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Abstract Background Toxoplasma gondii is an obligate intracellular protozoan parasite that can invade all mammalian cells. It is well established that natural killer (NK) cells have critical protective roles in innate immunity during infections by intracellular pathogens. In the current study, we conducted an in vitro experiment to evaluate NK cell differentiation and activation from human umbilical cord blood mononuclear cells (UCB‐MNCs) after infection with T. gondii tachyzoites. Methods UCB‐MNCs were infected by fresh tachyzoites of type I (RH) or type II (PTG) strains of T. gondii pre‐expanded in mesenchymal stem cells for 2 weeks in a medium enriched with stem cell factor, Flt3, IL‐2, and IL‐15. Flow cytometry analysis and western blot analysis were performed to measure the CD57+, CD56+, and Granzyme A (GZMA). Results Data revealed that incubation of UCB‐MNCs with NK cell differentiation medium increased the CD57+, CD56+, and GZMA. UCB‐MNCs cocultured with PTG tachyzoites showed a significant reduction of CD56+ and GZMA, but nonsignificant changes, in the levels of CD56+ compared to the control UCB‐MNCs (p > .05). Noteworthy, 2‐week culture of UCB‐MNCs with type I (RH) tachyzoites significantly suppressed CD57+, CD56+, and GZMA, showing reduction of NK cell differentiation from cord blood cells. Conclusion Our findings suggest that virulent T. gondii tachyzoites with cytopathic effects inhibit NK cell activation and eliminate innate immune responses during infection, and consequently enable the parasite to continue its survival in the host body.

Background: Current study was designed to provide a better insight into the circulating genotypes, genetic diversity, and population structure of Echinococcus spp. between southeast of Iran and Pakistan. Methods: From Jun 2020 to Dec 2020, 46 hydatid cysts were taken from human (n: 6), camel (n: 10), goat (n: 10), cattle (n: 10) and sheep (n: 10) in various cities of Sistan and Baluchestan Province of Iran, located at the neighborhood of Pakistan. DNA samples were extracted, amplified, and subjected to sequence analysis of cox1 and nad1 genes. Results: The phylogeny inferred by the Maximum Likelihood algorithm indicated that G1 genotype (n: 19), G3 genotype (n: 14) and G6 genotype (n: 13) assigned into their specific clades. The diversity indices showed a moderate (nad1: Hd: 0.485) to high hap- lotype diversity (cox1: Hd: 0.867) of E. granulosus s.s. (G1/G3) and low nucleotide diver- sity. The negative value of Tajima’s D and Fu’s Fs test displayed deviation from neutrali- ty indicating a recent population expansion. A parsimonious network of the haplotypes of cox1 displayed star-like features in the overall population containing IR9/PAK1/G1, IR2/PAK2/G3 and IR18/G6 as the most common haplotypes. A pairwise fixation index (Fst) indicated that E. granulosus s.s. populations are genetically moderate differen- tiated between southeast of Iran and Pakistan. The extension of haplotypes PAK18/G1 (sheep) and PAK26/G1 (cattle) toward Iranian haplogroup revealed that there is dawn of Echinococcus flow due to a transfer of alleles between mentioned populations through transport of livestock or their domestication. Conclusion: The current findings strengthen our knowledge concerning the evolution- ary paradigms of E. granulosus in southeastern borders of Iran and is effective in control- ling of hydatidosis.

Abstract Background Toxoplasmosis is a serious or life-threatening disease in immunosuppressed patients and pregnant women. This study examined the likely association between Toxoplasma gondii infection and COVID-19 patients with moderate illness. Methods Seventy blood samples were collected from patients at the Health Reference Laboratory of Tabriz, Northwest Iran from April 2021 to September 2021. In addition, 70 healthy subjects of the same age (37 ± 15 years) and sex distribution were ethnically matched. Sera samples were examined for the detection of anti-Toxoplasma antibodies using ELISA. Nested-PCR targets were amplified based on the B1 and GRA6 genes. GRA6 amplicons were subjected to sequencing and phylogenetic analysis. Results The seroprevalence of toxoplasmosis based on IgG titer was 35.7% in the COVID‑19 patients and 27.1% in the control group, representing not to be associated with the Toxoplasma seropositivity in COVID‑19 patients (P = 0.18) compared to healthy subjects. Anti-T. gondii IgM was not found in any of the patients and healthy individuals. According to PCR amplification of the B1 and GRA6 genes, the frequency of T. gondii in COVID-19 patients was 14.2% (10/70). However, no T. gondii infection was detected in the healthy group. The CD4+T cell count was relatively lower in toxoplasmosis-infected patients (430–450 cells/mm3) than in control group (500–1500 cells/mm3). High genetic diversity (Hd: 0.710) of the type I strain of T. gondii was characterized in the patients. Present results showed that consumption of raw vegetables and close contact with stray cats can increase the transmission of T. gondii to COVID-19 patients (P

Abstract Background Neutropenia is the most important cause of life-threatening invasive fungal infections (IFIs). Here, we studied the frequency and antifungal susceptibility profiles of Candida species that colonized or caused infections among neutropenic patients with solid or hematological malignancies. Methods A total of 362 clinical samples were collected from 138 patients. After initial isolation using a mix of mycological methods, isolates were screened using chromogenic culture media. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was applied for molecular identification. Positive or suspected cases were confirmed using the reference method of sequencing. Antifungal susceptibility testing for voriconazole and caspofungin was carried out using the microbroth dilution method. An in-silico assay was applied for phylogenetic analysis. Results Thirty-four Candida strains were isolated. C. albicans (47.06%) and C. glabrata (29.41%) were the most frequent strains. Antifungal treatment reduced the chance of Candida colonization by almost 76% in neutropenic patients (OR: 1.759; 95% CI: 1.349 to 2.390; p value: 0.000). An unusual and non-resistant strain, C. lambica, was reported from the bloodstream of a 56-year-old man with hematologic malignancy (HM). Eight isolates were non-susceptible, and one isolate was resistant to voriconazole. Also, four isolates were non-susceptible to caspofungin. Conclusion We can conclude that there is a cause-and-effect relationship between neutropenia, HM background, and Candida species separated from neutropenic patients, which can lead to possible infections. Further and repetitive studies are recommended using different molecular methods for better prediction and management of fungal infections in neutropenic patients.

