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Fasciolopsiasis caused by Fasciolopsis buski, is a significant cause of morbidity. In the present study F. buski detected during endoscopy of a 50-year-old male patient form Uttar Pradesh, India was used for phylogenetic analysis based on 28S rDNA and ITS2 regions. The parasite was genetically similar to an isolate of pig from Meghalaya. The intra-species genetic variation for 28S rDNA was 1-3% (India), 5% (Vietnam) and for ITS2, 1-2% (India) while 17 % from Vietnam. Thus, F. buski from humans or pigs from India were closely related than those from Vietnam and China which were grouped into separate clades., 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 Indian Association of Medical Microbiologists. Published by Elsevier B.V. All rights reserved.)

Parasites constitute a significant economic burden and highly impact environmental, public, and animal health. The emergence of many parasitic diseases is environmentally mediated and they share the same biogeography with humans and both domestic and wild animals. American liver fluke, Fascioloides magna - a trematode parasite of domestic and wild ungulates - is an example of the anthropogenic introduction of an "invasive alien species" in Italy and Europe. Multiple introductions to Europe have led to the biogeographical expansion of the parasite across the Danube region mainly provided by the presence of suitable habitats for all hosts involved in the parasite's life cycle, human-assisted transport, and drastic environmental events such as flooding. In Italy, it was introduced and established in La Mandria Regional Park (LMRP) near Turin in 1865 along with imported wapitis (Cervus elaphus canadensis) from North America (Bassi, 1875), but with no reported expansion to the surrounding areas. LMRP isolated F. magna focus, poses an important threat of possible expansion since the enclosed area is vulnerable to occasional bidirectional passage of roe deer. Additionally, tributary rivers to the Po river system, traversing the enclosed area, could further bolster the possibility of such spread. In this study, we developed a duplex qPCR assay for F. magna and its principal intermediate host Galba truncatula optimized for testing eDNA samples to meet the needs for surveillance of the parasite. Moreover, we validated the developed assay in natura by testing samples derived from filtered water and sediments collected inside and outside LMRP's fenced-off area. Our findings for the first time demonstrate the presence of F. magna's eDNA outside the park's internal fenced-off area., Competing Interests: Declaration of competing interest The authors have no competing interests to declare that are relevant to the content of this article., (Copyright © 2024. Published by Elsevier B.V.)

Human induced translocation and introduction of species have reshaped parasite fauna on a global scale. The introduction of the large American liver fluke Fascioloides magna from North America to Europe is an example of an invasive alien parasite causing significant ecological and economic damage. Recent genetic studies have shown that F. magna was introduced to Europe on multiple occasions forming three permanent foci of infection. This study primarily focuses on the poorly researched genetic structuring of F. magna flukes originating from Croatia and Serbia. Additional samples from USA and Italy are also included, thereby providing novel insights into F. magna's biogeography. Partial cox1 and nad1 genes were amplified from 216 F. magna flukes extracted from red deer, roe deer, fallow deer, white-tailed deer and wild boar. Seven cox1 and nine nad1 haplotypes were identified, of which two cox1 and four nad1 haplotypes have not been not previously found. Our analysis has expanded the knowledge about possible sources of F. magna introduction to Europe, by identifying a cox1 haplotype shared by flukes from the north-eastern parts of the USA and Italy and another cox1 haplotype shared by flukes also from north eastern parts of the USA and the Danube floodplains., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dean Konjevic reports financial support was provided by Croatian Science Foundation., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The foodborne intestinal trematode Fasciolopsis buski causes the neglected zoonotic disease fasciolopsiasis. We detected F. buski infection in 14 pediatric patients in Sitamarhi, Bihar, and in pigs in Sivasagar, Assam, India. Proper diagnostic methods and surveillance are urgently needed to accurately estimate the true burden of this disease in India.

