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Background and Aim: Cystic echinococcosis (CE), as a major zoonotic helminth infection, imposes remarkable socioeconomic burden on many endemic countries across the world, including Iran. Due to the high importance of free-roaming dogs in the transmission of CE, epidemiological and molecular studies in this type of hosts are required in the endemic regions. This study aimed to investigate the epidemiology and genotyping of Echinococcus granulosus isolated from stray dogs in Kerman, Southeast Iran. Materials and Methods: Eighty-four samples were isolated from stray dogs in the city and suburbs of Kerman in coordination with the health authorities and the municipality office for rabies control and dog population management. Dog demographic data, including age and sex were collected. The worm was isolated by necropsy and genomic DNA was extracted and partial cytochrome c oxidase subunit 1 gene was amplified using specific primers. Phylogenetic and Templeton-Crandall-Sing (TCS) network analyses were carried out on the sequence data. Results: The overall prevalence of CE in the surveyed dogs was 10.7% (9/84 cases). Out of 84 stray dogs, 33 (39.3%) and 51 (60.7%) cases were male and female, respectively. There was not a statistically significant difference between the infection and gender of dogs. However, infection is shown more in dogs under one year of age with a statistically significant difference (p

Our knowledge on the susceptibility of humans to different genotypes of the zoonotic tapeworm Echinococcus granulosus and the genetic make-up of the cysts in multi-organ involvements is li...

Background: Cystic echinococcosis, a major public health and economic concern, is a zoonotic helminth infection with worldwide distribution. This study was conducted to investigate the genetic characteristics of hydatid cysts isolated from human and livestock in Hamadan region, western Iran during 2016-2017. Methods: Ten human hydatid cysts and 40 animal hydatid cysts including 32 sheep, 5 cattle and 3 goats were genotyped by PCR amplification of two mitochondrial genes, cox1 and nad1. Genetic identification of the isolates was performed by using bioinformatics software and mtDNA nucleotide sequences of the parasite, available in GenBank database. Results: The PCR amplification was successfully carried out on 50 hydatid cyst isolates and then the nucleotide sequencing was conducted. The sequence analysis of the samples found that the isolates belonged to E. granulosus sensu stricto including G1 (42/50, 84), G2 (4/50, 8) and G3 (4/50, 8) genotype. The G1 genotype was detected in human (8/10, 80), sheep (26/32, 81), cattle (5/5, 100) and goat (3/3, 100) hydatid cysts. The G2 and G3 genotypes were found only in sheep and human isolates. Alignment analysis of the cox1 and nad1 gene sequences revealed thirteen and ten sequence types, respectively. Conclusion: G1 was the prevailing genotype of E. granulosus in the area and dogsheep transmission cycle should be considered when implementing hydatidosis control programs. In addition, high genetic diversity was detected among the hydatid cyst isolates. © 2019, Tehran University of Medical Sciences (TUMS). All rights reserved.

Echinococcosis is considered a cosmopolitan zoonosis caused by different species of small taeniid tapeworms of the genus Echinococcus and is regarded as a neglected zoonosis. Cystic and alveolar echinococcoses are endemic diseases of Tibetan, Pamir, and Iranian plateaus. All of the countries within the Iranian plateau are affected by echinococcosis. Pakistan, Turkey, and Iran are the three most populous countries of the region, in which echinococcosis is highly endemic. The three neighboring countries share strong cultural and socioeconomic ties. The present study aimed to provide a broad review of the status of cystic and alveolar echinococcosis, summarizing the current knowledge about geographical distribution, molecular epidemiology, and transmission dynamics of Echinococcus granulosus sensu lato and Echinococcus multilocularis in this region. Additionally, we aimed to understand disease burden and risk factors as basic requirements for establishing a surveillance system and planning prevention and control programs. A considerable body of information is available on different aspects of echinococcosis in this region; however, several information and research gaps need to be filled before planning control programs. None of the countries in the region have an elaborate echinococcosis control program. Effective control programs require multi/intersectoral coordination within a One Health approach with a long-term political and administrative commitment and enhanced international collaboration among the three countries.

