Prevalence and associated risk factors of human intestinal parasitic infections: a population-based study in the southeast of Kerman province, southeastern Iran.

BACKGROUND: Determination of the prevalence and distribution pattern of intestinal parasites is a fundamental step to set up an effective control program to improve the health status. This study aimed to determine the prevalence of intestinal parasitic infections and associated risk factors among inhabitants of Rudbar-e Jonub county, southeast of Kerman province, southeastern Iran. METHODS: In this cross-sectional study, 861 stool specimens were collected from inhabitants of Rudbar-e Jonub county through a multistage cluster sampling method in 2018. The collected specimens were examined by parasitological methods including, direct wet-mounting (for the fresh specimens with a watery consistency), formalin-ethyl acetate sedimentation and agar plate culture. RESULTS: The prevalence of intestinal parasites was 34.2% (95% CI 30.1 to 38.2). The prevalence of protozoan parasites 32.3% (95% CI 28.4 to 36.5) was significantly higher than helminthic parasites 3.2% (95% CI 2.1 to 4.7). Blastocystis sp. (13.3%), Entamoeba coli (11.4%) and Giardia lamblia (10.6%) as protozoan parasite and Hymenolepis nana (2.4%) as helminthic parasite were the most common detected intestinal parasites in the study. Entamoeba histolytica/dispar (1.5%), Iodamoeba bütschlii (1.0%), Chilomastix mesnili (0.5%), Entamoeba hartmanni (0.4%), Enterobius vermicularis (0.3%) and Ascaris lambercoides (0.3%) were other detected parasites. Multiple logistic regression revealed a significant association of intestinal parasitic infections with source of drinking water and residency status (rural/urban). Multiple infections with 2 or 3 parasitic agents constituted 22.7% of 295 infected cases. CONCLUSIONS: This study revealed a high prevalence of intestinal protozoan infections among inhabitants of Rudbar-e Jonub county. Intestinal parasites especially protozoans remain a challenging public health problem wherever sanitation and health measures are limited in Iran.

Molecular characterization of bacterial, viral and fungal endosymbionts of Acanthamoeba isolates in keratitis patients of Iran.

Free-living amoebae belong to the genus Acanthamoeba; can feed on microbial population by phagocytosis, and with the capability to act as a reservoir and a vehicle of microorganisms to susceptible host. Therefore, the role of endosymbiosis in the pathogenesis of Acanthamoeba is complex and not fully understood. The aim of the present study was to identify bacterial, fungal, and human adenovirus (HADV) endosymbionts as well as evaluating the endosymbionts role of such organisms in the pathogenesis of Acanthamoeba in keratitis patients living in Iran. Fifteen Acanthamoeba (T4 genotype) isolates were recovered from corneal scrapes and contact lenses of patients with keratitis. Cloning and purification was performed for all isolate. Gram staining was performed to identify bacterial endosymbionts. DNA extraction, PCR, and nested PCR was set up to identify endosymbiont of amoeba. Evaluation of pathogenicity was conducted by osmo-tolerance and thermo-tolerance assays and cell culture, and then CPE (cytopathic effect) was survey. Statistical analysis was used between Acanthamoeba associated endosymbionts and Acanthamoeba without endosymbiont at 24, 48, 72, and 96 h. A p value < 0.05 was considered as significant, statistically. A total of 9 (60%) Acanthamoeba (T4 genotypes) isolates were successfully cloned for detecting microorganism endosymbionts. The only isolate negative for the presence of endosymbiont was ICS9. ICS7 (Pseudomonas aeruginosa, Aspergillus sp., and human adenovirus endosymbionts) and ICS2 (Escherichia coli endosymbiont) isolates were considered as Acanthamoeba associated endosymbionts. ICS7 and ICS2 isolates were highly pathogen whereas ICS9 isolate showed low pathogenicity in pathogenicity evaluated. Positive CPE for ICS7 and ICS2 isolates and negative CPE for ICS9 isolate were observed in cell culture. The average number of cells, trophozoites, and cysts among ICS7, ICS2, and ICS9 isolates at 24, 48, 72, and 96 h was significant. This is the first survey on microbial endosymbionts of Acanthamoeba in keratitis patients of Iran, and also the first report of Aspergillus sp, Achromobacter sp., Microbacterium sp., Brevibacillus sp, Brevundimonas sp and Mastadenovirus sp in Acanthamoeba as endosymbionts. Our study demonstrated that microbial endosymbionts can affect the pathogenicity of Acanthamoeba; however, further research is required to clarify the exact pattern of symbiosis, in order to modify treatment protocol.

