determined 1.7% (5/296), 7.43% (22/296) and 7.78% (26/296) by microscopy, Seminested multiplex PCR and qRT-PCR-HRM, respectively. Out of 26 positive cases were detected by qRT-PCR-HRM, 21, 1 and 4 cases were detected P. falciparum, P. vivax and mixed infection of P. falciparum and P. vivax, respectively. Conclusion: Our data indicating on existence of significant number of asymptomatic reservoirs that assists in prolonged endemicity of the disease. On the other hand, the molecular methods are better alternatives for microscopy especially for monitoring of asymptomatic cases of malaria.

Background: KMP-11 (Kinetoplastid membrane protein-Π) exists in all species of kinetoplastid family. It is fully conserved and the protein produced by this gene can induce a very high cellular immune response. We aimed to design a suitable construction for a Leishmania major DNA vaccine and evaluate the protective efficacy of it as a candidate for DNA vaccine against cutaneous leishmaniasis in BALB/c mice. Methods: This experimental study was conducted in Tehran City, Iran, between April 20, 2015 and May 30, 2016. KMP-11 gene of L. major (MRHO/IR/75/ER, Iranian strain) and NT-GP96 of Xenopus GP96 DNA from a pBluescript-GP96 plasmid were amplified by PCR and the purified PCR products were cloned into the pJET1.2/blunt plasmid vector, then, subcloned into pEGFP-N1 plasmid as an expression vector. Finally, the KMP-11 gene was fused with GP96 and afterward the combination cloned in pEGFP-N1. All the cloned genes confirmed by enzyme digestions. Then, four groups of mice were immunized with PBS, pEGFP-N1, pEGFP-N1-KMP, and pEGFP-N1-fusion. Four weeks after immunization, all animals were challenged with L. major virulent promastigotes. Results: The constructed fusion potentially showed an ability to elicit Th1 responses that led to cutaneous lesion healing. Interestingly, the group received KMP11-GP96 –GFP showed the highest ratio of IFN- γ /IL-4 and IgG2a/IgG1 compare to other groups. No side effect was observed after using the fusion in the mice. Conclusion: The constructed fusion could well stimulate both the cellular and humoral immune systems that led to cutaneous lesion healing in mice.

Background and Objectives: Many plant extracts have antiparasitic properties and even some of them are effective for in vivo treatment. One of the medicinal plants is Peganum harmala, which can grow from 30 to 100 cm. The aim of the present study was to determine the effect of aqueous extract of Peganum harmala on trophozoites and cysts of Acanthamoeba in vitro. Methods: In this experimental study, a sample from a patient with amoebic keratitis, was cultured with Escherichia coli bacterium in non-nutrient agar medium (NNA) 1.5%. Then, the aqueous extract of Peganum harmala was prepared. The extract was affected on trophozoites and cysts of Acanthamoeba at three times (24, 48, and 72h) in various concentrations (1.25, 2.5, 5, and 10mg/ml). The percentage of live parasites, was evaluated using homocytometer lamel, MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], and flow cytometry method. The data were analyzed using one-way ANOVA, one sample Kolmogorov-Smirnov test, and repeated measures test The significance level was considered to be p

Background Congenital toxoplasmosis is an important cause of spontaneous abortion worldwide. However, there is limited information on detection and genotypic characterization of Toxoplasma gondii (T. gondii) in women with recurrent spontaneous abortion (RSA). The aim of this study is the molecular detection and genotypic characterization of T. gondii in formalin-fixed, paraffin-embedded fetoplacental tissues (FFPTs) of women with RSA that have referred to the Avicenna Research Institute in Tehran, Iran. Materials and Methods This experimental research was undertaken on 210 FFPTs of women with RSA. The information of the patients was collected from the archives of Avicenna Research Institute in Tehran, Iran. After DNA extraction, the presence of T. gondii was examined by nested polymerase chain reaction targeting the GRA6 gene. Genotyping was performed on positive samples using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) that targeted the GRA6 and SAG3 genes. Sequencing was conducted on two GRA6 positive samples. Results T. gondii DNA was detected in 3.8% (8/210) of the samples. Genotyping showed that all positive samples belonged to type III of the T. gondii genotype. Sequencing two genomic DNAs of the GRA6 gene revealed 99% similarity with each other and 99-100% similarity with T. gondii sequences deposited in GenBank. There were six patients with histories of more than three abortions; one patient had a healthy girl and another patient had two previous abortions. Abortions occurred in the first trimester of pregnancy in seven patients and in the second trimester of pregnancy in one patient. Conclusion The results of this study have indicated that genotype III is the predominant type of T. gondii in women with RSA in Tehran, Iran. Also, our findings suggest that toxoplasmosis may play a role in the pathogenesis of RSA. However, further studies are needed to elucidate a clear relationship between T. gondii infection and RSA.

