Design and expression of polytopic construct of cathepsin-L1, SAP-2 and FhTP16.5 proteins of Fasciola hepatica.

The enzyme-linked immunosorbent assay (ELISA) technique can play an important role in the early detection of fascioliasis. However, they have some diagnostic limitations, including cross-reaction with other helminths. It seems that the combination of recombinant parasite proteins as antigen can reduce these problems. Hence, the present study was aimed to design and confirm the antigenic recombinant multi-epitope (rMEP) construct of three protein epitopes (linear and conformational B-cell epitopes) of the parasite using immunoinformatic tools. For this purpose, the tertiary structures of Fasciola hepatica cathepsin-L1, saposin-like protein 2 and 16.5-kDa tegument-associated protein were predicted using the I-TASSER server. Validation of the modelled structures was performed by Ramachandran plots. The antigenic epitopes of the proteins were achieved by analysing the features of the IEDB server. The synthesized gene was cloned into the pET-22b (+) expression vector and transformed into the Escherichia coli BL21. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to verify and analyse the expression of the rMEP protein. Western blotting was utilized to confirm rMEP protein immunogenicity in two forms, one using an anti-His tag antibody and the other with human pooled sera samples (fascioliasis, non-fascioliasis and negative control sera). Our results demonstrated that the rMEP designed for the three proteins of F. hepatica was highly antigenic, and immune-detection techniques confirmed the antigen specificity. In conclusion, the presented antigenic multi-epitope may be very helpful to develop serodiagnostic kits such as indirect ELISA to evaluate the proper diagnosis of fascioliasis in humans and ruminants.

In vitro gene manipulation of spinal muscular atrophy fibroblast cell line using gene-targeting fragment for restoration of SMN protein expression.

The reduced level of survival motor neuron (SMN) protein, caused by homozygous deletions in the SMN gene, led to a common neurodegenerative disorder known as spinal muscular atrophy (SMA). In spite of extensive efforts to find a cure for SMA, there is currently no effective treatment available for this devastating disease. In this study, restoration of SMN expression through 'gene-targeting' method in SMA fibroblast cells was attempted. We designed a 2697-bp gene-targeting cassette; it consisted of an SMN1 open reading frame expressing 38 kD SMN protein and the upstream and downstream regions of exon 1 of SMN1 gene at the ends as the homology arms. SMA fibroblast cells were transfected by gene-targeting cassette using Lipofectamine LTX-PLUS reagent. Occurrence of homologous recombination in selected cells was investigated by PCR analysis. Increased expression of SMN protein was shown by real-time PCR and western blotting analysis. The immunofluorescence analysis results demonstrated that the number of SMN nuclear structures, Gems, was the same as or greater than the number of Gems found in normal fibroblasts. The results of this study indicate that gene-targeting methods do, in fact, present as an alternative for restoration of SMN expression in SMA patients-derived cells in vitro.

Decellularized human amniotic membrane: more is needed for an efficient dressing for protection of burns against antibiotic-resistant bacteria isolated from burn patients.

Human amniotic membranes (HAMs) have attracted the attention of burn surgeons for decades due to favorable properties such as their antibacterial activity and promising support of cell proliferation. On the other hand, as a major implication in the health of burn patients, the prevalence of bacteria resistant to multiple antibiotics is increasing due to overuse of antibiotics. The aim of this study was to investigate whether HAMs (both fresh and acellular) are an effective antibacterial agent against antibiotic-resistant bacteria isolated from burn patients. Therefore, a HAM was decellularized and tested for its antibacterial activity. Decellularization of the tissue was confirmed by hematoxylin and eosin (H&E) and 4,6-diamidino-2-phenylindole (DAPI) staining. In addition, the cyto-biocompatibility of the acellular HAM was proven by the cell viability test (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, MTT) and scanning electron microscopy (SEM). The resistant bacteria were isolated from burns, identified, and tested for their susceptibility to antibiotics using both the antibiogram and polymerase chain reaction (PCR) techniques. Among the isolated bacteria, three blaIMP gene-positive Pseudomonas aeruginosa strains were chosen for their high resistance to the tested antibiotics. The antibacterial activity of the HAM was also tested for Klebsiella pneumoniae (American Type Culture Collection (ATCC) 700603) as a resistant ATCC bacterium; Staphylococcus aureus (mecA positive); and three standard strains of ATCC bacteria including Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27833), and S. aureus (ATCC 25923). Antibacterial assay revealed that only the latter three bacteria were susceptible to the HAM. All the data obtained from this study suggest that an alternative strategy is required to complement HAM grafting in order to fully protect burns from nosocomial infections.