Significance of Nitric Oxide Level in Giardiasis.

BACKGROUND: Giardiasis is one of the most prevalent intestinal protozoa infections in humans. Nowadays, nitric oxide (NO) is known to be involved in the immune system against Giardia intestinalis. Therefore, the aim of the present study was to evaluate the level of NO in individuals with giardiasis in comparison to normal subjects. METHODS: This descriptive study was conducted among 49 Giadia positive and 39 age and gender matched healthy volunteers. Examination of stool samples was done by wet mount technique and formol-ether concentration method. Serum samples were obtained for laboratory examination. NO production was quantified by measuring nitrite, a stable end product of NO, using the Griess reaction based on ELISA method. By using the standard curve in Excel program, the concentration of NO2- in samples was obtained. Finally, all data were analyzed using SPSS version 17. RESULTS: Values obtained from NO assays were placed into 4 groups: ≤ 10 (decline), 10.01 - 15 (normal), 15.01 - 25 (increase), and more than 25 µM (sharp increase). The mean level of NO in patients with G. intestinalis was 32.19 ± 2.15 µM and in people without G. intestinalis was 17.1 ± 1.33 µM. Eight point two percent of patients with Giardiasis were in normal range, but 2%, 20.4%, and 69.4% were in decline, increase, and sharp increase ranges, respectively. In group 2 (without infection), 17.9% were in normal range, and 20.5%, 51.3%, and 10.3% were in decline, increase, and sharp increase ranges, respectively. There was a statistical difference in nitric oxide levels between positive and negative groups with a 95% confidence interval. (p-value = 0.001). CONCLUSIONS: In our study, the number of people who showed a sharp increase in NO levels was significantly higher in individuals with giardiasis as compared to the control group, and patients infected with giardiasis showed significant increase in NO levels. Therefore, we suggest that further studies are required to understand the exact function of NO in the immune system against giardiasis in humans. It will be important to offer a new therapeutic target for eliminating G. intestinalis.

First Paleoparasitological Report on the Animal Feces of Bronze Age Excavated from Shahr-e Sukhteh, Iran.

Shahr-e Sukhteh (meaning burnt city in Persian) in Iran is an archeological site dated back to around 3,200-1,800 BC. It is located in Sistan and Baluchistan Province of Iran and known as the junction of Bronze Age trade routes crossing the Iranian plateau. It was appointed as current study area for paleoparasitological investigations. Excavations at this site have revealed various archeological materials since 1967. In the present study, sheep and carnivore coprolites excavated from this site were analyzed by means of rehydration technique using TSP solution for finding helminth eggs. Dicrocoelium dendriticum, Capillaria sp., and Taenia sp. eggs were identified, while some other objects similar to Anoplocephalidae and Toxocara spp. eggs were also retrieved from the samples but their measured parameters did not match those of these species. The present paper illustrates the first paleoparasitological findings of Bronze Age in eastern Iran supporting the economic activities, peopling, and communication as well as the appropriate condition for zoonotic helminthiasis life cycle in Shahr-e Sukhteh archeological site.

Isolation and genotyping of Acanthamoeba strains (T4, T9, and T11) from amoebic keratitis patients in Iran.

The continuous increase in Acanthamoeba keratitis, a severe corneal infection, worldwide is mainly due to the increase in the number of soft contact lens users. In the present study, which involves a 5-year study, a total of 138 corneal scraps and contact lenses together with their paraphernalias were obtained from suspected amoebic keratitis patients. All samples were cultured using culture-enrichment method. Pathogenic assay, using thermotolerance and osmotolerance tests were also performed on the positive strains. Sequencing of the isolated strains was done by targeting the DF3 region of 18s rRNA gene. The results revealed that 18 (13 %) of patients were infected with Acanthamoeba spp. As expected, T4 genotype was the most common genotype among the clinical samples; however, in three cases, Acanthamoeba belonging to T11 and T9 were detected. Interestingly, T9 genotype, commonly classified as non-pathogenic amoebae, was identified as a causal agent of a patient with amoebic keratitis. From the pathogenic assay, four strains belonging to T4 genotypes were highly pathogenic. This is the first report of Acanthamoeba T9 genotypes isolated in Iran and the first report of T9 type occurring in amoebic keratitis patients worldwide. Due to the increasing trend of amoebic keratitis (AK) and the identification of new genotypes, such as T9 as the causative agent of AK, more researches in this field are necessary in the region and the world at large.