Antigens in hydatid cyst fluid (HCF) have been discovered to bear a significant resemblance to antigens present in cancer cells. MicroRNA-1 (miR-1) is a well-known member of the tumor inhibitor miRNA family and has been shown to have pro-apoptotic and tumor-inhibitory functions. This study aimed to evaluate the ability of HCF to prevent breast cancer and to explore the underlying mechanisms that affect cancer cells. For this study, MDA-MB-231 and MCF-7 breast cancer cells were cultured and divided into two groups: one group received HCF treatment and the other group was untreated and served as the control group. The cytotoxicity and cell viability of various HCF concentrations on breast cancer cells were evaluated using the MTT assay. In addition, the expression level of miR-1 in HCF-treated and untreated breast cancer cells was analyzed using qRT-PCR. The study found that HCF treatment reduced the growth of MDA-MB-231 and MCF-7 breast cancer cells, indicating that it was cytotoxic to the cells. Specifically, the IC50 concentration of HCF after 24 hours of treatment was 7.32 µg/mL for MDA-MB-231 cells and 13.63 µg/mL for MCF-7 cells. In addition, qRT-PCR analysis revealed that the expression level of miR-1 was significantly increased in HCF-treated MDA-MB-231 (P=0.0203) and MCF-7 (P=0.0394) cell lines compared to untreated controls. Although HCF has been shown to inhibit the growth of breast cancer cells and to upregulate miR-1, a key tumor suppressor in cancer cells, the specific mechanisms responsible for this effect remain unclear. Further studies are needed to fully understand the molecular pathways underlying HCF’s antitumor activity and its potential as a therapeutic agent in cancer therapy.

The reduced burden of helminth parasites in industrialized countries is probably one of the reasons for the increased prevalence of autoimmune disorders such as multiple sclerosis (MS). The current study aimed to evaluate the potential preventive effects of hydatid cyst fluid (HCF) on the disease severity in an EAE mouse model of MS. EAE-induced mice were treated with HCF before and after EAE induction. An RT-PCR-based evaluation of IFN-γ, IL-1β, TNF, T-bet, IL-4, GATA3, IL-17, RoRγ, TGF-β, and FOXP3 expression levels in splenocytes and an ELISA-based analysis of IFN-γ and IL-4 levels in cell culture supernatant of splenocytes were performed. Histopathological examinations of mice during the study were also conducted. The expression levels of T-bet, IL-4, GATA3, TGF-β, and FOXP3 in EAE + HCF mice were significantly higher compared to EAE + PBS mice. In the EAE + HCF group, the expression levels of IFN-γ, IL-1β, and TNF were significantly lower than in the EAE + PBS group. The histopathological results showed significantly reduced inflammation and demyelination in EAE + HCF mice compared to EAE + PBS mice. Our study provides proof-of-concept in the EAE mouse model of MS that helminth-derived products such as HCF have a potential prophylactic effect on MS development and present a novel potential therapeutic strategy.

Background: Hydatidosis is known as one of the most prevalent zoonotic diseases across the world. This complication is also endemic in Iran, followed by a higher risk of infection in rural areas. To our knowledge, there has been no study on the seroprevalence of hydatidosis in Sistan and Baluchistan Province, Southeast of Iran. The main objective of the current study was to examine the seroprevalence of hydatidosis and its risk factors in high-risk individuals (farmers and ranchers) living in Sistan and Baluchistan Province. Materials and Methods: This study included 500 serum samples, and the participants were requested to complete a researcher-made questionnaire. Subsequently, counter-current immunoelectrophoresis (CCIEP) and enzyme-linked immunosorbent assay (ELISA) methods were employed to analyze the anti-Echinococcus granulosus antibody. The analysis of the obtained data was conducted by logistic regression in SPSS software, version 22. Results: According to the results, four (0.8%) cases were found positive for anti-E. granulosus antibody by both CCIEP and ELISA tests. Seroprevalence of hydatidosis was more in rural people, compared to those in urban areas. It was also higher in illiterate people than in educated people. Nevertheless, seropositivity showed no significant differences with age, gender, occupational status, education level, place of residence, and contact with dogs (P>0.05). Conclusion: The prevalence rate of hydatidosis in Sistan and Baluchistan Province was similar to that in neighboring provinces. According to the findings, high-risk individuals offer remarkable information about the epidemiology of hydatidosis in Sistan and Baluchistan province in southeastern Iran. This could help to manage and prevent this infection.

Cystic echinococcosis (CE) is a life-threatening helminthic disease caused by the Echinococcus granulosus sensulato complex. Previous evidence indicates that the host's innate immune responses against CE can combat and regulate the growth rate and mortality of hydatid cyst in the host's internal organs. However, the survival mechanisms of CE are not yet fully elucidated in the human body. In the present study, the apoptotic effects of fertile and infertile hydatid fluid (HF) were tested on murine peritoneal cells in vivo mice model. Mice were divided into five groups including; control group, fertile HF-treated peritoneal cells, infertile HF-treated peritoneal cells, protoscolices (PSCs)-treated peritoneal cells and HF+PSCs-treated peritoneal cells group. Mice groups were intraperitoneally inoculated with PBS, HF, and/or PSCs. Afterwards, peritoneal cells were isolated and mRNA expression of STAT3, caspase-3, p73 and Smac genes were evaluated by quantitative Real-time PCR. After 48 hours of exposure, the protein levels of Smac and STAT3 was determined by western blotting technique. After 6 hours of exposure, Caspase-3 activity was also measured by fluorometric assay. The intracellular reactive oxygen species (ROS) production was examined in all groups. The mRNA expression levels of p73, caspase-3 and also Caspase-3 activity in HF+PSCs-treated peritoneal cells were higher than in the test and control groups (Pv

To examine the genetic diversity of Leishmania major, 100 Giemsa-stained positive slides were collected from endemic foci of Iran (Northeast, Central, and Southwest provinces) over two consecutive years during 2019-2021. The Leishmania ITS-rDNA gene was amplified and Leishmania sp. was recognized by PCR-RFLP and sequencing. In addition, 178 registered ITS-rDNA sequences from other geographical regions of Iran were retrieved from GenBank, including different host species (human, sandfly and rodent). A total of 40 new haplotypes were discovered using the ITS-rDNA sequence analysis. IR29 (20.6%) and IR34 (61%) were the two most common haplotypes, represented by a star-like feature in the overall population. Analysis of the molecular variance test revealed low genetic diversity of L. major in human cases (Haplotype diversity; 0.341), rodent (Hd; 0.387) and sandfly (Hd; 0.390) sequences. The lowest genetic diversity of L. major was observed in Southwest/Southeast Iran (Hd: 0.104-0.286). The statistically Fst value indicated that L. major is not genetically differentiated between geographic regions of Iran, except for the Northeast-Southwest (Fst: 0.29055) and Central-Southwest (Fst: 0.30294) population pairs. The current study as the first investigation discloses new perspectives for further evaluation in the identification local transmission paradigms and initiating effective prevention strategies.

Background: Antimalarial drug resistance is one of the important challenges for governments in the fight against malaria. Molecular surveillance of antimalarial drug resistance supports early detection of how the recommended treatments work. This allows immediate action to reduce any threat and prevent it from spreading. Therefore, the aim of this study was to evaluate the frequency of dihydrofolate reductase (dhfr) mutants in Plasmodium falciparum resistance to pyrimethamine in Iranian malaria patients. Materials and Methods: In 2020, 27 patients (21 males and 5 females) with imported P. falciparum cases were studied. The nested-PCR technique first confirmed the species in all samples and then amplification was done by the semi-nested-PCR method in order to detect single nucleotide polymorphisms (SNPs) in dhfr gene related to pyrimethamine resistance. Results: All samples in the 18S rRNA gene had species-specific bands for P. falciparum strains. In the sequence analysis of pfdhfr gene amplification after comparison with the standard strain (wild type), 21 patients had a double mutation (C59R+S108N) and six patients had a triple mutation (N51I+C59R+S108N) of pyrimethamine resistance. Conclusion: The results of this study showed that the susceptibility of P. falciparum to pyrimethamine in the treatment of malaria is significantly reducing. These findings can raise concerns about pyrimethamine resistance in P. falciparum. Due to the high emergence of double and triple mutants related to pyrimethamine resistance, the malaria surveillance and treatment systems in Iran, the use of pyrimethamine should be considered.

