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Cutaneous Leishmaniasis (CL) is one of the world's neglected diseases which is caused by Leishmania spp. The aim of this study was to assess molecular profile and antimony resistance of Leishmania isolated from human and rodent hosts. Samples were collected from suspected CL patients referred to health centres and wild rodent's traps in Gonbad-e-Qabus region, north-eastern Iran. Smears were subjected to PCR-RFLP to identify Leishmania species. In addition, ITS1-PCR products were sequenced for phylogenetic analysis. Clinical isolates and rodent samples were subjected to MTT assay to determine IC50 values and in vitro susceptibilities. Expression levels of antimony resistance-related genes were determined in CL isolates. Out of 1,949 suspected patients with CL and 148 rodents, 1,704 (87.4%) and 6 (4.05%) were positive with direct smear, respectively. Digestion patterns of BusRI (HaeIII) endonuclease enzyme were similar to what expected for Leishmania major. Phylogenetic analysis revealed that the highest interspecies similarity was found between current L. major sequences with L. major obtained from Russia and Uzbekistan. Out of 20 L. major samples tested, 13 (65%) were resistant to meglumine antimoniate (MA) treatment, with an activity index (AI) exceeding 4. The remaining 7 samples (35%) responded to MA treatment and were classified as sensitive isolates, with a confirmed sensitive phenotype based on their AI values. The comparison expression analysis of three major antimony resistance-associated genes in unresponsive clinical isolates demonstrated significant fold changes for TDR1 (4.78-fold), AQP1 (1.3-fold), and γ-GCS (1.17-fold) genes (P < 0.05). Herein, we demonstrate genetic diversity and antimony resistance of L. major isolated from human and reservoir hosts in north-eastern Iran, which could be the basis for planning future control strategies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: In recent years, cases of human visceral leishmaniasis (HVL) have been reported in some districts of Golestan Province, northeastern Iran, particularly in rural areas. Recent epidemiological evidence in Leishmania infantum endemic regions of in Iran indicates approximately 50%–80% of seropositive dogs are asymptomatic for Leishmania infection. Objectives: The goal in this study was to determine Leishmania species infecting domestic dogs in Golestan Province, Iran. Methods: Between 2015 and 2016, blood samples were obtained from 100 domestic dogs in rural regions of Golestan Province, northeastern Iran. All samples were tested for anti‐Leishmania antibodies using a direct agglutination test (DAT), and for Leishmania spp. kinetoplast DNA (kDNA) using PCR. Results: Seven (7%) dogs were antibody positive and 25 dogs (25%) were Leishmania spp. DNA positives by PCR positive for leishmaniasis. Four of the seven (71%) antibody‐positive dogs and 19 of the 25 (76%) PCR‐positive dogs were asymptomatic. The rate of infection detected by PCR was significantly higher in male dogs (21/75, 28%) than that in female dogs (4/25, 16%). The ITS1 PCR‐RFLP assay identified the presence of L. infantum, L. tropica or Crithidia spp. in the 25 PCR‐positive samples. Conclusions: The high proportion of asymptomatic dogs in the study areas represent they act as potential reservoirs in the transmission cycle of Leishmania spp. and also Crithidia fasciculata as an emerging agent for the first time. Moreover, our data showed that PCR is a more reliable assay than DAT for detecting Leishmania spp. infection among asymptomatic dogs. [ABSTRACT FROM AUTHOR]

Introduction: Echinococcosis or hydatid disease is one of the most important zoonosis across the world. Materials and Methods: The recombinant P29 protein is considered as a suitable candidate for evaluating diagnostic ELISA to reach a more accurate conclusion. Results: In this study the P29 sequence comprised with somewhat similar sequences registered in Gene Bank and showed 100% identity with P29 sequences derived from Echinococcus granolosus (E. granulosus) (XP_024351425.1), E. multilocularis (AHA85396.1), Endophilin B1 protein of E. granulosus (CDS21096.1). The primary and secondary structure of the peptide was analyzed due to its characterization of peptide motifs to be expressed on MHC Class HLA allels. Conclusion: The recombinant P29 derived peptides can be produced and analyzed due to be determined as vaccine design candidate against Hydatidosis. [ABSTRACT FROM AUTHOR]