Phenytoin effects on inflammatory mediator's production by gingival fibroblasts: a comparative study in children and adults.

BACKGROUND: About 30 to 50% of patients taking Phenytoin develop significant gingival alternations especially in buccal anterior part of oral cavity. This study was done to compare the synthesis of its inflammatory mediators and related gingival overgrowth in different ages. METHODS: Samples were collected from biopsy of a healthy gingival of four adults in 35-42 years old through crown lengthening surgery and four children in 4-11 years old through impact tooth surgery, after local anesthesia and from the keratinized soft tissues around the teeth. Gingival biopsies were transferred to a medium which containing DMEM and cultured on specific plates 25 cm2 and put on incubator containing CO2 with temperature of 37 °C. MTT was used to compare the Proliferation rate of fi broblasts. Supernatant of culture medium of test and control sinks were collected by sampler and concentration of IL1ß, PGE2, IL6, TGFß, TNFα and IL8 were analyzed by ELISA. RESULTS: Different proliferation rate of Phenytoin induced gingival fibroblasts in adults (0.073 ± 0.177) as compared to children (0.056 ± 0.028) was not significant. Production of PGE2, TGFß and IL6 by Phenytoin induced gingival fibroblasts in children was increased as compared to adults (p < 0.05). Production of IL8 by Phenytoin induced gingival fibroblasts in children was decreased compared to adults (P = 0.02). CONCLUSION: Phenytoin induced gingival fibroblasts of children produce more amounts of IL1ß, PGE2, IL6, TGFß and IL8 as compared to adults' fibroblasts. More Comprehensive studies with well-documented designs using other methods are recommended to verify these results.

Comparison of the RE and B1 gene for detection of Toxoplasma gondii infection in children with cancer.

Early, accurate and effective diagnosis of toxoplasmosis can make an important contribution to the prevention and control of disease, especially in people who are at risk. In this study, two commonly used genomic repeats of Toxoplasma gondii, RE (GenBank accession number AF146527) and B1, were compared to each other in nested-PCR assay. Five hundred and thirty-five blood samples from children with leukemia were tested for the presence of T. gondii antibodies using enzyme immunoassays. One hundred and ten DNA samples of these patients (50 IgM+, IgG+, 10 IgM-, IgG+, and 50 IgM-, IgG-) were analyzed by nested-PCR. The specificity of two nested PCR assays was determined using the DNA samples of other parasites and human chromosomal DNA. As a result, 82% (41/50) and 68% (34/50) of the IgM+, IgG+ samples were positive on duplicate RE and B1-nested PCR analyses, respectively. None of the 10 IgM-, IgG+ seropositive samples was detected positive after testing RE and B1-nested PCR assays in duplicate. One (2%) of the 50 seronegative samples was positive by duplicate RE-nested PCR but none of them were positive by duplicate B1-nested PCR. The detection limit of the RE-nested PCR assay was 640 fg of T. gondii DNA whereas this rate for B1-nested PCR was 5.12 pg of the DNA template. No cross-reactivity with the DNA of other parasites and human chromosomal DNA was found. The results indicate that an RE-based nested PCR assay is more sensitive than B1 genomic target, of those tested, for detection of T. gondii. It is noteworthy that in comparison with B1-nested PCR, RE-nested PCR could detect the T. gondii DNA in seronegative samples too.

Gene regulation of pteridine reductase 1 in leishmania promastigotes and amastigotes using a full-length antisense construct.

