Search

Abstract: Echinococcus is a genus of cestode parasites of paramount veterinary and medical importance globally. Two species, Echinococcus granulosus sensu lato and Echinococcus multilocularis, are endemic to North America and are the etiologic agents of cystic echinococcosis and alveolar echinococcosis, respectively. North America is currently experiencing an epidemiological shift in the state of transmission, distribution, and prevalence of E. multilocularis, following the introduction of the European variant of the parasite. The rapid expansion of the parasite into the northeastern United States has resulted in novel detections of spillover to humans, indicating the need for active surveillance to direct public health education and disease control measures. Our surveillance of the parasite in definitive hosts in New York State from 2019 to 2023 yielded 8 detections of the parasite, for a sample prevalence of 8.3% (8/96). Phylogenetic analysis of mitochondrial markers confirmed that all New York isolates belong to the European lineage and group in a well-supported clade with E. multilocularis isolates described from Slovakia and Poland. Investigation of the highly polymorphic EmsB microsatellite marker revealed several E. multilocularis genotypes circulating in New York, including 1 that is distinct from European isolates detected in western North America. These data are consistent with 1 or more unique introductions from Europe rather than a spread from western regions of Canada and the United States, where other European variants are found. Echinococcus multilocularis appears to have a complex epidemiology in North America that warrants further detailed surveillance to evaluate the risks for zoonotic transmission., (© American Society of Parasitologists 2024.)

Herein we describe a single nucleotide polymorphism-specific polymerase chain reaction (PCR) assay to rapidly detect and differentiate variants belonging to the European and North American lineages of Echinococcus multilocularis in clinical samples. This is an extremely relevant and applicable test in North America because the range of E. multilocularis continues to expand across the continent and because of a rise in prevalence in wildlife, domestic animals, and humans. The endemic North American (NA) and introduced European (EU) variants are believed to have different pathogenic potentials, with the EU variants being more infective and pathogenic than the NA variants. The rise of the EU variants of E. multilocularis increases the risk of spillover from wildlife to humans because of its increased potential for infectivity. Current PCR-based diagnostics can detect E. multilocularis deoxyribonucleic acid (DNA), but DNA sequencing is required to identify the specific variant. Our assay provides a straightforward conventional PCR method to differentiate the NA and EU variants, and we suggest this same approach could be used for the diagnosis of other parasites or variants that are genetically very similar. As surveillance continues for E. multilocularis across North America, identifying the different genetic variants from different geographic regions will become essential to understanding the current epidemiological shift that the parasite is experiencing, as well as informing public health decisions in affected areas., (© American Society of Parasitologists 2024.)

Alveolar echinococcosis (AE) is a rare but severe disease that affects more than 18,000 people worldwide per year. The complete sequencing of the mitochondrial genome of Echinococcus multilocularis has made it possible to study the genetic diversity of the parasite and its spatial and temporal evolution. We amplified the whole mitochondrial genome by PCR, using one uniplex and two multiplex reactions to cover the 13,738 bp of the mitogenome, and then sequenced the amplicons with Illumina technology. In total, 113 samples from Europe, Asia, the Arctic and North America were analyzed. Three major haplogroups were found: HG1, which clustered samples from Alaska (including Saint-Lawrence Island), Yakutia (Russia) and Svalbard; HG2, with samples from Asia, North America and Europe; and HG3, subdivided into three micro-haplogroups. HG3a included samples from North America and Europe, whereas HG3b and HG3c only include samples from Europe. In France, HG3a included samples from patients more recently diagnosed in a region outside the historical endemic area. A fourth putative haplogroup, HG4, was represented by only one isolate from Olkhon Island (Russia). The increased discriminatory power of the complete sequencing of the E. multilocularis mitogenome has made it possible to highlight four distinct geographical clusters, one being divided into three micro-haplogroups in France., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Introduction: Alveolar echinococcosis (AE) is a life-threatening disease in humans caused by the larval stage of Echinococcus multilocularis . Domestic animals, dogs, foxes, and small mammals constitute the circular chain of AE. To evaluate the infection, distribution, and genetic polymorphism of AE in the Ili Prefecture (Nilka, Xinyuan and Zhaosu), we conducted this survey., Methods: In June and July 2018, 267 small mammals were captured using water-infusion and mousetrap methods. Combined pathogenic and molecular biological methods were used to observe the histopathology of Echinococcus carried by rodents, amplify the mitochondrial nad 1 gene of the pathogen, and investigate the genotype and haplotype diversity of Echinococcus in rodents in Ili Prefecture., Results: Morphological identification revealed that these captured small mammals belonged to three species, with Microtus gregalis being the dominant species (183/267). Pathological and molecular biological results confirmed that E. multilocularis was the pathogen of echinococcosis in small mammals, with an infection rate of 15.73% (42/267). Among the three areas sampled, the highest infection rate of rodents was 25.45% (14/55) in Nilka County. However, there was no significant difference in the infection rates between regions (χ 2 = 5.119, p > 0.05). Of the three captured rodent species, M. gregalis had the highest infection rate of 17.49% (32/183), but there was no significant difference in infection rates between the rodent species (χ 2 = 1.364, p > 0.05). Phylogenetic analyses showed that the nad1 gene sequences obtained in this study clustered in the same clade as isolates from China. These isolates contained 21 haplotypes (Hap_1-21); Hap_2 was the most common haplotype (9/42). Furthermore, haplotype diversity (0.925 ± 0.027) and nucleotide diversity (0.01139 ± 0.00119) were higher in the Ili Prefecture than in other regions, indicating that population differentiation was high. Tajima's D and Fu's Fs tests were negative (p > 0.10), indicating that the population had expanded. The low fixation index (Fst) ranged from 0.00000 to 0.16945, indicating that the degree of genetic differentiation was different among different populations., Discussion: In summary, Ili Prefecture is a high incidence area of AE, and Microtus spp. may play an important role in the transmission of AE in this area. The results of this study provide basic data for further study of the molecular epidemiology, genetic differences, and control of E. multilocularis in the Ili Prefecture, Xinjiang., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Wang, Zhao, Ban, Zhang, Quan, Teliewuhan, He, Chen and Zhang.)