Background: Fascioliasis is a neglected zoonotic disease, caused by Fasciola species in human and livestock. We aimed to detect the seroprevalence of human fascioliasis Gorgan City, Golestan Province, northern Iran using ELISA method in 2017. Methods: Overall, 612 serum samples were analyzed. A relevant questionnaire for demographic data was obtained for all cases. An indirect ELISA test was used to detect IgG antibodies against Fasciolain the sera. The data analysis was performed employing SPSS program version 21. Results: Eleven cases (1.79%) were seropositive for fascioliasis. The seroprevaence of fascioliasis was 1.9% and 1.1% among males and females, respectively. There was no statistically significant association between the fascioliasis and ana-lyzed variables such as sex, age, residence, job, education, etc. Conclusion: This study was conducted only on the people referring to the Ref-erence Laboratory of Gorgan. It cannot be distributed to the whole city. Thus, due to importance of the disease, finding the seroprevalence of fascioliasis in a comprehensive survey in Golestan Province should be accounted in further studies. [ABSTRACT FROM AUTHOR]

Background: Canids are definitive hosts of Echinococcus multilocularis and Echinococcus granulosus. This study aimed to survey these two Echinococcus species in canids of North-Khorasan Province, northeastern Iran, using morphological criteria and genetic characterization of mitochondrial DNA. Methods: The carcasses of 106 canids, namely 61 jackals (Canis aureus), 23 foxes (Vulpes vulpes), 19 dogs (Canis familiaris) and three wolves (Canis lupus) were collected from the study area in 2013–2014 and examined for Echinococcus species. Morphological features were assessed by microscopy of adult worms. For molecular characterization, DNA was extracted, mostly from the adult worms but also from eggs. DNA fragments of the cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) mitochondrial genes were amplified and sequenced. Sequences were aligned and compared with reference sequences. Intraspecific and interspecific diversity were calculated and phylogenetic analysis was performed. Results: Overall, 9.4% of the canids (eight jackals and two foxes) were found infected with E. multilocularis by molecular methods, of which seven cases were also confirmed using morphological description of the adult worms. Echinococcus granulosus was found in 6.6% of the canines (four dogs, two jackals and one wolf) as determined by both molecular methods and adult cestode morphology. All E. granulosus isolates were identified as the G1 genotype. Comparative sequence analysis indicated 0–0.7% and 0% intraspecific divergence within E. granulosus isolates and 0% and 0–0.2% within E. multilocularis isolates for cox1 and nad1, respectively. Conclusions: This study revealed the presence of E. multilocularis and E. granulosus in canids of North-Khorasan Province of Iran. Jackals were found infected with both E. multilocularis and E. granulosus, but infection with the former species was higher. [ABSTRACT FROM AUTHOR]

Background: Hydatidosis is a neglected global zoonotic disease, caused by larval stage of the cestode Echinococcus granulosus in human and animal. Because of high economic and medical importance of the disease, this study was performed to find the seroprevalence of human hydatidosis in Gorgan City, Golestan Province, northern Iran. Methods: In this cross-sectional study, blood samples were collected from people referring to Reference laboratory of Golestan University of Medical sciences in 2017. A relevant questionnaire was completed for demographic data for each person. Echinococcus IgG antibody was investigated by ELISA using native antigen B. The data were analyzed using SPSS software applying logistic regression. Results: Overall, 612 blood samples were collected. Cut-off was considered 0.29. Sixteen cases (2.6%) were seropositive for hydatidosis. The seroprevalence of hydatidosis was 2.3% and 4.7% among males and females, respectively. There was no statistically significant correlation between the hydatidosis and investigated variables such as sex, age, tribes, residence, education, etc. Conclusion: The prevalence of human hydatidosis shows approximately the same range as other regions of Iran. Although due to the neighboring the Mazandaran Province reported as the highest seroprevalence of hydatidosis, we expected more rate of seropositivity. [ABSTRACT FROM AUTHOR]