BACKGROUND: Pteridine metabolic pathway is unusual features of Leishmania, which is necessary for the growth of parasite. Leishmania has evolved a complex and versatile pteridine salvage network which has the ability of scavenging a wide area of the conjugated and unconjugated pteridines especially folate and biopterin. In this study, we focus on the inhibition of ptr1 gene expression. METHODS: L. major ptr1 gene was cloned into pcDNA3 and digested using KpnI and BamHI. The gene was subcloned so that antisense will transcribe and called pcDNA-rPTR. Leishmania major was cultured and late logarithmic-phase promastigotes were harvested. The promastigotes were divided into two groups. One group was transfected with 50 µg of pcDNA-rPTR, whereas the other group was transfected with pcDNA3. Transfected cells were cultured and plated onto semi-solid media. Mouse pritonean macrophages were transfected using pcDNA-rPTR-tansfected promastigotes. Western blotting was performed on mouse transfected pritonean macrophages and extracts from transfected promastigotes of L. major using a L. major ptr1 antibody raised in rabbits. RESULTS: The PTR1 protein was not expressed in pcDNA-rPTR- tansfected promastigotes and mouse macrophage transfected with pcDNA-rPTR- tansfected promastigotes. CONCLUSION: This approach might be used to study the pteridine salvage pathway in Leishmania or to assess the possibility of using gene expression inhibition in the treatment of leishmaniasis.

Double-Stranded RNA Viral Infection in Tehran Trichomonas vaginalis Isolates.

BACKGROUND: Trichomonas vaginalis is a pathogenic protozoon and may be contaminated with dsRNA virus called Trichomonas vaginalis virus (TVV). The viral infection is an important factor for its pathogenesis and sensitivity to metronidazole. The presence of TVV is associated with qualitative and quantitative expression of cysteine proteinases and surface immunogenic; P270. The purpose of this study was to determine TVV frequency in T. vaginalis clinical isolates in Tehran, Iran. METHODS: The 46 T. vaginalis isolates were collected from Tehran Province and cultured in TYI-S-33 culture medium. Viral RNA was extracted and RT-PCR was done. RESULTS: Of 46 T. vaginalis isolates, 8 isolates (17.39%) were infected with TVV-1. There was not any association between patient age and TVV- infected T. vaginalis. There were 17.39% viral infection in T. vaginalis isolates which was lower than that reported by other researchers. CONCLUSION: This is the first report on T. vaginalis isolates infection by TVV-1 in Iran.

Inhibition of Leishmania major PTR1 Gene Expression by Antisense in Escherichia coli.

BACKGROUND: Protozoa related to Trypanosome family including Leishmania, synthesize enzymes to escape from drug therapy. One of them is PTR1 that its enzymatic activity is similar to dihydrofolate reductase (DHFR). Dihydrofolate reductase - thymidylate synthase has a major role in DNA synthesis, if it is inhibited, the result would be the death of parasite. Since PTR1 activity is similar to DHFR, causes the decrease of inhibition effect of drug. The aim of this study was inhibition of Iranian L. major PTR1 expression with mRNA antisense in prokaryotic system as an approach to appear of the drugs therapeutic effects more. METHODS: PTR1 gene was ligated to pACYCDuet-1 and pcDNA3 plasmids as sense and antisense plasmids, respectively. Simultaneously transfer of sense and antisense plasmids was done in E. coli strain M15. SDS-PAGE and western blot analysis were carried out to analyze the expression. RESULTS: Sense and antisense plasmids were prepared and confirmed by restriction analysis and PCR then simultaneously transfer of them was done. SDS-PAGE and western blot analysis showed PTR1 gene was inhibited by mRNA antisense in bacterial cells. CONCLUSION: Expression of PTR1 gene in sense plasmid was inhibited successfully by antisense plasmid.

Expression and Purification of P43 Toxoplasma gondii Surface Antigen.

BACKGROUND: Toxoplasma gondii is an obligate intracellular protozoan parasite, capable of infecting all species of mammals including man. Congenital toxoplasmosis is more important during pregnancy for the first time. In this study we expressed and purified P43 Toxoplasma gondii tachyzoite and bradyzoite specific surface antigen. METHODS: The recombinant pGEMEX-1 contained Toxoplasma P43 coding sequence was transformed into E. coli and mass cultured in LB medium contained 100 µg/ml ampicillin at 37°C over night. The T7 promoter was induced by 1mM isopropyl-1-thio-ß-D-galactopyranoside (IPTG. Recombinant protein was purified by affinity chromatography and confirmed by gel diffusion dot blot and western blot,-using specific anti Toxoplasma antibodies. RESULTS: Recombinant plasmid was induced by IPTG and analyzed by SDS-PAGE. Recombinant protein was confirmed by Western-blot and dot blot using anti human Toxoplasma antibody. CONCLUSION: Recombinant Toxoplasma P43 was produced successfully.

