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Abstract Fascioliasis is a zoonotic parasitic infection caused by Fasciola species in humans and animals. Despite significant advances in vaccination and new therapeutic agents, little attention has been paid to validating methods for the diagnosis of fascioliasis in humans. Serological techniques are convenient assays that significantly improves the diagnosis of Fasciola infection. However, a more sensitive method is required. The aim of this study was to compare the Real-Time PCR technique with the indirect-ELISA for the detection of Fasciola hepatica in human. Using a panel of sera from patients infected with Fasciola hepatica (n = 51), other parasitic infections (n = 7), and uninfected controls (n = 12), we optimized an ELISA which employs an excretory–secretory antigens from F. hepatica for the detection of human fascioliasis. After DNA extraction from the samples, molecular analysis was done using Real-Time PCR technique based on the Fasciola ribosomal ITS1 sequence. Of 70 patient serum samples, 44 (62.86%) samples were identified as positive F. hepatica infection using ELISA and Real-Time PCR assays. There was no cross-reaction with other parasitic diseases such as toxoplasmosis, leishmaniasis, taeniasis, hydatidosis, trichinosis, toxocariasis, and strongyloidiasis. The significant difference between the agreement and similarity of the results of patients with indirect ELISA and Real-Time PCR was 94.4% and 99.2%, respectively (Cohen’s kappa ≥ 0.7; P = 0.02). Based on the Kappa agreement findings, the significant agreement between the results of ELISA and Real-Time PCR indicates the accuracy and reliability of these tests in the diagnosis of F. hepatica in humans.

Abstract Background Fascioliasis, caused by the liver flukes Fasciola hepatica and Fasciola gigantica, is a global zoonotic helminthic disease. The livestock and human are the final hosts of the parasites. Northern Iran is an important endemic region for fascioliasis. Few studies have been conducted on the characterization of Fasciola isolates from eastern regions of the Caspian littoral of the country. Objective The aim of the present study was to identify F. hepatica, F. gigantica and intermediate/hybrid forms of Fasciola isolates from livestock in Golestan province, northern Iran, using morphometric and molecular tools. Methods Livestock livers naturally infected with Fasciola spp. were collected from Golestan slaughterhouse during 2019–2020. The worms were morphometrically studied using a calibrated stereomicroscope. Genomic DNA was extracted from all samples, and polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) was performed on internal transcribed spacer (ITS1) region using Rsa1 restriction enzyme. All the isolates were then analysed by multiplex PCR on Pepck region. Results A total of 110 Fasciola isolates were collected from the infected livers, including 94 sheep, 12 cattle and 4 goats. Morphometric analysis of 61 adult Fasciola isolates indicated that, 44 and 17 isolates belonged to F. hepatica and F. gigantica, respectively. Eighty‐one and 29 isolates belonged to F. hepatica and F. gigantica using ITS1‐RFLP, respectively. However, Pepck Multiplex PCR indicated 72 F. hepatica, 26 F. gigantica and 12 intermediate/hybrid forms. All 12 hybrid isolates were found in sheep host. Two isolates were identified as F. gigantica using morphometry and F. hepatica using both molecular methods. Conclusion The present study confirmed the existence of both F. hepatica and F. gigantica species and reported the first molecular evidence of hybrid Fasciola isolates in ruminants of Golestan province.

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: We aimed to compare semi-nested PCR with indirect ELISA to diagnose human fasciolosis. Methods: Overall, 70 serum samples were collected from different areas in Iran suspected for fascioliasis. Individuals were classified based on diagnostic of fascioliasis and habitat in endemic areas. Finally, all serum samples were tested by indirect ELISA (using secretory excretory antigen) and semi-nested PCR (using ITS1 gene). The study was conducted in the School of Publish Health, Tehran University of Medical Sciences, Iran in 2021. Results: Significant differences were found between agreement and similarity of patients' results of indirect ELISA and semi-nested PCR 94.46% and 98.4% respectively (Cohen's kappa ≥0.6; P-value≤0.05). No cross-reactions were observed with other parasitic diseases (toxocariasis, hydatidosis, strongyloidiasis, toxoplasmosis, cutaneous leishmaniasis, taeniasis and trichinosis). 69.84% of samples were positive by both techniques. In addition, the percentage of agreement and similarity between the results of the two techniques based on habitat in endemic areas was 88.9-100% and 97.7-100%, respectively (Cohen's kappa ≥0.6; P-value≤0.05). Conclusion: Semi-nested PCR could be a suitable method for following up on patients' treatment and a confirmatory method for ELISA as for diagnosis of human fascioliasis.