Little is known about the genetic and morphological characters of Taenia ovis. The purpose of the present study was to characterize sheep isolates of T. ovis using rostellar hook morphometry as well as mitochondrial genes sequence analysis. Ninety sheep specimens of Cysticercus ovis were collected from 18 slaughterhouses in Iran. The mean ± s.d. for total length of large and small hooks were 174.1 ± 6.4 and 116.7 ± 5.4 µ m, respectively. CO1 and 12S rRNA sequence analysis showed 11 and nine haplotypes, respectively. The level of pairwise nucleotide variations between individual haplotypes of CO1 and 12S rRNA genes were 0.3–1.1 and 0.2–1.0%, respectively. Level of nucleotide variation in CO1 and 12S rRNA between T. ovis haplotypes from present study and eight other Taenia species was found to be 11.3–17.8 and 5.3–16.3%, respectively. Phylogenetic analysis clustered all T. ovis isolates into a single clade comprised of the all CO1 and 12S rRNA haplotypes. CO1 nucleotide difference between T. ovis ovis and T. asiatica was 13.6% that is lesser than the corresponding difference between T. ovis ovis and T. ovis krabbei , warranting the designation of two separate species as T. ovis and T. krabbei. Interclass correlation coefficients showed that there was no significant association between rostellar hook length variation and the variability of the mitochondrial genes. [ABSTRACT FROM AUTHOR]

Background: The aim of the present study was genotyping of Echinococcus granulosus isolates from dogs and jackals in Mazandaran Province, northern Iran, and using partial sequence of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1). Methods: E. granulosus isolates (n = 15) were collected from 42 stray dogs and 16 jackals found in south of the Caspian Sea in northern Iran. After morphological study, the isolates were genetically characterized using consensus sequences (366bp) of the cox1 gene. Phylogenetic analysis of cox1 nucleotide sequence data was performed using a Bayesian Inference approach. Results: Four different sequences were observed among the isolates. Two genotypes [G1 (66.7%) and G3 (33.3%)] were identified among the isolates. The G1 sequences indicated three sequence profiles. One profile (Maz1) had 100% homology with reference sequence (AN: KP339045). Two other profiles, designated Maz2 and Maz3, had 99% homology with the G1 genotype (ANs: KP339046 and KP339047). A G3 sequence designated Maz4 showed 100% homology with a G3 reference sequence (AN: KP339048). Conclusion: The occurrence of the G1 genotype of E. granulosus sensu stricto as a frequent genotype in dogs is emphasized. This study established the first molecular characterization of E. granulosus in the province. [ABSTRACT FROM AUTHOR]

Nineteen hydatid cyst isolates collected from camels in central Iran were subjected to sequences analysis of mitochondrial cytochrome c oxidase subunit 1 ( cox1) and NADH dehydrogenase subunit 1 ( nad1) genes. A consensus sequence obtained containing 366 nucleotides for cox1 and 471 nucleotides for nad1 genes. Overall, the camel isolates indicated five different sequences in cox1 and nine in nad1 genes. The sequences analysis indicated that 26.3%, 42.1%, and 31.6% of isolates belonging to G1, G3, and G6 genotypes of Echinococcus granulosus, respectively. The isolates with G3 genotype indicated one cox1 sequence having 100% homology with reference G3 sequence (AN: M84663) and two different nad1 sequences, one having100% homology with reference G3 sequence (AN: AJ237634) and the other with a silent mutation (G to A) in position 279. The presence of G3 genotype (buffalo strain) of E. granulosus as dominant genotype in camels is emphasized. As G3 genotype has formerly been reported in human, the epidemiological role of camels is warranted in future surveys. [ABSTRACT FROM AUTHOR]