Ascariasis is an important and prevalent geo-helminth infection, especially in underdeveloped countries such as Afghanistan, where public health problems and devastation of hygienic infrastructures usually lead to the higher incidence of the infection. Here, we report two cases of intestinal obstruction that accompanied with biliary ascariasis due to Ascaris lumbercoides in pregnant women and a 6-year-old girl from Afghanistan. Biliary ascariasis can be associated with cystic duct obstruction, gallbladder dilation, acute cholecystitis, acute cholangitis, obstructive jaundice, and biliary colic.

Vaginal infections are one of the most common reasons a woman visits a gynecologist. The increased resistance to conventional antibiotics is one of the main reasons for searching and developing new antimicrobial agents, especially those of natural origin. In traditional Persian medicine, the gall of Quercus infectoria has been claimed to eliminate vagina and cervix from excessive discharge. So, the aim of the present study was to evaluate the antimicrobial activity of ethanolic extract of Quercus infectoria gall as well as its active constituent, gallic acid, against some vaginal pathogens. In this study, the ethanolic extract of Quercus infectoria gall was obtained by maceration and standardized based on amount of gallic acid. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of this extract as well as its active compound, gallic acid, were determined against Candida spp., Gardnerella vaginalis, Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Trichomonas vaginalis and Lactobacillus acidophilus. The results demonstrated remarkable activity of ethanolic extract of Quercus infectoria gall against investigated pathogens with MIC and MBC in the range between 0.125 mg/ml and 16 mg/ml. The most inhibitory and bactericidal activity was observed on Streptococcus agalactiae and Staphylococcus aureus. The effects of gall dried ethanolic extract on Trichomonas vaginalis showed 100 % inhibition of the parasitic growth with concentration of 800 µg/ml after 24 h incubation. The antimicrobial and anti-trichomonas activity of extract was more than gallic acid. It seems that ethanolic extract of Quercus infectoria gall could inhibit the growth of vaginal pathogens. Further preclinical and clinical studies are required to confirm the efficacy of this natural extract in vaginitis.

Toxoplasmosis is a zoonotic parasitic disease of global importance. It is widespread and endemic practically all over the world, with infection prevalence varying by geographic location. The parasite and the disease are neglected, which is illustrated by the lack of baseline information on the prevalence from many regions. Developed, peaceful regions are in better situation to address toxoplasmosis, while the neglected status is particularly pronounced in regions with other challenges. Due to the lack of baseline data, these regions are rarely mentioned in discussions about the neglected status of the disease. A dramatic manifestation of toxoplasmosis, congenital toxoplasmosis, is the dreaded outcome of vertical transmission of the infection from the mother to the unborn child. For this reason, pregnant women are a key target group for primary prevention of T. gondii infections, and baseline data on the prevalence in pregnant women is important. In this cross-sectional seroepidemiological study, we estimated Toxoplasma gondii seroprevalence and evaluated possible risk factors for seropositivity in pregnant women in Kabul, Afghanistan. Altogether 207 of the 431 women included in the study tested positive for immunoglobulin G antibodies against T. gondii, yielding an apparent seroprevalence estimate of 48.03% (95% CI 43.33-52.75). Based on the final multivariable model for T. gondii seropositivity, non-concrete floor in the house and well or river as water source were identified as risk factors for seropositivity, while residence in rural area was a protective factor. The majority of the participants (72.9%) reported that they did not know about T. gondii. Our study is the first to report an estimate of T. gondii seroprevalence in pregnant women in Afghanistan. The high seroprevalence indicates substantial infection pressure, and the results of the risk factor analysis suggest that the environmental route, infection from oocysts, might be the most relevant to address to prevent the infections in the region. Our results contribute to the global discussion on neglected status of toxoplasmosis.