Evaluation of Recombinant SAG1 Protein for Detection of Toxoplasma gondii Specific Immunoglobulin M by ELISA Test.

BACKGROUND: Toxoplasmosis is a serious disease in immunocompromised patients and pregnant women. Differentiation of acute and chronic infection is a major challenge in serodiagnosis of the disease. Since the aim of this study was to assess the diagnostic utility of recombinant SAG1 (rec-SAG1) for the detection of Toxoplasma-specific IgM antibodies in human sera, by an enzyme-linked immunosorbent assay (ELISA). METHODS: The purified recombinant protein SAG1 was applied in house ELISA test and the ability of it in binding to specific immunoglobulin M in 30 serum samples of acute infected patients was evaluated. The results obtained by assays with the recombinant SAG1 and standard commercial assays were compared. RESULTS: The sensitivity and specificity of in house ELISA compared to a standard commercial ELISA (com-ELISA) were 80% and 90%, respectively. CONCLUSION: It was concluded that the rec-SAG1 could be an alternative marker for detection of anti Toxoplasma-specific IgM and diagnosis of acute infection.

Identification of different Theileria species (Theileria lestoquardi, Theileria ovis, and Theileria annulata) in naturally infected sheep using nested PCR-RFLP.

Ovine theileriosis is an important hemoprotozoal disease of sheep and goats in tropical and subtropical regions that leads to economic losses in these animals. A nested PCR-restriction fragment length polymorphism (RFLP) was carried out to identification Theileria species in sheep in some area in western half of Iran (Sari, Rasht, Urmia, Ilam, and Ahvaz). Two hundred and fifty blood samples were taken from sheep during tick activating season (summer of 2008). Microscopic examination revealed that 9.2% (23/250) sheep were infected by Theileria spp. piroplasms. Parasitemia ranged from 0.011% to 0.015%. In nested PCR assessment of DNA samples, 32.8% (82/250) sheep were positive. The negative samples were confirmed by amplifying of ovine beta-actin gene as an internal control. The differentiation of Theileria species was based on RFLP patterns using three restriction enzymes: HpaII, Rsa1, and Bsh 1285I. Out of 82 positive samples, 54.8% (45/82) and 40.2% (33/82) were positive for Theileria lestoquardi and Theileria ovis respectively. Mixed infection was detected in 4.8% (4/82) cases. Based on their PCR product digestion pattern with HpaII (1178, 900, 278, and 106 bp), it seemed to be mixture of Theileria annulata and T. lestoquardi. The presence of T. annulata was supported by sequence analysis. This is the first report of naturally infected sheep with T. annulata in Iran. Geographical distribution of Theileria species in sheep is shown according to the result of microscopy and nested PCR and RFLP data.

Development of sensitive detection of cryptosporidium and giardia from surface water in iran.

BACKGROUND: The protozoan parasites Cryptosporidium spp. and Giardia are known to occur widely in both raw and drinking waters. They are two of the causative agents of waterborne outbreaks of gastroenteritis throughout the world. In the present study, a PCR assay and FA were developed for detection of Cryptosporidium oocysts and Giardia cyst in environmental samples. METHODS: We have detected Cryptosporidium spp. oocysts and Giardia cysts in seeded and unseeded environmental water samples by PCR method. Water samples were spiked with oocysts (50, 100,300,500) and filtrated with a 1.2-µm pore size cellulose nitrate and follow by DNA extraction and purification by QIAamp DNA mini kit. Nested-PCR assay amplified an 850 bp fragment of 18s rRNA gene specific for Cryptosporidium and 435 bp fragment of glutamate dehydrogenase (GDH) target gene for Giardia. Also many river water from north of Iran, be checked by these methods. RESULTS: Cryptosporidium and Giardia DNAs were detected in seeded water sample and Giardia was detected in all 5 water samples from river in north of Iran by nested- PCR and FA. Also in one river water sample, Cryptosporidium was detected. CONCLUSION: This protocol is effective for detection of these waterborne parasites in treated and untreated water samples. This study can also serve as a platform for further investigations and research water source in Iran.

Gene cloning, expression and serological evaluation of the 12-kDa antigen-B subunit from Echinococcus granulosus.