Objective: The aim of this study was to identify Echinococcus species by morphological and molecular means., Methods: A dead gray wolf (Canis lupus) was found near Erzurum province and brought to the parasitology laboratory. Sedimentation and counting technique (SCT) and polymerase chain reaction (PCR) analysis were conducted., Results: The SCT implications indicated that the wolf had a substantial worm burden (62,720 and 49,280 parasites) due to a co-infection of E. granulosus s.l. and E. multilocularis. Genus/species-specific PCR was used to analyze DNA extracted from adult worms and confirmed as E. granulosus s.s. and E. multilocularis, utilizing COI and 12S rRNA gene sequence analysis, respectively., Conclusion: This report presents the first co-detection of E. granulosus s.s. and E. multilocularis in a gray wolf found in an urban area in a highly endemic area for human echinococcosis in northeastern Turkey. The results emphasize that AE is not only a problem of rural areas, but also occurs in urban areas, which may pose a threat to public health. Therefore, surveillance in urban areas is crucial. The need to develop new control strategies for domestic and wildlife in the study area is also highlighted., (© 2024. The Author(s).)

Background: Alveolar echinococcosis (AE) is a clinically serious zoonosis caused by the fox tapeworm Echinococcus multilocularis. We studied the diversity and the distribution of genotypes of E. multilocularis isolated from foxes in Brandenburg, Germany, and in comparison to a hunting ground in North Rhine-Westphalia., Methods: Echinococcus multilocularis specimens from 101 foxes, 91 derived from Brandenburg and 10 derived from North Rhine-Westphalia, were examined. To detect potential mixed infections with different genotypes of E. multilocularis, five worms per fox were analyzed. For genotyping, three mitochondrial markers, namely cytochrome c oxidase subunit 1 (Cox1), NADH dehydrogenase subunit 1 (Nad1), and ATP synthase subunit 6 (ATP6), and the nuclear microsatellite marker EmsB were used. To identify nucleotide polymorphisms, the mitochondrial markers were sequenced and the data were compared, including with published sequences from other regions. EmsB fragment length profiles were determined and confirmed by Kohonen network analysis and grouping of Sammon's nonlinear mapping with k-means clustering. The spatial distribution of genotypes was analyzed by SaTScan for the EmsB profiles found in Brandenburg., Results: With both the mitochondrial makers and the EmsB microsatellite fragment length profile analyses, mixed infections with different E. multilocularis genotypes were detected in foxes from Brandenburg and North Rhine-Westphalia. Genotyping using the mitochondrial markers showed that the examined parasite specimens belong to the European haplotype of E. multilocularis, but a detailed spatial analysis was not possible due to the limited heterogeneity of these markers in the parasite population. Four (D, E, G, and H) out of the five EmsB profiles described in Europe so far were detected in the samples from Brandenburg and North Rhine-Westphalia. The EmsB profile G was the most common. A spatial cluster of the E. multilocularis genotype with the EmsB profile G was found in northeastern Brandenburg, and a cluster of profile D was found in southern parts of this state., Conclusions: Genotyping of E. multilocularis showed that individual foxes may harbor different genotypes of the parasite. EmsB profiles allowed the identification of spatial clusters, which may help in understanding the distribution and spread of the infection in wildlife, and in relatively small endemic areas., (© 2021. The Author(s).)

Human alveolar echinococcosis (AE) is a zoonotic cestode infection which is usually fatal in the absence of treatment. Treatment involves major surgery or indefinite antiparasitic therapy. The incidence is rising in Europe and Asia, with an increased risk observed in immunocompromised individuals. Previously, AE acquisition in North America was extremely rare, except for one remote Alaskan Island. Recent studies have demonstrated a new European-like strain of Echinococcus multilocularis (Em) in wildlife and in human AE in western Canada. We report the experience of all AE patients diagnosed in Alberta. Each was diagnosed by histopathology, serology, and PCR-confirmed by a reference laboratory. Seventeen cases of human AE, aged 19-78 years, nine females, were diagnosed between 2013 and 2020: all definitely or probably acquired in Alberta. Six lived in urban areas, and 14 had kept dogs. In eight, the lesions were found incidentally on abdominal imaging performed for other indications. Six were immunocompromised to varying degrees. Six were first diagnosed at surgery. All have been recommended benzimidazole therapy. One died of surgical complications. Clinicians should be aware of this diagnostic possibility in patients presenting with focal nonmalignant hepatic mass lesions. Greater urbanization of coyotes, the predominant definitive host of Em in Alberta, and growing numbers of immune suppressed individuals in the human population may lead to increasing recognition of AE in North America.

Echinococcus multilocularis is a parasite species of zoonotic importance which can be fatal to humans and causes Alveolar Echinococcosis (AE). This report describes the development of a cyst from the liver of a wild boar and molecular confirmation of its identification. The cyst material was obtained from the liver of a wild boar killed by hunters. Genomic DNA was extracted from the germinal layer of the cyst material, and 875 bp mitochondrial cytochrome c oxidase subunit I (COI) gene fragment was amplified by PCR and sequenced. A BLAST search matched 100% with published Echinococcus multilocularis sequences. This study confirms the occurrence of E. multilocularis in a wild boar for the first time in Turkey., (© American Society of Parasitologists 2020.)

Alveolar and cystic echinococcosis (AE, CE) caused by E. multilocularis and E. granulosus s.l., respectively, are considered emerging zoonotic diseases in Kyrgyzstan with some of the world highest regional incidences. Little is known regarding the molecular variability of both species in Kyrgyzstan. In this study we provide molecular data from a total of 72 parasite isolates derived from humans (52 AE and 20 CE patients) and 43 samples from dogs (23 infected with E. multilocularis and 20 with E. granulosus s.l.).Genetic variability in E. multilocularis was studied using the concatenated complete sequences of the cob, nad2 and cox1 mitochondrial genes adding a total of 3,558bp per isolate. The cob/nad2/cox1 A2 haplotype was identified in 63.4% of the human and in 65.2% of the dog samples. This haplotype was originally described in samples from Kazakhstan and St. Lawrence Island (Alaska, USA). We also describe here 16 non-previously defined variants of E. multilocularis (called A11-A26). All haplotypes cluster together within the Asian group in the haplotype network. Based on Fst values, low level of genetic differentiation was found between the populations of E. multilocularis isolated from different regions within the country. However, high degree of differentiation was found when all the concatenated sequences from Kyrgyzstan are considered as a single population and compared with the population of the parasite from the neighbouring country China. In the case of E. granulosus s.l. the analysis was based in 1,609bp of the cox1 gene. One isolate from a dog was identified as E. equinus, while all the other sequences were identified belonging to E. granulosus s.s. In total, 24 cox1 haplotypes of E. granulosus s.s. were identified including the already described variants: Eg01 (in 6 samples), Eg33 (in 4 samples), EgCl04 (in 2 samples), Eg03 (in 1 sample) and Eg32 (in 1 sample). From the twenty-five other isolates of E. granulosus s.s. a total of 19 non-previously described cox1 haplotypes were identified and named as EgKyr1 to EgKyr19. The most common haplotype infecting human is the EgKyr1 which was found in 5 isolates.The cob/nad2/cox1 A2 haplotype of E. multilocularis is responsible for the majority of human infections in Kyrgyzstan and is also found in the majority of dogs included in this study. Further similar studies in different parts of Asia could elucidate if it is also the most common variant infecting humans in other countries. It remains unknown if this particular haplotype presents differences in virulence which could have contributed to the emergency of alveolar echinococcosis in Kyrgyzstan. In the case of E. granulosus s.s. it seems that there is no dominant haplotype infecting humans in Kyrgzstan. Further characterization of biological or antigenic features of dominant mitochondrial haplotypes could help to elucidate if they present differences which could be relevant in the diagnostic, pathogenicity or in the host/parasite interaction when infecting humans., Competing Interests: The authors have declared that no competing interests exist.