A 4-yr-old male intact lesser spot-nosed guenon ( Cercopithecus petaurista ), housed at a North American zoological facility, presented with acute lethargy, inappetence, and mild neurologic signs. Physical examination revealed hemorrhagic pleural effusion in the right hemithorax. This guenon's condition improved over several days but then deteriorated, and the guenon presented with lethargy and weakness. A hemorrhagic pleural effusion was identified within the left hemithorax. The guenon developed respiratory and cardiac arrest while anesthetized. Gross examination revealed tract formation in the liver, adhesions of the liver to the diaphragm, hemorrhagic thoracic and abdominal effusion, and a single trematode within the right hemithorax. Morphologic features and species identification by PCR confirmed that the parasite was Fascioloides magna . Histologic examination revealed tract formation in the liver associated with biliary hyperplasia, fibrosis and hepatic necrosis, severe bile peritonitis, and pleuritis. This is the first report of an infection by F. magna in a primate.

Purpose: The giant liver fluke, Fascioloides magna, is an important parasite of white-tailed deer (Odocoileus virginanus), a commercially important game species in the northeastern United States. However, the parasite has never been genetically characterized from this region and so its genetic interrelationships with other enzootic populations on the continent are unknown. The purpose of this study was to genetically characterize F. magna from the Adirondack region of northern New York (NNY) and determine its relationship with its enzootic cohorts., Methods: Flukes were extracted from seven livers of O. virginanus at three localities in and around the Adirondack Park in northern New York. DNA was extracted from eight individuals using standard procedures, and a 440-bp fragment of the COI gene was amplified using species-specific primers. F. magna sequences generated from a previous phylogeographic study were obtained from the GenBank database and a mtDNA dataset was compiled, aligned, and edited for molecular analyses. Analyses based on eight mtDNA sequences included haplotype network reconstruction, along with hierarchical and pairwise (Ф ST ) AMOVA tests., Results: F. magna from the Adirondacks was found to be most genetically similar to specimens from Minnesota based on shared haplotypes and relatively low genetic differentiation (Ф ST = 0.331). East coast enzootic populations exhibited panmixia while their west coast cohorts were genetically distinct., Conclusion: Based on the strong genetic similarities between F. magna from northern New York and Minnesota, we propose that the GLR enzootic range of F. magna be extended to encompass the Adirondack region of NNY.

Fascioloides jacksoni (syn. Fasciola jacksoni, Cobbold, 1869) (Platyhelminthes: Echinostomatoidea), is a liver fluke that causes severe morbidity and mortality of Asian elephants (Elephas maximus maximus). Understandings on molecular diagnosis, epidemiology, genetics and evolution of this flatworm are limited. In this study, we present the complete mitochondrial DNA (mt) sequence of 14,952 bp obtained from an individual fluke and comparative characterization of mitogenomic features with fasciolids, primarily, Fascioloides magna and other taxa in the superfamily Echinostomatoidea. Taxonomic relationship within and between Echinostomatoidea, Opisthorchioidea and Paramphistomoidea in the order Plagiorchiida, are also taxonomically considered. The complete circular mt molecule of Fas. jacksoni contained 12 protein-coding, two ribosomal RNA, 22 transfer RNA genes, and a non-coding region (NCR) rich in tandem repeat units. As common in digenean trematodes, Fas. jacksoni has the usual gene order, the absence of atp8 and the overlapped region by 40 bp between nad4L and nad4 genes. The NCR located between tRNA Glu (trnE) and cox3 contained nine nearly identical tandem repeat units (TRs of 113 bp each). Special DHU-arm missing tRNAs for Serine were found for both, tRNA S1(AGN) and tRNA S2(UCN) . Base composition indicated that cox1 of Fas. jacksoni showed the lowest (11.8% to Fas. magna, 12.9 - 13.6% to Fasciola spp. and 18.1% to Fasciolopsis buski) and nad6 the highest divergence rate (19.2%, 23.8-26.5% and 27.2% to each fasciolid group), respectively. A clear bias in nucleotide composition, as of 61.68%, 62.88% and 61.54%, with a negative AT-skew of the corresponding values (-0.523, -0.225 and - 0.426) for PCGs, MRGs and mtDNA for Fas. jacksoni and likewise data for the fasciolids. Phylogenetic analysis confirmed the sister branch of Fas. jacksoni and Fas. magna with the nodal support of 100%, clearly separated from the taxonomically recognized Fasciola spp. With the previous studies, mitogenomic data presented in this study are strongly supportive for Fasciola jacksoni reappraisal as Fascioloides jacksoni in the Fascioloides genus., Competing Interests: Declaration of Competing Interest None, (Copyright © 2020. Published by Elsevier B.V.)

