Serological and Molecular Characteristics of the First Korean Case of Echinococcus multilocularis

In December 2011, we reported an autochthonous case of Echinococcus multilocularis infection in a 42-year-old woman in Korea. The diagnosis was based on histopathological findings of the surgically resected liver cyst. In the present study, we evaluated the serological and molecular characteristics of this Korean E. multilocularis case. The patient''s serum strongly reacted with affinity-purified native Em18 and recombinant Em18 antigens (specific for E. multilocularis) but negative for recombinant antigen B8/1 (reactive for Echinococcus granulosus). In immunoaffinity chromatography, the serum also strongly reacted with E. multilocularis and only weakly positive for E. granulosus. We determined the whole nucleotide sequence of cox1 (1,608 bp) using the paraffin-embedded cystic tissue which was compared with E. multilocularis isolates from China, Japan, Kazakhstan, Austria, France, and Slovakia. The Korean case showed 99.8-99.9% similarity with isolates from Asia (the highest similarity with an isolate from Sichuan, China), whereas the similarity with European isolates ranged from 99.5 to 99.6%.     

Genetic Diversity of Echinococcus granulosus in Center of Iran

Hydatid cyst caused by Echinococcus granulosus is one of the most important parasitic diseases around the world and many countries in Asia, including Iran, are involved with this infection. This disease can cause high mortality in humans as well as economic losses in livestock. To date, several molecular methods have been used to determine the genetic diversity of E. granulosus. So far, identification of E. granulosus using real-time PCR fluorescence-based quantitative assays has not been studied worldwide, also in Iran. Therefore, the aim of this study was to investigate the genetic diversity of E. granulosus from center of Iran using real-time PCR method. A total of 71 hydatid cysts were collected from infected sheep, goat, and cattle slaughtered in Isfahan, Iran during 2013. DNA was extracted from protoscolices and/or germinal layers from each individual cyst and used as template to amplify the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) (420 bp). Five cattle isolates out of 71 isolates were sterile and excluded from further investigation. Overall, of 66 isolates, partial sequences of the cox1 gene of E. granulosus indicated the presence of genotypes G1 in 49 isolates (74.2%), G3 in 15 isolates (22.7%), and G6 in 2 isolates (3.0%) in infected intermediate hosts. Sixteen sequences of G1 genotype had microgenetic variants, and they were compared to the original sequence of cox1. However, isolates identified as G3 and G6 genotypes were completely consistent with original sequences. G1 genotype in livestock was the dominant genotype in Isfahan region, Iran.     

Genetic Diversity and Population Genetic Structure Analysis of Echinococcus granulosus sensu stricto Complex Based on Mitochondrial DNA Signature

The genetic diversity and population genetics of the Echinococcus granulosus sensu stricto complex were investigated based on sequencing of mitochondrial DNA (mtDNA). Total 81 isolates of hydatid cyst collected from ungulate animals from different geographical areas of North India were identified by sequencing of cytochrome c oxidase subunit1 (coxi) gene. Three genotypes belonging to E. granulosus sensu stricto complex were identified (G1, G2 and G3 genotypes). Further the nucleotide sequences (retrieved from GenBank) for the coxi gene from seven populations of E. granulosus sensu stricto complex covering 6 continents, were compared with sequences of isolates analysed in this study. Molecular diversity indices represent overall high mitochondrial DNA diversity for these populations, but low nucleotide diversity between haplotypes. The neutrality tests were used to analyze signatures of historical demographic events. The Tajima’s D test and Fu’s FS test showed negative value, indicating deviations from neutrality and both suggested recent population expansion for the populations. Pairwise fixation index was significant for pairwise comparison of different populations (except between South America and East Asia, Middle East and Europe, South America and Europe, Africa and Australia), indicating genetic differentiation among populations. Based on the findings of the present study and those from earlier studies, we hypothesize that demographic expansion occurred in E. granulosus after the introduction of founder haplotype particular by anthropogenic movements.     

Detection of Echinococcus multilocularis in carnivores in Razavi Khorasan province, Iran using mitochondrial DNA

Background

Echinococcus multilocularis is the source of alveolar echinococcosis, a potentially fatal zoonotic disease. This investigation assessed the presence of E. multilocularis infection in definitive hosts in the Chenaran region of Razavi Khorasan Province, northeastern Iran.