Alveolar echinococcosis, caused by the larval stages of the tapeworm Echinococcus multilocularis (Leuckart, 1863), is of increasing concern in the northern hemisphere. Most cases of alveolar echinococcosis (excluding Alaska) appear to be linked with European and Asian genotypes that highlight the need for a more precise delimiting of their actual distribution and tracing historical episodes of their translocations and introductions into new areas. We have herein summarized previous available research studies, which mentioned firstly described geographic M1/M2 variants of E. multilocularis using molecular tools (established by sequencing of mitochondrial genes cox1, 366 bp and nad1, 471 bp), in an attempt to consolidate their correct affiliations with the geographic origin in sense of the original description from the early 1990´s. Since 2009, inverted designations (M1 named as M2 and vice versa) are being prevailing in research literature (we found ten erroneous vs. three correct classifications) that might bias genetic interpretation of comparative data in specific cases. When comparing M1/M2 profiles to those obtained from mitochondrial evidences over the last decades, the phylogenetic analysis revealed that the M1 strain (originally described from China, Alaska, North America) grouped with the Asian clade of E. multilocularis more recently established, whereas the M2 strain (described from the German vole) had a specific structure, in cox1 clustering with the North American clade. It is presumed that events of intercontinental expansion and isolation covering glacial and interglacial periods during the late Pleistocene have likely accounted for the transmission of this discrete genotype from Beringia into endemic area of western and central Europe via circumpolar movements of foxes., Competing Interests: Declaration of Competing Interest The authors state that they have no conflict of interest., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

Introduction: Echinococcus multilocularis is a very dangerous zoonotic parasite threatening human health. The red fox is the main definitive host, and cats and dogs less commonly. Rats can be intermediate hosts., Objective: The aim of the study was to determine the parasitofauna of Norway rats and some cats and dogs living on a farm near a forest., Material and Methods: A parasitological section on 15 Norway rats was conducted. The internal organs were examined by means of macroscopic and microscopic methods. For molecular examination, a QIAmp DNA Mini Kit (Qiagen) was used., Results: Based on necropsy, parasitological and molecular examinations, of the 15 examined rats, 1 was found to have larvae of E. multilocularis , while 3 others had eggs of Hymenolepis diminuta, H. nana and Syphacia obvelata . The faeces of the pets did not contain any developmental forms of parasites., Conclusions: This is the first case of Echinococcus multilocularis infestation in a rat in Poland.

Objective: To amplify and sequence Coxl and Nadl genes in Echinococcus multilocularis isolates from Qinghai Province, and to create phylogenetic trees and molecular clocks, so as to provide evidence for estimating the evolutionary relationships and origins of E. multilocularis in Qinghai Province., Methods: Twenty-two post-surgical specimens of patients with hepatic alveolar echinococcosis were sampled from Qinghai Provincial People's Hospital in 2017. The Coxl and Nadl genes were amplified from E. multilocularis samples and sequenced. Then, the gene sequences were aligned to the Coxl and Nadl genes of Echinococcus species in GenBank database. The intra-species variation was observed, and the phylogenetic tree and molecular clock were created., Results: All E. multilocularis samples shared more than 99% genetic homology with the sequences of Coxl and Nadl genes from the E. multilocularis Asian strain in the GenBank database. A total of 6 genotypes were identified, including 2 isolates that had no 100% homology with the sequences of known genes in the GenBank database. Phylogenetic tree analysis revealed remarkable clustering of the E. multilocularis samples with the E. multilocularis Asian strain, and the E. multilocularis isolates from Qinghai Province were estimated to date back to 94 000 years ago by the molecular clock., Conclusions: The present study characterizes 6 E. multilocularis genotypes in Qinghai Province, including 2 novel genotypes. Asian strain is the predominant strain of E. multilocularis in Qinghai Province, and the E. multilocularis isolates from Qinghai Province date back to 94 000 years ago.

Echinococcosis is a disease caused by the Echinococcus species that parasitizes in humans. Alveolar echinococcosis (AE) which is caused by Echinococcus multilocularis is harmful to humans. AE mainly occurs in the liver and can be transferred to retroperitoneal lymph nodes, lung, brain, bone, spleen and other organs through lymphatic and blood vessels. Cholangiocarcinoma can occur in the intrahepatic and extrahepatic bile ducts and is more common in the hilar. We reported a case of hilar bile duct alveolar echinococcosis which was originally misdiagnosed an cholangiocarcinoma.

