The Role of Some Free-Ranging Animals in the Transmission of Multi-Host Species of Cryptosporidium Spp.

Background: We aimed to characterize Cryptosporidium spp. in rats, cats, pigeons, and crows. Methods: Fifty-five animal origin Cryptosporidium spp. genome were identified, genotyped and confirmed by nested PCR and of RFLP-PCR analysis as well as sequenced based on 18s rRNA and gp60 genes in Tehran (2012-2019). Finally, the phylogenetic analysis was performed by MEGA software (version 7). Results: By the molecular method, Cryptosporidium spp. were detected in 24 (15.2%), 15 (15%), 2 (2%) and 13 (13%) cases of wild rats, cat, pigeon, and crow, respectively. Among the identified species by the RFLP pattern, most isolates were identified as C. parvum (24/157) 17.8% in rats, (15/100) 15% in cats, (13/100) 13%in crew and (2/100) 2% in pigeons; and the rest of the cases were C. muris and C. felis. The results of sequencing did not prove the existence of C. parvum, C. felis, C. muris, and rat genotype. Subtyping of C. parvum was indicated that the dominant subtype family belongs to the IId family and the subtype A20G1 was the most common subtype detected in all hosts while A19G1 was detected in one isolate of cat and pigeon. Conclusion: Free-ranging animals are infected by species/subtype of Cryptosporidium, which can infect humans. This shows by itself the hygienic importance of the free-ranging animals in urban ecosystems. In the transmission of human cryptosporidiosis, the multi-host Cryptosporidium species such as C. parvum, C. felis, and C. muris can be transferred potentially from these animals to humans.

Molecular characterization and genotyping of Toxoplasma gondii in free-living animals in Iran: Effect of One Health.

To understand the transmission of Toxoplasma gondii, this parasite's genetic diversity distribution in free-living hosts is essential. This research's objective is the molecular genotyping of T. gondii isolates from the brain and muscles of Columbidae, Corvidae, Rattus, and Felidae of Mianeh County, East-Azerbaijan Province, Northwest Iran. Three hundred fifty samples were taken. For the genotyping of T. gondii, the GRA6 gene was amplified and digested by the Tru1I (MseI) enzyme. Results of RFLP were confirmed by sequencing and phylogenetic analysis. In total, 52%, 34%, 24%, and 50% of Columbidae, Corvidae, Rattus, and Felidae were positive for T. gondii DNA, respectively. All isolated Columbidae were identified as genotype III (100%). Also, 94.1% and 5.9% of Corvidae isolates, 84.4% and 15.6% of the Rattus isolates, and 51.7% and 48.3% of the Felidae isolates belonged to genotypes III and II, respectively. This study is the first to evaluate genetic similarity and phylogenetic analysis between many definitive and intermediated hosts in northwestern Iran. The finding indicates that the T. gondii cycle is maintained among these hosts. As a result, their presence in the environment can be a risk factor for transmitting the infection to humans. Due to demographic and geographic differences in various regions, further studies are required to determine the genetic population structure.

In vitro anti-gastrointestinal cancer activity of Toxocara canis -derived peptide: Analyzing the expression level of factors related to cell proliferation and tumor growth.

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.

Exosomes secreted by Blastocystis subtypes affect the expression of proinflammatory and anti-inflammatory cytokines (TNFα, IL-6, IL-10, IL-4).

Background: Blastocystis 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. Methods: The 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. Results: Exosomes 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). Conclusion: To 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.

High occurrence of Blastocystis sp. subtype 3 in individuals referred to medical laboratories in Kermanshah, Iran.

Aim: The current study investigated the prevalence and genotypes of Blastocystis sp. in individuals who referred to medical laboratories in Kermanshah, Iran. Background: Blastocystis sp. is a common intestinal protozoan found in humans and a wide range of animals, and it is involved in the development of gastrointestinal disorders. Methods: A total of 950 stool samples were examined using the standard formalin-ether concentration technique. All specimens were cultured in Robinson xenic medium. Subsequently, DNA extraction and PCR amplification of subtype specific sequence-tagged site (STS) were conducted. Results: Microscopic examination showed that 86 out of 950 samples (9.05%) were infected with Blastocystis sp. Subsequently, 33 of 86 positive samples were cultured and molecularly confirmed by conventional PCR, indicating six subtypes (ST1-ST6). Of note, ST3 (45.0%) was the predominant subtype, followed by ST1 (15.15%) and ST5 (12%). Conclusion: Based on the current findings, ST3 was the most frequent subtype among all positive samples. Having a better understanding of Blastocystis sp. subtype distribution and risk factors would lead to improved preventive measures.