Understanding dynamics of free-roaming dog (FRD) population is critical for planning and implementation of dog population management programs. FRD population size estimation as well as dynamic modeling of dog population under different female dog neutering interventions were investigated in order to determine the most appropriate animal birth control approach. We performed population size estimate of dogs using sight-resight surveys by photography in a randomly selected 25 blocks of the city and all the suburbs of greater Kerman area. Main demographic features were characterized and the dog density distribution was mapped. A dynamic model was developed to predict free-roaming dog population variations after 5 and 10 years. Different scenarios based on 10, 30, 50, 60 and 70% female dog sterilization were considered to predict the effects of animal birth control measures. Free roaming dog population was estimated at 6781 dogs (65.3% males) in Kerman and suburbs with several major population hotspots. Analysis of the dog locations within the city showed that the largest proportion of the dogs were observed in the vacant lots (46.2%). Modeling predictions indicated that, in the absence of management, the free-roaming dog population could increase from a baseline of 6781 to 13,665 dogs (2.02 fold increase) in 5 years and to 19,376 dogs in 10 years (2.86 fold increase). Using a population dynamics model, we simulated five neutering coverages to explore the impact of female neutering on free-roaming dog population size. The 5-year projections of the model have shown that 50% annual female dog sterilization significantly reduced free-roaming dog population by 0.44 comparing to the baseline population. Findings of the present study improve our knowledge on the nature and extent of dog population dynamics in Iran. Effective population control and selection of the most appropriate neutering interventions require a comprehensive knowledge of the characteristics and dynamics of FRD population.

Cystic echinococcosis (CE) is an important public health challenge in Iran that requires a One Health approach to control. Filling information gaps and research needs on different aspects of the disease, including parasite biology, disease ecology, human/animal infection, and administrative issues, is essential to address challenges faced by CE control programs.

Little is known about the genetic and morphological characters ofTaenia ovis. The purpose of the present study was to characterize sheep isolates ofT. ovisusing rostellar hook morphometry as well as mitochondrial genes sequence analysis. Ninety sheep specimens ofCysticercus oviswere 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 ± 5.4µm, 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 betweenT. ovishaplotypes from present study and eight otherTaeniaspecies was found to be 11.3–17.8 and 5.3–16.3%, respectively. Phylogenetic analysis clustered allT. ovisisolates into a single clade comprised of the all CO1 and 12S rRNA haplotypes. CO1 nucleotide difference betweenT. ovis ovisandT. asiaticawas 13.6% that is lesser than the corresponding difference betweenT. ovis ovisandT. ovis krabbei, warranting the designation of two separate species asT. ovisandT. krabbei. Interclass correlation coefficients showed that there was no significant association between rostellar hook length variation and the variability of the mitochondrial genes.

Synanthropic fly species can be potential mechanical vectors of many infectious agents. The potential of the flies to carry Echinococcus granulosus eggs is not fully documented. The purpose of the present study was to determine the possible role of non-biting flies to carry taeniid eggs. A total of 210 flies were collected from seven selected sites in areas of Kerman city, southeastern Iran from November 2016 to May 2017. Adult flies were live-caught using sweeping nets. Flies were placed individually in small glass bottles and transported to the laboratory. All the flies were killed by deep freezing and then identified to the species level using both morphological and molecular methods. The flies were homogenized in test tubes and genomic DNA was extracted and amplified by PCR. PCR protocols were used both to identify the live-caught flies to the species level, and for the detection of E. granulosus. The laboratory reared second generation flies were experimentally exposed to dog feces manually spiked by Echinococcus eggs. Two runs of experiments with 1–3 h of exposure were designed. For each experiment 20 flies were selected from the stock colony and were starved for three days. After each experiment, the flies were frozen for further molecular studies. The dominant fly species were Musca domestica and Lucilia sericata. No eggs were found on the body surface and/or guts of live-caught flies. After the first hour of exposure, 60%, of the flies of both species were found to harbor Echinococcus eggs. However, in the case of L. sericata 50% of the flies harbored Echinococcus eggs after 3 h of exposure. Results of the present study indicate the probable role of synanthropic flies in harboring Echinococcus eggs and mechanical transmission of cystic echinococcosis. When the helminth eggs are susceptible to desiccation grooming flies can remove many of eggs from exterior surfaces of them. Despite this result the role of synanthropic flies in the transmission of certain helminthiases should not be discounted because of their vagility and feeding mechanisms.