Background: Dogs and cats can transmit zoonotic helminths to humans, e.g. Toxocara spp. and Echinococcus multilocularis. Strategic deworming may help minimize this risk. Studies in several European countries have shown that pets are dewormed less frequently against roundworms and tapeworms than recommended by the European Scientific Counsel Companion Animal Parasites (ESCCAP). The objective of this study was to identify percentages of dogs and cats falling into the different risk categories defined by the German ESCCAP guidelines and to evaluate whether deworming frequency and parasite monitoring in Germany follows these guidelines., Results: According to questionnaire results from 500 dog and 500 cat owners, deworming of dogs in Germany averages 2.07 times/year while for cats this average is 1.72 times/year. In contrast, evaluation of risk factors placed only 2% (10/500) of dogs in ESCCAP category A with a recommended deworming/examination frequency of 1-2 times per year, while 4.8% (24/500) were placed in category B (4 treatments/examinations per year recommended), 30.8% (154/500) in category C (12 treatments/examinations per year against tapeworms and 4 treatments/examinations per year against roundworms recommended) and 62.4% (312/500) in category D (12 treatments/examinations per year recommended). All cats were placed either in risk group A [52.8% (264/500)] or D [47.2% (236/500)]. Generalized linear models indicated that risk group D cats were treated significantly more often against helminths than risk group A cats. There were no significant differences in deworming frequency between risk groups in dogs. The most important factor influencing deworming frequency was the frequency of veterinary visits. Dogs and cats were treated significantly more often if owners visited their veterinarian more than once yearly., Conclusions: The percentage distribution of risk groups considerably varied between dogs and cats. Nevertheless, 62% of dogs and 47% of cats were assigned to category D for which monthly treatments/examinations are recommended by the ESCCAP guidelines. Veterinarians play a key role in instructing pet owners with regard to helminthoses and their prevention, and should take the time for adequate risk assessments. The reported low deworming frequencies despite the high potential parasite infection risk suggests that pet owner advice through veterinarians needs to be improved.

Efficient and sensitive diagnostic tools are essential for the study of the eco-epidemiology of Echinococcus species. We evaluated an automated magnetic bead-based DNA extraction commercial kit followed by qPCR (MB-qPCR), for the detection of Echinococcus multilocularis and Echinococcus canadensis in coyote (Canis latrans) fecal samples. The diagnostic sensitivity was determined by validating the method against the scraping, filtration, and counting technique (SFCT) for samples collected in Canada. From the 60 samples tested, 27 out of 31 SFCT positives samples for Echinococcus cestodes were positive in the MB-qPCR for E. multilocularis, with a sensitivity of 87.1% (95% CI 70.2 to 96.4%). Two samples were also positive for E. canadensis in the MB-qPCR and confirmed by morphological identification of adult worms. The agreement of the MB-qPCR and the SFCT was statistically significant with a kappa value of 0.67 (95% CI 0.48-0.85; p value < 0.001). The magnetic bead-based DNA extraction followed by qPCR proved to have a sensitivity comparable to the SFCT to detect E. multilocularis. Although the diagnostic sensitivity for E. canadensis was not estimated, MB-qPCR identified E. canadensis cases previously overlooked when using SFCT. We propose a combination of molecular and morphological identification using the MB-qPCR and the SFCT to detect both parasites, allowing for a more efficient large-scale surveillance, and detecting co-infections of Echinococcus species that can be difficult to identify when only based on morphology.

Coyotes (Canis latrans) are resilient, adaptable, cosmopolitan omnivores that are increasingly prevalent in urban environments, where they interact with both humans and domestic dogs. Coyotes potentially transmit zoonotic parasites, including the tapeworm Echinococcus multilocularis, which appears to be increasing in prevalence in western North America. In this study, we analysed the carcasses of 23 urban coyotes in Edmonton, Alberta, Canada. Focusing primarily on the helminth community, we recovered three tapeworm species (E. multilocularis, Taenia pisiformis, T. serialis), four nematodes (Toxascaris leonina, Uncinaria stenocephala, Capillaria sp., Physaloptera sp.), and two trematodes (Alaria arisaemoides and A. americana). Compared to previous studies of urban coyotes conducted in North America, we report one of the highest levels of E. multilocularis infection in North America: 65.2% infection prevalence. These results amplify concerns expressed by others about the increasing prevalence of this zoonotic parasite and the role coyotes may play in parasite transmission. More research is needed to better understand how various ecological factors, urbanization and wildlife management practices influence the transmission of potentially zoonotic parasites such as E. multilocularis.

The aim of this review was to assess our current knowledge on phylogeography and global genetic structure of Echinococcus multilocularis populations originating from rodents, wild canid hosts, and human. Six bibliographic databases were searched from 1990 to 2017, identifying a total of 110 publications. The cytochrome c oxidase subunit 1 (cox1) and cytochrome b (cytb) sequences of E. multilocularis from Asia, Europe, and North Americas were analyzed to estimate the diversity and neutrality indices, and genetic differentiation. A total of 69 (cox1, 36.7%) and 16 haplotypes (cytb, 19.2%) were grouped into various geographical clades. A parsimonious haplotype network demonstrated a star-like feature with haplo-groups Em2 (Asia: 36%), Em105 (Eastern Tibetan plateau: 4.8%), Em46 (Europe: 9.1%), Em73, (Europe: 2.7%) and Em92 (North Americas: 4.3%) as the most common haplotypes. A relatively high level of genetic diversity was detected in rodent-derived E. multilocularis isolates (Haplotype diversity: 0.944), wild canids (Hd: 0.912), and human origin (Hd: 0.704). The highest number of haplotypes (n = 59) and the highest haplotype diversity (0.969) were identified in the Asian and European populations, respectively. Cladistic phylogenetic tree indicated the European clade has a sister relationship with the Asian clade. However, some North American haplotypes were assigned to the European clade together with haplotypes from Poland. The statistically significant Fst values indicated that E. multilocularis populations of Asian-European, Asian-North American, and European-North American origins were genetically differentiated (Fst: 0.22624 to 0.43059). An occurrence of distinct parasite populations suggests that E. multilocularis derived from glacial refugia have been plausibly sustained by indigenous hosts during the Pleistocene Epoch., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Alveolar echinococcosis, caused by the tapeworm Echinococcus multilocularis, is one of the world's most dangerous zoonosis and an emerging disease with growing incidence in humans. The disease has been reported in new areas and host species in the last two decades, and the primary hosts of the parasite - red fox, golden jackal and grey wolf - are expanding their distribution in Europe. Here we report the morphological and molecular identification of Echinococcus multilocularis tapeworms in one of 29 carcasses of adult golden jackals in Croatia, where the only previous report of the parasite was in red foxes in 2016. These results suggest that alveolar echinococcosis should be treated as an emerging disease in Croatia.