A 12-kDa subunit of antigen B from Echinococcus granulosus has recently been cloned, expressed and used in diagnostic ELISA to test human sera for evidence of cystic echinococcosis. The performance of the ELISA based on the recombinant antigen (rAgB) was compared with that of similar assays based on native antigen B (nAgB) or hydatid-cyst fluid. For the preparation of the rAgB, total RNA was extracted from Ec. granulosus protoscoleces so that antigen-B complementary DNA could be synthesised, amplified by PCR, and then cloned into the pQE30 expression vector. The recombinant plasmid was transformed in Escherichia coli and induced using isopropyl-beta-D-thiogalactopyrano-side. Bacterial samples were collected, lysed and then analysed by SDS-PAGE and western blotting. The recombinant protein was purified by affinity chromatography. Although the performance of the ELISA based on cyst fluid appeared identical to that of the assay based on the recombinant antigen (with a sensitivity, specificity, positive predictive value and negative predictive value of 96.0%, 97.0%, 97.2% and 95.5%, respectively), the corresponding results for the ELISA based on nAgB (98.6%, 100%, 100% and 98.5%) were slightly better. Despite this difference (which was not statistically significant), the comparative ease with which large quantities of the recombinant antigen could be produced make the antigen a potentially useful tool in the diagnosis of cystic echinococcosis.

An efficient procedure for purification of recombinant human ß heat shock protein 90.

BACKGROUND AND THE PURPOSE OF THE STUDY: Heat Shock Protein 90 (Hsp90) is typically the most abundant chaperone in the eukaryotic cell cytoplasm, and its expression is essential for loading immunogenic peptides onto major histocompatibility complex molecules for presentation to T-cells. Therefore, it may act as a good candidate as an adjuvant molecule in vaccine technology. METHODS: Initially the human Hsp90ß gene was cloned into the heat inducible expression vector pGP1-2 and then the recombinant protein was isolated by ion exchange chromatography. After intradermal injection of confirmed purified band of protein to rabbits and isolation of the serum IgG antibody, for its affinity purification, the rabbit's purified Hsp90 specific IgG was coupled to the cyanogen bromide-activated Sepharose 4B. RESULTS: The recovery of the purified protein of interest by affinity chromatography was 50%. CONCLUSION: This research enabled purification of human heat shock protein by a laboratory prepared column chromatography.

Cloning and expression of human vascular endothelial growth factor gene and inhibition of its expression by antisense in prokaryotic system.

BACKGROUND AND THE PURPOSE OF THE STUDY: Angiogenesis is an important process in physiology and disease pathogenesis and is controlled in a healthy body by a number of stimulatory and inhibitory factors. The aim of this study was to determine the effect of antisense transcript on the sense transcript of the endothelial growth factor (EGF) gene in bacterial system as an approach for the gene regulation in tumors. METHODS: The hepatoma cell line (HepG2) was stimulated by PMA. VEGF mRNA was used for RT-PCR. VEGF cDNA was synthesised and cloned into T-vector pTZ57R, then sense fragment of VEGF subcloned into pACYC Duet-1 expression vector and antisense VEGF subcloned into pCDNA3 expression vector. Recombinant plasmids were transforemed into BL21 bacterial cells. Expression of recombinant plasmid was analysed by western blot technique. RESULTS: The recombinant pCDNA3-VEGF (pYZantiVEGF) was successfully expressed in BL21 cells. Western blot analysis showed that the expression of VEGF decreased significantly in the cells transfected with VEGF antisense RNA compared with the pACYCDUET-1-VEGF (pYZsenseVEGF) transfected and control. MAJOR CONCLUSIONS: The expression of VEGF in BL21 cells was strong. In vitro, antisense of VEGF inhibited VEGF expression significantly in BL21 cells.

Serological Evaluation of EgAgB16 kDa, a Recombinant Antigen from Echinococcus granulosus for Diagnosis of Human Hydatidosis.