Background: In Iran, both forms of cutaneous (CL) and visceral leishmaniasis (VL) have been re-ported; so the accurate species identification of the parasite(s) and the analysis of genetic diversity are necessary. Methods: The investigation was conducted from 2014 to 2015 in the northwest and south of Iran, where VL is endemic (7 provinces). Blood samples of patients and infected dogs were collected and sera separated for serologic examinations (DAT, rK39). Spleen or bone marrow samples from infected dogs were also collected to confirm the infection. DNAs of 70 samples amplified by targeting a partial sequence of ITS (18S rRNA–ITS1–5.8S rRNA–ITS2) gene. All the amplicons were sequenced and analyzed with restriction fragment length polymorphism (RFLP) using the TaqI enzyme. Results: The cause of all 70 VL cases, were L. infantum, so, the dominant specie is L. infantum. The sequencing results of all VL cases and RFLP analysis corroborate each other. Discrimination of Iranian Leishmania isolates using ITS gene gives us this opportunity to detect, identify and construct the phylogenetic relationship of Iranian isolates. In addition, detection and differentiation of Leishmania spp. DNA was confirmed by amplification of variable area of the minicircle kDNA (conserved sequence blocks (CSB)). Conclusion: Low divergence and high likelihood were seen among L. infantum isolates of human and dogs from Iran with a very slight divergence was seen between isolates from northwest and south of Iran, thus grouped in a unique clad. No correlation was observed between intraspecies divergence and geographic distribution of the isolates.

Background: In Iran, both forms of cutaneous (CL) and visceral leishmaniasis (VL) have been reported; so the accurate species identification of the parasite(s) and the analysis of genetic diversity are necessary. Methods: The smears were collected from lesions samples of 654 patients with CL, who attended local health centers in 12 provinces of Iran during 2013-2015. The smears were checked for the presence of amastigotes by light microscopy. DNA of 648 Leishmania isolates, amplified by targeting a partial sequence of ITS (18S rRNA–ITS1–5.8S rRNA–ITS2) gene. Twenty-five of all the amplicons were sequenced and analyzed with restriction fragment length polymorphism (RFLP) using the Taq1 enzyme. Results: All the smears were positive microscopically. The PCR-RFLP analysis revealed that 176 (27%) CL patients were infected with L. tropica and, 478 (73%) with L. major. The dominant species in all over Iran is L. major. The sequencing results of all CL patients and RFLP analysis confirmed each other. Based on our phylogenetic tree, 25 ITS DNA sequences were grouped into two clusters representing L. major and L. tropica species. Phylogenetic tree derived from the ITS sequences supports a clear divergence between L. major from the other species. Conclusion: Discrimination of Iranian Leishmania isolates using ITS gene gives us this opportunity to detect, identify, and construct the phylogenetic relationship of Iranian isolates.

Background: Cystic echinococcosis (CE), as a zoonotic disease cause to health threat and economic losses. Despite implemented control programs, few countries have been able to decrease or eliminate this infection. Vaccination of the intermediate host offers an additional strategy to control the parasite transmission and EG95 antigen is considered more than the others in the vaccine issue. According to the high protection induced by the EG95 recombinant vaccine, this study was designed to construct recombinant plasmid formulation of EG95 antigen. Methods: In 2015, the Echinococcus granulosus eggs were recovered from an infected dog in Parasitological laboratory of Tarbiat Modares University in Tehran, Iran. Following hatching, the oncospheres of E. granulosus were activated to increase the presence of the desired mRNA. The extracted mRNA was transcribed to the cDNA which used as template in RT-PCR. Then the EG95 gene cloned into pET28a vector and the recombinant plasmids expression was investigated in prokaryotic and eukaryotic cells. Results: The recombinant plasmid encoding EG95 antigen was successfully constructed and identified by PCR, restriction enzyme digestion and sequencing. In vitro expression of the EG95 antigen was confirmed in prokary­otic and eukaryotic systems by SDS-PAGE and western blotting analysis. Conclusion: Because of potential advantages of DNA vaccines, including ability to induce long-term immune responses, low production cost and stability in different temperatures, this study carried out to construct the EG95 gene into a vector. This recombinant vector can be evaluated in further studies as a DNA vaccine may provide new prospects for the development of a vaccine against cystic hydatid disease.