Echinococcus multilocularis is a zoonotic tapeworm of medical and veterinary importance that occasionally infects accidental intermediate hosts causing severe disease or even death. In dogs, alveolar echinococcosis has been reported mainly in central Europe and Canada. The paper presents clinical, biochemical and histological signs of alveolar echinococcosis in a dog from Slovakia and the results of mitochondrial nad1 gene analysis. In 2016, 11-years old Siberian husky female suffering from inappetence was presented to the Veterinary Clinic in Žilina. Biochemical blood examination and blood count showed only minor changes, but abdominal ultrasonography showed the hepatomegaly and the presence of nonhomogeneous hypodense lobulated formation in the left liver lobe. Histological and molecular examinations of excided tissue confirmed the diagnosis of alveolar echinococcosis of the liver. BLAST analysis of E. multilocularis nad1 gene revealed that the nucleotide sequence did not exactly match the previously identified M1 (AJ237639) and/or M2 genotype (AJ237640). In total, two single nucleotide polymorphisms (SNPs) occurred within overlapping region of nad1 gene. In the discussion, clinical and laboratory findings of the infection in other dog patients are compared and the possibilities of diagnosis and therapy of the disease are discussed.

In Western Europe, the Echinococcus multilocularis lifecycle is predominantly sylvatic, typically involving red foxes (Vulpes vulpes) as the main definitive hosts with Microtus spp. and Arvicola spp. as intermediate hosts. During a 4-year surveillance study (2012-2015), Danish red foxes and raccoon dogs (n = 1345) were examined for E. multilocularis. Moreover, 134 insectivores and rodents collected in South Jutland during spring and summer 2016 were examined for the presence of metacestodes. The sedimentation and counting technique and molecular typing were used to identify E. multilocularis infections in the carnivores, while the rodent livers were examined macro- and microscopically for parasite lesions. Following morphological identification of E. multilocularis adult worms, the identity was verified by sequence analysis of the 12S rRNA gene in most cases (n = 13). Echinococcus multilocularis infection was demonstrated in 19 red foxes (Vulpes vulpes) originating from only two specific areas of South Jutland, namely Højer and Grindsted, and in two raccoon dogs (Nyctereutes procyonoides), originating from Højer. In Højer, 28.5% (CI 95% 11.7-45.3) of the examined red foxes were E. multilocularis positive per year. Moreover, positive red foxes were identified each year from 2012 to 2015, while E. multilocularis positive red foxes were only identified in Grindsted in 2013 (4.0%) and 2014 (6.4%). In contrast, all collected rodents were negative for E. multilocularis. We conclude that E. multilocularis is locally endemic in South Jutland with a high local prevalence in Højer.

Human alveolar echinococcosis (AE) is a highly pathogenic zoonotic parasitic disease caused by Echinococcus multilocularis. An ultrasound study in southern Kyrgyzstan during 2012 revealed a prevalence of 4.2% probable or confirmed AE and an additional 2.2% possible AE, representing an emerging situation. The risk for probable or confirmed AE was significantly higher in dog owners.

Echinococcus multilocularis has been spreading through Europe but has not yet been reported in Croatia. We report the results of a surveillance programme to detect E. multilocularis in red foxes (Vulpes vulpes) in different parts of Croatia. PCR-based screening of faecal samples from 238 red foxes in 2015 and 150 in 2016 indicate prevalences of 7.5% in 2015 and 6.6% in 2016 (overall 7.2%, CI 4.9 to 10.3). Positive samples were confirmed by sequencing parts of the nad1 gene and the gene encoding mitochondrial 12S rRNA. Geographic locations of all examined and positive cases were mapped to provide data on the distribution of E. multilocularis. Our results provide the first detection of E. multilocularis in Croatia and extend the southern boundary of this parasite's endemic area.

Alveolar echinococcosis is a severe zoonotic disease caused by the parasite Echinococcus multilocularis. In Europe, the lifecycle of this cestode is mainly sylvatic based on a prey-predator interaction between the red fox and small rodents as definitive and intermediate hosts, respectively. National surveillance of E. multilocularis in red foxes in Poland has reported a clear distinction between low endemic areas (from 2 to 5.7%) in the western half and high endemic areas (11.8 to 50.0%) in the eastern half of the country. A drastic increase of prevalence has been observed in the eastern half of Poland since the 2000's. Microsatellite EmsB genotyping was performed on 301 E. multilocularis worms from 87 foxes sampled throughout Poland, leading to identification of 29 EmsB profiles. The main profile, Pol19, was identified across the country and accounted for 44.9% of the worms collected. The conformity of 18 Polish profiles was established by comparison with previous profiles identified in Europe, but none corresponded to the most common European profiles. Poland was confirmed as a peripheral area of the main European focus, with more recent colonization by the parasite. The sharing of common profiles mainly by neighboring provinces was confirmed by a clustering analysis identifying four main groups. Expansion of the parasite in Poland in these four groups appears to be influenced by the situation in neighboring countries. Acquiring EmsB genotyping data from eastern European countries, for which very few data are reported, is necessary to understand the expansion of the parasite in the whole of Europe., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Alveolar echinococcosis (AE) is a life-threatening human disease caused by Echinococcus multilocularis transmitted between rodents and dogs/foxes in the Northern Hemisphere. The study aims to identify the genetic variation of the parasite in AE patients from China., Methods: E. multilocularis isolates were collected from wild small mammals (n = 6) and AE patients (n = 56) from western China. Genomic DNA was extracted from different tissue samples including paraffin tissue blocks, ethanol fixed tissues and frozen tissues surgically removed. Two mitochondrial gene fragments (526 bp for cob and 474 bp for nad2) of E. multilocularis were amplified and sequenced., Results: The parasite fragment sequences of cob fragments from AE patients showed two haplotypes, and nad2 gene fragment sequences had four haplotypes. The gene sequences from Microtus sp. were 100% identical to the sequences of some isolates from AE patients. These haplotypes were distributed in both Qinghai and Xinjiang provinces. Alignment analysis with the sequences from the GenBank databases showed five genotypes including three Asian genotypes, one from Europe and one from North America., Conclusions: Most AE patients harbored the Asian genotype 1 which may be an indication of its relative frequency in the definitive hosts and the environment or of its pathogenicity to humans, which calls for further research.