Chitosan nanoparticles improve the effectivity of miltefosine against Acanthamoeba.

BACKGROUND: Acanthamoeba keratitis (AK) is a corneal sight-threatening infection caused by the free-living amoebae of the genus Acanthamoeba. Early and appropriate treatment significantly impacts visual outcomes. Mucoadhesive polymers such as chitosan are a potential strategy to prolong the residence time and bioavailability of the encapsulated drugs in the cornea. Regarding the recent administration of miltefosine (MF) for treating resistant AK, in the present study, we synthesized miltefosine-loaded chitosan nanoparticles (MF-CS-NPs) and evaluated them against Acanthamoeba. METHODOLOGY/PRINCIPAL FINDINGS: Chitosan nanoparticles (CNPs) were prepared using the ionic gelation method with negatively charged tripolyphosphate (TPP). The zeta-potential (ZP) and the particle size of MF-CS-NPs were 21.8±3.2 mV and 46.61±18.16 nm, respectively. The release profile of MF-CS-NPs indicated linearity with sustained drug release. The cytotoxicity of MF-CS-NPs on the Vero cell line was 2.67 and 1.64 times lower than free MF at 24 and 48 hours. This formulation exhibited no hemolytic activity in vitro and ocular irritation in rabbit eyes. The IC50 of MF-CS-NPs showed a significant reduction by 2.06 and 1.69-fold in trophozoites at 24 and 48 hours compared to free MF. Also, the MF-CS-NPs IC50 in the cysts form was slightly decreased by 1.26 and 1.21-fold at 24 and 48 hours compared to free MF. CONCLUSIONS: The MF-CS-NPs were more effective against the trophozoites and cysts than free MF. The nano-chitosan formulation was more effective on trophozoites than the cysts form. MF-CS-NPs reduced toxicity and improved the amoebicidal effect of MF. Nano-chitosan could be an ideal carrier that decreases the cytotoxicity of miltefosine. Further analysis in animal settings is needed to evaluate this nano-formulation for clinical ocular drug delivery.

Eimeria leuckarti in equid coprolites from the Sassanid Era (2nd-6th century CE) excavated in Chehrabad Salt Mine archaeological site, Iran.

OBJECTIVE: This study reports coccidian oocysts in an equid coprolite dated to the Sassanid Empire (2nd-6th century CE) recovered in Chehrabad Salt Mine archaeological site, Iran. METHODS: Between 2015 and 2017, an archaeoparasitological investigation led to the discovery of an equid coprolite in the Chehrabad Salt Mine archeological site, (Douzlakh), western Iran. Samples were rehydrated using trisodium phosphate solution and were examined by light microscopy. RESULTS: Seven oocysts of Eimeria leuckarti (Flesch, 1883) were identified; they were in various stages of sporulation. CONCLUSION: This is the first report of ancient coccidian oocysts from equids. The importance of this observation is discussed, and current knowledge of eimeriid oocysts at archaeological sites is reviewed. SIGNIFICANCE: The observations of E. leuckarti increases current knowledge of parasite biodiversity in ancient Iran when it rested along the Silk Road, a network of trade routes connecting the East and West that was central to economic, cultural, political, and religious interactions between these regions, and to livestock movement that could contribute to the transmission of the parasites from/to other regions. LIMITATIONS: The contextual information about animal species present in and around the Salt Mine during its working periods, including Achaemenid dynasty (6th to 4th century BCE) and Sassanid era (2nd to 6th century CE), is very limited and does not allow secure conclusions regarding the host origin of the coprolites. SUGGESTIONS FOR FURTHER RESEARCH: Application of molecular biology tools to identify the correct host origin of the coprolites and to detect more parasite species is advocated.

Gastrointestinal Parasites of Domestic Mammalian Hosts in Southeastern Iran.

Gastrointestinal parasites (GIP) are a major cause of disease and production loss in livestock. Some have zoonotic potential, so production animals can be a source of human infections. We describe the prevalence of GIP in domestic mammals in Southeastern Iran. Fresh fecal samples (n = 200) collected from cattle (n = 88), sheep (n = 50), goats (n = 23), camels (n = 30), donkeys (n = 5), horse (n = 1), and dogs (n = 3) were subjected to conventional coprological examination for the detection of protozoan (oo)cysts and helminth ova. Overall, 83% (166/200) of the samples were positive for one or more GIP. Helminths were found in dogs, donkeys, sheep (42%), camels (37%), goats (30%), and cattle (19%), but not in the horse. Protozoa were found in cattle (82%), goats (78%), sheep (60%), and camels (13%), but not in donkeys, dogs, or the horse. Lambs were 3.5 times more likely to be infected by protozoa than sheep (OR = 3.5, 95% CI: 1.05-11.66), whereas sheep were at higher odds of being infected by helminths than lambs (OR = 4.09, 95% CI: 1.06-16.59). This is the first study assessing the prevalence of GIP in domestic mammals in Southeastern Iran.

