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The Chinese liver fluke, Clonorchis sinensis, causes the disease clonorchiasis, affecting ~35 million people in regions of China, Vietnam, Korea and the Russian Far East. Chronic clonorchiasis causes cholangitis and can induce a malignant cancer, called cholangiocarcinoma, in the biliary system. Control in endemic regions is challenging, and often relies largely on chemotherapy with one anthelmintic, called praziquantel. Routine treatment carries a significant risk of inducing resistance to this anthelmintic in the fluke, such that the discovery of new interventions is considered important. It is hoped that the use of molecular technologies will assist this endeavour by enabling the identification of drug or vaccine targets involved in crucial biological processes and/or pathways in the parasite. Although draft genomes of C. sinensis have been published, their assemblies are fragmented. In the present study, we tackle this genome fragmentation issue by utilising, in an integrated way, advanced (second- and third-generation) DNA sequencing and informatic approaches to build a high-quality reference genome for C. sinensis, with chromosome-level contiguity and curated gene models. This substantially-enhanced genome provides a resource that could accelerate fundamental and applied molecular investigations of C. sinensis, clonorchiasis and/or cholangiocarcinoma, and assist in the discovery of new interventions against what is a highly significant, but neglected disease-complex.

The revolution in genomics has enabled large-scale population genetic investigations of a wide range of organisms, but there has been a relatively limited focus on improving analytical pipelines. To efficiently analyse large data sets, highly integrated and automated software pipelines, which are easy to use, efficient, reliable, reproducible and run in multiple computational environments, are required. A number of software workflows have been developed to handle and process such data sets for population genetic analyses, but effective, specialized pipelines for genetic and statistical analyses of nonmodel organisms are lacking. For most species, resources for variomes (sets of genetic variations found in populations of species) are not available, and/or genome assemblies are often incomplete and fragmented, complicating the selection of the most suitable reference genome when multiple assemblies are available. Additionally, the biological samples used often contain extraneous DNA from sources other than the species under investigation (e.g., microbial contamination), which needs to be removed prior to genetic analyses. For these reasons, we established a new pipeline, called Escalibur, which includes: functionalities, such as data trimming and mapping; selection of a suitable reference genome; removal of contaminating read data; recalibration of base calls; and variant-calling. Escalibur uses a proven gatk variant caller and workflow description language (WDL), and is, therefore, a highly efficient and scalable pipeline for the genome-wide identification of nucleotide variation in eukaryotes. This pipeline is available at https://gitlab.unimelb.edu.au/bioscience/escalibur (version 0.3-beta) and is essentially applicable to any prokaryote or eukaryote.

Background Echinococcus tapeworms cause a severe helminthic zoonosis called echinococcosis. The genus comprises various species and genotypes, of which E. granulosus (sensu stricto) represents a significant global public health and socioeconomic burden. Mitochondrial (mt) genomes have provided useful genetic markers to explore the nature and extent of genetic diversity within Echinococcus and have underpinned phylogenetic and population structure analyses of this genus. Our recent work indicated a sequence gap (> 1 kb) in the mt genomes of E. granulosus genotype G1, which could not be determined by PCR-based Sanger sequencing. The aim of the present study was to define the complete mt genome, irrespective of structural complexities, using a long-read sequencing method. Methods We extracted high molecular weight genomic DNA from protoscoleces from a single cyst of E. granulosus genotype G1 from a sheep from Australia using a conventional method and sequenced it using PacBio Sequel (long-read) technology, complemented by BGISEQ-500 short-read sequencing. Sequence data obtained were assembled using a recently-developed workflow. Results We assembled a complete mt genome sequence of 17,675 bp, which is > 4 kb larger than the complete mt genomes known for E. granulosus genotype G1. This assembly includes a previously-elusive tandem repeat region, which is 4417 bp long and consists of ten near-identical 441–445 bp repeat units, each harbouring a 184 bp non-coding region and adjacent regions. We also identified a short non-coding region of 183 bp, which includes an inverted repeat. Conclusions We report what we consider to be the first complete mt genome of E. granulosus genotype G1 and characterise all repeat regions in this genome. The numbers, sizes, sequences and functions of tandem repeat regions remain to be studied in different isolates of genotype G1 and in other genotypes and species. The discovery of such ‘new’ repeat elements in the mt genome of genotype G1 by PacBio sequencing raises a question about the completeness of some published genomes of taeniid cestodes assembled from conventional or short-read sequence datasets. This study shows that long-read sequencing readily overcomes the challenges of assembling repeat elements to achieve improved genomes.