The larval stages of the cestodes Echinococcus multilocularis and Echinococcus granulosus cause the important zoonoses known as larval echinococcoses. These larvae are protected by a unique, massive, mucin-based structure known as the laminated layer. The mucin glycans of the E. granulosus laminated layer are core 1- or core 2-based O-glycans in which the core Galpβ1-3 residue can initiate a chain comprising one to three additional Galpβ1-3 residues, a motif not known in mammalian carbohydrates. This chain can be capped with a Galpα1-4 residue, and can be ramified with GlcNAcpβ1-6 residues. These, as well as the GlcNAcpβ1-6 residue in core 2, can be decorated with the Galpα1-4Galpβ1-4 disaccharide. Here we extend our analysis to the laminated layer of E. multilocularis, showing that the non-decorated cores, together with Galpβ1-3(Galpα1-4Galpβ1-4GlcNAcpβ1-6)GalNAc, comprise over 96% of the glycans in molar terms. This simple laminated layer glycome is exhibited by E. multilocularis grown either in vitro or in vivo. Interestingly, all the differences with the complex laminated layer glycome found in E. granulosus may be explained in terms of strongly reduced activity in E. multilocularis of a putative glycosyltransferase catalysing the elongation with Galpβ1-3. Comparative inter-species analysis of available genomic and transcriptomic data suggested a candidate for this enzyme, amongst more than 20 putative (non-core 1) Gal/GlcNAc β1-3 transferases present in each species as a result of a taeniid-specific gene expansion. The candidate gene was experimentally verified to be transcribed at much higher levels in the larva of E. granulosus than that of E. multilocularis., (Copyright © 2016 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Echinococcus multilocularis is a cestode that causes human alveolar echinococcosis, a lethal zoonosis of public health concern in central Asia and western China. In the present study, one of 42 Eastern mole voles (Ellobius tancrei) caught in Sary Mogol (Alay valley, southern Kyrgyzstan) presented liver lesions with E. multilocularis from which the EmsB target was amplified. The Asian profile obtained was almost identical to one amplified from domestic dog faeces collected in a nearby village. This observation adds additional information to the potential role of E. tancrei in the transmission of E. multilocularis, and to the known distribution range of E. multilocularis (Asian strain) in central Asia.

Alveolar echinococcosis, caused by the tapeworm Echinococcus multilocularis, is one of the most severe parasitic diseases in humans and represents one of the 17 neglected diseases prioritised by the World Health Organisation (WHO) in 2012. Considering the major medical and veterinary importance of this parasite, the phylogeny of the genus Echinococcus is of considerable importance; yet, despite numerous efforts with both mitochondrial and nuclear data, it has remained unresolved. The genus is clearly complex, and this is one of the reasons for the incomplete understanding of its taxonomy. Although taxonomic studies have recognised E. multilocularis as a separate entity from the Echinococcus granulosus complex and other members of the genus, it would be premature to draw firm conclusions about the taxonomy of the genus before the phylogeny of the whole genus is fully resolved. The recent sequencing of E. multilocularis and E. granulosus genomes opens new possibilities for performing in-depth phylogenetic analyses. In addition, whole genome data provide the possibility of inferring phylogenies based on a large number of functional genes, i.e. genes that trace the evolutionary history of adaptation in E. multilocularis and other members of the genus. Moreover, genomic data open new avenues for studying the molecular epidemiology of E. multilocularis: genotyping studies with larger panels of genetic markers allow the genetic diversity and spatial dynamics of parasites to be evaluated with greater precision. There is an urgent need for international coordination of genotyping of E. multilocularis isolates from animals and human patients. This could be fundamental for a better understanding of the transmission of alveolar echinococcosis and for designing efficient healthcare strategies., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A 12-yr-old female Goeldi's monkey (Callimico goeldii) in British Columbia, Canada was diagnosed with alveolar echinococcosis (AE) on postmortem examination. Echinococcus multilocularis has been identified in several species of nonhuman primates, most frequently Old World primates, in zoos and research facilities in Europe and Asia. The strain affecting this monkey was identified as a European haplotype, indistinguishable from E. multilocularis recently identified in several canids in British Columbia. The animal is suspected to have been exposed while living in a zoological institution in Alberta, where E. multilocularis has also been reported in urban coyotes. Alveolar echinococcosis is a zoonotic disease of increasing concern in the United States and Canada, and this disease should be considered on the differential list of any nonhuman primate exhibiting signs of abdominal pain or distension, along with diagnostic imaging consistent with cystic structures of the liver or other organs.

EmsB is a molecular marker applied to Echinococcus multilocularis genotyping studies. This marker has largely been used to investigate the epidemiology of the parasite in different endemic foci. The present study has lifted the veil on the genetic structure of this microsatellite. By in silico analysis on the E. multilocularis genome the microsatellite was described in about 40 copies on the chromosome 5 of the parasite. Similar structure was found in the relative parasite Echinococcus granulosus, where the microsatellite was firstly described. The present study completes the first investigations made on the EmsB microsatellite origins and confirms the reliability of this highly discriminant molecular marker., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Alveolar echinococcosis is the most serious helminthic zoonosis of the northern hemisphere affecting humans. The causative agent, Echinococcus multilocularis, occurs in geographically distinct strains that can be distinguished based on sequence variations of mitochondrial genes and nuclear microsatellite targets. This report finds that the human case of Alveolar echinococcosis occurring in Minnesota in 1977 was caused by the N2 haplotype described previously; the N2 haplotype is distinct from European and Asian isolates of the parasite and is restricted to the central region of North America., (© The American Society of Tropical Medicine and Hygiene.)

Alveolar echinococcosis, caused by the cestode Echinococcus multilocularis, is the most serious parasitic disease for humans in Europe, with a sylvatic life cycle generally between small rodents and red foxes. General expansion of the range of E. multilocularis has been observed across Europe over the last 15years. In France, a westward spread of the known endemic areas of the parasite was described recently. For genotyping, the microsatellite EmsB was used to trace expansion in five French areas. A total of 22 EmsB profiles were identified, with five similar to those previously described in other parts of Europe. An imbalance of genetic diversity was observed between the five areas which also revealed their interconnection with the presence of common profiles, notably the two main profiles both present in all regions except one in the North. These two findings are similar to those described at the European level, highlighting transmission of the parasite by a mainland-island system. A spatio-temporal scenario of the expansion of E. multilocularis can be proposed with spread from the French historical focus in eastern France to the Lorraine, the Champagne-Ardenne and finally the North, while simultaneously another expansion has occurred from the historical focus into the West. The colonization by the parasite into the West and North areas from the historical focus was probably due to the migration of foxes several decades ago. Recent detection of the parasite in new endemic "départements" may be due to more active research rather than a recent spread of the parasite. Regarding the numerous data obtained by the different EmsB analyses, principally across Europe, centralization of all the profiles described in a public databank appears necessary in order to obtain a precise understanding of transmission of the parasite from one country to another., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In 2009, a haplotype closely related to European strains of Echinococcus multilocularis was detected in a dog from the Quesnel region of British Columbia, Canada. We now report the establishment of this same haplotype in 7 coyotes (Canis latrans) trapped within 40 km of Quesnel, BC. In addition, 3 coyotes and 1 red fox (Vulpes vulpes) harboured adult cestodes morphologically compatible with that of E. multilocularis (overall prevalence 33% in 33 carnivores). None of 156 potential intermediate hosts, including 131 representatives of two highly suitable rodent species, Peromyscus maniculatus and Microtus pennsylvanicus, trapped from a region 120-210 km south of Quesnel were infected. This report confirms the establishment and local transmission of a European-type strain of E. multilocularis (the causative agent of human alveolar hydatid disease), in wildlife in a forested region of North America where this cestode had not been previously detected, with significance for public and animal health.