Many members of Fasciolidae are common trematodes in cattle, buffaloes, sheep, elephants, pigs, with some capable of infecting humans also. In this study, the complete or near-complete sequences of ribosomal transcription unit (rTU or rDNA), each of Fasciola hepatica (Australia), Fascioloides jacksoni (Sri Lanka), Fasciolopsis buski (Vietnam) and three isolates of F. gigantica (Vietnam), were obtained and characterized. The full length of rDNA for each F. hepatica, 'hybrid' Fasciola sp., Fas. jacksoni and Fa. Buski, was 7657 bp, 7966 bp, 7781 bp and 8361 bp, with the complete intergenic spacer region (IGS) (862 bp, 1170 bp, 987 bp and 561 bp), respectively. The rDNA of two 'pure' F. gigantica isolates from Vietnam was 6794 bp with unsequenced IGS. For 28S rRNA genes the Fasciola spp. are equal, 1958 bp for 18S, 160 bp for 5.8S, 3863 bp and 454 bp for ITS1 but ITS2 differ by one nucleotide (Thymine) (359 or 360 bp). The ITS1 of the sensu lato Fa. buski has some distinguishable features, 286 bp for ITS2, 3862 bp for 28S and four repeat units of 356-361 bp each found in ITS1. The 28S rDNA analysis showed the lowest level of divergence (0-0.57%) between F. hepatica and F. gigantica and higher (2.23-2.62%) and highest (6-6.42%) for Fas. jacksoni and Fasciolopsis, respectively. The tree of 43 strains/species clearly produced a well-supported phylogeny, where 18 fasciolids consistently grouped, forming a discrete Fasciolidae clade, distinct from Philophthalmidae, Echinostomatidae and Echinochasmidae in Echinostomatoidea. Fascioloides jacksoni is outside Fasciola spp.: basal with Fas. magna, as previously demonstrated.

Liver and intestinal flukes of the family Fasciolidae cause zoonotic food-borne infections that impact both agriculture and human health throughout the world. Their evolutionary history and the genetic basis underlying their phenotypic and ecological diversity are not well understood. To close that knowledge gap, we compared the whole genomes of Fasciola hepatica, Fasciola gigantica, and Fasciolopsis buski and determined that the split between Fasciolopsis and Fasciola took place ∼90 Ma in the late Cretaceous period, and that between 65 and 50 Ma an intermediate host switch and a shift from intestinal to hepatic habitats occurred in the Fasciola lineage. The rapid climatic and ecological changes occurring during this period may have contributed to the adaptive radiation of these flukes. Expansion of cathepsins, fatty-acid-binding proteins, protein disulfide-isomerases, and molecular chaperones in the genus Fasciola highlights the significance of excretory-secretory proteins in these liver-dwelling flukes. Fasciola hepatica and Fasciola gigantica diverged ∼5 Ma near the Miocene-Pliocene boundary that coincides with reduced faunal exchange between Africa and Eurasia. Severe decrease in the effective population size ∼10 ka in Fasciola is consistent with a founder effect associated with its recent global spread through ruminant domestication. G-protein-coupled receptors may have key roles in adaptation of physiology and behavior to new ecological niches. This study has provided novel insights about the genome evolution of these important pathogens, has generated genomic resources to enable development of improved interventions and diagnosis, and has laid a solid foundation for genomic epidemiology to trace drug resistance and to aid surveillance., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Fasciolopsiasis is rarely known as the parasitic disease in Nepal. Herein, we report a case of fasciolopsiasis in a 22-year-old man who was admitted in the hospital with abdominal pain, distension and loss of appetite for a month. He had previously diagnosed with acute viral hepatitis but, his abdominal pain was not resolving despite improvement in his liver function and general condition. During endoscopy an adult digenean worm was seen in the first part of the duodenum. After isolation, the worm was identified morphologically as Fasciolopsis buski. Microscogic examination of the patient's stool revealed eggs with a morphology consistent with F. buski. Eggs were yellow-brown, ellipsoidal, unembmbryonated, operculated, filled with yolk cells, with thin shell and ranging 118-130 µm in length and 60-69 µm in width. The abdominal pain of the patient was resolved after treatment with praziquantel. By the present study, it was confirmed for the first time that fasciolopsiasis is indigenously transmitted in Nepal. Accordingly, the epidemiological studies in humans and reservoir host animals should be performed intensively in near future.