Long non-coding, tandem-repetitive regions in mitochondrial (mt) genomes of many metazoans have been notoriously difficult to characterise accurately using conventional sequencing methods. Here, we show how the use of a third-generation (long-read) sequencing and informatic approach can overcome this problem. We employed Oxford Nanopore technology to sequence genomic DNAs from a pool of adult worms of the carcinogenic parasite, Schistosoma haematobium, and used an informatic workflow to define the complete mt non-coding region(s). Using long-read data of high coverage, we defined six dominant mt genomes of 33.4 kb to 22.6 kb. Although no variation was detected in the order or lengths of the protein-coding genes, there was marked length (18.5 kb to 7.6 kb) and structural variation in the non-coding region, raising questions about the evolution and function of what might be a control region that regulates mt transcription and/or replication. The discovery here of the largest tandem-repetitive, non-coding region (18.5 kb) in a metazoan organism also raises a question about the completeness of some of the mt genomes of animals reported to date, and stimulates further explorations using a Nanopore-informatic workflow.

Many freshwater snails of the genus

Many freshwater snails of the genus Bulinus act as intermediate hosts in the life-cycles of schistosomes in Africa and adjacent regions. Currently, 37 species of Bulinus representing four groups are recognised. The mitochondrial cytochrome c oxidase subunit 1 (cox1) gene has shown utility for identifying and differentiating Bulinus species and groups, but taxonomic relationships based on genetic data are not entirely consistent with those inferred using morphological and biological features. To underpin future systematic studies of members of the genus, we characterised here the mitochondrial genome of Bulinus truncatus (from a defined laboratory strain) using a combined second- and third-generation sequencing and informatics approach, enabling taxonomic comparisons with other planorbid snails for which mitochondrial (mt) genomes were available. Analyses showed consistency in gene order and length among mitochondrial genomes of representative planorbid snails, with the lowest and highest nucleotide diversities being in the cytochrome c oxidase and nicotinamide dehydrogenase subunit genes, respectively. This first mt genome for a representative of the genus Bulinus should provide a useful resource for future investigations of the systematics, population genetics, epidemiology and/or ecology of Bulinus and related snails. The sequencing and informatic workflow employed here should find broad applicability to a range of other snail intermediate hosts of parasitic trematodes.

International audience; 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.

Cystic echinococcosis (CE) is a severe parasitic disease caused by the species complexEchinococcus granulosussensu lato. Human infections are most commonly associated withE. granulosussensu 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 withinE. 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.

The larval stage of the species complex Echinococcus granulosus sensu lato (s.l.) is the cause of a widespread zoonotic disease known as cystic echinococcosis (CE). The disease is highly prevalent in southern Italy and represents a serious public health issue. The main aim of this study was to characterize E. granulosus s.l. genotypes from wild boar on a continental area of Italy (Campania region), using recently developed mtDNA markers of nad2 and nad5 for reliable identification of different genotypes. Here, nad5 (680 bp) allowed for a clear identification of G1 and G3, whereas a combination of nad2 (714 bp) and nad5 (1394 bp in total) did the same for genotype G7 and its haplogroups G7a and G7b. The results of this study revealed for the first time the presence of genotype G7 in continental Italy. While haplogroup G7b was previously shown to be restricted to the islands of Corsica and Sardinia, here we demonstrate that haplogroup G7b is also present on the mainland of Italy. This work has implications in designing future strategies to reduce CE in Italy.

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.

International audience; 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.