IgG avidity ELISA test for diagnosis of acute toxoplasmosis in humans.

Serum samples, 100 in the total number, were collected from different laboratories in Tehran, Iran and tested for anti-Toxoplasma specific IgG and IgM antibodies using indirect immunofluorescent antibody test (IFAT). Using the IgG (chronic) and IgM (acute) positive samples, the IgG avidity test was performed by ELISA in duplicate rows of 96-well microtiter plates. One row was washed with 6 M urea and the other with PBS (pH 7.2), then the avidity index (AI) was calculated. Sixteen out of 18 (88.9%) sera with acute toxoplasmosis showed low avidity levels (AI ≤ 50), and 76 out of 82 (92.7%) sera in chronic phase of infection showed high avidity index (AI>60). Six sera had borderline ranges of AI. The results showed that the IgG avidity test by ELISA could distinguish the acute and chronic stages of toxoplasmosis in humans.

Genomic palaeoparasitology traced the occurrence of Taenia asiatica in ancient Iran (Sassanid Empire, 2th cent. CE-6th cent. CE).

Palaeoparasitology investigates parasitological infections in animals and humans of past distance by examining biological remains. Palaeofaeces (or coprolites) are biological remains that provide valuable information on the disease, diet, and population movements in ancient times. Today, advances in detecting ancient DNA have cast light on dark corners that microscopy could never reach. The archaeological site of the Chehrabad salt mine of Achaemenid (550-330 BC) and Sassanid (third-seventh century AD) provides remains of various biotic and abiotic samples, including animal coprolites, for multidisciplinary studies. In the present work, we investigated coprolites for helminth eggs and larvae by microscopy and traced their biological agents' DNA by Next Generation Sequencing. Our results revealed various helminths, including Taenia asiatica, the species introduced in the 1990s. Implementing advanced modern molecular techniques like NGS gives a paramount view of pathogenic agents in space and time.

Giardia intestinalis: DNA extraction approaches to improve PCR results.

Difficulty in disrupting cysts of Giardia intestinalis, a cosmopolitan protozoan parasite, decreases the yield of DNA extracted and reduces the effectiveness of the polymerase chain reaction (PCR). To improve the detection of the Giardia Glutamate Dehydrogenase (gdh) gene, we re-evaluated the effects of deoxyribonucleic acid (DNA) extraction methods. Purified and concentrated cysts from 33 fecal samples were disrupted using conventional methods, and DNA extraction was conducted using two protocols: the QIAamp Stool Mini Kit and phenol/chloroform/isoamyl alcohol (PCI). PCR amplification was successful for 12 extracted DNA samples (36%) using PCI following a glass bead and freeze/thaw pretreatment and for all 33 samples (100%) using the QIAamp Stool Mini Kit following the aforementioned pretreatment. Consequently, the pretreatment of cysts with glass beads and freeze/thaw cycles followed by extraction of DNA with the QIAamp Stool Mini kit was the more effective protocol.

First molecular identification of Sarcocystis miescheriana (Protozoa, Apicomplexa) from wild boar (Sus scrofa) in Iran.

Sarcocystis isolate obtained from the thigh muscle of a wild boar (Sus scrofa), captured from Gilan Province, northern Iran, was subjected to molecular analysis. Genomic DNA was obtained using a DNA extraction tissue kit and Polymerase chain reaction (PCR) for amplification of the 18S ribosomal DNA region yielded an 842 bp DNA band on agarose gel. Analysis of DNA sequencing by BLAST confirmed the isolate as Sarcocystis miescheriana and the sequence was deposited in GenBank by Accession No. GU395554. This is the first molecular identification of an isolate of S. miescheriana in Iran.

Trichomonas vaginalis: investigation of a novel diagnostic method in urine samples using cysteine proteinase 4 gene and PCR technique.