Methodology/Principal Findings

Fecal samples from 77 domestic and stray dogs and 14 wild carnivores were examined using the flotation/sieving method followed by multiplex PCR of mitochondrial genes. The intestinal scraping technique (IST) and the sedimentation and counting technique (SCT) revealed adult Echinococcus in the intestines of five of 10 jackals and of the single wolf examined. Three jackals were infected only with E. multilocularis but two, and the wolf, were infected with both E. multilocularis and E. granulosus. Multiplex PCR revealed E. multilocularis, E. granulosus, and Taenia spp. in 19, 24, and 28 fecal samples, respectively. Echinococcus multilocularis infection was detected in the feces of all wild carnivores sampled including nine jackals, three foxes, one wolf, one hyena, and five dogs (6.5%). Echinococcus granulosus was found in the fecal samples of 16.9% of dogs, 66.7% of jackals, and all of the foxes, the wolf, and the hyena. The feces of 16 (21.8%) dogs, 7 of 9 (77.8%) jackals, and all three foxes, one wolf and one hyena were infected with Taenia spp.

Conclusions/Significance

The prevalence of E. multilocularis in wild carnivores of rural areas of the Chenaran region is high, indicating that the life cycle is being maintained in northeastern Iran with the red fox, jackal, wolf, hyena, and dog as definitive hosts.     

High endemicity of alveolar echinococcosis in Yili Prefecture,Xinjiang Autonomous Region,the People’s Republic of China: Infection status in different ethnic communities and in small mammals

Alveolar echinococcosis (AE) is a life-threatening disease in humans caused by the larval stage of Echinococcus multilocularis. The tapeworm is transmitted between small mammals and dogs/foxes in the Northern Hemisphere. In this study 286 AE cases were reported from eight counties and one city in Yili Prefecture, Xinjiang Autonomous Region, the People’s Republic of China from 1989 to 2015 with an annual incidence (AI) of 0.41/100,000. Among the patients, 73.08% were diagnosed in the last 11 years. Four counties in the high mountainous areas showed higher AI (0.51–1.22 cases/100,000 residents) than the four counties in low level areas (0.19–0.29/100,000 residents). The AI of AE in Mongolian (2.06/100,000 residents) and Kazak (0.93/100,000 residents) ethnic groups was higher than the incidence in other ethnic groups indicating sheep-farming is a risk for infection given this activity is mainly practiced by these two groups in the prefecture. A total of 1411 small mammals were captured with 9.14% infected with E. multilocularis metacestodes. Microtus obscurus was the dominant species in the mountain pasture areas with 15.01% of the voles infected, whereas Mus musculus and Apodemus sylvaticus were the dominant small mammals in the low altitude areas. Only 0.40% of A. sylvaticus were infected with E. multilocularis. PCR amplification and sequencing analysis of the mitochondrial cox1 gene showed that E. multilocularis DNA sequences from the small mammals were identical to isolates of local human AE cases. The overall results show that Yili Prefecture is a highly endemic area for AE and that the high-altitude pasture areas favorable for M. obscurus may play an important role in its transmission in this region.     

Serodiagnosis of Echinococcus spp. Infection: Explorative Selection of Diagnostic Antigens by Peptide Microarray

Background

Production of native antigens for serodiagnosis of helminthic infections is laborious and hampered by batch-to-batch variation. For serodiagnosis of echinococcosis, especially cystic disease, most screening tests rely on crude or purified Echinococcus granulosus hydatid cyst fluid. To resolve limitations associated with native antigens in serological tests, the use of standardized and highly pure antigens produced by chemical synthesis offers considerable advantages, provided appropriate diagnostic sensitivity and specificity is achieved.

Methodology/Principal Findings

Making use of the growing collection of genomic and proteomic data, we applied a set of bioinformatic selection criteria to a collection of protein sequences including conceptually translated nucleotide sequence data of two related tapeworms, Echinococcus multilocularis and Echinococcus granulosus. Our approach targeted alpha-helical coiled-coils and intrinsically unstructured regions of parasite proteins potentially exposed to the host immune system. From 6 proteins of E. multilocularis and 5 proteins of E. granulosus, 45 peptides between 24 and 30 amino acids in length were designed. These peptides were chemically synthesized, spotted on microarrays and screened for reactivity with sera from infected humans. Peptides reacting above the cut-off were validated in enzyme-linked immunosorbent assays (ELISA). Peptides identified failed to differentiate between E. multilocularis and E. granulosus infection. The peptide performing best reached 57% sensitivity and 94% specificity. This candidate derived from Echinococcus multilocularis antigen B8/1 and showed strong reactivity to sera from patients infected either with E. multilocularis or E. granulosus.