Background: Echinococcosis or Hydatid disease is a zoonotic disease that is caused by Echinococcus granulosus. The disease is a high public health concern in Iran, but there is little known about the genetic diversity and epidemiology of Echinococcus spp. in Iranian shepherd dogs. Materials and Methods: Fifty shepherd dogs were investigated for the adult worm of E. granulosus from May 2020 to April 2021 in Sistan and Baluchestan Province, the southeastern border of Iran. DNA extraction of samples and amplifying was done, and sequence analysis of mitochondrial genes (Cox1 and Nad1) was performed. Results:Out of 50 shepherd dogs, 11 cases (22%) were infected with E. granulosus. No significant difference was observed regarding demographic factors (P>0.05). The phylogenetic analyses of Cox1 and Nad1 sequences demonstrated G1 genotype (sheep strains) in all isolates. Based on sequence analyses, a low (Cox1, Hd [haplotype diversity: 0.200; Hn [number of haplotypes]: 2) to moderate (Nad1, Hd: 0.533; Hn: 4) genetic (haplotype) diversity of E. granulosus G1 genotype and low nucleotide diversity (π: 0.00052-0.00243) were observed. Conclusion: The first identification of a sheep strain (G1) in the final host in Sistan and Baluchestan Province indicates that potential intermediate hosts play a secondary role in preserving the biology of the dog-sheep cycle. The present study’s findings enrich our knowledge about the prevalence of E. granulosus, the classification of strains, and the genetic diversity of the parasite in Iranian herding dogs. This information helps develop strategies and programs for monitoring and controlling infection in stray dogs in the region

Background: We aimed to detect the genetic diversity of samples identified morphologically as Fasciola spp. from sheep, cattle and goat from Lorestan Province, western Iran using PCR-RFLP method. Besides, we evaluated the genetic diversity indices, sequencing and phylogenetic analysis using mitochondrial gene (ND1 and CO1). Methods: PCR-RFLP analysis of ribosomal ITS1 fragment by RsaI restriction enzyme to investigate the genetic characteristics of Fasciola species obtained from different hosts (18 sheep, 21 cattle, and 17goats) was conducted. The samples were sequenced. Sequences were evaluated using BLAST software and the parasite species were identified with similarity percentage and overlap with the species registered in the gene bank. Then similarity and diversity of intra-species and intra-species diversity of Fasciola species were calculated. Results: In Lorestan, based on RFLP pattern, 93% (52) of the Fasciola spp. isolates had a RFLP pattern related to F. hepatica and 7% (4) were F. gigantica. No hybrid forms were detected. The CO1 gene could clarify 19 haplotypes against ND1 gene that found 22 haplotypes among livestock. Sequencing results of the mtDNA showed intra-species identity 98. 5%-100% and Intra-species-diversity: 0-1.5% compared to the GenBank sequences. Conclusion: Using PCR-RFLP method, two species of F. hepatica and F. gigantica, were present in Lorestan Province, but F. hepatica was more prevalent. Mitochondrial genes could better test variability indices in different hosts than ribosomal genes, consequently among mitochondrial genes, the ND1 gene could better examine differences and similarities than CO1.

Background and Aim: Giardia duodenalis is one of the most common enteric protozoan parasites in vertebrates, such as humans, domestic and wild animals, causing giardiasis. To the best of our knowledge, little is known about the genetic diversity of G. duodenalis assemblages. This study aimed to identify genetic diversity of G. duodenalis assemblages in Iranian stray dogs. Materials and Methods: A total of 450 fecal samples were collected from 2015 to 2016 from stray dogs of Northwest Iran. All specimens were observed microscopically following concentration and flotation techniques. Subsequently, DNA samples were extracted, amplified, and sequenced targeting the glutamate dehydrogenase gene. Results: The overall prevalence of G. duodenalis in infected dogs was estimated at 1.6%, based on microscopic and molecular diagnoses. Sequencing and phylogenetic analyses indicated a high level of genetic diversity of assemblage C (haplotype diversity; 0.802). Conclusion: The pairwise sequence distances between the identified isolates of assemblage C showed an intradiversity of 0.3%-1.3% and identity of 98.7%-100%. Current findings indicate that a significant genetic diversity of G. duodenalis assemblage C haplotypes is unequivocally circulates among stray dogs in Northwest Iran.

Abstract Cystic echinococcosis (CE) and alveolar echinococcosis (AE) are life-threatening parasitic infections worldwide caused by Echinococcus granulosus (sensu lato) and E. multilocularis, respectively. Very little is known about the factors affecting innate susceptibility and resistance to infection with Echinococcus spp. Although benzimidazolic drugs against CE and AE have definitively improved the treatment of these cestodes; however, the lack of successful control campaigns, including the EG95 vaccine, at a continental level indicates the importance of generating novel therapies. This review represents an update on the latest developments in the regulatory functions of innate immune pathways such as apoptosis, toll-like receptors (TLRs), and inflammasomes against CE and AE. We suggest that apoptosis can reciprocally play a bi-functional role among the host-Echinococcus metabolite relationships in suppressive and survival mechanisms of CE. Based on the available information, further studies are needed to determine whether the orchestrated in silico strategy for designing inhibitors and interfering RNA against anti-apoptotic proteins and TLRs would be effective to improve new treatments as well as therapeutic vaccines against the E. granulosus and E. multilocularis.

Background: This study is an attempt to investigate the effect of nano-liposome containing voriconazole on voriconazole-resistant A. flavus strains on the one hand, and to consider the expression of cyp51A and MDR1genes, regarded as important genes involved in the development of resistance to triazoles before and after voriconazole and voriconazole-loaded nano-liposomes exert their effects, on the other hand.Methods: Strains of A. flavus isolated from patients were investigated and their susceptibility to voriconazole was determined. Next, having applied a slight modification to the thin film hydration-sonication technique, the liposomal formulation of voriconazole was produced. After that, the voriconazole-loaded nano-liposome was subjected to in-vitro antifungal susceptibility testing to obtain minimum inhibitory concentration against fungal isolates. Cyp51A and MDR1 mRNA levels were amplified by qRT-PCR instrument.Results: The effect of nano-liposome containing voriconazole on the reduction of MIC in A. flavus isolates were considered to be significant. After using MIC50 concentration of VCZ, the cyp51A gene expression in voriconazole-susceptible A. flavus strains and voriconazole-resistant strains 10folds and 7folds depicted a downregulation, respectively, which was more pronounced in the expression of a liposomal formulation of VCZ (13folds and 15folds respectively). Identically, the same procedure was applied to MDR1, even though it induced 1, 2, 3, 4-fold reductions.Conclusion: Considering the benefits of liposome-containing voriconazole formulation, such as the reduction of the side effects of the pure drug as well as minimizing the drug's toxicity coupled with the enhanced drug bioavailability and stability, the formulation can be used in drug-sensitive and drug-resistant species.

