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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: 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]

A polycephalic larva of Taeniidae family isolated from abdominal cavity of a great gerbil (Rhombomys opimus) from Golestan province, northern Iran, was subjected to molecular analysis. Genomic DNA from the larva was obtained using a DNA extraction tissue kit. Polymerase chain reaction was performed for amplification of the partial 12S rRNA, cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase 1 (nad1) mitochondrial genes. BLAST analysis of DNA sequencing indicated 99.00% homology in 12S rRNA and cox1 genes and 98.00% homology in nad1 gene with Hydatigera krepkogorski (accession No. AB731762). The sequences of current isolate were deposited in GenBank by accession Nos. MF281971, MF281972 and MF281973 for 12 SrRNA, cox1 and nad1 genes, respectively. This study was the first report of molecular characterization of H. krepkogorski from Iran. Isolation and characterization of the adult stage from definitive host will help to better clarify incomplete life cycle and morphology data of this species in the world. [ABSTRACT FROM AUTHOR]

Background: Cryptosporidium is an intestinal protozean parasite causing water-borne and foodborne outbreaks of diarrheal diseases. The present study was performed in order to find prevalence and subtypes of Cryptosporidium among children with diarrhea in Gonbad Kavoos City, Northern Iran. Methods: Diarrheic samples were collected from 547 children. The initial parasitological diagnosis was made based on detection of oocysts using the modified Ziehl-Neelsen acid-fast staining method. The positive microscopically samples were selected for sequence analysis of partial 60 kDa glycoprotein (gp60) gene. Results: Out of 547 collected samples, 27 (4.94%) were positive for Cryptosporidium oocysts. Fifteen from 27 positive samples successfully amplified in PCR. Sequences analysis of gp60 gene in 15 Cryptoporidium isolates revealed that all of them (100%) were C.parvum. The results showed three subtypes of IIa subtype family (7 cases) including IIaA16G2R1, IIaA17G1R1, IIaA22G3R1 and one subtype of IId subtype family (8 cases). The most common allele was IId A17G1d (53.3%). Conclusion: The predominance of zoonotic subtype families of C.parvum species (IIa, IId) in the present study is in concordance with previous studies in Iran and emphasizes the significance of zoonotic transmission of cryptosporidiosis in the country. [ABSTRACT FROM AUTHOR]

Aim: The present study was performed in order to differentiate E. histolytica and E. dispar in children from Gorgan city, using a PCR method. Background: Differential detection of two morphologically indistinguishable protozoan parasites Entamoeba histolytica and E. dispar has a great clinical and epidemiological importance because of potential invasive pathogenic E. histolytica and non-invasive parasite E. dispar. Patients and methods: One hundred and five dysentery samples were collected from children hospitalized in Taleghani hospital in Gorgan city. The fecal specimens were examined by light microscopy (10X then 40X) to distinguish Entamoeba complex. A single round PCR amplifying partial small-subunit rRNA gene was performed on positive microscopy samples to differentiate E. histolytica/ E. dispar and E. moshkovskii from each other. Results: Twenty-five specimens (23.8%) were positive for Enramoeba complex in direct microscopic examination. PCR using positive controls indicated E. histolytica and E. dispar in two (2/25, 8%) and three (3/25, 12%) samples, respectively. Conclusion: There is a warrant to performing molecular diagnosis for stool examination at least in hospitalized children in order to prevent incorrect reports from laboratories and consequently mistreating by physicians. [ABSTRACT FROM AUTHOR]

Background: Toxoplasma gondii is one of the most prevalent parasites of human and warm- blooded animals. Toxoplasmosis is important especially in two groups: pregnant women and immunocompromised patients. If women acquire the primary infection during the pregnancy, it would be life threatening or remains severe disorders for the fetus. This study was performed to evaluate the seroprevalence of T. gondii infection in pregnant women referred to Health Center in Gorgan City, Golestan Province, northern Iran. Methods: Serum samples were collected from pregnant women referred to Health Center in Gorgan City, south eastern Caspian Sea. Anti- Toxoplasma IgG and IgM antibodies were determined by commercially ELISA kits and the relation of infection with socio-demographic and risk factors such as age, education, occupation, cat ownership, soil contact and some other factors was studied. Results: From 555 tested sera of pregnant women referred to Health Center in Gorgan, 39.8% had IgG antibodies against T. gondii and 3.4% were positive for IgM antibodies. A significant correlation was seen between T. gondii infection with age and soil contact. Conclusion: About 60% of pregnant women in Gorgan City are seronegative against T. gondii, so they should considered as at risk persons. [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]

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]