Background: Cystic Echinococcosis (CE) is one of the most common parasitic zoonosis caused by Echinococcus granulosus worldwide. This study investigated genotype diversity of Echinococcus granulosus isolated from stray dogs and golden jackals in Ilam province, West of Iran. Methods: Adult worms were collected from the small intestine of the stray dogs and golden jackals from Ilam Province roads during 2012-2014. DNA was extracted from the adult worms and the partial mitochondrial NADH dehydrogenase subunit1 (nad1) was amplified by PCR then the products were digested by using HpaII, Rsa1, and Alu1 restriction enzymes. In order to confirm RFLP results, a number of PCR products were bi-directionally sequenced. Results: Totally, 20 stray dogs out of 75 (26.66%) and two out of 73 (2.74%) golden jackal showed infection with E. granulosus. Amplified PCR product for all isolates was a band of approximately 550bp. Alu1 digested the product into two bands of approximately 160bp and 390bp fragments, while the Rsa1 cut the product into 320bp and 230bp fragments and the HpaII had no effect on the PCR product for both dog and jackal samples. The isolate sequences of mtDNAnad1 gene indicated 100% homology with references G1, G2 and G3 sequences in the GenBank database. Conclusion: The genotype of adult E. granulosus was similar to larval stage genotypes of parasite (G1-G3 complex).

Background: Rhipicephalus sanguineus is the most widely distributed tick in the world, which is partly due to its biological flexibility and the global distribution of its major host, the domestic dog. In Mediterranean region it could be principal reservoir host for Leishmania infantum, usually transmitted by the phlebotomine sand flies. In this study, we evaluated the vector potential of R. sanguineus in transmitting L. infantum to uninfected dogs. Methods: During 2014, five dogs with clinical manifestations of canine visceral leishmaniasis (CVL), high anti-Leishmania antibody titers and tick infestation, were selected from CVL endemic areas (Tehran and Alborz provinces). At least, twenty live ticks were removed from each infected dog. After morphological identification, the ticks were divided into two groups; ticks belonging to the first group were dissected for parasitological examinations and semi-nested PCR assay, and those of the second group were selected for the transmission of CVL caused by L. infantum to uninfected dogs. Following tick infestation, all uninfected dogs were kept for 9 months and examined monthly for clinical and serological tests. Results: Nearly, 67% of ticks were infected by L. infantum using the semi-nested PCR. All other parasitological tests of ticks were negative. Clinical examinations and serological tests of the investigated dogs revealed negative results. Nested-PCR test results performed on splenic biopsy samples of dogs were also negative. Conclusion: L. infantum-positive R. sanguineus ticks were unable to transfer L. infantum from infected dogs to healthy ones. The detection of L. infantum DNA in ticks collected from naturally infected dogs by semi-nested PCR does not prove their vectorial competence.

Cutaneous leishmaniasis is a common parasitic disease that is endemic in some parts of Iran. The drugs of choice used for leishmaniasis therapy are associated with a risk of recurrence and serious adverse effects. Therefore, finding a safe and effective treatment is of great importance. In the present study, the effect of aloe-emodin on the growth of Leishmania major amastigotes was evaluated under in vitro conditions. In addition, the efficacy of a topical of aloe-emodin ointment was investigated in BALB/c mice with cutaneous leishmanial ulcers. Different concentrations (40 μg/mL, 80 μg/mL, 120 μg/mL, and 160 μg/mL) of aloe-emodin were tested on Leishmania amastigotes twice: 24 hours and 48 hours. The induced apoptosis and necrotic effects of two concentrations (40 μg/mL and 120 μg/mL) of aloe-emodin on promastigotes were investigated by flow cytometry. Under the in vivo condition, aloe-emodin ointment efficacy was evaluated at two concentrations (i.e., 0.1% and 1%). Serum indicator factors of the test and control groups were tested to evaluate the toxic effects of this compound on the liver and kidney. Results showed that aloe-emodin inhibited the growth of Leishmania amastigotes and induced apoptosis in promastigotes. Topical application of aloe-emodin ointment likewise reduced the ulcer size. No significant differences in biochemical analysis were observed between the control and treated groups. In conclusion, aloe-emodin showed antileishmanial effects under in vitro and in vivo conditions and may be used in clinical trials.