Trichomonas vaginalis is the agent of a highly prevalent sexually transmitted disease that leads to vaginitis, urethritis, ectocervicitis and has been associated with human immunodeficiency virus (HIV). Detection of T. vaginalis based on wet-mount microscopy and culture methods is insensitive and time consuming, respectively. Thus the quest for reliable PCR techniques of T. vaginalis in vaginal discharge and urine sample is more importance. In this study, 500 urine and vaginal-discharge samples were collected from women referred to Sexual Transmitted Disease Clinic of Mirzakuchakkhan Hospital in Tehran, Iran between May 2008 and March 2009. Wet-mount and culture methods were done on the vaginal discharges, and PCR assay targeting cysteine proteinase 4 (CP4) was performed on the urine samples. The present study demonstrated 16 (3.2%) of patients were infected with T. vaginalis using culture and wet-mount, whereas PCR assay using CP4 could detect 12 (2.4%) positivity. Sensitivity and specificity of urine PCR assay compared to culture were 80% (95% CI, 54-96) and 99.6% (95% CI, 98.96-100), respectively. These results indicate that using urine-based detection method for T. vaginalis may not be appropriate in women.

Echinococcus granulosus genotypes in livestock of Iran indicating high frequency of G1 genotype in camels.

In this study, 112 Echinococcus granulosus isolates from different livestock of Iran were genotyped by PCR amplification of ribosomal DNA-internal transcribed spacer 1 (rDNA-ITS1) region followed by restriction fragment length polymorphism (RFLP) with the enzyme RsaI. The possibility of intra-genotype variation was also investigated using randomly amplified polymorphic DNA (RAPD) analysis. Isolates from sheep, goats, cattle and the majority of camels (12 of 18; 66.7%) were identified as the G1 genotype and a few camel isolates (6 of 18; 33.3%) belonged to the G6 genotype. Overall G1 and G6 genotypes were identified in 94.6% (106 of 112) and 5.3% (6 of 112) of all isolates, respectively. RAPD analysis based on 15 separate primers showed 7-14 bands of 200-3000bp for strain G1. Considering each individual primer, no differences observed among isolates from different hosts and between livers and lungs. This study confirmed the existence of G1 and G6 genotypes in Iran. Moreover, G1 is much more prevalent even in camels, indicating the importance of sheep-dog cycle in public health. Studying intra-genotypic variation of E. granulosus warrants more research using other primers and methods.

First report of a mixed infection due to Acanthamoeba genotype T3 and Vahlkampfia in a cosmetic soft contact lens wearer in Iran.

Acanthamoeba keratitis cases have emerged in the recent years in Iran. In this case, an amoebic keratitis due to a mixed infection with Acanthamoeba and Vahlkampfia species is reported. Corneal scrapes, contact lenses and contact lens cases obtained from the patient were analysed and were positive for cysts of Acanthamoeba and Vahlkampfia genera. Genus-specific PCR was carried out for both genera, confirming the microscopic observations. To our knowledge, this is the first reported case of a possible mixed amoebic infection due to Acanthamoeba and Vahlkampfia and raises awareness within contact lens wearers in Iran.

Genotyping of Acanthamoeba isolates from clinical and environmental specimens in Iran.

In this study, 15 Acanthamoeba isolates from AK patients and 10 environmental samples (water, soil and animal-origin samples) were classified at the genotype level based on the sequence analysis of the Diagnostic Fragment 3 (DF3) of Acanthamoeba small subunit rRNA gene. The obtained results revealed that most of these Acanthamoeba strains belonged to genotype T4 both in clinical and environmental samples. The presence T11 genotype in clinical samples was also revealed after the genotyping analysis and to our knowledge this is the first report of T11 genotype in Iran. Moreover, the isolation of T4 genotype from cow faeces in this study highlights a possible transmission of Acanthamoeba through animal faeces in Iran. Overall, the widespread distribution of pathogenic Acanthamoeba T4 across the environmental sources and the increasing number of contact lens wearers in Iran, demands more awareness within the public and health professionals as this pathogen is emerging as a risk for human health in Iran and worldwide.

Isolation of Balamuthia mandrillaris from urban dust, free of known infectious involvement.

The free-living amoeba Balamuthia mandrillaris can cause fatal encephalitis in humans and other mammals. The organism is associated with soils, and soil exposure has been identified as a risk factor for this pathogen. However, B. mandrillaris has been isolated only once from soils believed to be the source of the infection in child from California, USA who died of Balamuthia amoebic encephalitis and once from another unrelated soil source. We report for a third time the isolation of B. mandrillaris from the environment and for the second time its isolation from a sample not known to be involved with pathogenicity. We have established the new clonal B. mandrillaris strain (ID-19) in axenic media. The identity of our isolate was originally by morphology using a light microscope and this has been confirmed by 16S rRNA gene PCR. The new strain ID-19 groups with others of the species. The fact that our isolate came from dust particles deposited on surfaces from the air in an urban environment may suggest that it is not just soil exposure that constitutes a risk factor for Balamuthia infection. This is the first report of this organism from Iran.