Echinococcosis/hydatidosis is one of the most important zoonotic diseases commonly found in different regions of Iran with a major economic and public health importance. In the current study, Echinococcus granulosus isolates were collected from hospitalized patients in Isfahan, central Iran. The genotypes of 30 samples were determined by polymerase chain reaction amplification of internal transcribed spacer-1 region of ribosomal DNA, followed by restriction fragment length polymorphism (RFLP) with two restriction enzymes namely AluI and MspI. As expected, each isolate yielded an approximately 1-kbp DNA fragment on the electrophoresis gel. According to RFLP results for both enzymes, all isolates had an equal pattern indicating the G1 genotype. Our findings confirmed that G1 is the dominant genotype of cystic echinococcosis in human in central Iran, with predilection to different organs including liver, lung, and brain, and warrants the importance of sheep dog cycle in public health. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Taenia multiceps is a widely distributed zoonotic tapeworm of canids. The larval stage of the parasite (Coenurus) occurs in sheep, goat and cattle and has been rarely reported from humans. This study investigated genetic variability of two mitochondrial genes in 102 isolates of T. multiceps. Metacestodes were collected from brains and hearts of sheep in Tehran and Qom provinces of Iran. DNA of each isolate was extracted and used for PCR amplification of cytochrome c oxidase subunit I (CO1) and 12S ribosomal DNA (12S rRNA) genes. All amplicons were sequenced and the sequence data were analyzed using NCBI Blast and BioEdit. Phylogenetic trees and pairwise calculations were obtained by using Mega5 software. In total 7 and 25 representative haplotypes were differentiated for CO1 and 12S rRNA genes, respectively. For CO1 sequences 11 segregation sites within 7 haplotypes were observed. For 12S rRNA sequences a total of 32 segregation sites were observed in 25 haplotypes. CO1 gene displayed lower diversity than 12S rRNA gene with an overall nucleotide variation of 3.0% for CO1 vs. 7.2% for 12S rRNA. Pairwise comparisons among 7 haplotypes in CO1 and 12S rRNA genes showed the level of nucleotide differences 0.3–2.5% and 0.2–4.0%, respectively. A high degree of genetic variation was found in the isolates of T. multiceps in Iran. Additional molecular studies are required on the parasite from other intermediate hosts. [Copyright &y& Elsevier]

Abstract: Cystic echinococcosis is a zoonotic infection caused by the dog tapeworm, Echinococcus granulosus. In the present study, adults of E. granulosus (n=20) were collected from 71 dogs from Western Iran and were genetically characterized using DNA sequencing of the partial mitochondrial cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase 1 (nad1). Consensus sequences were obtained for cox1 (366) and nad1 (471) genes. Phylogenetic analysis of concatenated nad1 and cox1 nucleotide sequence data was performed using Bayesian Inference approach. Overall, the dog isolates indicated nine different sequences in cox1 and seven in nad1 genes. Three genotypes (G1 [75%], G2 [10%] and G3 [15%]) were identified from the isolates. The G2 sequences indicated 100% homology with reference G2 sequence in both cox1 (Genbank accession number M84662) and nad1 (AJ237633) genes. G3 sequences showed 100% homology with G3 reference sequence in nad1 (AJ237633), but displayed two different cox1 profiles, each having 99% homology with reference G3 sequence (M84663). In the phylogenetic tree all of the isolates were grouped into a distinct cluster corresponding to the G1–G3 complex with relevant reference sequences. The presence of G1 genotype (sheep strain) of E. granulosus sensu stricto as dominant genotype in dogs is emphasized. To the best of our knowledge, this study established the first record of E. granulosus sensu stricto, G2 genotype in Iran. [Copyright &y& Elsevier]

