Inimesele ohtliku paelussi Echinococcus granulosus sensu stricto globaalne geneetiline mitmekesisus ja fülogeograafia

Väitekirja elektrooniline versioon ei sisalda publikatsiooneTsüstiline ehhinokokoos (CE) on haigus, mida põhjustavad Echinococcus granulosus sensu lato (s. l.) kompleksi kuuluvad paelussid. Sellesse kompleksi kuuluvate liikide hulgas on E. granulosus sensu stricto (s. s.) eriti oluline, olles ülemaailmselt kõige laialdasemalt levinud ning ka kõige sagedasem CE tekitaja inimesel. Kuigi liigi geneetilise struktuuri mõistmiseks oli läbi viidud mitmeid uurimustöid, esines olulisi lünki, kuna siiani oli kasutatud lühikesi mitokondriaalseid (mtDNA) järjestusi. Esiteks oli üheks kõige olulisemaks probleemiks E. granulosus s. s. mitokondriaalsete genotüüpide olemasolu ning eristamine. Teiseks, kuigi oli välja pakutud, et G1–G3 on mitogenoomi geneetilise sarnasuse alusel üks liik, ei olnud seda teaduslikult näidatud. Kolmandaks, senised geneetilise mitmekesisuse ning fülogeograafia analüüsid ei olnud võimaldanud mõista detailseid globaalseid mustreid vähese fülogeneetilise eristusvõime tõttu. Käesolevas doktoritöös analüüsiti ülemaailmselt kogutud parasiidiproove sekveneerides mitokondri genoomi pea täies pikkuses ning kaasates analüüsi ka mitmed tuumalookused. Esiteks tegime kindlaks, et E. granulosus s. s. koosneb kahest mitokondriaalsest genotüübist G1 ning G3 ja näitasime, et G2 ei ole eraldi genotüüp. Teiseks tegime kindlaks, et G1 ning G3 võib lugeda üheks liigiks. Kolmandaks näitasime, et genotüüpide G1 ja G3 geneetiline mitmekesisus on kõrge ning globaalsed alampopulatsioonid on geneetiliselt vähe diferentseerunud. Seda on suure tõenäosusega mõjutanud intensiivne ülemaailmne loomakaubandus ning -transport. Fülogeograafilise analüüsi tulemused näitasid, et E. granulosus s. s. üheks olulisemaks ekspansiooni keskpunktiks on olnud Lähis-Ida piirkond, mis on hästi tuntud lamba, veise ja kitse kodustamise piirkonnana – kõik need liigid on ka olulised E. granulosus s. s. vaheperemehed. Antud doktoritöö on aluseks tulevastele E. granulosus s. s. genotüüpide leviku, geneetilise mitmekesisuse ning fülogeograafia alastele analüüsidele.Cystic echinococcosis (CE) is a disease caused by tapeworms within the species complex Echinococcus granulosus sensu lato (s. l.). Within this complex, E. granulosus sensu stricto (s. s.) is associated with the majority of human CE cases globally and thus merits particular attention. Although extensive research had been carried out on the genetic structure of this species, significant gaps still existed due to the use of relatively short mitochondrial DNA sequences so far. Firstly, one of the most pressing questions was the existence and distinction of E. granulosus s. s. mitochondrial genotypes. Secondly, despite the assumptions that the mitochondrial genotypes can be regarded as a distinct species, further analysis was required. Thirdly, analyses of the patterns of genetic diversity and phylogeography of E. granulosus s. s. had lacked sufficient phylogenetic power to reveal detailed insight. The present thesis addressed these issues by sequencing and analysing near-complete mtDNA sequences and several nuclear loci for a large panel of globally distributed E. granulosus s. s. samples. Firstly, we determined that E. granulosus s. s. comprises two clearly distinct mitochondrial genotypes G1 and G3, whereas G2 is not a valid genotype. Secondly, we confirmed that G1 and G3 can indeed be regarded as a single species. Thirdly, the analyses of the global patterns of genetic diversity and phylogeography of E. granulosus s. s. revealed high genetic variation within genotypes G1 and G3, coupled with low genetic differentiation between G1 and G3 subpopulations globally, which is likely the consequence of extensive global animal transport and trade. The phylogeographic analysis revealed the Middle East as the origin of a large-scale expansion of both genotypes. The new data that the present thesis presents underpins our fundamental understanding of the genetic make-up of E. granulosus s. s. and provides a strong basis for future research on this parasite