Fascioliasis is a zoonotic disease caused by Fasciola spp. We report five serologically and molecularly confirmed cases in an emerging region in Iran. A retrospective, case series study, performed in Lorestan Province, west of Iran between January 2015 and June 2016. From 1256 patients examined, 16 patients had positive serum ELISA. Five cases were approved as infected with fasciolosis using stool exam and PCR. Age ranged from 24 to 80 yr with mean age of 45 years. All of patients were adults and four of them had abdominal and back pain. Other symptoms included fever and chills, coughing and sore throat, weight loss, cutaneous manifestations. All patients lived in the rural environment, and four reported the ingestion of raw aquatic plants such as watercress. In fecal examination for fluke eggs, four samples were positive for F. hepatica eggs. Conventional PCR analysis showed that five human stools were positive for F. hepatica. All of 5 patients were treated with the usual dose of triclabendazole. A history of recent consumption of raw aquatic plants (in 4 out of 5 patients) is an important finding, but in one patient the source of infection remained unclear. Lorestan should be considered as an emerging region for this disease and further research in this province should be carried out.

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.

Background: We aimed to detect Fasciola specific deoxyribonucleic acid (DNA) by nested-PCR assay on human stool and urine samples and compare the results with the respective ELISA diagnostic assay. Methods: Overall, 206 clinically suspected cases of fascioliasis were enrolled in the study. Blood samples were collected from all the patients, and serum samples were isolated. ELISA assay, using Fasciola somatic antigen (SA), was carried out to detect anti Fasciola antibodies for the collected sera. DNA was randomly extracted from 25 stool and 10 urine samples of seropositive individuals and was evaluated by conventional PCR and nested PCR methods. The nested-PCR results were confirmed by sequencing the 430 bp region of ribosomal ITSI gene. Stool and urine samples from patients with different parasitic diseases and 25 stool samples from healthy individuals served as controls. Urine samples were collected from 10 healthy controls as well. Results: Fascioliasis was detected by ELISA in 24.8% of the individuals. Of these, 25 seropositive patients were randomly assigned to the study. Fasciola DNA was identified in the stool samples of 96% of seropositive patients by nested PCR but ova of Fasciola was detected by parasitology methods in only 20% of seropositive cases. Fasciola DNA was identified in 90% of the urine samples by nested PCR. No cross-reactions were observed with other parasites. Conclusion: Detection of cfDNA in stool and urine samples has high accuracy and thus can be used for the diagnosis of Fasciola infection in human.

Background: It is difficult to make an exact morphological distinction between Fasciola hepatica and Fasciola gigantica. We used High Resolution Melting analysis (HRM) method to differentiate the F. hepatica species from F. gigantica in order to differentiate them. Methods: Overall, 80 adult liver flukes were collected from infected slaughtered animals including cattle, sheep and goats from Lorestan Province, western Iran from Sep 2015 to Aug 2017. Genomic DNA was extracted using commercial DNA extraction kit. The multilocus sequences of mDNA including COX1, COX3 and ND6 were amplified employing real-time PCR & HRM analysis. Specific and universal primer pairs were designed for differentiation Fasciola spp. Results: Universal primers cannot be used to distinguish between these two species, but in the contrary, specific primer pairs of each species could differentiate them properly. Molecular identification using specific primer pairs were consistent. Conclusion: HRM is a simple, fast and reliable method for detecting and differentiating F. hepatica from F. gigantica and can be used for diagnostic and epidemiological purposes.