Abstract Background The larval stage of the tapeworm Echinococcus granulosus is the causative agent of hydatid disease in humans. This zoonotic parasitic infection remains a major health problem in certain areas of the world where is still endemic. In view of the ineffectiveness of some drug treatments, the surgical removal of cysts remains the preferred treatment option together with the administration of albendazole and mebendazole. However, severe side effects of these drugs have been reported which demands developing new scolicidal agents that confer suitable efficacy and fewer side effects during surgery. Methods To that purpose, in the present work we assessed the effectiveness of ivermectin (IVM), a macrocyclic lactone endectocide that has shown to be an effective nematocidal drug against other important parasitic infections. To overcome the limitations observed in some drug formulations and resistance, we used nano lipid carriers (NLCs) as a targeted and sustained drug delivery system for IVM. We evaluated the in vitro cestocidal and apoptotic effects of NLCs-loaded IVM versus IVM by quantifying the expression of caspase-3 mRNA. Results We found that after 60 and 120 min of administration, 800 μg/ml and 400 μg/ml NLCs-loaded IVM induced 100% mortality, respectively. On the other hand, the 800 μg/ml of IVM induced 100% mortality rate 150 min after administration. Additionally, we found that NLCs-loaded IVM induced higher mRNA caspase-3 expression suggesting a more potent apoptotic effect on the parasite. Conclusions These data suggest that NLCs-loaded IVM may be a promising alternative to current treatments although in vivo studies are needed.

Abstract Background Cryptosporidium spp. are causative agents of gastrointestinal diseases in a wide variety of vertebrate hosts. Mortality resulting from the disease is low in livestock, although severe cryptosporidiosis has been associated with fatality in young animals. Methods The goal of this systematic review and meta-analysis was to review the prevalence and molecular data on Cryptosporidium infections in selected terrestrial domestic and wild ungulates of the families Bovidae (bison, buffalo, cattle, goat, impala, mouflon sheep, sheep, yak), Cervidae (red deer, roe deer, white-tailed deer), Camelidae (alpaca, camel), Suidae (boar, pig), Giraffidae (giraffes) and Equidae (horses). Data collection was carried out using PubMed, Scopus, Science Direct and Cochran databases, with 429 papers being included in this systematic analysis. Results The results show that overall 18.9% of ungulates from the investigated species were infected with Cryptosporidium spp. Considering livestock species (cattle, sheep, goats, pigs, horses and buffaloes), analysis revealed higher Cryptosporidium infection prevalence in ungulates of the Cetartiodactyla than in those of the Perissodactyla, with cattle (29%) being the most commonly infected farm animal. Conclusions Overall, the investigated domestic ungulates are considered potential sources of Cryptosporidium contamination in the environment. Control measures should be developed to reduce the occurrence of Cryptosporidium infection in these animals. Furthermore, literature on wild populations of the named ungulate species revealed a widespread presence and potential reservoir function of wildlife.

Background and purpose: Toxoplasma gondii (T. gondii) is an obligatory intracellular protozoan parasite. Toxoplasmosis is highly prevalent and has a great effect on public health, therefor, there is a need for vaccine and sensitive diagnostic procedures. This study aimed at cloning and investigating the expression of ROP13 gene of T. gondii. Materials and methods: In this study, the gene was cloned in pTG19-T vector and transferred to a TOP10 strain of E.coli following ROP13 gene amplification using PCR. Then the ROP13 gene was sub cloned in expression plasmid pcDNA3. Also, pcROP13 was transferred to CHO cells and the expression level was evaluated by IFA method. Results: Cloning and sub cloning of ROP 13 gene were confirmed by PCR, sequencing and enzymatic digestion. The gene sequencing showed complete homology with a recorded sequence in the gene bank. Moreover, the expression of ROP13 gene in CHO eukaryotic cells was confirmed by IFA method. Conclusion: The results showed that ROP13 gene was successfully sub cloned into the pcDNA3 expression vector and expressed in CHO cells. Therefore, it can be used in the development of vaccines and in diagnostic tests.

Objective: To identify serodiagnosis and quantification of Toxoplasma gondii (T. gondii) infection among pregnant women in Salmas, northwest of Iran. Methods: In this cross-sectional study, 276 blood samples were collected from pregnant women referred to the health care centers in Salmas city. The demographic variables were also recorded. Titers of anti-Toxoplasma IgM and IgG antibodies (Ab) were determined using the chemiluminescence immunoassay. Quantitative real-time PCR targeting the T. gondii repeated element gene was also performed on the blood sample. Results: Out of all, 19.92% (55/276) and 2.17% (6/276) patients were seropositive for anti-Toxoplasma IgG and IgM Ab, respectively. Moreover, the presence of T. gondii DNA was observed in 12.31% (34/276) blood samples. A significant relationship was observed between the IgG Ab seropositivity and contact with the cat as a risk factor (P=0.022). Conclusions: The seroprevalence rate of T. gondii infection in pregnant women is relatively low. Consequently, the seronegative pregnant women are at risk, and a considerable rate of positive blood samples for the presence of parasite's DNA should not be ignored. Besides, quantitative real-time PCR could be considered as an accurate method for diagnosis of acute toxoplasmosis especially when the precise results are of the most importance in pregnancy. Limiting contact with cats is also suggested for pregnant women.

Background: Human Echinococcosisis a cyclo-zoonotic infection caused by tapeworms of the Echinococcus granulosus sensu stricto complex. The detection of mitochondrial genome data of genus Echinococcus can reflect the taxonomic status, genetic diversity, and population structure genetics. Methods: Totally, 52 formalin-fixed paraffin-embedded (FFPE) tissue samples from patients with histologically confirmed CE were collected from Mazandaran province, Iran in the period of Mar 1995 to May 2018. All extracted DNAs from (FFPE) tissue samples were subjected to amplify by polymerase chain reactions method targeting cytochrome c oxidase subunit 1 (cox1) gene. All PCR amplicons were sequenced to phylogenetic analysis and genetic diversity. Results: Molecular analysis showed that 50(96.1%) and 2 (3.84%) isolates were identified as G1 andG3 E. granulosus genotypes, respectively. DNA sequence analyses indicated a high gene diversity for G1 (Haplotype diversity: 0.830) and G3 genotypes (Hd: 1.00). Based on multiple sequence alignment analyses, 7 (13.46%; G1 genotype) and 2 (3.84%; G3 genotype) new haplotypes were unequivocally identified. Conclusion: G3 genotype (Buffalo strain) was identified from two human hydatidosis isolates in the region. Present study strengthens our knowledge about taxonomic status, transmission patterns of Echinococcus parasite to human and heterogeneity aspects of this parasite in clinical CE isolates of Northern Iran.