Conclusions/Significance

This study provides proof of principle for the discovery of diagnostically relevant peptides by bioinformatic selection complemented with screening on a high-throughput microarray platform. Our data showed that a single peptide cannot provide sufficient diagnostic sensitivity whereas pooling several peptide antigens improved sensitivity; thus combinations of several peptides may lead the way to new diagnostic tests that replace, or at least complement conventional immunodiagnosis of echinococcosis. Our strategy could prove useful for diagnostic developments in other pathogens.     

Echinococcus granulosus: variability of the host-protective EG95 vaccine antigen in G6 and G7 genotypic variants

Cystic hydatid disease in humans is caused by the zoonotic parasite Echinococcus granulosus. As an aid to control transmission of the parasite, a vaccine has been produced for prevention of infection in the parasite’s natural animal intermediate hosts. The vaccine utilizes the recombinant oncosphere protein, EG95. An investigation into the genetic variability of EG95 was undertaken in this study to assess potential antigenic variability in E. granulosus with respect to this host-protective protein. Gene-specific PCR conditions were first established to preferentially amplify the EG95 vaccine-encoding gene (designated eg95-1) from the E. granulosus genome that also contains several other EG95-related genes. The optimized PCR conditions were used to amplify eg95-1 from several parasite isolates in order to determine the protein-coding sequence of the gene. An identical eg95-1 gene was amplified from parasites showing a G1 or G2 genotype of E. granulosus. However, from isolates having a G6 or G7 genotype, a gene was amplified which had substantial nucleotide substitutions (encoding amino acid substitutions) compared with the eg95 gene family members. The amino acid substitutions of EG95 in the G6/G7 genotypes may affect the antigenicity/efficacy of the EG95 recombinant antigen against parasites of these genotypes. These findings indicate that characterization of eg95 gene family members in other strains/isolates of E. granulosus may provide valuable information about the potential for the EG95 hydatid vaccine to be effective against E. granulosus strains other than the G1 genotype.     

Assessment of a 10-year dog deworming programme on the transmission of Echinococcus multilocularis in Tibetan communities in Sichuan Province,China

Human alveolar echinococcosis (AE) is considered a neglected zoonotic disease by the World Health Organization (WHO). The causative pathogen, Echinococcus multilocularis, lives as an adult tapeworm in the intestinal tract of canines. AE was identified as an emerging public health issue in Tibetan communities of Shiqu County 20 years ago. On St. Lawrence Island, Alaska (USA), in the 1980s peri-domestic transmission of E. multilocularis was controlled by regular deworming of owned dogs over a 10-year period. In Tibetan communities, on the Tibetan Plateau, control of E. multilocularis transmission is challenging due to the continental setting, complex epidemiology, disease ecology, geography, and socio-cultural factors. However, a control programme based on deworming owned dogs using praziquental (PZQ) has been carried out since 2006. Assessment was conducted in townships where baseline data were available 10 years prior. Purging of dogs by oral administration of arecoline was used to measure E. multilocularis prevalence, trapping small mammals around communities was employed to assess the change in infection of pikas and voles, and analysis of human AE abdominal ultrasound-based data was used to understand the change in prevalence in the past decade. In all three evaluated townships, the E. multilocularis prevalence in owned dogs was significantly (P < 0.01) reduced from 7.23% (25/346) during 2000–2003 to 0.55% (1/181) in 2016. Human AE ultrasound-based prevalence (adjusted for age and sex) in five evaluated townships decreased significantly (P < 0.01) from 6.25% (200/3,198) during 2000–2002 to 3.67% (706/19,247) during 2015–2017. The 2016 prevalence of E. multilocularis metacestodes in small mammal intermediate hosts was not significantly different from the prevalence in 2008. The control programme was effective in reducing E. multilocularis infection in owned dogs and human AE prevalence, but did not significantly impact infection in wildlife intermediate hosts.     