Human Alveolar Echinococcosis (HAE) is a potentially fatal parasitic disease caused by Echinococcus multilocularis, a cestode characterized by a sylvatic life-cycle involving several species of rodents and lagomorphs as intermediate hosts and canids as definitive hosts. Despite the wide distribution of the parasite in North America, the number of competent intermediate host species identified to date is still relatively small, and mainly includes the northern vole (Microtus oeconomus), brown lemming (Lemmus sibiricus), northern red-backed vole (Myodes rutilus), deer mouse (Peromyscus maniculatus) and meadow vole (Microtus pennsylvanicus). By monitoring the infections in rodents in the city of Calgary (Alberta, Canada), we have detected a case of severe alveolar echinococcosis in a southern red-backed vole (Myodes gapperi), a species never reported before as an intermediate host for this parasite. Observation of protoscolices in the intra-abdominal multilocular cysts indicates that M. gapperi could act as a competent intermediate host for the transmission of E. multilocularis. Since M. gapperi can be found in close proximity to, and within metropolitan areas, this species could play a role in the establishment and maintenance of the sylvatic life-cycle of E. multilocularis in urban landscapes, where the potential for zoonotic transmission is higher. The new intermediate host reported needs to be taken into account in future surveys and transmission models for this parasite., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

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.

To investigate echinococcosis in co-endemic regions, three polymerase chain reaction (PCR) assays based on the amplification of a fragment within the NADH dehydrogenase subunit 1 (ND1) mitochondrial gene were optimized for the detection of Echinococcus shiquicus, Echinococcus granulosus G1, and Echinococcus multilocularis DNA derived from parasite tissue or canid fecal samples. Specificity using parasite tissue-derived DNA was found to be 100% except for E. shiquicus primers that faintly detected E. equinus DNA. Sensitivity of the three assays for DNA detection was between 2 and 10 pg. Ethanol precipitation of negative PCR fecal samples was used to eliminate false negatives and served to increase sensitivity as exemplified by an increase in detection from 0% to 89% of E. shiquicus coproDNA using necropsy-positive fox samples.

Echinococcus multilocularis is a threatening cestode involved in the human alveolar echinococcosis. The parasite, mainly described in temperate regions of the Northern hemisphere was described for the first time in 1999 in the High Arctic Svalbard archipelago, Norway. The origin of this contamination could be due to an anthropogenic introduction from mainland Europe by domestic dogs or with the introduction of the sibling vole, perhaps from mainland Russia (St. Petersburg area), or with roaming Arctic foxes, known as the main definitive host of the parasite in Arctic regions. The genetic diversity of E. multilocularis in Svalbard was investigated here for the first time by genotyping using EmsB microsatellite and compared to other genotyped populations in the main worldwide endemic areas. We found low polymorphism amongst the 27 metacestode isolates from sibling voles trapped in the core of the distribution area of the vole on Svalbard. E. mutilocularis Arctic populations, using the Arctic fox as the definitive host, were genetically separated from European temperate populations that use the red fox, but closely related to St. Lawrence Island samples from Alaska. The result is inconsistent with the hypothesis of an anthropogenic introduction from mainland Europe, but can be seen as consistent with the hypothesis that Arctic foxes introduced E. multilocularis to Svalbard., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The parasitic diseases which we know today as cystic and alveolar echinococcosis are zoonoses known since antique times, and 1855, respectively. Whether the two clinically and morphologically distinct diseases were caused, according to a "unicistic" and a "dualistic" theory, by only one or two different cestode species was the subject of a fierce, 100 years long debate involving scientists from many countries. The natural life cycle of Echinococcus granulosus was fully clarified in 1855 after successful animal experiments. In contrast, the natural final and intermediate hosts of Echinococcus multilocularis remained unknown, and the advocates of either theory had to draw on a number of surrogate arguments to defend their positions. The seesaw of reasoning and mutual defeats of the two theories, and the final recognition of E. multilocularis as an independent species in the 1950s are described in this article., (2010 Elsevier B.V. All rights reserved.)

Intraspecific genetic variation of Echinococcus multilocularis, the etiologic agent of human alveolar echinococcosis, has been evaluated among 76 geographic isolates from Europe, Asia and North America by using sequence data of mitochondrial and nuclear DNA. Relatively low genetic variation was found only in the mitochondrial DNA sequence consisting of 3 protein-coding genes. Pairwise divergence among the resultant 18 haplotypes ranged from 0.03 to 1.91%. Phylogenetic trees and parsimony network of these haplotypes depicted a geographic division into European, Asian and North American clades, but 1 haplotype from Inner Mongolia was unrelated to other haplotypes. The coexistence of the Asian and North American haplotypes could be seen, particularly on the St. Lawrence Island in the Bering Sea. These data suggest an evolutionary scenario in which distinct parasite populations derived from glacial refugia have been maintained by indigenous host mammals. The nuclear DNA sequence for the immunodominant B cell epitope region of ezrin/radixin/moesin-like protein (elp) was extremely conservative, indicating that the elp antigen is available for immunodiagnosis in any endemic areas.