Leishmania parasites as the causative agent of leishmaniasis belong to Trypanosomatidae family. Parasite, vector, vertebrate host and environment are major factors in pathogenesis of Leishmania. Parasite dependent factors are virulence factors which exist in Leishmania species such as LPG, GP63. In recent years, the importance of these factors in the field of vaccine and drug has been considered by researchers. Sand fly biting behavior and salivary gland proteins are vector dependent factors which are effective in the Leishmania pathogenesis. Age, gender, nutrition, immune system, infectious diseases, genetic, occupation, socio-economic characteristics, and habitat are vertebrate host mediated factors. Temperature, rainfall, wind and its speed, soil, and continuous changes in climate are also environmental factors. Therefore the aim of this study was to evaluate the pathogenesis of Leishmania parasites.

Background: Leishmania major is a causative agent of zoonotic cutaneous leishmaniasis in the center of Iran, Abarkouh district. Molecular characterization and precise incrimination of Leishmania species was carried out to perform controlling measurements and to design treatment programs for zoonotic cutaneous leishmaniasis. Methods: All smears isolated from ulcers of suspected patients were examined under a light microscope and graded for amastigotes frequency. Extraction of DNA, PCR, RFLP and sequencing of ITS-rDNA genotype were done to increase the efficacy of Leishmania parasites identification at their species-specific level and to detect any Leishmania infections within. Results: Humans were found to be infected with L. major with high infection frequency and also Leishmania tropica was identified with low occurrence for the first time as non-native species using molecular analyses. The rates of infections was considerable with microscopic observation (n = 65, 73%) out of 89 smears prepared from suspected patients. Molecular analyses showed that the density of L. major was significantly higher (n = 48, 53.93%) than L. tropica (n = 4, 4.49%) (Mann–Whitney U test: p

Cystic hydatid disease (CHD) is a zoonotic disease with different clinical stages caused by the larval stage of the cestode Echinococcus granulosus. It is important to highlight as a public health problem in various regions of the world. In the current study, the efficacy and apoptotic activity of the liposomal system containing juglone (5-hydroxy-1,4-naphthoquinone) were assessed against protoscoleces (PSCs) in vitro. To this aim, firstly, liposomal vesicles were prepared by the thin-film method. Their physico-chemical features were assessed using Zeta-Sizer and Scanning Electron Microscope (SEM). Subsequently, various concentrations (50, 100, 200, 400, and 800 μg/mL) of juglone nanoliposomes at different exposure times (15, 30, 60, and 120 min) were used against PSCs. Results showed that juglone nanoliposomes at all tested concentrations induced scolicidal effect, however, 800 μg/mL and 400 μg/mL of juglone nanoliposomes could reach 100% mortality in 60 and 120 min, respectively. Additionally, we found that caspase-3 mRNA expression was higher in PSCs treated with juglone nanoliposomes compared to control groups (p < 0.001). Therefore, juglone nanoliposomes are suggested to have a more potent apoptotic effect on PSCs. Generally, optimized doses of juglone nanoliposomes could display significant scolicidal effects. Moreover, further in vivo studies are required to evaluate the efficacy of this nanoliposome.

Aim: One of the main diagnostic problems of conventional polymerase chain reaction (PCR) is indiscrimination of low parasitic loads in soil samples. The aim of this study is to determine the genetic diversity and identification of Toxocara spp. from public areas soil inferred by loop-mediated isothermal amplification (LAMP) assay. Materials and Methods: A total of 180 soil samples were collected from various streets and public parks of northwest Iran. The DNA of recovered Toxocara eggs were extracted and amplified by PCR and LAMP following ZnSO4 flotation technique. The amplicons of internal transcribed spacer-2 gene were sequenced to reveal the heterogeneity traits of Toxocara spp. In addition, Toxocara canis sequences of southwest Iran were directly retrieved to compare gene flow between two distinct populations. Results: Toxocara spp. eggs were found in 57, 14 and 77 of soil samples using the microscopy, PCR and LAMP (detection limit 1-3 eggs/200 g soil), respectively. 7.7% of isolates were identified as T. canis by PCR method, while LAMP was able to detect 27.2%, 15.5% and 12.2% as Toxocara cati, T. canis and mixed infections, respectively. The kappa coefficient between LAMP and microscopy indicated a strong agreement (0.765) but indicated a faint agreement among LAMP-PCR (0.203) and PCR-microscopy (0.308) methods. A pairwise fixation index (Fst) as a degree of gene flow was generally low (0.02156) among Toxocara populations of northwest and southwest Iran. Conclusions: The statistically significant Fst value indicates that the T. canis populations are not genetically well differentiated between northwest and southwest Iran. This shows that here is possibly an epidemiological drift due to the transfer of alleles. The LAMP assay because of its shorter reaction time, more sensitivity, and simultaneous detection of environmental contamination to be appears as valuable field diagnosis compared to PCR. Therefore, the detection of low Toxocara spp. loads from public area soils will help to expand epidemiological understanding of toxocariasis and establishing preventive strategies in resource-limited endemic of Iran.

Background: Cutaneous leishmaniasis (CL) as a public health concern is increasingly circulating by causative agents of Leishmania tropica and L. major in Iran. As regard to recent treatment failure and controlling problems, the accurate elucidation of heterogeneity traits and taxonomic status of Leishmania spp. should be broadly addressed by policymakers. This study was designed to determine the genetic variability and molecular characterization of L. major and L. tropica from Iranian CL patients. Methods: One hundred positive Giemsa-stained slides were taken from clinical isolates of CL at Pol-e-Dokhtar County, Southwest Iran, from May 2014 to Sep 2016. DNAs were directly extracted and amplified by targeting ribosomal internal transcribed spacer (ITS) gene following microscopic observation. To identify Leishmania spp. amplicons were digested by restriction enzyme HaeIII subsequent PCR-RFLP technique. To reconfirm, the isolates were directly sequenced to conduct diversity indices and phylogenetic analysis. Results: Based upon the RFLP patterns, 84 and 16 isolates were explicitly identified to L. tropica and L. major respectively. No co-infection was found in clinical isolates. The high genetic diversity of L. tropica (Haplotype diversity 0.9) was characterized compared to L. major isolates (Hd 0.476). The intra-species diversity for L. tropica and L. major isolates corresponded to 3%-3.9% and 0%-0.4%, respectively. Conclusion: Findings indicate the L. tropica isolates with remarkable heterogeneity than L. major are predominantly circulating at Pol-e-Dokhtar County. Occurrence of high genetic variability of L. tropica may be noticed in probable treatment failure and/or emerging of new haplotypes; however, more studies are warranted from various geographic regions of Southwest Iran, using large sample size.