Genetic Characterization of Human-Derived Hydatid Cysts of Echinococcus granulosus Sensu Lato in Heilongjiang Province and the First Report of G7 Genotype of E. canadensis in Humans in China

Cystic echinococcosis (CE) caused by the larval stage of Echinococcus granulosus sensu lato (s.l.) is one of the most important zoonotic parasitic diseases worldwide and 10 genotypes (G1–G10) have been reported. In China, almost all the epidemiological and genotyping studies of E. granulosus s.l. are from the west and northwest pasturing areas. However, in Heilongjiang Province of northeastern China, no molecular information is available on E. granulosus s.l. To understand and to speculate on possible transmission patterns of E. granulosus s.l., we molecularly identified and genotyped 10 hydatid cysts from hepatic CE patients in Heilongjiang Province based on mitochondrial cytochrome c oxidase subunit I (cox1), cytochrome b (cytb) and NADH dehydrogenase subunit 1 (nad1) genes. Two genotypes were identified, G1 genotype (n = 6) and G7 genotype (n = 4). All the six G1 genotype isolates were identical to each other at the cox1 locus; three and two different sequences were obtained at the cytb and nad1 loci, respectively, with two cytb gene sequences not being described previously. G7 genotype isolates were identical to each other at the cox1, cytb and nad1 loci; however, the cytb gene sequence was not described previously. This is the first report of G7 genotype in humans in China. Three new cytb gene sequences from G1 and G7 genotypes might reflect endemic genetic characterizations. Pigs might be the main intermediate hosts of G7 genotype in our investigated area by homology analysis. The results will aid in making more effective control strategies for the prevention of transmission of E. granulosus s.l.     

A survey of Echinococcus species in wild carnivores and livestock in East Africa

We examined 71 faecal samples of carnivores from Queen Elizabeth National Park (QENP), Uganda, for eggs of Echinococcus species. Thirty-nine faecal samples contained taeniid eggs. For species diagnosis, DNA was isolated from a total of 1984 individual taeniid eggs. To differentiate eggs of Echinococcus felidis from other taeniid taxa (including the closely related Echinococcus granulosus sensu stricto), a restriction fragment length polymorphism (RFLP)-PCR of the mitochondrial nad1 gene was developed. As the faecal samples were taken from the environment, the host species was determined for all samples, except for one, by RFLP-PCR of the cob gene. Seven hundred and ninety-one of the 1984 eggs yielded a suitable PCR product. E. felidis was present in 34 of 47 samples from lions, none of 18 samples from leopards, and one of five samples from spotted hyenas. No Echinococcus taxon other than E. felidis was found, but three samples from lions contained eggs of Taenia regis. Two hydatid cysts of warthog origin from QENP were available for this study; molecular examination showed that one belonged to E. felidis, the other to E. granulosus (G1 strain). As a comparison of methods demonstrated that molecular diagnostic tools used for previous surveys of Echinococcus isolates in eastern Africa are not suitable to discriminate between E. felidis and E. granulosus sensu stricto, we re-examined 412 hydatid cyst samples of human, sheep, cattle, camel and goat origin from Kenya. Previous results were confirmed, as E. granulosus sensu stricto and Echinococcus canadensis G6/7 strain, but no E. felidis was found among these samples. In conclusion, we provide evidence that E. felidis is a frequent parasite of lions in Uganda, and possibly also occurs in hyenas. Additionally, we show that warthogs interact as intermediate hosts for E. felidis. We did not find evidence that E. felidis is present in eastern Africa outside conservation areas.     

Sequence Analysis of cytb Gene in Echinococcus granulosus from Western China

Echinococcus granulosus is the causative agent of cystic echinococcosis with medical and veterinary importance in China. Our main objective was to discuss the genotypes and genetic diversity of E. granulosus present in domestic animals and humans in western China. A total of 45 hydatid cyst samples were collected from sheep, humans, and a yak and subjected to an analysis of the sequences of mitochondrial cytochrome b (cytb) gene. The amplified PCR product for all samples was a 1,068 bp band. The phylogenetic analysis showed that all 45 samples were identified as E. granulosus (genotype G1). Ten haplotypes were detected among the samples, with the main haplotype being H1. The haplotype diversity was 0.626, while the nucleotide diversity was 0.001. These results suggested that genetic diversity was low among our samples collected from the west of China based on cytb gene analysis. These findings may provide more information on molecular characteristics of E. granulosus from this Chinese region.     

