New mitogenome and nuclear evidence on the phylogeny and taxonomy of the highly zoonotic tapeworm Echinococcus granulosus sensu stricto

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 coxl 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. (C) 2017 Elsevier B.V. All rights reserved.Peer reviewe

Serological Evidence and Associated Risk Factors for Toxoplasma gondii in Gorgan and a Review of Recent Studies in Golestan Province

Background and purpose: Toxoplasma gondii is one of the most common parasitic infections in humans and other vertebrates worldwide. This study aimed at determining the seroprevalence of Toxoplasma infection and associated risk factors in individuals attending a Reference Laboratory in Gorgan, Iran. Moreover, recent studies in Golestan province were reviewed. Materials and methods: A descriptive cross-sectional survey was conducted in 551 blood samples obtained from February-June 2017. Serum samples were isolated and examined for IgG and IgM antibodies against T. gondii using ELISA. Data were analyzed in SPSS applying logistic regression. Results: Total prevalence of T. gondii was 63.5% (350 out of 551). Among the samples, 306 (55.5%), 12 (2.17%), and 32 (5.8%) were found to be positive for IgG, IgM and both IgG and IgM against T. gondii, respectively. There were significant associations between T. gondii infection and age and soil contact (P0.05). Conclusion: About two thirds of individuals were infected with T. gondii. Accordingly, a high number of subjects have been exposed to T. gondii in this area, which their seropositivity was seen to increase by age as a risk factor. High seropositivity indicates appropriate conditions for the life cycle of the parasite in this province. Therefore, preventive measurements at the community level, particularly in high-risk groups, are warranted

Echinococcus granulosus sensu lato GENOTYPES IN DOMESTIC LIVESTOCK AND HUMANS IN GOLESTAN PROVINCE, IRAN

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

Prevalence and Genetic Characterization of Cryptosporidium Spp. In Diarrheic Children from Gonbad Kavoos City, Iran

Background: Cryptosporidium is an intestinal protozean parasite causing water­borne 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 Cryptosporidium 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

Echinococcus granulosus Sensu Stricto in Dogs and Jackals from Caspian Sea Region, Northern Iran

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

Seroprevalence of Toxoplasma gondii Infections in Pregnant Women in Gorgan City, Golestan Province, Northern Iran-2012

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 re-mains severe disorders for the fetus. This study was performed to evaluate the seroprevalence of T. gondii infection in pregnant women referred to Health Cen-ter 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 rela-tion 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

Molecular identification of ovine Babesia spp. in north of Iran

Background: Babesia is blood-tissue parasite, which are transmitted by hard ticks from Ixodidae family. The parasite is the cause of Babesiosis among ruminants. Babesia is as one of main fatal factors among livestock in endemic regions such as Iran. The aim of this study was to investigate Babesia spp infection using microscopic and molecular methods among small ruminants in Mazandaran and Golestan provinces, northern Iran, in 2011-2012. Materials and Methods: In this cross-sectional study, 22 flocks of total 220 sheep and goats were selected from different regions of these provinces. Subsequently, ten suspected animals were selected randomly and blood sample were taken from their ear. To diagnose thick and thin smears were prepared and stained with Geimsa stain and then examined under light microscope. Standard PCR and Semi nested PCR was performed to differentiate genus of Theileria and Babesia, also identify the species of Babesia. Results: A total of 220 blood samples (160 sheep and 60 goats), 34 cases (15.4 %) had Babesia infection using microscopic. Whereas, 11 cases (5%) were found positive through Babesia spp using standard PCR and also among them, two cases were found mixed infection through Theileria spp. In addition, two microscopic negative were positive by PCR assay. Totally, using semi-nested PCR, Babesia ovis (n=10) and Babesia motasi (n=1) were detected. Conclusion: Our results showed ovine Babesiosis was common in the Iranian Northern provinces .Moreover, Babesia ovis is the main causative agent of ovine Babesiosis in this areas. The relatively high prevalence of Babesia infection in livestock indicates the epizootic stability status of babesiosis in the northern part of Iran

Domestic dogs carriers of Leishmania infantum, Leishmania tropica and Crithidia fasciculata as potential reservoirs for human visceral leishmaniasis in northeastern Iran

Abstract 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

Genotyping of Echinococcus granulosus Isolates from Human Clinical Samples Based on Sequencing of Mitochondrial Genes in Iran, Tehran

  Background: The present study was aimed to investigate molecular diversity of Echino-coccus 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 de-posited 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