Giardia duodenalis is an important intestinal parasite responsible for diarrhea in humans and animals worldwide. Up to now, G. duodenalis infections in cattle have been reported in many studies around the world. Hence, the aim of the present study is to report on the distribution of G. duodenalis in cattle at global scale and to evaluate the global prevalence, risk factors and genetic characterization of G. duodenalis infection among cattle worldwide. International databases were systematically searched to identify relevant studies. A random-effects meta-analysis model was used to estimate the overall and the subgroup-pooled prevalence of G. duodenalis across studies, and the variance between studies (heterogeneity) was quantified by I 2 index. One hundred and fifty-eight articles (including 195 datasets), from 48 countries met eligibility criteria for analysis. Considering detection methods, the pooled prevalence was estimated to be 24% (95% confidence interval (CI), 19–30%) using copro-antigen techniques, 22% (95% CI, 17–28%) using molecular, and 16% (95% CI, 12–20%) using microscopic detection. Molecular methods showed that the highest number of reports were associated with assemblage E (45/46; 97.83% studies), assemblage A (33/46; 71.74% studies) and assemblage A+E (10/46; 21.74% studies). The pooled prevalence different of subgroups (WHO regions, countries, and type of cattle) were analyzed separately. Moreover, a significant association was observed between G. duodenalis infection with cattle suffering from diarrhea (odds ratio (OR), 2.61; 95% CI, 1.50–4.55) and pre-weaned calves (OR, 1.79; 95% CI, 1.08–2.95). These results suggest that the corresponding control scheme and effective management measures should be formulated to reduce the transmission of G. duodenalis infection according to the difference of geographical conditions in different areas. [ABSTRACT FROM AUTHOR]

Cystic echinococcosis, a zoonotic disease caused by Echinococcus granulosus sensu lato (s. l.), is a significant global public health concern. Echinococcus granulosus s. l. is currently divided into numerous genotypes (G1–G8 and G10) of which G1–G3 are the most frequently implicated genotypes in human infections. Although it has been suggested that G1–G3 could be regarded as a distinct species E . granulosus sensu stricto (s. s.), the evidence to support this is inconclusive. Most importantly, data from nuclear DNA that provide means to investigate the exchange of genetic material between G1–G3 is lacking as none of the published nuclear DNA studies have explicitly included G2 or G3. Moreover, the commonly used relatively short mtDNA sequences, including the complete cox1 gene, have not allowed unequivocal differentiation of genotypes G1–G3. Therefore, significantly longer mtDNA sequences are required to distinguish these genotypes with confidence. The main aim of this study was to evaluate the phylogenetic relations and taxonomy of genotypes G1–G3 using sequences of nearly complete mitogenomes (11,443 bp) and three nuclear loci (2984 bp). A total of 23 G1–G3 samples were analysed, originating from 5 intermediate host species in 10 countries. The mtDNA data demonstrate that genotypes G1 and G3 are distinct mitochondrial genotypes (separated by 37 mutations), whereas G2 is not a separate genotype or even a monophyletic cluster, but belongs to G3. Nuclear data revealed no genetic separation of G1 and G3, suggesting that these genotypes form a single species due to ongoing gene flow. We conclude that: (a) in the taxonomic sense, genotypes G1 and G3 can be treated as a single species E . granulosus s. s.; (b) genotypes G1 and G3 should be regarded as distinct genotypes only in the context of mitochondrial data; (c) we recommend excluding G2 from the genotype list. [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]

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]

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]

Tapeworms of the species complex of Echinococcus granulosus sensu lato (s. l.) are the cause of a severe zoonotic disease – cystic echinococcosis, which is listed among the most severe parasitic diseases in humans and is prioritized by the World Health Organization. A stable taxonomy of E. granulosus s. l. is essential to the medical and veterinary communities for accurate and effective communication of the role of different species in this complex on human and animal health. E. granulosus s. l. displays high genetic diversity and has been divided into different species and genotypes. Despite several decades of research, the taxonomy of E. granulosus s. l. has remained controversial, especially the species status of genotypes G6–G10. Here the Bayesian phylogeny based on six nuclear loci (7387 bp in total) demonstrated, with very high support, the clustering of G6/G7 and G8/G10 into two separate clades. According to the evolutionary species concept, G6/G7 and G8/G10 can be regarded as two distinct species. Species differentiation can be attributed to the association with distinct host species, largely separate geographical distribution and low level of cross-fertilization. These factors have limited the gene flow between genotypic groups G6/G7 and G8/G10, resulting in the formation of distinct species. We discuss ecological and epidemiological differences that support the validity of these species. [ABSTRACT FROM AUTHOR]