Background: The detection of Fasciola species in various geographical regions is essential for health policymaking. Here, we aimed to identify livestock (cattle and sheep) related Fasciola genotypes by restriction fragment length polymorphism PCR. Methods: Seventy adult Fasciola flukes were collected from 70 infected livers of 35 cattle and 35 sheep slaughtered in Zabol abattoir, outh-east Iran (Jan-Jul 2017). Fasciola species were determined based on molecular features. For molecular detection, Fasciola ITS1 region was amplified and sequenced. A 700 bp fragment was amplified. These were digested with RasΙ enzyme. F. hepatica specific fragments were 47, 59, 68, 104, and 370, while those related to F. gigantica had 45, 55, 170, 370. Results: The two main species of F. hepatica and F. gigantica are responsible for fasciolosis in sheep and cattle in our region. From 35 Fasciola isolated from cattle, 3 and 32 were F. hepatica and F. giagantica respectively. From 35 Fasciola isolated from sheep, 4 were F. hepatica and 31 were F. gigantica. Conclusion: All Seventy Fasciola samples from two different hosts (cattle and sheep) were identified as either F. hepatica or F. gigantica by PCR-RFLP. Genotypic variability of Fasciola species was high in our region. It is recommended to assess molecular variation of Fasciola isolates in other host livestock.

Background: We evaluated the genetic diversity of samples identified morphologically as Fasciola spp. from sheep, cattle and goat from Kermanshah Province, western Iran using PCR-RFLP method.Methods: We used PCR-RFLP analysis of ribosomal ITS1 fragment using RsaI restriction enzyme to investigate the genetic characteristics of Fasciola species obtained from different hosts (16 sheep, 28 cattle, 4 goats). The species of Fasciola were confirmed by sequencing the 700 bp region of ribosomal ITS1 gene.Results: In Kermanshah, F. hepatica was present in 96% of the samples, F. gigantica was found only in two cattle sample. No hybrid forms were detected in the present study. Conclusion: Our results contribute to clarify the dark spots of Fasciola genotyping in different parts of Iran. Keywords: Fasciola hepatica, Fasciola gigantica, Genotype, PCR, Iran

Response Letter to the Editor in Reference to Letter to the Editor about “Molecular Characterization of Animal Fasciola spp. Isolates from Ker-manshah, Western Iran”

Background: We aimed to determine the prevalence of fasciolosis in the definitive hosts (human and livestock) and intermediate (Lymnaea snails) hosts in Kermanshah Province, western Iran from 2014-2016. Methods: The study on animals was descriptive and retrospective one. All daily records of animals slaughtered in the abattoirs were analyzed. For the study of human fascioliasis, 975 serum samples were collected from different parts of Kermanshah Province and analyzed using ELISA based on excretory-secretory antigens. Moreover, 4400 Lymnaea snails were collected from 25 habitats. The snails were identified and examined for presence of cercariae by shedding method. Results: Fasciolosis was diagnosed in 1.7% of slaughtered animals, which was significantly greater than the other species (P

Fascioliasis is a foodborne zoonotic disease caused by the two parasite species Fasciola hepatica and F. gigantica. In spite of the presence of both species of Fasciola in the livestock, to our knowledge, to date, no cases of human F. gigantica infection have been reported in Iran officially. Here, we report such a case in a 25 yr old woman referred to The Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran in 2016. CT imaging and MRCP revealed an ill-defined lesion of segments of liver. Specific ELISA produced a positive result besides detecting egg of the parasite via stool exam. The identification of parasite species was performed by the DNA extracted from the eggs and sequencing ITS-1, in addition to comparison to GenBank retrieved sequences, using the BLAST search tool. The sample showed 100% identity with F. gigantica. She was treated for fasciolosis with a single dose of Egaten® 10 mg/kg with positive response. This is the first case of human fasciolosis due to F. gigantica reported in Iran.