To investigate the genetic variability and population structure of Echinococcus granulosus complex, 79 isolates were sequenced from different host species covering human, dog, camel, goat, sheep and cattle as of various geographical sub-populations of Iran (Northwestern, Northern, and Southeastern). In addition, 36 sequences of other geographical populations (Western, Southeastern and Central Iran), were directly retrieved from GenBank database for the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. The confirmed isolates were grouped as G1 genotype (n=92), G6 genotype (n=14), G3 genotype (n=8) and G2 genotype (n=1). 50 unique haplotypes were identified based on the analyzed sequences of cox1. A parsimonious network of the sequence haplotypes displayed star-like features in the overall population containing IR23 (22: 19.1%) as the most common haplotype. According to the analysis of molecular variance (AMOVA) test, the high value of haplotype diversity of E. granulosus complex was shown the total genetic variability within populations while nucleotide diversity was low in all populations. Neutrality indices of the cox1 (Tajima's D and Fu's Fs tests) were shown negative values in Western-Northwestern, Northern and Southeastern populations which indicating significant divergence from neutrality and positive but not significant in Central isolates. A pairwise fixation index (Fst) as a degree of gene flow was generally low value for all populations (0.00647-0.15198). The statistically Fst values indicate that Echinococcus sensu stricto (genotype G1-G3) populations are not genetically well differentiated in various geographical regions of Iran. To appraise the hypothetical evolutionary scenario, further study is needed to analyze concatenated mitogenomes and as well a panel of single locus nuclear markers should be considered in wider areas of Iran and neighboring countries.

Blastocystis is a parasite that colonizes in the human intestine. Its clinical features include diarrhea, abdominal pain, or urticarial and irritable bowel syndrome (IBS). Spite of being significant genetic diversity and numerous subtypes within the genus there were no associations between its subtypes and symptomatology. Aim of this project was subtyping of the protozoa in 184 Iranian people with history of IBS/IBD (n = 74) or chronic urticaria (n = 59) and individuals referred to general clinic (n = 51). Microscopic and molecular examinations used for identifying and subtyping of Blastocystis. Overall, frequency of the parasite was 24.46% while, 29.41% of people who referred to general clinic, 20.27%, and 25.42% of IBS/IBD and urticarial cases were infected, respectively. Subtyping result showed that 28.89% of all people were infected with Blastocystis sp. while the prevalence of ST3, ST2 and ST1 were 22.22%, 22.22%, and 17.78%, respectively. Blastocystis sp., was identified in most IBS/IBD cases (46.7%) followed with ST2 and ST3 (13.3 and 13.3, respectively). Whereas, in chronic urticaria group ST2(33.3%) was the major subtype and most individuals in control group were infected with ST3 (33.3%). Pearson’s Chi Square test showed no significant differences between the parasite or subtype prevalence and diseases (p > 0.05). Given significant factors have effect on clinical signs including host or parasite genetics, microbiota, as well as environmental factors, it seems that further studies are needed to find out different markers of host susceptibility to diverse parasite genotypes in patients with irritable bowel syndrome or urticaria.

Background: In medicine, ozone therapy is effectively used in a broad spectrum of diseases. Reviews have shown that ozone gas demonstrates potent antimicrobial effects against a wide range of pathogenic microorganisms, such as oral bacteria, fungi, viruses, and parasite even in resistant strains. The present investigation was designed to assess the protoscolicidal effects of ozone gas on hydatid cysts protoscoleces in vitro and in vivo. Methods: Hydatid cyst protoscoleces were acquired from sheep livers that were slaughtered at Kerman slaughterhouse, Iran. The viability of protoscoleces was assessed by the eosin exclusion examination after exposure with ozone gas for 1 to 14 min in vitro and ex vivo. Results: In this study, in vitro assay showed that ozone gas at the concentration of 20 mg/L killed 85 and 100% of hydatid cyst protoscoleces after 4 and 6 min of treatment, respectively. However, in the ex vivo analysis, a longer time was needed to confirm a potent protoscolicidal activity such that ozone gas after an exposure time of 12 min, 100% of the protoscoleces were killed within the hydatid cyst. Conclusion: : In conclusion, the findings of the present study showed that ozone gas at low concentrations (20 mg/L) and short times (4-6 min) might be used as a novel protoscolicidal drug for use in hydatid cyst surgery. However, more clinical surveys are required to discover the precise biological activity of ozone gas in animal and human subjects.

Aim: Fascioliasis is one of the most zoonotic diseases with global extension. As the epidemiological distribution of Fasciola may lead to various genetic patterns of the parasite, the aim of this study is to identify Fasciola hepatica based on spermatogenesis, and phylogenetic analysis using mitochondrial (nicotiamide adenine dinucleotide dehydrogenase subunit I ND1 and cytochrome oxidase subunit I) gene marker. Materials and Methods: In this study, 90 F. hepatica collected from 30 cattle at slaughterhouse located in three different geographical locations in the North-East of Iran were evaluated based on spermatogenetic ability and internal transcribed spacer 1 gene restriction fragment length polymorphism pattern. Genetic diversity and phylogenetic relationship using mtDNA gene marker for the isolates from the North-East of Iran, and other countries were then analyzed. Results: Partial sequences of mtDNA showed eight haplotypes in both genes. The phylogenic analysis using neighbor joining as well as maximum likelihood methods showed similar topologies of trees. Pairwise fixation index between different F. hepatica populations calculated from the nucleotide data set of ND1 gene are statistically significant and show the genetic difference. Conclusion: F. hepatica found in this region of Iran has different genetic structures through the other Fasciola populations in the world. © Reaghi, et al. Open Access.