Abstract Cystic echinococcosis (CE) is a zoonotic disease caused by the larval stage of the species complex Echinococcus granulosus sensu lato. Within this complex, genotypes G6 and G7 have been frequently associated with human CE worldwide. Previous studies exploring the genetic variability and phylogeography of genotypes G6 and G7 have been based on relatively short mtDNA sequences, and the resolution of these studies has often been low. Moreover, using short sequences, the distinction between G6 and G7 has in some cases remained challenging. The aim here was to sequence complete mitochondrial genomes (mitogenomes) to obtain deeper insight into the genetic diversity, phylogeny and population structure of genotypes G6 and G7. We sequenced complete mitogenomes of 94 samples collected from 15 different countries worldwide. The results demonstrated that (i) genotypes G6 and G7 can be clearly distinguished when mitogenome sequences are used; (ii) G7 is represented by two major haplogroups, G7a and G7b, the latter being specific to islands of Corsica and Sardinia; (iii) intensive animal trade, but also geographical isolation, have likely had the largest impact on shaping the genetic structure and distribution of genotypes G6 and G7. In addition, we found phylogenetically highly divergent haplotype from Mongolia (Gmon), which had a higher affinity to G6. Graphical abstract Unlabelled Image Highlights • Complete mitogenomes allow correct identification and differentiation of G6 and G7. • Genotype G7 is represented by two major haplogroups: G7a and G7b. • Complete mitogenomes provide deep insight into the population structure of G6 and G7. • A phylogenetically highly divergent haplotype, Gmon, was identified in Mongolia [ABSTRACT FROM AUTHOR]

Cystic echinococcosis (CE) is a severe parasitic disease caused by the species complex Echinococcus granulosus sensu lato. Human infections are most commonly associated with E. granulosus sensu stricto (s.s.), comprising genotypes G1 and G3. The objective of the current study was to provide first insight into the genetic diversity and phylogeography of genotype G3. Despite the epidemiological importance of the genotype, it has remained poorly explored due to the ambiguity in the definition of the genotype. However, it was recently demonstrated that long sequences of mitochondrial DNA (mtDNA) provide a reliable method to discriminate G1 and G3 from each other. Therefore, we sequenced near-complete mtDNA of 39 G3 samples, covering most of the known distribution range and host spectra of the genotype. The phylogenetic network revealed high genetic variation within E. granulosus s.s. G3 and while G3 is significantly less prevalent worldwide than G1, the genetic diversity of both of the genotypes is equally high. We also present the results of the Bayesian phylogeographic analysis, which yielded several well-supported diffusion routes of genotype G3 originating from Turkey and Iran, suggesting the Middle East as the origin of the genotype. [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]

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. [ABSTRACT FROM AUTHOR]

The present study investigated the possible role of Leishmania RNA virus 2 (LRV2) in the severity of dermal lesions and treatment failure due to Leishmania major. The drug susceptibility of 14 clinical isolates of L.major , including resistant (n = 7) and sensitive (n = 7) isolates, was checked in the J774A.1 macrophage cell line. The presence of LRV2 among isolates was investigated by the RdRp gene and semi-nested PCR. Moreover, 1 × 106 sensitive L. major LRV2+ and LRV2- promastigotes were inoculated subcutaneously into the base tails of the 40 BALB/c mice divided into 4 groups (n = 10 in each group), including clinical LRV2+, clinical LRV2-, positive control LRV2+ and negative control LRV2-. The groups were infected with a unique isolate. The lesion size and parasite burden were evaluated. Sensitive and resistant isolates were determined by the drug susceptibility method. A higher presence of LRV2 was observed among MA-resistant isolates (6/7) compared with susceptible isolates (4/7), which was not statistically significant (P = 0.237). On the other hand, a comparison of the lesion sizes between the LRV2+ and LRV2- BALB/c mice groups revealed that the mean size of the lesion in the LRV2+ groups was significantly higher than the LRV2- (P = 0.034). In the same direction, there was an increased parasite burden in mice inoculated with LRV2+ groups compared with the LRV2- BALB/c mice groups (P = 0.002). Our findings showed that the presence of LRV2 could be one of the factors contributing to exacerbating CL. Although we found a higher presence of LRV2 in the resistant isolates, it seems that further investigations are recommended to determine the detailed association between lesions' aggravation and being comparatively unresponsive to treatment. [Display omitted] • The presence of LRV2 among these isolates was detected by semi nested-PCR. • The presence of the LRV2 was observed more among Glucantime-resistant isolates (6/7) compared with susceptible isolates (4/7). • Comparison of the size of the lesions between the LRV2+ and LRV2- BALB/c mice groups revealed that the mean size of lesions in the LRV2+ groups was significantly higher than the LRV2-. [ABSTRACT FROM AUTHOR]