Background Mitochondrial genomes provide useful genetic markers for systematic and population genetic studies of parasitic helminths. Although many such genome sequences have been published and deposited in public databases, there is evidence that some of them are incomplete relating to an inability of conventional techniques to reliably sequence non-coding (repetitive) regions. In the present study, we characterise the complete mitochondrial genome—including the long, non-coding region—of the carcinogenic Chinese liver fluke, Clonorchis sinensis, using long-read sequencing. Methods The mitochondrial genome was sequenced from total high molecular-weight genomic DNA isolated from a pool of 100 adult worms of C. sinensis using the MinION sequencing platform (Oxford Nanopore Technologies), and assembled and annotated using an informatic approach. Results From > 93,500 long-reads, we assembled a 18,304 bp-mitochondrial genome for C. sinensis. Within this genome we identified a novel non-coding region of 4,549 bp containing six tandem-repetitive units of 719–809 bp each. Given that genomic DNA from pooled worms was used for sequencing, some variability in length/sequence in this tandem-repetitive region was detectable, reflecting population variation. Conclusions For C. sinensis, we report the complete mitochondrial genome, which includes a long (> 4.5 kb) tandem-repetitive region. The discovery of this non-coding region using a nanopore-sequencing/informatic approach now paves the way to investigating the nature and extent of length/sequence variation in this region within and among individual worms, both within and among C. sinensis populations, and to exploring whether this region has a functional role in the regulation of replication and transcription, akin to the mitochondrial control region in mammals. Although applied to C. sinensis, the technological approach established here should be broadly applicable to characterise complex tandem-repetitive or homo-polymeric regions in the mitochondrial genomes of a wide range of taxa., Author summary In the present study, we characterised the complete mitochondrial genome of Clonorchis sinensis—a carcinogenic liver fluke. To do this, we sequenced from total genomic DNA from multiple adult worms using a new method (Oxford Nanopore technology) to obtain data for long stretches of DNA, and then assembled these data to construct a mitochondrial genome of 18,304 bp, containing a > 4.5 kb-long tandem-repetitive region—not previously detected in this species. The results demonstrate that this method is effective at sequencing long and complex non-coding elements—not achievable using conventional techniques. The discovery of this long tandem-repetitive region in C. sinensis provides an opportunity to now explore its origin(s) and length/sequence diversity in populations of this species, and also to characterise its function(s). The technological approach employed here should have broad applicability to characterise previously-elusive non-coding mitochondrial genomic regions in a wide range of taxa.

International audience; Echinococcus granulosus sensu stricto (s.s.) is the major cause of human cystic echinococcosis worldwide and is listed among the most severe parasitic diseases of humans. To date, numerous studies have investigated the genetic diversity and population structure of E. granulosus s.s. in various geographic regions. However, there has been no global study. Recently, using mitochondrial DNA, it was shown that E. granulosus s.s. G1 and G3 are distinct genotypes, but a larger dataset is required to confirm the distinction of these genotypes. The objectives of this study were to: (i) investigate the distinction of genotypes G1 and G3 using a large global dataset; and (ii) analyse the genetic diversity and phylogeography of genotype G1 on a global scale using near-complete mitogenome sequences. For this study, 222 globally distributed E. granulosus s.s. samples were used, of which 212 belonged to genotype G1 and 10 to G3. Using a total sequence length of 11,682 bp, we inferred phylogenetic networks for three datasets: E. granulosus s.s. (n = 222), G1 (n = 212) and human G1 samples (n = 41). In addition, the Bayesian phylogenetic and phylogeographic analyses were performed. The latter yielded several strongly supported diffusion routes of genotype G1 originating from Turkey, Tunisia and Argentina. We conclude that: (i) using a considerably larger dataset than employed previously, E. granulosus s.s. G1 and G3 are indeed distinct mitochondrial genotypes; (ii) the genetic diversity of E. granulosus s.s. G1 is high globally, with lower values in South America; and (iii) the complex phylogeographic patterns emerging from the phylogenetic and geographic analyses suggest that the current distribution of genotype G1 has been shaped by intensive animal trade.

International audience; Cystic echinococcosis (CE), a zoonotic disease caused by tapeworms of the species complex Echinococcus granulosus sensu lato, represents a substantial global health and economic burden. Within this complex, E. granulosus sensu stricto (genotypes G1 and G3) is the most frequent causative agent of human CE. Currently, there is no fully reliable method for assigning samples to genotypes G1 and G3, as the commonly used mitochondrial cox1 and nad1 genes are not sufficiently consistent for the identification and differentiation of these genotypes. Thus, a new genetic assay is required for the accurate assignment of G1 and G3. Here we use a large dataset of near-complete mtDNA sequences (n = 303) to reveal the extent of genetic variation of G1 and G3 on a broad geographical scale and to identify reliable informative positions for G1 and G3. Based on extensive sampling and sequencing data, we developed a new method, that is simple and cost-effective, to designate samples to genotypes G1 and G3. We found that the nad5 is the best gene in mtDNA to differentiate between G1 and G3, and developed new primers for the analysis. Our results also highlight problems related to the commonly used cox1 and nad1. To guarantee consistent identification of G1 and G3, we suggest using the sequencing of the nad5 gene region (680 bp). This region contains six informative positions within a relatively short fragment of the mtDNA, allowing the differentiation of G1 and G3 with confidence. Our method offers clear advantages over the previous ones, providing a significantly more consistent means to distinguish G1 and G3 than the commonly used cox1 and nad1.