Blastocystis is a unicellular, anaerobic, eukaryotic protist, a common parasite found in the intestinal tracts of animals and humans. During the last few years, the host fecal DNA analysis by nucleic acid-based method has led to significant advances in Blastocystis diagnostics and enabled subtypes (STs). The zoonotic transmission of Blastocystis to humans is not well understood, therefore the present study was conducted to identify Blastocystis subtypes in Iran from different animal hosts from northwest of Iran. A total of 427 fresh fecal specimens were collected from cattle, sheep, poultry and pigeon (40,150,132,105 respectively). To detect the Blastocystis sp., each fecal specimen was examined microscopically. Total DNA from the samples that were positive for Blastocystis sp. was isolated, and the barcoding region of the small subunit of ribosomal rRNA (18S rRNA) was amplified and sequenced. Subsequently, sequence analyses, genetic diversity indices and evolutionary relationships of Blastocystis subtype populations were carried out. In total, 14.98% of the analyzed samples were positive for Blastocystis sp. and the subtypes detected were ST3,7,10 and 14. Among these, the ST10 was the main subtype that was found only in the cattle, sheep and poultry and the zoonotic subtype ST3 was present only from cattle. Our study shows the presence of Blastocystis subtypes in the sheep in north west of Iran and also demonstrated that the genetic approaches are crucial to understand the host specify of subtypes and the mode of infection. The study suggests that the genetic approaches will help us to understand the host specificity of subtypes and their role in infection if they are obtained from human and animals from the same geographical locations. Therefore, it is important to study the zoonotic aspects of this parasite with large number of samples from different groups of animals and from different geographical locations.

Fasciolosis is a zoonotic disease caused by Fasciola hepatica and Fasciola gigantica. Over the last decade, diagnostic tools to detect and differentiate Fasciola species have improved, but our knowledge of the distribution of haplotypes and gene flow of this parasite is not comprehensive yet. The purpose of this study was to investigate this gap in the epidemiology of F. hepatica in different provinces of Iran between 2015 and 2017. Isolated Fasciola were collected from abattoirs in 9 provinces. The partial sequence of mitochondrial NADH dehydrogenase subunit 1 (ND1) gene was used for the identification and molecular analysis of F. hepatica isolates. The amplified PCR products were purified and subjected to direct sequencing for subsequent construction of phylogenetic tree and network analysis. In the 130 subjects analyzed, 37 ND1 haplotypes were detected. This is the first study in Iran which investigates F. hepatica population and its genetic structure, based on mitochondrial ND1 marker in different geographical regions of Iran.

Background: Cystic echinococcosis is a zoonotic infection and considered as a major economic and public health concern worldwide. This research was conducted to determine genotypic characteristics of livestock and human hydatid cyst isolates from Hamadan area, western Iran. Methods: Sampling was conducted in Hamadan industrial slaughterhouse and Beast Hospital of Hamadan City, western Iran, from 2015 to 2016. Overall, 74 livestock isolates including 69 sheep, 3 cattle and 2 goats and 9 human hydatid cysts were genotyped by PCR amplification of the rDNA ITS1 region and followed restriction fragment length polymorphism (RFLP) analysis with four restriction endonuclease enzymes, RsaI, HpaII, AluI, and TaqI, and sequencing. Results: The PCR amplicon size of each isolate was approximately 1 kb which was the same with that of sheep strain. According to the RFLP patterns, the isolates belonged to a single species, E. granulosus sensu stricto (G1–G3 complex). Furthermore, sequencing of representative amplicons confirmed that the RFLP-genotyped isolates corresponded to E. granulosus sensu stricto. Conclusion: E. granulosus sensu stricto is the prevailing species of E. granulosus sensu lato in the region and pointed out the importance of sheep/dog cycle in human transmission.