Echinococcus P29 Antigen: Molecular Characterization and Implication on Post-Surgery Follow-Up of CE Patients Infected with Different Species of the Echinococcus granulosus Complex

The protein P29 is a potential serological marker for post-treatment monitoring of cystic echinococcosis (CE) especially in young patients. We now have demonstrated that P29 is encoded in the Echinococcus genus by a single gene consisting of 7 exons spanning 1.2 kb of DNA. Variability of the p29 gene at inter- and intra-species level was assessed with 50 cDNA and 280 genomic DNA clones isolated from different E. granulosus s.l. isolates (E. granulosus sensu stricto (G1), E. equinus (G4), E. ortleppi (G5), E. canadensis (G6), E. canadensis (G7) and E. canadensis (G10)) as well as four E. multilocularis isolates. Scarce interspecies polymorphism at the p29 locus was observed and affected predominantly E. granulosus s.s. (G1), where we identified two alleles (A1 and A2) coding for identical P29 proteins and yielding in three genotypes (A1/A1, A2/A2 and A1/A2). Genotypic frequencies expected under Hardy-Weinberg equilibrium revealed a high rate of heterozygosity (47%) that strongly supports the hypothesis that E. granulosus s.s. (G1) is predominantly outbreeding. Comparative sequence analyses of the complete p29 gene showed that phylogenetic relationships within the genus Echinococcus were in agreement with those of previous nuclear gene studies. At the protein level, the deduced P29 amino acid (AA) sequences exhibited a high level of conservation, ranging from 97.9% AA sequence identity among the whole E. granulosus s.l. group to 99.58% identity among E. multilocularis isolates. We showed that P29 proteins of these two species differ by three AA substitutions without implication for antigenicity. In Western-blot analyses, serum antibodies from a human CE patient infected with E. canadensis (G6) strongly reacted with recombinant P29 from E. granulosus s.s. (G1) (recEg(G1)P29). In the same line, human anti-Eg(G1)P29 antibodies bound to recEcnd(G6)P29. Thus, minor AA sequence variations appear not to impair the prognostic serological use of P29.     

Immunomodulatory mechanisms during Echinococcus granulosus infection

The pathologic events that ensue after humans ingest the eggs of Echinococcus granulosus and continue while cystic echinococcosis develops, provide an excellent example illustrating the evasive strategies helminth parasites use to develop, progress and cause chronic disease. The hydatid cyst secretes and exposes numerous immunomodulatory molecules to the host’s immune system. By characterizing these molecules we can understand the mechanisms that E. granulosus uses for increasing the efficiency and persistency of infection in the host. These molecules modulate both the innate and adaptive arms of the immune response and appear to target cellular and humoral responses. In this review, we discuss recent advances in the immunobiology of host-E. granulosus interactions that provide intriguing insights into the complex interplay between host and parasite that ultimately facilitates parasite survival.     

Molecular Identification of Echinococcus multilocularis Infection in Small Mammals from Northeast,Iran

Background

Alveolar echinococcosis is a zoonotic disease caused by the metacestode of Echinococcus multilocularis. Many species of small mammals, including arvicolid rodents or Ochotona spp., are natural intermediate hosts of the cestode. The main aim of this study was to identify natural intermediate hosts of E. multilocularis in Chenaran County, Razavi Khorasan Province, northeastern Iran, where the prevalence of infected wild and domestic carnivores is high.

Methodology/Principal Findings

A program of trapping was carried out in five villages in which this cestode was reported in carnivores. The livers of 85 small mammals were investigated for the presence of E. multilocularis infection using multiplex PCR of mitochondrial genes. Infections were identified in 30 specimens: 23 Microtus transcaspicus, three Ochotona rufescens, two Mus musculus, one Crocidura gmelini, and one Apodemus witherbyi.

Conclusions/Significance

A range of small mammals therefore act as natural intermediate hosts for the transmission of E. multilocularis in Chenaran County, and the prevalence suggested that E. multilocularis infection is endemic in this region. The existence of the life cycle of this potentially lethal cestode in the vicinity of human habitats provides a significant risk of human infection.     

Morphological and biological characterization of cell line developed from bovine <Emphasis Type="Italic">Echinococcus granulosus</Emphasis>

The taeniid tapeworm Echinococcus granulosus is the causative agent of echinococcal disease, a major zoonosis with worldwide distribution. Several efforts to establish an in vitro model of E. granulosus have been undertaken; however, many of them have been designed for Echinococcus multilocularis. In the present study, we have described and characterized a stable cell line obtained from E. granulosus bovine protoscoleces maintained 3 yr in vitro. Growth characterization, morphology by light, fluorescent and electronic microscopy, and karyotyping were carried out. Cell culture origin was confirmed by immunofluorescent detection of AgB4 antigen and by PCR for the mitochondrial cytochrome c-oxidase subunit 1 (DCO1) gene. Cells seeded in agarose biphasic culture resembled a cystic structure, similar to the one formed in secondary hosts. This cell line could be a useful tool to research equinococcal behavior, allowing additional physiological and pharmacological studies, such as the effect of growth factors, nutrients, and antiparasitic drugs on cell viability and growth and on cyst formation.     