Background: Fascioliasis is one of important zoonotic disease caused by Fasciola gigantica and F. hepatica. The final hosts of this parasite are ruminants and humans. Iran is one of the endemic areas in the world, about six million people at risk of infection. The aim of this study was to identify and determine the genetic diversity of Fasciola species in cattle after distinguish of their species. Methods: One hundred and seventeen liver specimens collected from naturally infected cattle in 5 geographical regions in 2014-2017. Flukes stained with Hematoxylin-Carmine dye to examine for the existence of sperm within seminal vesicles. DNA was extracted from each individual, and ITS1, ND1and CO1 genes were amplified using specific primers. For discrimination of Fasciola species, ITS1 PCR-RFLP was used based on digestion pattern of RsaI enzyme. Genetic analyses and diversity and neutrality indices estimated by Dnasp5 based on NDI. Results: Six nonspermic and 111 spermic flukes were diagnosed. All of nonspermic specimens were F. gigantica and collected from South East, South West and North West of Iran. Genetic haplotype diversity has been observed in F. gigantica based on ND1. Fst value analysis showed that minimum and maximum genetic difference between Iranian F. gigantica with Bangladesh (Fst = 0.01414) and Egypt (Fst = 0.36653) respectively. Conclusion: It is the first report of existing of nonspermic Fasciola. High haplotype and nucleotide diversity could be due to ecological factors in life cycle, animal migration and coexisting of the final host of this parasite. Haplotype and nucleotide diversity of spermic F. gigantica in Iran and other countries in the world led to creating a variety of haplogroups.

Background: Intestinal parasitic infections are major causative agents of wildlife health complications among different parts of the world. This study aimed to investigate the gastro-intestinal parasites in feces of the zoo animals based on parasitological and morphometric criteria. Methods: One hundred fresh fecal samples were collected from 35 species of animal lived in Eram park zoo, Tehran, Central Iran during Oct 2015 to Jun 2015. All collected samples were examined by microscopic observation following direct wet mount preparation (normal saline and Lugol's iodine), formalin-ether concentration, and permanent staining. The morphometric aspects of the recovered eggs were surveyed with the aid of Camera Lucida (×400). Results: 65.7% (23/35) of zoo animal species were infected with intestinal parasites. The superfamily Trichostrongyloidea (6/16) and Strongylus sp. (16/4) were the most prevalent helminthic infections, while Blastocystis sp. (6/14), Entamoeba cyst (3/14) and Eimeria sp. (3/14) were the common protozoan parasites. For the first time, Bivitellobilharzia nairi egg was identified an elephant at Iran. Intestinal parasitic infections were apparently circulating among animals of the Eram park zoo. Conclusion: Identified parasitic infections can consider as a threatening source to visitors and workers' health that have contact with animals or their feces. Therefore, the effectual preventive strategies should be addressed to determine the risk factors, mechanisms of cross-transmission of parasite, the importance of applying the hygienic practices and well adjusting deworming programs for the animals, zoo workers and visitors.

Aim: Fascioliasis is one of the most zoonotic diseases with global extension. As the epidemiological distribution of Fasciola may lead to various genetic patterns of the parasite, the aim of this study is to identify Fasciola hepatica based on spermatogenesis, and phylogenetic analysis using mitochondrial (nicotiamide adenine dinucleotide dehydrogenase subunit I [ND1] and cytochrome oxidase subunit I) gene marker. Materials and Methods: In this study, 90 F. hepatica collected from 30 cattle at slaughterhouse located in three different geographical locations in the North-East of Iran were evaluated based on spermatogenetic ability and internal transcribed spacer 1 gene restriction fragment length polymorphism pattern. Genetic diversity and phylogenetic relationship using mtDNA gene marker for the isolates from the North-East of Iran, and other countries were then analyzed. Results: Partial sequences of mtDNA showed eight haplotypes in both genes. The phylogenic analysis using neighbor joining as well as maximum likelihood methods showed similar topologies of trees. Pairwise fixation index between different F. hepatica populations calculated from the nucleotide data set of ND1 gene are statistically significant and show the genetic difference. Conclusion: F. hepatica found in this region of Iran has different genetic structures through the other Fasciola populations in the world.

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.

We treated a homeless man in Iran with a history of squamous cell carcinoma who had ophthalmomyiasis caused by Chrysomya bezziana parasites. This case highlights a much-neglected condition and describes measures to prevent it.

Background: We employed a highly sensitive loop-mediated isothermal amplification (LAMP) by targeting 18S rRNA gene to identify the rapid mass screening of Leishmania infections in captured sand flies of southwest Iran and In vitro culture. Methods: One hundred fifty sand flies were collected from 11 sites adjacent to Iraqi’s borders in southern parts of Khuzestan Province by using sticky sheets of paper and CDC miniature light traps during late May 2014 to Nov 2015. Following morphological identification of sand flies species, the DNA of infected samples was extracted and amplified by PCR and LAMP assays by targeting ITS-rDNA and 18S rRNA genes. The PCR amplicons were directly sequenced to conduct the phylogenetic analysis Results: Ten (6.6%) Leishmania infections were identified by LAMP assay (detection limit 0.01 parasites DNA) among infected Sergentomyia baghdadis, S. sintoni and Phlebotomus papatasi sand flies that was more sensitive than PCR (n=6.4%; (detection limit 101parasites DNA). LAMP can identify 101-106promastigotes/100 µl RPMI 1640 while PCR recognized104-106 promastigotes. The majority infection rate of sand flies was confirmed to L. major inferred by phylogenetic analysis. Conclusion: This is the first exploration characterized the Old World Leishmania infections by LAMP technique in both infected sand flies and In vitro conditions. The LAMP method because of its shorter reaction time, robustness, more sensitivity, lack of requirement of complicated equipment and visual discriminatory of positivity can be appeared a promising tool instead of PCR to identify low Leishmania loads and entomological monitoring of leishmaniasis in resource-limited endemic of the world.

Abstract Background Hydatidosis is considered to be a neglected cyclo-zoonotic disease in Middle East countries particularly northwestern Iran which is caused by metacestode of tapeworm Echinococcus granulosus sensu lato. Human hydatidosis is a high public health priority in the area, however there is little known from a morphometric and phylogenetic perspective on molecular epidemiology of adult Echinococcus spp. in Iranian stray dogs. Methods 80 dogs (38 males and 42 females) were collected during June 2013 to April 2014 in northwestern Iran. The isolated parasites from each dog were distinguished by morphometric keys including small, large hook length and blade length. Subsequently, isolates were confirmed by sequencing of mitochondrial cytochrome oxidase subunit 1 gene. Results 16 (8 males and 8 females) (Prevalence 20 %) out of 80 dogs were infected to genus Echinococcus. With regard to demographic factors, the frequency of parasitism in both male, female adults and their age groups showed no difference (P > 0.05). The phylogenetic analyses of cox1 sequences firmly revealed the 13 sheep strains (G1), one buffalo strain (G3), one camel strain (G6) and one mixed infection. The findings of rostellar hook morphology show an intraspecies variation range among G1 isolates. However, hook measurements in Echinococcus derived from G1 (sheep strain) were not a significant difference from those G6 and G3 strains. Six unique haplotypes were identified containing a high range of diversity (Haplotype diversity 0.873 vs. Nucleotide diversity 0.02). Conclusions First presence of camel strain (G6) in this region seems to indicate that potential intermediate hosts play a secondary role in the maintenance of camel-dog biology. Current findings have heightened our knowledge about determination of Echinococcus prevalence, strains of taxonomy and genotypic trait of parasite in Iranian stray dogs which will also help in the development of strategies for monitoring and control of infected stray dogs in the area.