Background: The aim of this study was to conduct a sero-epidemiological survey in Alborz Province, central Iran to detect the rate of hydatidosis in the city and nearby villages. Methods: Overall, 680 serum samples were collected from 536 male and 127 female subjects referred to different health centers of Karaj, Alborz Province, central Iran and nearby villages in 2014-15. All patients filled out a questionnaire and an informed consent. Sera were analyzed using indirect-ELISA test with AgB. Ten μg /ml antigens (Proceeded hydatid fluid), serum dilutions of 1:500 and conjugate anti-human coombs with 1:10000 dilutions were utilized to perform the test. Data analysis was conducted using SPSS software ver. 11.5. Results: Twenty-three cases (3.4%) were positive for hydatidosis by ELISA test. The prevalence of hydatidosis among females and males was 3.1% and 4.7%, respectively. The rate of the disease was significantly higher in areas where dogs were higher (P0.05). Conclusion: Because of the specific situation of Alborz Province, and availability of many stray dogs, obtained rate of hydatidosis shows that the authorities should be cautious to monitor the disease.

Background: The aim of this study was the seroepidemiological survey for detecting the status of human fasciolosis in Lorestan Province, western Iran. Methods: This cross-sectional study was conducted in 2015-16. Based on statistical estimations, 1256 serum samples were collected from different parts of Lorestan Province, western Iran, and stored at -20 °C until use. The collected serum samples were analyzed at Tehran University of Medical Sciences, Tehran, Iran using indirect ELISA method. Results: Anti-Fasciola antibodies were detected in 16 (1.3%) individuals. Regarding the seropositivity to fasciolosis, no significant differences were found between age groups, sex, level of education and occupation; however significant differences were observed regarding location, consuming local freshwater plants and water resources (P

Background: We aimed to describe morphological and morphometrical characteristics of Fasciola spp. in livestock from Ardabil Province, Northwest Iran. Methods: Forty adult flukes were collected from different definitive hosts (cattle and sheep). Previously specimens were identified as F. hepatica or F. gigantica based on PCR-RFLP of the ITS-1 region with RsaI enzyme. We identified Fasciola spp. based on morphological and metric assessment of external features of fresh adults, morphological and metric assessment of internal anatomy of stained mounted worms. Statistical analysis was conducted using the Student’s t-test implemented in SPSS 15.0 (SPSS, Chicago, Illinois). Then the morphometric criteria of Fasciola samples were compared with PCR-RFLP data. The results of PCR-RFLP were confirmed by COI gene sequence. Results: The differences between the body length, area of the body, peripheral of the body, succer area, cone length, cone width, in two species were significant (

Background: On the continuation of sorting the puzzle of the situation of hydatid disease in Iran and considering that so far no survey was conducted in this context in Arak City, Markazi Province central Iran, the present study was conducted to determine the seroprevalence of human hydatidosis using AgB-ELISA test.Methods: Totally 578 serum samples randomly were collected from patients referred to hospitals and different health centers in the city and 3 nearby villages of Arak. All sera were examined by ELISA tests using AgB. Before sampling, a questionnaire was filled out for each case. Data were analyzed using Chi-square test and multivariate logistic regression for risk factors analysis. P

Background: The current study was designed to evaluate immune responses induced by DNA vaccines encoding 8-kDa subunit of antigen B (HydI) of Echinococcus granulosus and murine interleukin 12 (IL-12) as genetic adjuvants in BALB/c mice. Methods: Expression plasmid pcDNA3.1 containing HydI (pcHyd1) as vaccine along with the murine interleukin 12 (pcMIL12) as adjuvant were used. Thirty-five mice in the five experimental groups received PBS, empty pcDNA3.1, pcHydІ, pcMIL-12, and pcHydІ+ pcMIL-12 in days zero, 14th and 28th. Two weeks after the last immunization, evaluation of the immune response was performed by evaluating the proliferation of splenic lymphocytes, IFN-γ and IL-4, determination of IgG isotyping titer. Results: Mice that received the pcHydI+pcMIL12 exhibited higher levels of lymphocyte proliferation compared to mice that received the pcHydI alone (P