Many trematode parasites cause infection in humans and are thought to be a major public health problem. Their ecological diversity in different regions provides challenging questions on evolution of these organisms. In this report, we perform transcriptome analysis of the giant intestinal fluke, Fasciolopsis buski, using next generation sequencing technology. Short read sequences derived from polyA containing RNA of this organism were assembled into 30,677 unigenes that led to the annotation of 12,380 genes. Annotation of the assembled transcripts enabled insight into processes and pathways in the intestinal fluke, such as RNAi pathway and energy metabolism. The expressed kinome of the organism was characterized by identifying all protein kinases. A rough draft genome assembly for Fasciolopsis buski is also reported herewith with SRA accessions for crosschecking the findings in the analyzed transcriptome data. Transcriptome data also helped us to identify some of the expressed transposable elements. Though many Long Interspersed elements (LINEs) were identified, only two Short Interspersed Elements (SINEs) were visible. Overall transcriptome and draft genome analysis of F. buski helped us to characterize some of its important biological characteristics and provided enormous resources for development of a suitable diagnostic system and anti-parasitic therapeutic molecules., Competing Interests: The authors have declared that no competing interests exist.

In last few years, a great effort has been made to understand genetic interrelationships of European and North American populations of giant liver fluke Fascioloides magna (Trematoda, Fasciolidae). In Europe, spatial distribution of this parasite is evidently dynamic and ongoing process since new F. magna populations have constantly been emerging. Most recently, occurrence of F. magna in red deer (Cervus elaphus), sika deer (Cervus nippon), fallow deer (Dama dama), roe deer (Capreolus capreolus), and wild boar (Sus scrofa) was reported from north-eastern Bavaria in Germany. Fascioloides magna specimens collected from those hosts were genotyped using two mitochondrial regions; cytochrome c oxidase (cox1) and nicotinamide dehydrogenase (nad1). Results were compared with reference mitochondrial haplotypes of previously characterized European F. magna populations from northern Italy, Czech Republic/Poland, and the Danube floodplain forests. The study revealed genetic uniformity of F. magna specimens from Germany with flukes from the Czech focus, what indicated that fascioloidosis has been spreading beyond well established Czech focus into neighbouring countries.

The large American liver fluke, Fascioloides magna, is an economically relevant parasite of both domestic and wild ungulates. F. magna was repeatedly introduced into Europe, for the first time already in the 19th century. In Austria, a stable population of F. magna has established in the Danube floodplain forests southeast of Vienna. The aim of this study was to determine the genetic diversity of F. magna in Austria. A total of 26 individuals from various regions within the known area of distribution were investigated for their cytochrome oxidase subunit 1 (cox1) and nicotinamide dehydrogenase subunit 1 (nad1) gene haplotypes. Interestingly, all 26 individuals revealed one and the same haplotype, namely concatenated haplotype Ha5. This indicates a homogenous population of F. magna in Austria and may argue for a single introduction. Alternatively, genetic homogeneity might also be explained by a bottleneck effect and/or genetic drift., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Fasciolopsis buski is a zoonotic intestinal fluke infecting humans and pigs, but it has been seriously neglected. It is yet to know whether there is any genetic diversity among F. buski from different geographical locations, particularly in sequences of nuclear ribosomal DNA (rDNA) and mitochondrial (mt) DNA. Therefore, we determined the sequences of partial 18S, the complete internal transcribed spacer (ITS) rDNA and the complete mt genome of F. buski from China, compared the rDNA and mtDNA sequences with those of isolates from India and Vietnam, and assessed the phylogenetic relationships of this fluke and related fasciolid trematodes based on the mtDNA dataset., Results: The complete mt genome sequence of F. buski from China is 14,833 bp, with 36 genes, including 12 protein-coding genes (PCGs), 22 tRNA genes, and two rRNA genes (rrnL and rrnS). The AT content of F. buski from China is 65.12%. The gene content and arrangement of the F. buski mt genome is similar to that of Fascioloides magna. Genetic distances between isolates of F. buski from China and India were high (28.2% in mtDNA, 13.2% in ITS-1 and 9.8% in ITS-2) and distinctly higher than the interspecific differences between Fasciola hepatica and Fasciola gigantica. The rDNA and mtDNA datasets for F. buski from China (isolate from pigs) and Vietnam (isolates from humans) were identical. The intergeneric differences in amino acid and nucleotide sequences among the genera Fasciolopsis, Fascioloides and Fasciola ranged between 24.64-25.56% and 26.35-28.46%, respectively., Conclusions: Our results indicate that F. buski from China and India may represent distinct taxa, while F. buski in Vietnam and China represent the same species. These findings might have implications for the implementation of appropriate control strategies in different regions. Further studies are needed to decode mtDNA and rDNA sequences of F. buski from various geographical isolates for the better understanding of the species complex of F. buski.