Background: Leishmaniasis as an emerging and reemerging disease is increasing worldwide with high prevalence and new incidence in recent years. For epidemiological investigation and accurate identification of Leishmania species, three nuclear and mitochondrial genes (ITS-rDNA, Hsp70, and Cyt b) were employed and analyzed from clinical samples in three important Zoonotic Cutaneous Leishmaniasis (ZCL) foci of Iran. Methods: In this cross-sectional/descriptive study conducted in 2014-15, serous smears of lesions were directly prepared from suspected patients of ZCL in Turkmen in northeast, Abarkouh in center and Shush district in southwest of Iran. They were directly prepared from suspected patients and DNA was extracted. Two nuclear genes of ITS-rDNA, Hsp70 and one mitochondrial gene of Cyt b within Leishmania parasites were amplified. RFLP was performed on PCR-positive samples. PCR products were sequenced, aligned and edited with sequencher 4.1.4 and phylogenic analyses performed using MEGA 5.05 software. Results: Overall, 203 out of 360 clinical samples from suspected patients were Leishmania positive using routine laboratory methods and 231 samples were positive by molecular techniques. L. major L. tropica, and L. turanica were firmly identified by employing different molecular genes and phylogenic analyses. Conclusion: By combining different molecular genes, Leishmania parasites were identified accurately. The sensitivity and specificity three genes were evaluated and had more advantages to compare routine laboratory methods. ITS-rDNA gene is more appropriate for firm identification of Leishmania species.

Background: Acanthamoeba is an opportunistic amphizoic protozoan found in different fresh water sources. The aim of this study was to identify and characterize Acanthamoeba isolates from surface resting waters, in Northwest Iran. Methods: Samples were collected from twenty-two different areas, between May and Sep 2014. After filtration, samples were cultivated on non-nutrient agar. The extracted DNAs were amplified and sequenced using partial 18S rRNA in order to genotype and phylogenetic analyses. Results: Thirty-four (68%) out of 50 collected samples were positive for free-living amoebae based on both culture and morphological characterizations but 28 samples were identified as Acanthamoeba spp. by PCR. Sequentially, one isolate was identified as A. lenticulata, (T5) (AN: KP940443, identity 99.7%-100%, and divergence 0.3%) whilst other sequenced isolates identified Acanthamoeba spp. (AN: KP940444-45) as very similar to A. rhysodes and A. royreba with identity 100% and divergence 0%. Conclusion: Surface resting waters in Northwest Iran, were potentially contaminated with pathogenic amphizoic protozoan. Further studies will be required to determine other Acanthamoeba species and genotypes in the region.

Background: Visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) are important public health problems in Iran. We aimed to evaluate the diagnostic potential of Western blot (WB) compared with indirect immunofluorescence test (IFAT) to serodiagnosis of leishmaniasis. Methods: This study was performed from 2010-2014 and participants were different parts of Iran. Serum samples were obtained from 43 patients with proven CL, 33 patients with proven VL, 39 patients with other parasitic diseases and 23 healthy individuals. Results: WB sensitivity for CL and VL was 100% and 91%, compared to IFA 4.6% and 87.8%, respectively. Sera from patients with CL and VL recognized numerous antigens with molecular weights ranging from 14 to 68 kDa and 12 to 94 kDa, respectively. The most sensitive antigens were 14 and 16 kDa for CL recognized by 100% of the sera from patients with proven CL and 12, 14 and 16 kDa for VL, recognized by 63.6%, 100% and 63.6% of the sera from patients with proven VL respectively. WB analysis is more sensitive than IFAT for the diagnosis of leishmaniasis particularly in cases of cutaneous leishmaniasis. The 12, 14 and 16 kDa can be valuable diagnostic molecules for serodiagnosis of leishmaniasis because at least two immunogenic molecules were simultaneously detected by all patient sera, as well as produced antibodies against these antigens have no cross-reactivity with other control groups. Conclusion: WB could be useful for screening and serodiagnosis of CL and VL in epidemiologic studies in endemic areas.

Introduction Leishmania major is the causative agent of zoonotic cutaneous leishmaniasis (ZCL), and great gerbils are the main reservoir hosts in Iran. Abarkouh in central Iran is an emerging focal point for which the reservoir hosts of ZCL are unclear. This research project was designed to detect any Leishmania parasites in different wild rodent species. Methods All rodents captured in 2011 and 2012 from Abarkouh district were identified based on morphological characteristics and by amplification of the rodent cytochrome b (Cyt b) gene. To detect Leishmania infection in rodents, deoxyribonucleic acid (DNA) of each ear was extracted. Internal transcribed spacer-ribosomal deoxyribonucleic acid (ITS-rDNA), microsatellites, kinetoplast deoxyribonucleic acid (kDNA) and cytochrome b genes of Leishmania parasites were amplified by polymerase chain reaction (PCR). Restriction fragment length polymorphism (RFLP) and sequencing were employed to confirm the Leishmania identification. Results Of 68 captured rodents in the region, 55 Rhombomys opimus were identified and nine Leishmania infections (9/55) were found. In addition, eight Meriones libycus and two Tatera indica were sampled, and one of each was confirmed to be infected. Two Meriones persicus and one Mus musculus were sampled with no infection. Conclusions The results showed that all 11 unambiguously positive Leishmania infections were Leishmania major. Only one haplotype of L. major (GenBank access No. EF413075) was found and at least three rodents R. opimus, M. libycus and T. indica—appear to be the main and potential reservoir hosts in this ZCL focus. The reservoir hosts are variable and versatile in small ZCL focal locations.

Summary: Background: One of the well-known foci of zoonotic cutaneous leishmaniasis (ZCL) in Iran is Turkemen Sahara, which is located in north eastern Iran. ZCL is a disease of mammals, and humans can become infected as accidental hosts. Many researchers have argued that Rhombomys opimus is the only main reservoir host of ZCL in this region of the Golestan province. No other rodents or mammals are thought to host or have been reported to host Leishmania parasites in this region. This research was designed and developed to isolate, detect and firmly identify Leishmania parasites in mammals and rodents other than R. opimus. Methods: Wild mammals were caught from gerbil burrows. Leishmania parasites were detected to assess the infection of reservoir hosts in 2010. Each genomic DNA sample was screened for Leishmania infection via nested PCR and sequencing using the internal transcribed spacer ribosomal DNA (ITS-rDNA) identification protocol for parasites. Results: The greatest number of infections (8/19) were found in Meriones libycus. One in three infections was found in Hemiechinus auritus, and this is the first report of infection in this species. Only Leishmania major was definitively identified and unambiguously typed in M. libycus and H. auritus. The infection rates in these two wild mammals were not significantly different, and no other gerbil parasites were detected in M. libycus or H. auritus at our study site. Conclusions: Recent findings of Leishmania turanica in R. opimus and failures to detect L. turanica in M. libycus may be attributable to unidentified Leishmania infections in two M. libycus due to unreadable sequences. These cases may represent mixed infections by L. major and L. turanica. The assumptions that gerbil parasites can be co-infectors provide a starting point for the identification of the causative and potential parasites responsible for the frequent infections that are mainly mediated via sandfly vectors. Keywords: Nested PCR, Hemiechinus auritus, ITS-rDNA, Leishmania major, Meriones libycus, Iran