Background: The present study was aimed to investigate molecular diversity of Echinococcus granulosus isolates collected from human clinical samples using two mitochondrial genes cox1 and nad1 in Iran. Methods: Forty seven human hydatid cysts were collected through surgery from two hospitals in Tehran during 2010-2012. To determine the fertility of protoscoleces, the cyst fluids were subjected to morphological microscopic examinations. Protoscoleces were removed from each cyst and their total genomic DNAs were extracted. PCR was performed to amplify fragments of 450 and 400 base pair (bp) for cox1 and nad1 genes, respectively. Genotype diversity and sequence variation of the strains were studied by bioinformatics software and in comparison with those mtDNA sequences already deposited in GenBank. Results: Sixteen, (53.3%), 13 (43.3%), and 1 (3.3%) samples were related to lung, liver, and spleen, respectively. The remained 17 unfertile samples were excluded from the study. From the 29 isolates, 86.7% (n=26) and 10% (n=3) were related to G1, and G3 genotypes, respectively. The sole isolate with G6 genotype was obtained from lung sample. Analysis of concatenated sequences of cox1+nad1 indicated the presence of 11 haplotypes among our strains that were related to genotypes G1 (n=9), G3 (n=1) and G6 (n=1). Conclusion: In consistent to other reports from Iran, genotypes G1, G3, and G6 were observed in our human isolates. The rate of G3 genotype was however higher than other studies implying that human can be considered as a new appropriate host for G3 genotype. Further studies with more sample size from different geographic areas of Iran are needed for E. granulosus mapping. [ABSTRACT FROM AUTHOR]

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]

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. [ABSTRACT FROM AUTHOR]

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]

Abstract: 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 &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]

• Microsporidiosis in pet and stray cats is an emerging zoonotic threat with public health significance worldwide. • Microsporidia infection demonstrated higher prevalence rates using microscopy 29.7 % (19.7–42.2 %), followed by serology and molecular techniques with 11 % (4.6–24.2 %) and 8.2 % (5.9–11.4 %), respectively. • Findings of molecular methods revealed that the highest rate of infection belong to Enterocytozoon bieneusi genotype D. Microsporidiosis in pet and stray cats is an emerging zoonotic threat with public health significance worldwide. However, the epidemiological patterns of feline microsporidiosis is still neglected around the world. Hence, current systematic review and meta-analysis aimed at characterizing the prevalence estimates and genotypes of microsporidian parasites among cats of the world. Several databases (PubMed, Web of Science, Scopus, and Google scholar) were systematically explored to find relevant studies. Evaluation of the weighted prevalences among included studies was done using random-effects model. Totally, 30 studies (34 datasets) reported from 19 countries were included in the present work. Microsporidia infection demonstrated higher prevalence rates using microscopy 29.7 % (19.7–42.2 %), followed by serology and molecular techniques with 11 % (4.6–24.2 %) and 8.2 % (5.9–11.4 %), respectively. Moreover, molecular data showed Enterocytozoon bieneusi as the most dominant reported species with 7.4 % (5.1–10.5 %). Also, investigations (11 studies) mostly isolated D genotype among all E. bieneusi genotypes. These results highlight cats as a potential reservoir for acquisition of microsporidia infection in humans, and surveillance programs should be implemented in high-risk areas. [ABSTRACT FROM AUTHOR]

The larval stages of tapeworms in the species complex Echinococcus granulosus sensu lato cause a zoonotic disease known as cystic echinococcosis (CE). Within this species complex, genotypes G6 and G7 are among the most common genotypes associated with human CE cases worldwide. However, our understanding of ecology, biology and epidemiology of G6 and G7 is still limited. An essential first step towards this goal is correct genotype identification, but distinguishing genotypes G6 and G7 has been challenging. A recent analysis based on complete mitogenome data revealed that the conventional sequencing of the cox1 (366 bp) gene fragment mistakenly classified a subset of G7 samples as G6. On the other hand, sequencing complete mitogenomes is not practical if only genotype or haplogroup identification is needed. Therefore, a simpler and less costly method is required to distinguish genotypes G6 and G7. We compared 93 complete mitogenomes of G6 and G7 from a wide geographical range and demonstrate that a combination of nad2 (714 bp) and nad5 (680 bp) gene fragments would be the best option to distinguish G6 and G7. Moreover, this method allows assignment of G7 samples into haplogroups G7a and G7b. However, due to very high genetic variability of G6 and G7, we suggest to construct a phylogenetic network based on the nad2 and nad5 sequences in order to be absolutely sure in genotype assignment. For this we provide a reference dataset of 93 concatenated nad2 and nad5 sequences (1394 bp in total) containing representatives of G6 and G7 (and haplogroups G7a and G7b), which can be used for the reconstruction of phylogenetic networks. • Analysis of nad2 and nad5 is required for identification of E. granulosus s.l. genotypes G6 and G7 • Constructing a phylogenetic network is also required for genotype assignment • We provide reference sequences of G6-G7 for helping to build phylogenetic networks [ABSTRACT FROM AUTHOR]