Background: Leishmania tropica, the causative agent of anthroponotic cutaneous leishmaniasis (ACL), and Leishmania major, which causes zoonotic cutaneous leishmaniasis (ZCL), are endemic in Iran. Methods: Cross-sectional study was de­signed to identify Leishmania species in cutaneous leishmaniasis patients who referred to Mashhad Health Centers from 2013 to 2014 using ITS-PCR-RFLP technique. First, physical examinations were performed in all suspected patients and CL cases were confirmed with microscopical examinations. A questionnaire was prepared and complet­ed for each confirmed patient and DNA from each lesion smear was extracted, separately. The ribosomal internal transcribed spacer was amplified with appropriate primers and PCR products were digested by enzyme Taq1 restrict enzyme. Results: From all patients, 51 cases (54.3%) were men and 43 of them (45.7%) were women. The most frequent age group was 20–29 years old (27.2%). Hands, face and feet were the most common sites for appearance of skin lesions. All of the 94 cases (100%) tested found to be positive by ITS-PCR-RFLP. Overall, Leishmania species were identified in all of the 94 lesion smears which 33 (35%) of them were L. major and 61 (65%) of the remained isolates were iden­tified L. tropica. Conclusion: Characterization of Leishmania isolates collected from different parts of Khorasan-Razavi Province showed that L. tropica is predominant agents of CL, especially in large and medium sized cities such as Mashhadand Shandiz. Moreover, this study revealed that ITS-PCR-RFLP based on our designed primers is a suitable method for species characterization.

Is Hair Lice Still a Public Health Problem?

Background: The aim of present study was to evaluate the protective efficacy of live recombinant L. tarentolae expressing KMP11-NTGP96-GFP fusion as candidates for live engineered recombinant vaccine against visceral leishmaniasis in BALB/c mice. Methods: KMP-11 and NT-GP96 genes cloned into the pJET1.2/blunt cloning vector and then into pEGFP-N1 expression vector. The KMP-11, NT-GP96 and GFP fused in pEGFP-N1 and subcloned into Leishmanian pLEXSY-neo vector. Finally this construct was transferred to L. tarentolae by electroporation. Tranfection was confirmed by SDS-PAGE, WESTERN blot, flowcytometry and RT-PCR. Protective efficacy of this construct was evaluated as a vaccine candidate against visceral leishmaniasis. Parasite burden, humoral and cellular immune responses were assessed before and at 4 weeks after challenge. Results: KMP- NT-Gp96-GFP Fusion was cloned successfully into pLEXSY -neo vector and this construct successfully transferred to L. tarentolae. Finding indicated that immunization with L. tarentolae tarentolae-KMP11-NTGP96-GFP provides significant protection against visceral leishmaniasis and was able to induce an increased expression of IFN-γ and IgG2a. Following challenge, a reduced parasite load in the spleen of the KMP11-NTGP96-GFP immunized group was detected. Conclusion: The present study is the first to use a combination of a Leishmania antigen with an immunologic antigen in live recombinant L. tarentolae and results suggest that L. tarentolae-KMP11-NTGP96-GFP could be considered as a potential tool in vaccination against visceral leishmaniasis and this vaccination strategy could provide a potent rout for future vaccine development.

Little is known about latent infection and molecular characterisation of Neospora caninum in sheep (Ovis aries). In this study, 330 sheep samples (180 hearts and 150 brains) were analysed for N. caninum DNA by nested polymerase chain reaction (PCR) targeting the Nc-5 gene. Neospora caninum DNA was detected in 3.9% (13/330) of sheep samples. The parasite’s DNA was detected in 6.7% of heart samples (12/180) and 0.7% (1/150) of brain samples. No clinical signs were recorded from infected or uninfected animals. Sequencing of the genomic DNA revealed 96% – 99% similarity with each other and 95.15% – 100% similarity with N. caninum sequences deposited in GenBank. To our knowledge, this is the first report on the use of PCR to identify latent neosporosis in sheep in Iran. The results of this study have the potential to contribute to our understanding of the role of N. caninum-infected sheep in the epidemiology of neosporosis.