Cystic echinococcosis (CE), caused by the metacestode of Echinococcus granulosus, is highly endemic over large parts of Iran. The purpose of this study was to investigate the prevalence rate of hydatidosis and mitochondrial cox1 real-time PCR with high-resolution melting curve (HRM) analysis of E. granulosus isolated from human and livestock. In this cross-sectional study, 61 formalin-fixed paraffin-embedded tissue isolates were collected from human CE cases and 83 hydatid cysts from the liver and lung lesions of the livestock in Khorasan Razavi province, Northeast Iran. DNA was extracted from each isolate and amplified by real-time PCR and analyzed using the HRM method. The HRM analysis using the cox1 gene of 40 E. granulosus human isolates showed that 35 (87.5%), 4 (10%), and 1 (2.5%) of the isolates were categorized as G1, G3, and G6 genotypes, respectively. Out of the total 1342 livestock inspected, 39 (4%) goats and 44(12%) cattle were found harboring hydatid cysts all belonging to E. granulosus sensu stricto. The results confirmed that the high prevalence of E. granulosus sensu stricto in intermediate hosts is remarkable in northeast of Iran coupled with the high prevalence of infection in livestock, which reinforced the need for hydatidosis control programs in this region.

OBJECTIVE: The purpose of the present study was morphometric and molecular characterization of Fasciola isolates from ruminants in Iran.METHODS: Flukes were collected from the livers of 54 naturally infected sheep and cattle. The proportion of body length to width (L/W) of each fresh fluke was measured using a digital caliper. We employed receiver operating characteristic (ROC) curve analysis to explore the reliability of L/W for differentiating the two species. Polymerase chain reaction (PCR)-sequencing was performed on ribosomal Internal Transcribed Spacers (ITS) and mitochondrial cytochrome c oxidase subunit 1 (cox1) genes. The sequences were then analyzed and phylogenetic relationships were investigated.RESULT: Forty-eight out of 54 isolates (88.9%) were identified as F. hepatica and four isolates (7.4%) as F. gigantica. All the sheep isolates were F. hepatica, while 4 out of 10 cattle were infected with F. gigantica. The morphometric study revealed an L/W ratio of 1.2 to 6.5 in Fasciola isolates with significantly higher L/W ratio in F. gigantica (p

Background: Dirofilariasis is a serious and potentially deadly condition in dogs and one of the zoonotic filarial infections, which inadvertently affects the humans. The objectives of this study were to determine the seroprevalence and the molecular identity of dirofilariasis in Kerman Province, southeastern Iran between Jul and Aug 2013. Methods: A hundred and forty-nine domestic dogs were randomly selected and five ml blood samples were taken from each dog. One ml of anticoagulant (EDTA) was used for each test in the parasitological study (modified Knott´s test) and sera samples were examined, using ELISA kit to detect Dirofilaria immitis antigen. Extracted DNA of all positive blood samples was used for molecular characterization and sequencing. Results: Four (2.7%) domestic dogs of the total 149 domestic dogs were infected with micofilariae of D. immitis, while the serological study showed 8 (5.4%) domestic dogs were infected with D. immitis. No signifi­cant difference, however, was found between dirofilariasis infec­tion and gender. On the other hand, a signifi­cant difference was observed between dirofilariasis infec­tion and age (P

The main objective of this study was to determine the prevalence of taeniid parasites and the specific detection of Echinococcus granulosus using copro-DNA polymerase chain reaction (PCR) analysis in the stray dogs of Kerman, south-eastern Iran. From September 2013 to May 2014, faecal samples of stray dogs were collected from different parts of the city of Kerman and its suburbs. Faecal samples from dogs were collected randomly within 24 h of defecation. All samples were transferred to the research lab and coprological examinations were conducted by the formalin–ether concentration method. In the microscopically positive samples, mitochondrial cytochrome c oxidase subunit 1 (cox1) specific primers were used to determine the taeniid identity of the infection. In addition, another set of primers was used for the specific diagnosis of E. granulosus sensu lato. In total, 307 faecal samples from stray dogs were examined for the presence of the parasites. Taeniidae eggs were detected in 34 dogs (11.07%). All 34 taeniid-positive specimens were PCR positive for cox1 (444 bp). Of all taeniid-positive specimens, 21 samples (6.8% of all dog specimens) were positive according to primers specific for E. granulosus. The findings of the present study revealed that canine echinococcosis is prevalent in the stray dogs in Kerman. The findings of the present study have important implications for hydatid control programmes in the area.