A Novel Chimeric Antigen as a Vaccine Candidate against Leishmania major: In silico Analysis.

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.

Effect of Imiquimod on Tachyzoites of Toxoplasma gondii and Infected Macrophages in vitro and in BALB/c Mice.

Treatment for toxoplasmosis is not completely successful because of their unwanted side effects, and new treatments are needed. Imiquimod has ability to moderate immune response and used to treat a wide variety of infections and tumors. The aim of the present study was to evaluate the effect of imiquimod on the tachyzoites of T. gondii and infected macrophages in vitro and in BALB/c mice. The viability of T. gondii was assessed in the presence of various concentrations of imiquimod by direct counting after 6 and 24 h. The MTT assay was used to identify the viability of uninfected macrophages. The apoptotic effects were determined with flow cytometry on the tachyzoites and infected macrophages. For evaluation of parasite load in pre-treatment or post-treatment of macrophages Quantitative real time PCR (qPCR) was performed. For in vivo experiments, BALB/c mice received imiquimod before and after challenge with parasites. The mortality rate of mice, parasite numbers in spleen, and the INF-γ and IL-4 cytokine levels in spleen lymphocytes were evaluated. Imiquimod demonstrated anti-Toxoplasma effects by reducing the number of tachyzoites. The results of flow cytometry for drug-treated tachyzoites showed that apoptosis did not rise significantly relative to the control group (p < 0.05). Moreover, apoptosis was enhanced in infected macrophages as the concentration of imiquimod was reduced. The parasitic burden in imiquimod pretreated macrophages was significantly lower than those treated after infection (p < 0.01). A marked reduction was observed in survival rate, parasite load and INF-γ level in BALB/c mice that received imiquimod before parasitic challenge relative to those received drug after parasitic challenge (p < 0.01). Overall, imiquimod in the pretreated group had greater anti-Toxoplasma effects than imiquimod in posttreated group in vitro and in vivo. imiquimod may be considered as a candidate for use against Toxoplasmosis both therapeutically and prophylactically.

An unlabelled probe-based real time PCR and modified semi-nested PCR as molecular tools for analysis of chloroquine resistant Plasmodium vivax isolates from Afghanistan.

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.

Evaluation of Asymptomatic Malaria Status in Eastern of Afghanistan Using High Resolution Melting Analysis.

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.

Toxoplasma gondii: Preventive and therapeutic effects of morphine and evaluation of treatment parameters of tachyzoites and infected macrophages in vitro and in a murine model.

Common medicines for the treatment of toxoplasmosis have limited efficacy and unwanted side effects. Opiates can effect both innate and cell-mediated immunity and stimulate the immune responses in different parasitic infections. In this work, preventive and therapeutic effects of morphine were evaluated on the tachyzoites of Toxoplasma gondii and infected macrophages in vitro and in a murine model. Different concentrations of morphine (0.1 and 0.01 µg/ml) were evaluated on mortality rate of T. gondii by direct counting after 3 and 24 hours. The cytotoxic and apoptotic effects of these drugs were measured by the MTT assays and flow cytometry analysis, respectively. The same procedures were assessed in T. gondii-infected macrophages. The parasite loads were determined using quantitative polymerase chain reaction (qPCR). For in vivo assessment, BALB/c mice treated with morphine before or after infection with tachyzoites. The survival rate of animals, parasite load in the spleen, and the IFN-γ and IL-4 cytokines levels were measured. Morphine was effective on tachyzoites of T. gondii and had a reverse relationship with its concentration. The results of flow cytometry showed that the toxic effects of morphine on tachyzoites after 3 hours was not statistically significant (p<0.05). Also, apoptosis in infected MQs rose with a decreasing concentration of morphine. The parasitic load in MQs treated with morphine before infection was lower than that in cells treated after infection and the differences were statistically significant (p<0.01). In mice that received morphine before infection, survival rate, parasite load and the IFN-γ level were significantly lower than in mice treated after infection (p<0.01). The results of this study have shown that morphine in the pre-treatment group had higher anti-Toxoplasma activity than morphine in post-treatment in vitro and in murine model.