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.

SUMMARYEchinococcus granulosusis the causative agent of cystic echinococcosis. The disease is a significant global public health concern and human infections are most commonly associated withE. granulosussensu stricto (s. s.) genotype G1. The objectives of this study were to: (i) analyse the genetic variation and phylogeography ofE. granulosuss. s. G1 in part of its main distribution range in Europe using 8274 bp of mtDNA; (ii) compare the results with those derived from previously used shorter mtDNA sequences and highlight the major differences. We sequenced a total of 91E. granulosuss. s. G1 isolates from six different intermediate host species, including humans. The isolates originated from seven countries representing primarily Turkey, Italy and Spain. Few samples were also from Albania, Greece, Romania and from a patient originating from Algeria, but diagnosed in Finland. The analysed 91 sequences were divided into 83 haplotypes, revealing complex phylogeography and high genetic variation ofE. granulosuss. s. G1 in Europe, particularly in the high-diversity domestication centre of western Asia. Comparisons with shorter mtDNA datasets revealed that 8274 bp sequences provided significantly higher phylogenetic resolution and thus more power to reveal the genetic relations between different haplotypes.

Echinococcus granulosus is a taeniid cestode and the etiological agent of an infectious zoonotic disease known as cystic echinococcosis (CE) or hydatid disease. CE is a serious public health concern in many parts of the world, including the Americas, where it is highly endemic in many regions. Echinococcus granulosus displays high intraspecific genetic variability and is divided into multiple genotypes (G1-G8, G10) with differences in their biology and etiology. Of these, genotype G1 is responsible for the majority of human and livestock infections and has the broadest host spectrum. However, despite the high significance to the public and livestock health, the data on genetic variability and regional genetic differences of genotype G1 in America are scarce. The aim of this study was to evaluate the genetic variability and phylogeography of G1 in several countries in America by sequencing a large portion of the mitochondrial genome. We analysed 8279bp of mtDNA for 52 E. granulosus G1 samples from sheep, cattle and pigs collected in Argentina, Brazil, Chile and Mexico, covering majority of countries in the Americas where G1 has been reported. The phylogenetic network revealed 29 haplotypes and a high haplotype diversity (Hd=0.903). The absence of phylogeographic segregation between different regions in America suggests the importance of animal transportation in shaping the genetic structure of E. granulosus G1. In addition, our study revealed many highly divergent haplotypes, indicating a long and complex evolutionary history of E. granulosus G1 in the Americas.