Objective: Echinococcus granulosus is a globally important cestode parasite causing remarkable medical and economical losses in the world. Ten genotypes (G1-G10) have been identified within this complex species. Protoscoleces rostellar hook characters e.g. total large and small hook lengths may be useful to differentiate genotypes. This study investigates the value of rostellar hook morphometry on genetically identified isolates of E. granulosus using mitochondrial cox1 and nad1 sequencing. Methods: In total, 24 hydatid cyst samples of livestock and human origin were collected. The isolates were then sequenced for the mitochondrial cox1 and nad1 genes and total large and small rostellar hook lengths of protoscoleces were measured. Results: Total large and small hook lengths could differentiate between G1 and G6 genotypes; however, G1 and G3 were not distinguishable by hook morphometry. Only large hook length was significantly different between the G3 and G6 isolates. Conclusion: The G6 genotype is readily distinguishable from G1 by using both small and large hook lengths; however, only total large hook length was significantly different between the G3 and G6 genotypes. (Turkiye Parazitol Derg 2012; 36: 215-8)

Cystic echinococcosis (CE) is a globally parasitic zoonosis caused by larval stages of Echinococcus granulosus. This study investigated E. granulosus genotypes isolated from livestock and humans in the Golestan province, northern Iran, southeast of the Caspian sea, using partial sequencing data of the cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase 1 (nad1) mitochondrial genes. Seventy E. granulosus isolates were collected from animals in slaughterhouses: 18 isolates from sheep, 40 from cattle, nine from camels, two from buffaloes and one from a goat, along with four human isolates (formalin-fixed, paraffin-embedded tissues) from CE patients of provincial hospitals. All isolates were successfully analysed by PCR amplification and sequencing. The sequence analysis found four E. granulosus genotypes among the 74 CE isolates: G1 (78.3%), G2 (2.7%), G3 (15%) and G6 (4%). The G1-G3 complex genotype was found in all of the sheep, goat, cattle and buffalo isolates. Among the nine camel isolates, the frequency of G1-G3 and G6 genotypes were 66.7% and 33.3%, respectively. All four human CE isolates belonged to E. granulosus sensu stricto. This study reports the first occurrence of the G2 genotype in cattle from Iran and confirms the previously reported G3 genotype in camels in the same country.

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.

Objective To find importance of morphometric criterion of larval rostellar hook of Echinococcus granulosus (E. granulosus) and the easy and reliable method for distinguish sheep and camel strains in epidemiologic studies. Methods Larval rostellar hooks ( n =1860) of 31 camel and sheep isolates in Iran, which already had been characterized by PCR, were carefully processed by computerized imagime analysis system (CIAS) and acquired data about rostellar hooks were analyzed using software SPSS. Results Measurement analysis of rostellar hooks [mean length (24.23±3.12) μ m] indicated that length of the large hook was a remarkable parameter for strain differentiation. Data analysis demonstrated that CIAS could be used as a reliable tool to distinguish camel from sheep strains with high sensitivity (95.2%) and specificity (91.5%). Conclusions CIAS as a specific, sensitive, economic, fast, and reliable means might be used for differentiation of E. granulosus strains. Although perimeter and area were measured by digital technology, they were not shown as discriminative criterion as total hook length did.

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)).

Background: Cutaneous leishmaniasis (CL) caused by Leishmania tropica is endemic in Kerman, southeastern Iran. While dogs have long been implicated as the main domestic reservoirs of L. infantum, etiological agent of zoonotic visceral leishmaniasis (ZVL), they can also carry L. tropica infection. The objective of the present study was to determine molecular identity and to evaluate histopathological changes due to CL in dogs in a well-known focus of anthroponotic CL (ACL) in Kerman, southeastern Iran. Methods: This study was carried out in three prospective series from 1994 to 2013 on dogs. Tissue samples were taken from 471 stray dogs. Pathological specimens including skin, spleen, liver and lymph nodes were prepared for paraffin blocks, sectioning and staining for further histopathological examination. PCR amplification of kDNA was performed to identify the causative agent and sequencing. Overall, two out of 471 stray dogs were infected with L. tropica. Hyperplasia of red pulp by the proliferation of histiocytes, lymphocytes, plasma cells and cytoplasm of histiocytes collection of amastigotes was noted. Results: Based on the results of PCR products and sequencing analysis, the parasites isolated from the lesions of two dogs were characterized as L. tropica, corresponding to a band of 830 bp Conclusion: This finding revealed infection with L. tropica in stray dogs in the city and suburbs of Kerman. This information is essential for public health concerns and planning effective future control programs. The role of dogs as potentional reservoir in the epidemiology of ACL needs further investigation.