Protein Detection of Excretory-Secretory Products and Somatic Extracts from Fasciola hepatica and F. gigantica Using Two-Dimensional Electrophoresis.

BACKGROUND: The aim of this research was to compare excretory-secretory and somatic extract materials of Fasciola hepatica and F. gigantica to detect protein maps of two species. METHODS: Twenty infected livers were collected from sheep in industrial slaughterhouse in Tehran, 2017-2019. Worms were detached from bile ducts, then recognized according to morphologic and morphometric criteria. After three times washing, worms were incubated in RPMI culture media and excretory-secretory products were collected. Worms were crushed and homogenized for preparation of somatic extract. Two Dimensional Electrophoresis gels were accomplished for both excretory-secretory material and somatic extracts. Gels were scanned with densitometer and analyzed with Same Spots software and protein spots were identified with Expasy database. RESULTS: For both excretory-secretory products and somatic extract, protein spots were appeared with two-dimensional electrophoresis technique. Quantitative analysis showed 40 and 28 protein spots for excretory-secretory of F. hepatica and F. gigantica respectively. For somatic extract 19 and 12 protein spots were recognized for F. hepatica and F. gigantica in that order. CONCLUSION: The rate of expression of some proteins were more in F. hepatica while expression of other proteins was high in F. gigantica. The expression of protease enzyme was higher in F. gigantica than F. hepatica. These data could be considered for biochemical differentiation of Fasciola species and subsequently to design and prepare of antigens for diagnosis/vaccine development.

Intestinal Protozoa in Domestic Cats (Carnivora: Felidae, Felis catus) in Northwestern Iran: A Cross-Sectional Study with Prevalent of Microsporidian and Coccidian Parasites.

BACKGROUND: In this study, some microsporidial and coccidian parasites were isolated from 103 domestic cats in the Meshkin Shahr area, northwestern Iran during the Jun 2014 to Jun 2015, and their genera were identified using parasitological methods with emphasis on their zoonotic importance. METHODS: One hundred and three fecal samples of domestic cats were collected and preserved in formalin (10%) and conserved in phosphate buffer saline solution, finally examined by microscopy after formalin-ether concentration and specific staining. Preservation in dichromate potassium (2.5%) was performed for all coccidian positive samples and then sporulated coccidian oocysts were investigated. RESULTS: The detected parasites were Isospora spp. 6/103(5.8%). Microsporidian spores were identified in 46/103 (44.6%) of all samples post-stained by the aniline blue staining method. CONCLUSION: Microsporidial infections were more prevalent in domestic cats. Further studies are needed in the identification of microsporidial spores isolated from infected cats.

Molecular Detection and Genotyping of Intestinal Microsporidia from Stray Dogs in Iran.

BACKGROUND: Microsporidia as one of the most important pathogens in veterinary and agricultural settings, have emerged in immunocompromised patients in Iran. To date, different Enterocytozoon bieneusi genotypes have been identified in humans and animals, supporting the possibility of zoonotic zoonosis transmission potential. The aim of this study was to evaluate the distribution of E. bieneusi genotypes among overpopulated stray dogs in vicinity of Tehran, the capital city of Iran. METHODS: Totally, 75 stool and 75 urine samples were obtained from 75 stray dogs during the time period from Mar 2015 to Oct 2015. DNA extraction was performed on all the samples and specific fragment of small subunit ribosomal RNA gene of E. bieneusi and Encephalitozoon spp. was amplified. Furthermore, specific primers targeting the internal transcribed spacer region of E. bieneusi were applied to determine the genotype of the microorganism. RESULTS: Microsporidia was detected in 5.3% of stool samples, while none of the urine samples was positive for microsporidia species. Overall, 440 bp fragment of E. bieneusi was amplified in all the samples and there was no amplification for Encephalitozoon spp. The results of sequencing of 410 bp fragment of internal transcribed spacer region showed that all the E. bieneusi were genotype D. CONCLUSION: E. bieneusi was the most prevalent microsporidian species in the stray dogs and all the positive isolates were characterized as genotype D.