To the Editor: Alveolar echinococcosis, which is caused by the fox tapeworm Echinococcus multilocularis, is an emerging disease in Europe that shows a high mortality rate (1). Humans can become infected after ingesting parasite eggs (e.g., through direct contact with dogs and red foxes [Vulpes vulpes] or with their contaminated feces). E. multilocularis tapeworm eggs are extremely resistant and can remain viable in the environment for years (2). Numbers of red foxes have increased in many countries in Europe in recent decades, and the E. multilocularis tapeworm has also expanded its range. This tapeworm has recently been reported in 17 countries in Europe, including Lithuania, Latvia, and Estonia (1). Foxes and associated tapeworms are also increasingly found in urban areas, prompting considerable public health concern (1,3). Foxes began to colonize urban areas in Estonia in 2005, and they have since been reported in 33 of 47 towns nationwide (L. Plumer, et al. unpub. data). Because ≈30% of foxes are infected with the E. multilocularis tapeworm in natural habitats in Estonia (4), it is essential to monitor parasite spillover into urban areas, where it could become a serious public health risk. Consequently, there is an acute need for methods that can effectively detect the parasite and thereby help prevent human infection. Although immunologic (2) and genetic methods (5–7) are available for identifying Echinococcus spp. parasites, a sensitive molecular diagnostic method that detects tapeworms and identifies their host species from degraded fecal samples would be useful. The purposes of this study were to develop a sensitive, noninvasive, genetic method to identify the host species by discriminating between feces of red foxes and dogs; detect E. multilocularis tapeworms in feces and distinguish them from the related parasite E. granulosus; and collect carnivore feces in an urban area in Estonia to identify this tapeworm. Fecal samples suspected to be from red foxes were collected during January–March 2012 and January–March 2013 from streets and grassy areas of Tartu, Estonia. Tartu is a relatively small city (area 39 km2) with 98,000 human inhabitants. We surveyed 14 transects, each ≈4 km in length, that included all major districts in the city (Figure). Each transect was searched weekly during the study period (total ≈850 km surveyed). Figure Location of Tartu in northeastern Europe, Estonia, and red fox feces sampling area in Tartu. The Tartu City boundary is indicated by a solid black line, survey transects are indicated by dashed lines, and fox fecal samples (n = 28) are indicated by circles. ... A total of 137 fecal samples were collected and stored at −80°C for ≥1 week to avoid risk of infection from any Echinococcus spp. eggs present (2) because E. multilocularis (4) and E. granulosus (8,9) tapeworms have been found in Estonia. Samples of ≈250 mg were placed into 2-mL tubes, heated at 65°C for 15 min, and stored at −80°C. The heating and cooling procedure helps to break the parasite egg shells, enabling more efficient DNA extraction. DNA was extracted by using the QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Species-specific primers (Technical Appendix Table) were designed to amplify short sequences of mitochondrial DNA. On the basis of primer specificity and amplicon size, we determined host and parasite species (Technical Appendix Figure 1). DNA extraction and PCR were performed in a laboratory dedicated to environmental samples (for a complete description of methods, see Technical Appendix). DNA was successfully extracted and amplified from 119 (86.9%) of 137 fecal samples. Of usable samples, 28 (23.5%) were from red foxes and 91 (76.5%) were from dogs. Two fox fecal samples (7.1%; 95% binomial CIs 0.9%–23.5%) were infected with E. multilocularis tapeworms; none of the dog samples were infected. To verify parasite identification, we amplified DNA from the 2 E. multilocularis–positive samples with E. multilocularis–specific primers and sequenced the amplification products. To verify host species identification, we used primers that produced longer amplification products (327 and 197 bp) than the corresponding PCR primers and sequenced amplification products from 5 fox samples and 5 dog samples. Sequencing procedures were performed according to the methods of Saarma et al. (10). Sequences from both E. multilocularis–positive samples showed 100% identity with an E. multilocularis tapeworm sequence (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AB018440","term_id":"6138994","term_text":"AB018440"}}AB018440) (Technical Appendix Figure 2). All sequenced fox and dog samples also belonged to the corresponding species. To estimate the sensitivity of this noninvasive genetic method, we determined the number of E. multilocularis eggs necessary to obtain a positive PCR result (Technical Appendix Figure 3). One egg was sufficient to give an E. multilocularis tapeworm–specific result. In summary, we developed a noninvasive genetic method that identifies E. multilocularis tapeworms and their host species in carnivore fecal samples found in urban environments. Furthermore, these tapeworms can even be detected in fecal samples from red foxes when only 1 parasite egg is present. Thus, this method is highly sensitive and discriminatory and can be used with degraded fecal samples to monitor E. multilocularis tapeworms and their hosts.

Background Echinococcus granulosus and E. multilocularis are tapeworm parasites of major medical and veterinary importance, causing cystic and alveolar echinococcosis, respectively. Both diseases are listed among the most severe parasitic diseases in humans, representing 2 of the 17 neglected diseases prioritised by the World Health Organisation. However, little is known about the role of urban animals in transmission of both parasite species. Findings A sensitive non-invasive genetic method was used to monitor E. granulosus and E. multilocularis infection among dog faecal samples collected from an urban area in Estonia in 2012–13. Out of 181 dog faecal samples analysed, 2.2% tested positive for E. granulosus, determined by sequencing as genotype G1. None of the samples tested positive for E. multilocularis. Conclusions We report contamination of an urban environment with highly pathogenic E. granulosus G1 disseminated by dogs, and a potential risk to human health.