Background: Fascioliasis is a zoonotic disease of livestock and human caused by Fasciola species. Here in, the results of serological evaluation of fascioliasis in peo­ple referring to the School of Public Health, Tehran University of Medical Sci­ences during 2008-2014 are presented. Methods: Demographic characterizations, symptoms and eosinophil rate were registered for every patient. Using somatic antigen of Fasciola, ELISA was per­formed and the results were analyzed. Data of questioners were analyzed as well. Results: Among 206 applicants, 24.8% were seropositive for fascioliasis, included 21% female and 28.3% male. Mean range of age of patients was between 13 to 67 yr. The highest rate of seropositivity was found among 20-30 yr old patients. Most of the patients had hypereosinophilia. All patients had history of eating raw vegeta­bles, or drinking unsafe water. Patients were referring from different provinces of Iran, including Gilan, Mazandaran, Tehran, Ardabil, Khuzestan, Lorestan, North Khorasan, Kermanshah, Azerbaijan, Fars, Kordestan, Hamedan and Markazi. Conclusion: During recent years, variety of provinces in Iran, where patients with fascioliasis are referred, has been increased. Patients coming from Gilan and Mazanda­ran provinces were referred early after the onset of their symptoms. Most probably, physicians in Gilan and Mazandaran are more alert on fascioliasis than other provinces. Previous wrong diagnosis was more common among patients refer­ring from other provinces than Gilan and Mazandaran provinces.

Background: Echinococcosis or hydatidosis is a chronic, zoonotic worldwide infection caused by the larval stage of the dog taeniid tapeworm Echinococcus granulosus. Vaccination has been considered as one of the ways to prevent of hy-datidosis in recent decades. The aim of this study was to construct a pcDNA3.1 eukaryotic expression vector contain-ing the subunit 8-kDa antigen B (Hyd1) of E. granulosus (G1 strain) and investigate its capability to induce protein ex-pression in mammalian cell line, as a basis toward developing a DNA vaccine against hydatidosis. Methods: The coding sequence of HydI was amplified by PCR with the specific PCR primers from pQE/HydI, and then was sub-cloned into pcDNA3.1 plasmid as expression vector. The pcHyd1 plasmid was digested by restriction enzymes and amplified with the specific PCR primers to confirm cloning of this gene in pcDNA3 plasmid. In last step, the sub-cloned gene was expressed in mammalian cell line (NIH 3T3 cells). Result: The subunit 8-kDa antigen B (Hyd1) was successfully sub-cloned in pcDNA3.1 and Hyd1 protein was ex-pressed in eukaryotic cell confirmed by SDS-PAGE and Western blot. Conclusion: Recombinant plasmid of pcDNA3.1 was successfully constructed and express of recombinant Hyd1 protein was confirmed. That is promising step for forthcoming measures on providing vaccine against human and animal hydatidosis.

Background: The aim of this study was to conduct a sero-epidemiological survey in Isfahan City and suburb areas, central Iran to detect the rate of human hydatidosis using ELISA test. Methods: Overall, 635 serum samples were collected from subjects referred to different health centers in urban and rural regions of the city. Sera were analyzed using Ag-B ELISA test. Ten μg/ml antigens, serum dilutions of 1:500 and conjugate anti-human coombs with 1:10000 dilutions were utilized to perform the test. All subjects filled out a questionnaire and an informed consent. Data analysis was conducted using SPSS 18 software. Cut-off was calculated as X+3 SD. Results: Cut-off value was calculated 0.19. Seven cases (1.1%) were seropositive for hydatidosis by ELISA test. The sero-prevalence of hydatidosis was 0.27% among females and 2.24% among males (P=0.019). Age group of 60-69 years old, with 2.59% as prevalence had the highest rate of positivity. There was no significant difference as regards age groups, job, residency, contact by dog and literacy. According to job, self-employed people had the highest rate of infection as 3.05%. The sero-prevalence of infection was 1.14% in diploma and 1.13% in illiterates. As regards residency, urban life (1.49%) showed no significant difference with rural life. Conclusion: The rate of prevalence in this region showed that necessary cautions should be taken into account to monitor the spread of human hydatidosis in this region. In comparison with other studies, the rate of infection was roughly less than other regions.