• Human toxocarosis is a neglected zoonosis with a worldwide distribution. • The overall seroprevalence of Toxocara spp. infection was found 2.85 % (11/386). • Toxocara spp. infection was high for some risk factors, but there were no statistically significant differences. • Toxocara spp. infection in children with eosinophilia (20 %) was high, but the difference was not statistically significant. The World Health Organization has categorized toxocarosis as a neglected tropical disease despite its significant impact on high-risk groups such as children. This study aimed to investigate the seroprevalence, risk factors, and clinical symptoms of Toxocara spp. infection among children 3–15 years old in northern Iran. A total of 386 children were enrolled in the study. All serum samples were tested for the presence of IgG antibodies against Toxocara spp. infection using an enzyme-linked immunosorbent assay. Moreover, relevant risk factors and clinical symptom data were obtained using questionnaires. Data analysis was performed using the SPSS software version 24. The overall seroprevalence of Toxocara spp. infection was found 2.85 % (11/386). However, Toxocara spp. infection was high for some risk factors, including eating soil (14.3 %), contacting cats (6.7 %), and consuming raw vegetables (3.7 %). However, there were no statistically significant differences regarding the risk factors and socio-demographic characteristics. Considering the clinical symptoms, Toxocara spp. infection was different in children with eosinophilia (20 %), ocular disorders (8.3 %), skin disorders (7.7 %), liver disorders (4.5 %), and stomach ache (4.2 %), although not statistically significant. The results revealed that the seroprevalence of Toxocara spp. infection was relatively low in children in northern Iran. It is suggested to conduct more studies in different parts of Iran to gain a deeper understanding of the toxocarosis seroprevalence and its status in high-risk groups such as children with asthma, hypereosinophilic syndrome, allergic skin disorders, and epilepsy. [ABSTRACT FROM AUTHOR]

Abstract: 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 842bp 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. [Copyright &y& Elsevier]

Ovine theileriosis as a critical agent in small ruminant production, can cause lethal infections. Different species of Theileria have been reported in various parts of the world, and each species causes different diseases in the host. This is the first molecular study to investigate the prevalence of ovine theileriosis and identify the dominant Theileria species in northern Iran. A number of 220 small ruminants, including sheep and goats, were randomly sampled from 22 flocks. Peripheral blood smears were stained by the Giemsa staining method. As well as for species identification, all samples were examined by PCR. From 220 samples, 160 and 60 were sheep and goat, respectively. By the Giemsa staining method, Theileria parasite was observed in 20 (9%) samples. But by polymerase chain reaction (PCR) method, 30 (13.6%) samples were positive for Theileria species. Theileria lestoquardi was the most common species found in these animals. The high prevalence of theileriosis in small ruminants demonstrates the emergence of ovine theileriosis in Mazandaran and Golestan provinces in northern Iran.

Previous marine biology studies found that the concentration of heavy metals in some parasites of fish such as acanthocephalans can be a proper bioindicator. Therefore, we attempted to measure five heavy metal concentrations in the tissues of the fish Gasterosteus aculeatus (G. aculeatus) and its acanthocephalan parasites, Corynosoma caspicum (C. caspicum) from the Southern Caspian Sea, northern Iran. G. aculeatus (three-spined stickleback) was collected from the south of the Caspian Sea, Mazandaran Province, northern Iran. After tissue preparation, the heavy metal concentrations in fishes and acanthocephalans were obtained using the tissue dissolution technique and an atomic absorption spectrophotometer. The concentrations of chromium (Cr), cadmium (Cd), lead (Pb), zinc (Zn), and copper (Cu) in the skin, liver, muscle, and intestine tissues of the fish and its parasites, C. caspicum, were measured and compared. Eighty (32%) of 250 collected fish were infected by at least one acanthocephalan parasite. The Cr indicated the highest concentration (5.329±3.275) of the heavy metals in acanthocephalan, even more than the skin, liver, and muscle of infected fishes. Cd had the lowest concentration (0.0333±0.0075) of heavy metals in acanthocephalan, but it was still higher than the concentration in the infected fishes' skin, liver, muscle, and intestine tissues. Our findings indicated that C. caspicum parasites can be considered extremely sensitive early-alert bioindicators, particularly in sensitive and under-threat environments with low pollution levels.