BACKGROUND: Regarding that accurate diagnosis of human hydatidosis still needs more investigations, the present study was conducted to clone, express, and evaluate the gene encoding AgB subunits (EgAgB16 kDa) from Echinococcus granulosus (Iranian G1 strain) and its evaluation by ELISA test. METHODS: DNA was extracted from protoscoleces and was utilized by PCR for strain identification. Total RNA was prepared with RNeasy protect mini kit from E. granulosus (Iranian G1 strain) protoscoleces collected from naturally infected sheep with hydatid cyst. Recombinant AgB16 kDa was produced using pETDuet as vector and evaluated by ELISA method. A panel of sera including hydatid cyst-infected individuals (n=72), healthy individual (n=48), toxoplasmosis (n=4), strongyloidosis (n=4), kala-azar (n=5) and tuberculosis (n=5) were examined using this recombinant antigen. RESULTS: Recombinant protein was purified by affinity chromatography using His-Tag column. After purification, recombinant protein was confirmed by western blot analysis using His Tag monoclonal antibody or hydatid positive human serum. The sensitivity, specificity; positive and negative predictive values were calculated as 93.5%, 95.6%, 96% and 92.9%, in that order. The cut-off point was detected 0.3 for rAgB16. CONCLUSION: While the produced recombinant AgB16 kDa showed promising results in diagnosing human hydatidosis, but more investigations should be implemented to reach an accurate gold standard.

Study on ITS1 Gene of Iranian Trichomonas vaginalis by Molecular Methods.

BACKGROUND: Trichomoniasis is a worldwide protozoan parasitic disease and metronidazole is a choice drug for its treatment. Because of disease importance in public health and its controversial ideas about the prevalence of drug resistance, this study was carried out. METHODS: Fifty-two suspected vaginal samples were collected from 2006 to 2007 in Gynecology Maryam Hospital, Tehran, Iran. All isolates were examined by microscopic, culture and PCR techniques. The PCR products were analyzed by RFLP and CSGE methods and two suspected samples were sequenced. RESULTS: Trichomonas vaginalis was identified from all 52 samples. Of 52 isolates, 45 samples were successfully cultured and amplified by PCR except one. Seven were positive only by PCR. Finally, ITS1 fragment was successfully amplified in 51 of 52. CSGE analysis and PCR products digestion by MspI followed by sequencing showed nucleotide mutation at position 209 (C209T) of the ITS1 fragment in two (3.9%) of them. CONCLUSION: The results showed mutation in ITS1 fragment of T. vaginalis in two (3.9%) of Iranian isolates which may be related to metronidazole resistance.

Non-radioactive labeled probe preparation for hbs gene detection.

Among some Bacillus species, a protein highly homologous to HU, classified HB and coded by hbs gene. According to the recent studies, the sequence of hbs gene just in one strain of Bacillus subtilis exists in gene bank (ATCC 23857). In this study, DNA from Bacillus subtilis ATCC 6633 was extracted and investigated by PCR. The PCR product was sequenced and shown to differ in just one nucleotide from B. subtilis ATCC 23857. Hence, it was chosen as reference and for the first time, used for non-radioactive labeled probe preparation. The PCR product in Bacillus subtilis with ATCC 6633 was labeled using non-radioactive DIG-labeled nucleotides and conditions of probe preparation and hybridization were optimized and checked it by Southern blotting.

Molecular identification of ovine Theileria species by a new PCR-RFLP method.

Theileria spp. infect wild and domestic ruminants in the tropical and subtropical regions of the world. Two species, T. lestoquardi and T. ovis, are suspected to cause ovine theileriosis in Iran. The epidemiological aspects of ovine theileriosis in Iran are poorly understood, and further investigations by sensitive and precise techniques are required. In this study, the use of a nested PCR for amplification of a fragment of the 18S ribosomal DNA from virtually all species of Theileria is described. For differentiation of various Theileria spp. a RFLP assay was developed as a diagnostic tool enabling direct, concurrent, highly specific and sensitive identification of Theileria spp. The sensitivity of the nested PCR for Theileria species was 10(-5)% parasitemia. Restriction fragment length polymorphism (RFLP) of the PCR products allowed differentiation between three different Theileria species (T. annulata, T. lestoquardi and T. ovis) and seems to be useful for differentiation of other species such as T. separata and Theileria spp. china. From 100 field blood samples obtained from sheep in East and South-East Iran, 56% were positive for Theileria spp. by nested-PCR compared with 21% by microscopic examination. Out of 56 positive samples, 12.5% (7/56) were positive for T. ovis and 87/5% (49/56) were positive for T. lestoquardi. This is the first report in which T. ovis has been detected in Iran using molecular identification techniques.