Genotype and Phenotype of Echinococcus granulosus Derived from Wild Sheep (Ovis orientalis) in Iran

The aim of the present study is to determine the characteristics of genotype and phenotype of Echinococcus granulosus derived from wild sheep and to compare them with the strains of E. granulosus sensu stricto (sheep-dog) and E. granulosus camel strain (camel-dog) in Iran. In Khojir National Park, near Tehran, Iran, a fertile hydatid cyst was recently found in the liver of a dead wild sheep (Ovis orientalis). The number of protoscolices (n=6,000) proved enough for an experimental infection in a dog. The characteristics of large and small hooks of metacestode were statistically determined as the sensu stricto strain but not the camel strain (P=0.5). To determine E. granulosus genotype, 20 adult worms of this type were collected from the infected dog. The second internal transcribed spacer (ITS2) of the nuclear ribosomal DNA (rDNA) and cytochrome c oxidase 1 subunit (COX1) of the mitochondrial DNA were amplified from individual adult worm by PCR. Subsequently, the PCR product was sequenced by Sanger method. The lengths of ITS2 and COX1 sequences were 378 and 857 bp, respectively, for all the sequenced samples. The amplified DNA sequences from both ribosomal and mitochondrial genes were highly similar (99% and 98%, respectively) to that of the ovine strain in the GenBank database. The results of the present study indicate that the morpho-molecular features and characteristics of E. granulosus in the Iranian wild sheep are the same as those of the sheep-dog E. granulosus sensu stricto strain.     

Natural Infection of the Ground Squirrel (Spermophilus spp.) with Echinococcus granulosus in China

Background

Echinococcus granulosus is usually transmitted between canid definitive hosts and ungulate intermediate hosts.

Methodology/Principal Findings

Lesions found in the livers of ground squirrels, Spermophilus dauricus/alashanicus, trapped in Ningxia Hui Autonomous Region, an area in China co-endemic for both E. granulosus and E. multilocularis, were subjected to molecular genotyping for Echinococcus spp. DNA. One of the lesions was shown to be caused by E. granulosus and subsequently by histology to contain viable protoscoleces.

Conclusions/Significance

This is the first report of a natural infection of the ground squirrel with E. granulosus. This does not provide definitive proof of a cycle involving ground squirrels and dogs or foxes, but it is clear that there is active E. granulosus transmission occurring in this area, despite a recent past decline in the dog population in southern Ningxia.     

Genotyping Echinococcus granulosus from dogs from Western Iran

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.     

Using mitochondrial and nuclear markers to evaluate the degree of genetic cohesion among Echinococcus populations

Based on the distinctiveness of their mitochondrial haplotypes and other biological features, several recent publications have proposed that some Echinococcus granulosus strains should be regarded as separate species. However, the genetic cohesion of these species has not been extensively evaluated using nuclear markers. We assess the degree of polymorphism of the partial mitochondrial cox1 (366 bp), the nuclear mdh (214 bp) and EgAgB4 (281-283 bp) genes of E. granulosus sensu lato isolates collected from areas where different strains occur sympatrically. Five distinct mitochondrial haplotypes were determined by direct sequencing (G1, G2, G5, G6 and G7). The mdh genotypes were first screened by SSCP: three alleles were identified (Md1-Md3), which were further confirmed by nucleotide sequencing. For EgAgB4, which was analysed by direct sequencing the PCR products, two groups of sequences were found: EgAgB4-1 and EgAgB4-2. No haplotype-specific mdh or EgAgB4 sequences occur. Nevertheless, alleles Md1 and Md2 and type 1 sequences of EgAgB4 showed a higher frequency within the group of haplotypes G1-G2, while allele Md3 and EgAgB4-2 are most frequent in the G5-G7 cluster. By AMOVA it is shown that 79% of the total genetic variability is found among haplotype groups. These findings are compatible with two not mutually exclusive evolutionary hypotheses: (a) that haplotypes share an ancestral polymorphism, or (b) that the reproductive isolation between parasites with distinct haplotypes is not complete, leading to gene introgression. The biologic and epidemiologic consequences of our findings are discussed.