Cystic echinococcosis (CE), caused by the larval stage of Echinococcus granulosus, presents an important medical and veterinary problem globally, including that in Iran. Different genotypes of E. granulosus have been reported from human isolates worldwide. This study identifies the genotype of the parasite responsible for human hydatidosis in three provinces of Iran using formalin-fixed paraffin-embedded tissue samples. In this study, 200 formalin-fixed paraffin-embedded tissue samples from human CE cases were collected from Alborz, Tehran, and Kerman provinces. Polymerase chain reaction amplification and sequencing of the partial mitochondrial cytochrome c oxidase subunit 1 gene were performed for genetic characterization of the samples. Phylogenetic analysis of the isolates from this study and reference sequences of different genotypes was done using a maximum likelihood method. In total, 54.4%, 0.8%, 1%, and 40.8% of the samples were identified as the G1, G2, G3, and G6 genotypes, respectively. The findings of the current study confirm the G1 genotype (sheep strain) to be the most prevalent genotype involved in human CE cases in Iran and indicates the high prevalence of the G6 genotype with a high infectivity for humans. Furthermore, this study illustrates the first documented human CE case in Iran infected with the G2 genotype., (© The American Society of Tropical Medicine and Hygiene.)

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

Accurate morphological differentiation between the liver fluke species Fasciola hepatica and Fasciola gigantica is difficult. We evaluated PCR-restriction enzyme profiles of internal transcribed spacer 1 (ITS1) that could aid in their identification. Fifty F. hepatica and 30 F. gigantica specimens were collected from different hosts in three provinces of Iran. For DNA extraction, we crushed fragments of the worms between two glass slides as a new method to break down the cells. DNA from the crushed materials was then extracted with a conventional phenol-chloroform method and with the newly developed technique, commercial FTA cards. A primer pair was selected to amplify a 463-bp region of the ITS1 sequence. After sequencing 14 samples and in silico analysis, cutting sites of all known enzymes were predicted and TasI was selected as the enzyme that yielded the most informative profile. Crushing produced enough DNA for PCR amplification with both the phenol-chloroform and commercial FTA card method. The DNA extracted from all samples was successfully amplified and yielded a single sharp band of the expected size. Digestion of PCR products with TasI allowed us to distinguish the two species. In all samples, molecular identification was consistent with morphological identification. Our PCR-restriction enzyme profile is a simple, rapid and reliable method for differentiating F. hepatica and F. gigantica, and can be used for diagnostic and epidemiological purposes., (Copyright 2009 Elsevier Inc. All rights reserved.)

In the present study, we have extended earlier taxonomic, biochemical and experimental investigations to characterize Echinococcus granulosus from various hosts in Iran utilizing DNA regions (designated pcox1 and pnad1) within the cytochrome c oxidase subunit 1 and NADH dehydrogenase 1 mitochondrial genes, respectively. An emphasis was placed on the characterization of E. granulosus isolates (cyst material) from humans, sheep, goats, cattle and camels, and on assessing their genetic relationships. PCR-based SSCP analysis of pcox1 and pnad1 amplicons derived from individual isolates (n=148) of E. granulosus revealed five (pc1-pc5) and nine (pn1-pn9) electrophoretic profiles, respectively. Sequencing of pcox1 and pnad1 amplicons representing unique SSCP profiles demonstrated that each profile was linked unequivocally to a particular sequence and that single point mutations were readily detectable by SSCP. Phylogenetic analyses of pcox1 and/or pnad1 nucleotide sequence data were conducted using Bayesian inference and maximum likelihood tree-building methods. Following the phylogenetic analyses of concatenated pcox1+pnad1 sequence data, including representatives of all presently recognized Echinococcus species/genotypes as well as Taenia saginata (as the outgroup), the majority of cyst isolates (142 of 148; 95.9%) from humans, ruminants (sheep, goats and cattle) and camels were assigned to the G1-G3 complex of E. granulosus (or E. granulosus sensu stricto), whereas some E. granulosus cysts (6 of 19; 31.6%) from camels were assigned to the G6-G10 complex (or E. canadensis). The present study reinforces the advantages of the mutation scanning-sequencing-phylogenetic approach to explore variation in multiple mitochondrial loci within and among Echinococcus populations, which provides a platform for future, detailed studies of the molecular epidemiology of E. granulosus in Iran and other countries. (Note: The sequences determined in the present study have been deposited in the GenBank database under accession numbers: FJ796203-FJ796207 (pcox1) and FJ796208-FJ796216 (pnad1)).