High-resolution melting (HRM) is a reliable and sensitive scanning method to detect variation in DNA sequences. We used this method to better understand the epidemiology and transmission of Echinococcus granulosus. We tested the use of HRM to discriminate the genotypes of E. granulosus and E. canadensis. One hundred forty-one hydatid cysts were collected from slaughtered animals in different parts of Isfahan-Iran in 2013. After DNA extraction, the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene was amplified using PCR coupled with the HRM curve. The result of HRM analysis using partial the sequences of cox1 gene revealed that 93, 35, and 2 isolates were identified as G1, G3, and G6 genotypes, respectively. A single nucleotide polymorphism (SNP) was found in locus 9867 of the cox1 gene. This is a critical locus for the differentiation between the G6 and G7 genotypes. In the phylogenic tree, the sample with a SNP was located between the G6 and G7 genotypes, which suggest that this isolate has a G6/G7 genotype. The HRM analysis developed in the present study provides a powerful technique for molecular and epidemiological studies on echinococcosis in humans and animals.

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.

Taenia saginata is an important tapeworm, infecting humans in many parts of the world. The present study was undertaken to identify inter- and intraspecific variation of T. saginata isolated from cattle in different parts of Iran using two mitochondrial CO1 and 12S rRNA genes. Up to 105 bovine specimens of T. saginata were collected from 20 slaughterhouses in three provinces of Iran. DNA were extracted from the metacestode Cysticercus bovis. After PCR amplification, sequencing of CO1 and 12S rRNA genes were carried out and two phylogenetic analyses of the sequence data were generated by Bayesian inference on CO1 and 12S rRNA sequences. Sequence analyses of CO1 and 12S rRNA genes showed 11 and 29 representative profiles respectively. The level of pairwise nucleotide variation between individual haplotypes of CO1 gene was 0.3–2.4 % while the overall nucleotide variation among all 11 haplotypes was 4.6 %. For 12S rRNA sequence data, level of pairwise nucleotide variation was 0.2–2.5 % and the overall nucleotide variation was determined as 5.8 % among 29 haplotypes of 12S rRNA gene. Considerable genetic diversity was found in both mitochondrial genes particularly in 12S rRNA gene.

Most of cutaneous leishmaniasis cases occur in only 7 countries, including Iran. Leishmania tropica is the main cause of anthroponotic cutaneous leishmaniasis in Iran. In order to study the heterogeneity and phylogeny of L. tropica in southern Iran, a total of 61 isolates were obtained from Bam district and the cities Kerman and Shiraz. The internal transcribed spacer (ITS) from the ribosomal DNA locus was amplified and then analysed by sequencing. Analysis of the ITS sequences showed four haplotypes in the isolates, including 3 haplotypes among the 58 isolates from the south eastern region, including Bam district and Kerman city, and 2 haplotypes among the 3 isolates from Shiraz city. The results showed a monophyletic structure for the south eastern population. In comparison to GenBank sequences of L. tropica from different countries, most of the southeast Iranian and Indian isolates are comprised in one cluster, while isolates from other countries and few other Iranian isolates group in a different cluster. Analysis of ITS sequences of south eastern L. tropica showed a homogeneous population which could be the basis for other molecular epidemiology studies using more discriminative markers and tracing possible changes in the population structure of L. tropica.

AlthoughTaenia hydatigenais one of the most prevalent taeniid species of livestock, very little molecular genetic information exists for this parasite. Up to 100 sheep isolates ofT. hydatigenawere collected from 19 abattoirs located in the provinces of Tehran, Alborz and Kerman. A calibrated microscope was used to measure the larval rostellar hook lengths. Following DNA extraction, fragments of cytochromecoxidase 1 (CO1) and 12S rRNA genes were amplified by the polymerase chain reaction method and the amplicons were subjected to sequencing. The mean total length of large and small hooks was 203.4 μm and 135.9 μm, respectively. FortyCO1and 39 12S rRNA sequence haplotypes were obtained in the study. The levels of pairwise nucleotide variation between individual haplotypes of CO1 and 12S rRNA genes were determined to be between 0.3–3.4% and 0.2–2.1%, respectively. The overall nucleotide variation among all theCO1haplotypes was 9.7%, and for all the 12S rRNA haplotypes it was 10.1%. A significant difference was observed between rostellar hook morphometry and bothCO1and 12S rRNA sequence variability. A significantly high level of genetic variation was observed in the present study. The results showed that the 12S rRNA gene is more variable thanCO1.

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.