Background & objectives: In the well-known zoonotic cutaneous leishmaniasis (ZCL) focus in Turkmen Sahara, border of Iran and Turkmenistan, ZCL has increased among humans in the past five years. The present study was undertaken to incriminate vectors of ZCL in the region, and to find molecular variation in Leishmania parasites. Methods: The sandflies were sampled using CDC light-traps and sticky papers. All the sandflies were identified using morphological characters of the head and abdominal terminalia. DNA was extracted from the dissected thorax and attached anterior abdomen of individual female sandfly. Leishmania detection and identification of sandflies were performed using PCR, digestion of BsuRI restriction enzyme and sequencing of ITS-rDNA gene and also by semi-nested PCR to amplify minicircle kinetoplast (k) DNA of Leishmania. Results: Leishmania infections were detected in 26 out of 206 female sandflies. Of the infected sandflies, 18 were Phlebotomus papatasi while eight were P. caucasicus/P. mongolensis. Two infections of L. turnica were detected, one in P. papatasi and other in P. caucasicus/P. mongolensis and the rest of the sandflies were found infected with L. major. Conclusion: Our finding showed that L. major had low diversity with only one common haplotype (GenBank Access No. EF413075). The novel haplotypes were discovered in L. major (GenBank Access No. KF152937) and in L. turanica (GenBank Access No. EF413079) in low frequency. These Leishmania parasites are circulating to maintain infections in the P. papatasi and P. caucasicus/P. mongolensis in Turkmen Sahara.

Background: The aim of this study was to identify the Trichomonas vaginalis strains/haplotypes based on identifying their probable variations in asymptomatic patients referred to Tabriz health centers, northwestern Iran. Methods: Sampling was taken from 50-suspected women to T. vaginalis in northwestern Iran. The obtained samples were smeared and cultured. Fifty DNA samples were extracted, amplified and identified by nested polymerase chain reaction and PCR-RFLP of actin gene using two endonuclease enzymes: MseI and RsaI. To reconfirm, the amplicons of actin gene were directly sequenced in order to identify the strains/haplotypes. Results: PCR-RFLP patterns, sequencing and phylogenetic analyses revealed definitely the presence of the G (n=22; 73.4%) and E (n=8; 26.6%) strains. Multiple alignments findings of genotype G showed five haplotypes and two amino acid substitutions in codons 192 and 211 although, no remarkable unique haplotype was found in genotype E. Conclusion: The accurate identification of T. vaginalis strains based on discrimination of their unknown haplotypes particularly those which are impacted on protein translation should be considered in parasite status, drug resistance, mixed infection with HIV and monitoring of asymptomatic trichomoniasis in the region.

Background & objectives: Hydaticosis is a zoonotic helminthic disease of human and other intermediated hosts in which larval stages of the tapeworm Echinococcus granulosu transfect human. The liver and lung are the host tissues for the hydatid cyst . It is unknown which mechanisms are involved in infertility of the cyst and suppression of the fertile cyst. This study was aimed to evaluate the expression of the apoptosis inducing-ligands such as TRAIL and Fas-L in germinal layer of the cyst and human normal tissue surrounding the cyst that is one of the unknown host innate immunity mechanisms against the hydatid cyst. Methods: In this study, four isolated hydatid cysts were used which had been diagnosed in patients by radiography and parasitological examination in Mashhad Ghaem hospital. Furthermore, the germinal layer of the cyst and accompanied normal peripheral tissues were separated by scalpel in sterile conditions. After homogenization, expression of TRAIL and Fas-L genes were studied by semi-quantitive RT-PCR method. Results: The TRAIL and Fas-L showed significant higher level expression in germinal layer of infertile cyst than the fertile cyst and host normal tissues. Conclusion: The host tissue-induced apoptosis of germinal layer of the fertile cysts is probably one of the infertility mechanism in patients with hydaticosis

Background:Haemonchosis has a negative effect on the farming industry throughout the world, especially in the tropic and sub-tropic countries. The present study was carried out to differentiate Haemonchus species from its main hosts in Iran, including sheep, goat and camel. Methods:The identification took place based on the morphometrics of the spic-ules and molecular characters. Two hundred seventy adult male nematodes were collected from the abomasums of different ruminants (90 samples from each ani-mal) at the slaughterhouses from different localities in Iran. Samples were morpho-logically identified according to the spicules’ morphometric measurements. In the section on molecular study, 10 samples of each Haemonchus isolates were genetically examined. A simple PCR-restriction fragment length polymorphism (PCR-RFLP) assay of the second internal transcribed spacer of ribosomal DNA (ITS2-rDNA) were described to confirm the PCR results. Results:PCR-RFLP profile obtained from the restriction enzyme HPa1 in H. con-tortus and H. longistipes indicated 1 (278 bp) and 2 (113 and 135 bp) different frag-ments, respectively. The morphological parameters clearly distinguish H. contortus from H. longistipes. Moreover, regarding the ITS2-rDNA, sequences of 295 bp and 314 bp were obtained from H. contortus and H. longistipes, respectively. Conclusion:High similarity of cloned PvMSP-142 kDa gene in comparison to reference sequence and other sequences could be beneficial as a remarkable mo-lecular marker for serological diagnostic.

ABSTRACT Background Leishmania major is a causative agent of zoonotic cutaneous leishmaniasis in the center of Iran, Abarkouh district. Molecular characterization and precise incrimination of Leishmania species was carried out to perform controlling measurements and to design treatment programs for zoonotic cutaneous leishmaniasis. Methods All smears isolated from ulcers of suspected patients were examined under a light microscope and graded for amastigotes frequency. Extraction of DNA, PCR, RFLP and sequencing of ITS-rDNA genotype were done to increase the efficacy of Leishmania parasites identification at their species-specific level and to detect any Leishmania infections within. Results Humans were found to be infected with L. major with high infection frequency and also Leishmania tropica was identified with low occurrence for the first time as non-native species using molecular analyses. The rates of infections was considerable with microscopic observation (n= 65, 73%) out of 89 smears prepared from suspected patients. Molecular analyses showed that the density of L. major was significantly higher (n= 48, 53.93%) than L. tropica (n= 4, 4.49%) (Mann-Whitney U test: p< 0.05) and two samples (2.25%) remained ambiguous after several sequencing. L. major did not have diversity with two common haplotypes but L. tropica were found to exhibit high diversity with three novel haplotypes. Conclusion L. major was considered the causative agent of leishmaniasis in the region, but the identification of a non-native L. tropica revealed the importance of further isolation of Leishmania parasites following molecular analyses and confirmation, and also revealed the importance of further isolation of Leishmania parasites from patients of the field areas who do not have easily access to health care centers for specialized treatment strategies.