In vitro toxicity evaluation of short cationic antimicrobial peptide (CM11) on Blastocystis sp.

Blastocystis infection accounts for one of the causes of gastrointestinal problems with the prevalence rate of 3-100% worldwide. There is a wide range of drugs examined for the treatment of infected patients, among them metronidazole (MTZ) has been introduced as one of the efficient drugs. Besides to the suitable clinical effects, the administration of MTZ has some reported side-effects which emphasize on the identification of putative alternates. To this end, we aimed to evaluate the cytotoxicity effect of a newly-introduced synthetic antimicrobial peptide (AMP) named CM11 on in vitro cultured Blastocystis. Our results exhibited that CM11 treatment affected the viability of parasites in two cultural conditions including culturing alone and in co-culture with the Caco-2 cell line. The time- and dose-dependent effect of CM11 was consistent with the effect of MTZ which was used as control positive. The highest toxicity effect of CM11 was observed at the concentration of 24 µg/ml, leading to 28.7% and 25% viable parasites after 24 h and 48 h incubation times, respectively. Interestingly, the disruption of the Blastocystis cell membrane could be observed in the treated parasites. Therefore, CM11 can be suggested as a potential treatment for Blastocystis-infected patients after further in vitro and in vivo assessments.

New prevalence surveillance of Toxoplasma gondii among rodents and stray cats by ELISA avidity and nested PCR methods, Northeast of Iran.

Rodents and stray cats are the sources of many parasitic infections including T. gondii, for other animals and human. Toxoplasmosis has a wide range of laboratory factors in its intermediate and definite hosts. Regarding the importance of rodents and stray cats as the hosts that spread the Toxoplasma gondii, it is necessary to obtain comprehensive information about these animals in the life cycle of T. gondii. The objective was to investigate the new prevalence of toxoplasmosis among target animals in Iran, using GRA6 gene in combinacion with ELISA avidity. In this study, 286 rodents and 210 stray cats were collected and their heart tissues extracted to obtain DNA, blood samples and IgG Ab of T.gondii parasite. We detected the positive tissue samples in our study by the nested-PCR method. Then, we examined T. gondii IgG ELISA avidity for assessment of toxoplasmosis among rodents and stray cats. This study, was conducted in January to March 2017, based on the prevalence study. The findings revealed that 246/286 (86.01%) of rodents and 180/210 (85.71%) of stray cats were positive by IgG ELISA avidity methods. moreover, 68 rodents samples and 38 stray cats samples were positive concerning the GRA6 Toxoplasma gene; and these positive samples were at intermediate levels for IgG avidity. We concluded that the new prevalence of toxoplasmosis among rodents and stray cats was at high levels, using the serologic method in Northeast of Iran and the results of quantitative ELISA avidity were as the same as those of the nested-PCR for detecting recent toxoplasmosis in these hosts.

First molecular identification and subtype distribution of Blastocystis sp. isolated from hooded crows (Corvus cornix) and pigeons (Columba livia) in Tehran Province, Iran.

Blastocystis is a common intestinal parasite among humans and animals such as non-human primates, pigs, cattle, birds, amphibians, and less frequently, rats, reptiles and insects. Since Blastocystis is a widely transmissible parasite between humans and mammals or birds, it is prominent to determine whether newly secluded non-human isolates are zoonotic. There are no comprehensive studies in Iran assessing the prevalence and molecular identification of Blastocystis infection in birds, especially in pigeons and crows. So, the aim of this study was to identify Blastocystis subtypes (STs) in crows and pigeons in Tehran province, Iran, using Nested PCR-RFLP and sequencing. Overall, 300 Blastocystis isolates from birds (156 pigeons and 144 crows) were subtyped by PCR, and the homology among isolates was then confirmed by RFLP analysis of the 18S rRNA gene. The prevalence of Blastocystis infection was detected 42.9% in pigeons and 44.4% in crows. All positive pigeons were owned by ST13 (100%). Among crows, 46 samples (71.8%) like pigeons were ST13, and 13 samples (20.3%) were ST14. Five samples (7.9%) remained unknown. This study was the first report of ST13 and ST14 of Blastocystis from birds. In the present study, our data revealed a high prevalence of Blastocystis sp. in pigeon's and crow's samples and the isolates from these birds were classified into two genetically distinct STs. Therefore, birds appear to be infected with various STs. It is important to determine the phylogenetic relationships between unknown STs from these birds and the multiple STs of Blastocystis.