Cystic echinococcosis in Iceland: a brief history and genetic analysis of a 46-year-old Echinococcus isolate collected prior to the eradication of this zoonotic disease

Cystic echinococcosis (CE) is considered the most severe parasitic disease that ever affected the human population in Iceland. Before the start of eradication campaign in the 1860s, Iceland was a country with very high prevalence of human CE, with approximately every fifth person infected. Eradication of CE from Iceland by 1979 was a huge success story and served as a leading example for other countries on how to combat such a severe One Health problem. However, there is no genetic information on Echinococcus parasites before eradication. Here, we reveal the genetic identity for one of the last Echinococcus isolates in Iceland, obtained from a sheep 46 years ago (1977). We sequenced a large portion of the mitochondrial genome (8141 bp) and identified the isolate as Echinococcus granulosus sensu stricto genotype G1. As G1 is known to be highly infective genotype to humans, it may partly explain why such a large proportion of human population in Iceland was infected at a time . The study demonstrates that decades-old samples hold significant potential to uncover genetic identities of parasites in the past

Mitochondrial genome of Bulinus truncatus (Gastropoda: Lymnaeoidea): implications for snail systematics and schistosome epidemiology

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 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 vectors of parasitic trematodes

First report of highly pathogenic Echinococcus granulosus genotype G1 in dogs in a European urban environment

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

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

Cystic echinococcosis, a zoonotic disease caused by Echinococcus granulosus sensu lato (s.l.), is a significant global public health concern. Echinococcus granulosus s. l. is currently divided into numerous genotypes (G1-G8 and G10) of which G1-G3 are the most frequently implicated genotypes in human infections. Although it has been suggested that G1-G3 could be regarded as a distinct species E. granulosus sensu stricto (s. s.), the evidence to support this is inconclusive. Most importantly, data from nuclear DNA that provide means to investigate the exchange of genetic material between G1-G3 is lacking as none of the published nuclear DNA studies have explicitly included G2 or G3. Moreover, the commonly used relatively short mtDNA sequences, including the complete coxl gene, have not allowed unequivocal differentiation of genotypes G1-G3. Therefore, significantly longer mtDNA sequences are required to distinguish these genotypes with confidence. The main aim of this study was to evaluate the phylogenetic relations and taxonomy of genotypes G1-G3 using sequences of nearly complete mitogenomes (11,443 bp) and three nuclear loci (2984 bp). A total of 23 G1-G3 samples were analysed, originating from 5 intermediate host species in 10 countries. The mtDNA data demonstrate that genotypes G1 and G3 are distinct mitochondrial genotypes (separated by 37 mutations), whereas G2 is not a separate genotype or even a monophyletic cluster, but belongs to G3. Nuclear data revealed no genetic separation of G1 and G3, suggesting that these genotypes form a single species due to ongoing gene flow. We conclude that: (a) in the taxonomic sense, genotypes G1 and G3 can be treated as a single species E. granulosus s. s.; (b) genotypes G1 and G3 should be regarded as distinct genotypes only in the context of mitochondrial data; (c) we recommend excluding G2 from the genotype list. (C) 2017 Elsevier B.V. All rights reserved.Peer reviewe

First detection of zoonotic tapeworm Echinococcus granulosus sensu lato genotype G7 in continental Italy.

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

Nanopore Sequencing Resolves Elusive Long Tandem-Repeat Regions in Mitochondrial 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.Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/)

High-resolution phylogeography of zoonotic tapeworm Echinococcus granulosus sensu stricto genotype G1 with an emphasis on its distribution in Turkey, Italy and Spain

Echinococcus granulosus is the causative agent of cystic echinococcosis. The disease is a significant global public health concern and human infections are most commonly associated with E. granulosus sensu stricto (s. s.) genotype G1. The objectives of this study were to: (i) analyse the genetic variation and phylogeography of E. granulosus s. 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 91 E. granulosus s. 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 of E. granulosus s. 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.Peer reviewe