Background: Alveolar echinococcosis (AE) is a severe helminth disease affecting humans, which is caused by the fox tapeworm Echinococcus multilocularis. AE represents a serious public health issue in larger regions of China, Siberia, and other regions in Asia. In Europe, a significant increase in prevalence since the 1990s is not only affecting the historically documented endemic area north of the Alps but more recently also neighbouring regions previously not known to be endemic. The genetic diversity of the parasite population and respective distribution in Europe have now been investigated in view of generating a fine-tuned map of parasite variants occurring in Europe. This approach may serve as a model to study the parasite at a worldwide level., Methodology/principal Findings: The genetic diversity of E. multilocularis was assessed based upon the tandemly repeated microsatellite marker EmsB in association with matching fox host geographical positions. Our study demonstrated a higher genetic diversity in the endemic areas north of the Alps when compared to other areas., Conclusions/significance: The study of the spatial distribution of E. multilocularis in Europe, based on 32 genetic clusters, suggests that Europe can be considered as a unique global focus of E. multilocularis, which can be schematically drawn as a central core located in Switzerland and Jura Swabe flanked by neighbouring regions where the parasite exhibits a lower genetic diversity. The transmission of the parasite into peripheral regions is governed by a "mainland-island" system. Moreover, the presence of similar genetic profiles in both zones indicated a founder event.

A cat excreting Echinococcus multilocularis eggs was recently identified in Hokkaido, representing the first such observation in Japan. The cat was raised free-range and frequently ate rodents. Fecal egg examination revealed eggs of taeniids (EPG: 440) and Spirometra spp. (EPG: >1000). PCR targeting part of the mitochondrial cytochrome c oxidase subunit I gene of E. multilocularis was positive with DNA from 3 single isolated taeniid eggs, and sequence analysis of one amplicon confirmed E. multilocularis. The results indicated that the eggs of E. multilocularis distributed in Hokkaido can be excreted in cat feces, and suggested the necessity of further studies to clarify whether the eggs excreted in cat feces are infective and thus whether cats can serve as infectious source to humans in Japan.

To date, only a single proven case of autochthonous human alveolar echinococcosis has been recorded in Minnesota in 1977. At that time, echinococcal lesions removed from the patient were experimentally inoculated into voles, and the parasite materials obtained from the voles were preserved as histopathologic specimens for 30 years. In this study, retrospective genetic analysis of larval Echinococcus multilocularis originating in the human case was performed using the histopathologic specimens. DNA was extracted from the hematoxylin and eosin-stained specimens, and mitochondrial cytochrome c oxidase subunit 1 gene (cox1) was amplified by polymerase chain reaction. Subsequently, 20 small fragments (100-216 bp) covering almost the entire sequences (97%) of the cox1 were successfully amplified, and the nucleotide sequence analysis showed that the E. multilocularis isolate from Minnesota was almost identical to an isolate from South Dakota rather than isolates from contiguous Alaska.

A new cost-effective method using silicon dioxide- and guanidine isothiocyanate-containing buffers, after previous alkaline lysis, was established for the DNA extraction from taeniid eggs isolated from canine faeces. The purified DNA can be used to amplify the species-specific 12S mitochondrial DNA of Echinococcus multilocularis in direct and nested polymerase chain reaction in order to differentiate between E. multilocularis and Taenia spp.

Alveolar echinococcosis (AE)--caused by the cestode Echinococcus multilocularis--is a severe zoonotic disease found in temperate and arctic regions of the northern hemisphere. Even though the transmission patterns observed in different geographical areas are heterogeneous, the nuclear and mitochondrial targets usually used for the genotyping of E. multilocularis have shown only a marked genetic homogeneity in this species. We used microsatellite sequences, because of their high typing resolution, to explore the genetic diversity of E. multilocularis. Four microsatellite targets (EmsJ, EmsK, and EmsB, which were designed in our laboratory, and NAK1, selected from the literature) were tested on a panel of 76 E. multilocularis samples (larval and adult stages) obtained from Alaska, Canada, Europe, and Asia. Genetic diversity for each target was assessed by size polymorphism analysis. With the EmsJ and EmsK targets, two alleles were found for each locus, yielding two and three genotypes, respectively, discriminating European isolates from the other groups. With NAK1, five alleles were found, yielding seven genotypes, including those specific to Tibetan and Alaskan isolates. The EmsB target, a tandem repeated multilocus microsatellite, found 17 alleles showing a complex pattern. Hierarchical clustering analyses were performed with the EmsB findings, and 29 genotypes were identified. Due to its higher genetic polymorphism, EmsB exhibited a higher discriminatory power than the other targets. The complex EmsB pattern was able to discriminate isolates on a regional and sectoral level, while avoiding overdistinction. EmsB will be used to assess the putative emergence of E. multilocularis in Europe.

The Xinjiang plateau of western China has been shown to have a high prevalence for human cystic echinococcosis (CE) caused by Echinococcus granulosus, and human alveolar echinococcosis (AE) caused by Echinococcus multilocularis. The domestic dog is suspected to be the primary definitive host for the transmission of both E. granulosus and E. multilocularis to humans in this locality. Seventeen of 30 stray dogs from Hejing County of Xinjiang were found positive for E. granulosus post mortem, and one double infection was suspected. Worm samples were collected, dyed by carmine, and observed microscopically. Carmine staining examination clearly revealed the differences in number of proglottids and appearance of uterine branches and lateral genital pore for those two species of Echinococcus. Furthermore, gene target DNA fragments were amplified for formal identification of the two parasite species, based on 12s rRNA mitochondrial gene. The PCR products were purified and sequenced. Compared with NCBI GenBank, the DNA sequences demonstrated 100% identity with E. granulosus (sheep strain, G1 genotype) and E. multilocularis.

West-central China is an important endemic focus of both alveolar and cystic echinococcosis where several species of intermediate host are commonly infected with Echinococcus granulosus and E. multilocularis . Isolates of E. granulosus were collected from humans and other animals from different geographical areas of Qinghai, Ningxia, Gansu and Sichuan, and genotyped using the mitochondrial DNA marker ATP synthase subunit 6 gene (atp6). The sheep strain (G1 genotype) of E. granulosus was shown to be the only genotype present in sheep, cattle, goats, yaks and humans in the study areas. However, some heterogeneity in the atp6 sequence was evident in a number of the isolates with the most frequent change being a silent substitution (G/A) at position 360 compared with the G1 reference sequence representing isolates collected from the majority of hosts except humans. Two E. multilocularis isolates examined also had sequences that varied from each other and from the reference E. multilocularis atp6 sequence. The genotypic variation we report may reflect phenotypic differences with important consequences in terms of increased host infectivity for hosts by local Echinococcus strains, possibly impacting on the epidemiology and control of echinococcosis. Such adaptations may also result in different sensitivity to drugs or increased virulence for hosts that will impede control efforts and even affect vaccination strategies against Echinococcus.