Reliable and rapid genotyping of large number of Echinococcus granulosus sensu lato isolates is crucial for understanding the epidemiology and transmission of cystic echinococcosis. We have developed a method for distinguishing and discriminating common genotypes of E. granulosus s.l. (G1, G3, and G6) in Iran. This method is based on polymerase chain reaction coupled with high resolution melting curve (HRM), ramping from 70 to 86 °C with fluorescence data acquisition set at 0.1 °C increments and continuous fluorescence monitoring. Consistency of this technique was assessed by inter- and intra-assays. Assessment of intra- and inter-assay variability showed low and acceptable coefficient of variations ranging from 0.09 to 0.17 %. Two hundred and eighty E. granulosus s.l. isolates from sheep, cattle, and camel were used to evaluate the applicability and accuracy of the method. The isolates were categorized as G1 (93, 94, and 25 %), G3 (7, 4, and 4 %), and G6 (0, 2, and 71 %) for sheep, cattle, and camel, respectively. HRM results were completely compatible with those obtained from sequencing and rostellar hook measurement. This method proved to be a valuable screening tool for large-scale molecular epidemiological studies.

Echinococcus granulosus, the aetiologic agent of cystic echinococcosis (CE), is one of the most important zoonotic helminthes worldwide. Isolates of the parasite show considerable genetic variation in different intermediate hosts. Several genotypes and species are described in different eco-epidemiological settings. This study investigated E. granulosus genotypes existing in livestock and humans from the province of Kerman, located in south-eastern Iran, using sequencing data of cox1 and nad1 mitochondrial genes. Fifty-eight E. granulosus isolates, including 35 from sheep, 11 from cattle, 9 from camels and 3 from goats, were collected from slaughterhouses throughout Kerman. One human isolate was obtained from a surgical case of CE. Mitochondrial cox1 and nad1 regions were amplified using polymerase chain reaction (PCR) and 38 isolates were sequenced. Genotypes G1 (73.7%), G3 (13.2%) and G6 (13.1%) were identified from the isolates. G1 was the most common genotype from sheep (86.7%), cattle (80%), camels (44.4%) and goats (100%). Sheep, cattle and camels were also found to be infected with the G3 genotype (buffalo strain). The human isolate was identified as the G6 genotype. Results showed that the G3 genotype occurred in different animal hosts in addition to G1 and G6 genotypes.

To describe a new emerging focus of anthroponotic cutaneous leishmaniasis (ACL) due to Leishmania tropica in rural areas of Dehbakry county, south-eastern Iran, after the earthquake of 2003.House-to-house survey of 3884 inhabitants for active leishmaniasis lesions or scars. The diagnosis was confirmed by smears, cultures and identification of the parasite by polymerase chain reaction (PCR).All age groups were affected, although patients ≤10 years of age showed the highest rate of infection (P = 0.0001). The overall prevalence rate was 5.3%; 6.3% in females and 4.3% in males. Of 204 cases, 1.8% had active sores and 3.5% had scars, with a significant difference between the sexes (P = 0.005). 47% of the lesions were on the face and 77.9% had one lesion. The incidence rose gradually 2004-2005, but grew exponentially 2006-2008. Electrophoresis of PCR products indicated that L. tropica was the causative agent.The current emergence was unexpected in this rural locality, where no previous history of CL was recorded. According to our knowledge this is the first report of a gradually establishing new ACL focus in rural communities after the 2003 earthquake.

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.

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.

Background: Toxoplasma gondii infection is widely prevalent in many species of warm-blooded animals including human. The aim of this study was to determine the prevalence of antibodies to T. gondii from slaughtered sleep and goats by mod­ified agglutination test (MAT) in Kerman region, southeastern Iran. Methods: Altogether 1340 blood samples were collected from 562 sheep and 778 goats from April to September 2005 in Kerman slaughterhouse. The sera were examined for T. gondii antibodies by MAT using an antibody titer of 1:20 or higher considered positive. The statistical analysis was performed by chi-square test and logistic regressions to analyze the influ­ence of all examined factor (age, sex and type of animals) on seroprevalence of toxoplasmosis. Results: Antibodies were found in sera of 262 out of 1340 (19.6%) samples. of 262 seropositive sera, 139 sheep (24.7%) and 123 goats (15.8%) were infected. Seropositive animals more than one year were 1.6 times more likely to be seropositive than the others were. Sheep were 1.5 times more likely to be infected than goats were (OR=1.53, 95% CI=1.15-2.04, p=0.004). Conclusion: Serological results indicated a widespread exposure to T. gondii among sheep and goats slaughtered in Kerman region and suggest that consumption of raw and undercooked meat of these animals can be a probable source of human tox­oplasmosis.