In-vitro and In-vivo Antileishmanial Activity of a Compound Derived of Platinum, Oxaliplatin, against Leishmania Major.

This study aimed to evaluate the antileishmanial efficacy of oxaliplatin against Leishmania major (L. major) both in-vitro and in-vivo. The IC50, CC50, and SI of oxaliplatin against promastigotes, murine macrophages, Raw 264.7 cells, and intramacrophage amastigotes of L. major were investigated in-vitro. The effects of this drug on intracellular amastigotes were also assayed, and the percentage of infectivity and IIR were calculated. Flow cytometry was performed to assay apoptosis, using 50 and 100 µg/mL of oxaliplatin in the promastigotes and macrophages. In-vivo, the BALB/c mice were classified into three groups, receiving oxaliplatin, glucantime, and phosphate-buffered saline for one month, respectively. The lesion size, IFN-γ, and IL-4 levels, and parasite burden were also evaluated in the animals. After 72 h, the IC50 and CC50 of oxaliplatin against promastigotes and macrophages were respectively 0.5 and 66.78 µg/mL. The apoptosis of promastigotes and macrophages using 50 µg/mL of oxaliplatin was 7.25% and 2.14%, respectively, while apoptosis induced at 100 µg/mL was 15.48% and 2.80%, respectively. Similar to the glucantime group, the mice treated with oxaliplatin showed a lower parasite burden and smaller lesions, compared with the PBS group (p < 0.01). Furthermore, higher IFN-γ levels were reported in mice receiving oxaliplatin in comparison with those receiving PBS (p < 0.01). The current findings indicated the efficacy of oxaliplatin against promastigote and amastigote forms of Leishmania and L. major-induced leishmaniasis.

Isolation of Encephalitozoon intestinalis from crows living in urban parks of Tehran, Iran: an investigation with zoonotic aspect.

Microsporidia are eukaryotic, intracellular obligate parasites that widely involve many organisms including insects, fish, birds, and mammals. One of the genera of Microsporidia is Encephalitozoon, which contains several opportunistic pathogens. Since Encephalitozoon spp. are zoonotic and opportunistic pathogens, it is important to find their reservoir hosts; hence, the current study aimed at isolating and identifying Encephalitozoon spp. in the crows by the light microscopy observations and molecular methods. For this purpose, 36 samples were collected by the dropping method; however, due to the low volume of samples, the total samples were collected in a sterile stool container and the polymerase chain reaction (PCR) was performed to detect Encephalitozoon spp. Accordingly, 300-bp bands, specific to Encephalitozoon spp., were observed and by sequencing E. intestinalis was identified. Crows can be considered as the hosts of E. intestinalis.

Molecular assessment of Neospora caninum and Toxoplasma gondii in hooded crows (Corvus cornix) in Tehran, Iran.

Neospora caninum and Toxoplasma gondii are two closely related protozoan parasites that have been detected from various species of bird hosts. However, little is known about the prevalence of N. caninum and T. gondii in crows. Hence, we examined the molecular frequency of N. caninum and T. gondii in the brain samples of hooded crows (Corvus cornix) that collected from different public parks of Tehran, Iran by nested-PCR method. We used the primers targeting the Nc5 and GRA6 genes for detection of N. caninum and T. gondii, respectively. From a total of 55 brain samples, 5 (9.9%) and 9 (16.36%) samples were positive for N. caninum and T. gondii, respectively. Sequencing of a N. caninum isolate revealed 95%-100% identity with the deposited N. caninum in GenBank. Genotyping of T. gondii isolates by PCR-RFLP analysis of the GRA6 gene revealed type III genotype in 8 isolates. The results of this study indicate that hooded crows may have a putative role in transmission of N. caninum and T. gondii to canines and felines definitive hosts, respectively.

A modified PCR-RFLP method to determine genetic diversity of Giardia lamblia human isolates based on triosephosphate isomerase (TPI) gene.