Background: The aim of this study was to detect the seroprevalence of human fascioliasis in Isfahan County, central Iran in 2013. Methods: Overall, 471 sera samples were collected from people recalled randomly to 20 health centers in the city of Isfahan and 10 related villages in 2014. Sera were examined using ELISA test. A questionnaire was filled out for each participant. Results: Altogether eight cases (1.7%) were seropositive which had the OD absorbance in ELISA test more than the calculated cutoff of 0.36. All of them were female. One positive subject had a history of consuming Delar (Local dish) and three seropositive cases with history of eating Zeitoon-Parvadeh (Proceeded olive). Conclusion: Isfahan County might be considered as one area apt for fascioliasis.More studies in terms of veterinary investigation and verifying the risk factors are necessary.

The aim of this study was to detect the genotype of Fasciola spp. in Meshkin-Shahr, Ardabil Province, northwestern Iran in different hosts using PCR-RFLP.The parasite hosts included cattle, and sheep. Overall, 70 adult flukes from livers of slaughtered animals were collected from the abattoirs of aforementioned area. The included 35 samples from infected sheep and 35 samples from 35 infected cattle. PCR-RFLP and sequence analysis of the first nuclear ribosomal internal transcribed spacer (ITS 1) region from Fasciola species were used to conduct the study.The fragment of approximately 700bp in all of the Fasciola samples was amplified. PCR products of ITS 1 were subjected for digestion by restriction enzyme. RsaI restriction enzyme was selected for RFLP method that caused the separation specifically of Fasciola species. Amplicons with the sequences of F. hepatica had a pattern of about 360, 100, and 60 bp band size, whereas F. gigantica worms had a profile of 360, 170, and 60 bp in size, respectively. Results based on PCR-RFLP analysis were confirmed by sequence analysis of representative ITS 1 amplicons. No hybrid forms were detected in the present study. All sheep were infected with F. hepatica but cattle were infected with both species.Both species of Fasciola are present in Ardabil. The method described here can be valuable for identification of Fasciola species in endemic parts for fasciolosis, regions with intermediate species and in that overlapping distribution area.

Background: The aim of this study was to conduct a seroprevalence survey in Meshkin-Shahr, Ardabil Province, north western Iran to detect the rate of human fascioliasis in the city and nearby villages. Literature shows that no such study has been conducted so far. Methods: Overall, 458 serum samples were collected by randomized cluster sam-pling method from 153 males and 305 females referred to different health centers of the region after recalling by staff in those centers in 2012. All cases filled out a questionnaire and an informed consent. Sera were analyzed using indirect-ELISA test. Ten μg /ml antigens (Liver Fluke Homogenate), serum dilutions of 1:500 and conjugate anti-human coombs with 1:10000 dilutions were utilized to perform the test. Data analysis was conducted using SPSS software ver. 18. Results: Nine cases (1.96%) were positive for fascioliasis by ELISA test. The sero-prevalence of fascioliasis among females was 1.63% and 2.6% in males. There was no significant difference as regards age groups, sex, job, residency, literacy and con-suming row vegetable. According to job, unemployment subjects had the highest rate of infection as 5.9%. The seroprevalence of infection was 1.52% in illiterate people. As for residency, urban life showed no significant difference with rural life (2.4% vs. 1.42). Age group of 40-49 yr old, with 3.3% seropositivity had the highest rate. Conclusion: Obtained seroprevalence of fascioliasis shows immediate attention of health authorities to the diseases in the area. The adjacent of Ardabil Province to endemic areas of fasciolosis accentuates this attention.