An infection of digestive system, Giardiasis, caused by tiny parasites called Giardia lamblia (also known Giardia intestinalis or Giardia duodenalis). Giardia sp. is the most common intestinal parasite of humans and other animals throughout the world. Isolates of G. lamblia are classified into eight assemblages based on isoenzyme and DNA analyses. Assemblages A and B infect humans and a broad range of other hosts. The purpose of this study was to genotype human isolates of G. lamblia by PCR-RFLP in Karaj city. 60 positive fecal samples of G. lamblia were collected. DNA extraction and amplification of TPI gene were successfully conducted by nested-PCR. Subsequently, all samples were positive. Sequencing on 5 samples was conducted to determine genetic differences. The presence of 2 genotypes of G. lamblia (A and B) was revealed by the alignment of the TPI sequences obtained with reference sequences. The results of RFLP technique show that 35 of 60 (58.3%) isolates belonged to assemblage A, and 17 of 60 (28.3%) belonged to assemblage B but 1(1.7%) sample was not determined. Whereas, 7 (11.6%) specimens were detected as mixed infections. The latter RFLP was carried out to identify subtypes.The final results were 100% (35/35) AII, 82.3% (14/17) BIII, and 17.7% (3/17) BIV. This study suggests that the modified RFLP method is favorably time saving and easily achievable and highly economical. Hence, the sub-assemblage AII might be dominant in Karaj city.

Detection of Cryptosporidium spp. in free ranging animals of Tehran, Iran.

Cryptosporidium is a world widely distributed parasite which comparatively has a high prevalence in developing countries. The zoonotic potential of some Cryptosporidium species has made the cryptosporidiosis a significant concern to physicians and veterinarians. The occurrence and zoonotic potential of Cryptosporidium species in probable reservoir hosts for man infections was determined by examining faeces of symptomatic and asymptomatic animals. The aim of this study is to screen the presence of Cryptosporidium in fecal sample of free ranging animals in Tehran using Ziehl-Neelsen staining method. The findings indicate that Cryptosporidium are present in 9/50 (18 %) stray cat (Felis catus), 12/50 (24 %) hooded crows (Corvus cornix), 23/180 (12.7 %) rat (Rattus norvegicus and R. rattus) and 1/40 (2.5 %) pigeons (Columba livia). This investigation confirms the potential role of rats, cats, crows and pigeons for zoonotic transmission of human cryptosporidiosis and they must be considered as reservoir hosts which can endanger public health.

Trichomoniasis in older individuals: a preliminary report from Iran.

Infection with Trichomonas vaginalis is one of the most common sexually transmitted diseases (STDs) in humans. The prevalence of infection in Iran has been reported 0.009-8 % depending on deferent socio-cultural conditions. This study aimed to determine the frequency of T. vaginalis according to age in patients referred to clinics, hospitals and medical diagnostic laboratories in Karaj city, Iran. For this purpose, fifty positive samples were collected from July 2012 to June 2014 from clinics, medical diagnostic laboratories and hospitals, then transferred to laboratory of parasitology and cultured in TYM medium. The results showed that all isolates were successfully cultured. Among 50 positive specimens, 43 cases were female and 7 cases male. The most positive cases (34 %) belonged to the ages over 50 year's group. The lowest positive cases (2 %) belonged to the ages of less than 20 years group. In Conclusion, unlike other STDs, which have a higher prevalence among adolescents and young adults, the rates of trichomoniasis are more evenly distributed among sexually active women of all age groups. However, frequency of this infection in women aged over 50 years (age of menopause) is notable and complementary studies are needed.

A Pictorial Key for Culex pipiens Complex (Diptera: Culicidae) In Iran.

BACKGROUND: The aim of this study was to design pictorial key and taxonomic literature of Culex pipiens complex in Iran. METHODS: Larvae were collected using standard dipping methods in 13 randomly selected areas of Bushehr, Hamedan, Kerman, Khorasan-e-Razavi, Khuzistan, Mazandaran, Tehran, Sistan and Baluchistan and Yazd Provinces from April 2009 to October 2010. The data were analyzed using SPSS Ver. 11.5. RESULTS: Culex pipiens larvae were identified based on the Seta 1 of the abdominal segments III-IV in north and central parts of Iran. This diagnostic character had some variation among the Cx. quinquefasciatus collected from south of the country. The identification value of intersection of costa, subcosta and bifurcation of R2+3 of female veins, was calculated as 90-100 % for Cx. pipiens. This diagnostic character was varied among the Cx. quinquefasciatus specimens. The male genitalia found as the main characters to distinguish of Cx. quinquefasciatus from Cx. pipiens. CONCLUSION: It is necessary more studies on the behavior and genetic variations of Cx. pipiens complex in Iran.