The veterinary important parasite of ruminants, giant liver fluke Fascioloides magna(Trematoda: Fasciolidae), isolated from liver of farmed fallow deer (Dama dama) from Podkarpackie Province (southeastern Poland) was genotypized by mitochondrial cytochrome c oxidase (cox1) and nicotinamide dehydrogenase (nad1) markers. The data on this newly emerged population were compared with mitochondrial haplotypes of recently detected Polish population of F. magna from Lower Silesian Wilderness (southwestern Poland) and with European populations of the parasite from all three natural foci; northern Italy, Czech Republic and the Danube floodplain forests. The flukes from Podkarpackie Province were found to be genetically identical with flukes from Czech Republic and Lower Silesian Wilderness in Poland. It is evident that central and southwestern Czech Republic, recognized as one of the endemic area of F. magna in Europe, has been enlarging and parasite has been invading several novel localities in Poland.

Background: Fascioloides magna (Trematoda: Fasciolidae) is an important liver parasite of a wide range of free-living and domestic ruminants; it represents a remarkable species due to its large spatial distribution, invasive character, and potential to colonize new territories. The present study provides patterns of population genetic structure and admixture in F. magna across all enzootic regions in North America and natural foci in Europe, and infers migratory routes of the parasite on both continents., Methods: In total, 432 individuals from five North American enzootic regions and three European foci were analysed by 11 microsatellite loci. Genetic data were evaluated by several statistical approaches: (i) the population genetic structure of F. magna was inferred using program STRUCTURE; (ii) the genetic interrelationships between populations were analysed by PRINCIPAL COORDINATES ANALYSIS; and (iii) historical dispersal routes in North America and recent invasion routes in Europe were explored using MIGRATE., Results: The analysis of dispersal routes of the parasite in North America revealed west-east and south-north lineages that partially overlapped in the central part of the continent, where different host populations historically met. The exact origin of European populations of F. magna and their potential translocation routes were determined. Flukes from the first European focus, Italy, were related to F. magna from northern Pacific coast, while parasites from the Czech focus originated from south-eastern USA, particularly South Carolina. The Danube floodplain forests (third and still expanding focus) did not display relationship with any North American population; instead the Czech origin of the Danube population was indicated. A serial dilution of genetic diversity along the dispersion route across central and eastern Europe was observed. The results of microsatellite analyses were compared to previously acquired outputs from mitochondrial haplotype data and correlated with past human-directed translocations and natural migration of the final cervid hosts of F. magna., Conclusions: The present study revealed a complex picture of the population genetic structure and interrelationships of North American and European populations, global distribution and migratory routes of F. magna and an origin of European foci.

Background: Representatives of the trematode family Fasciolidae are responsible for major socio-economic losses worldwide. Fascioloides magna is an important pathogenic liver fluke of wild and domestic ungulates. To date, only a limited number of studies concerning the molecular biology of F. magna exist. Therefore, the objective of the present study was to determine the complete mitochondrial (mt) genome sequence of F. magna, and assess the phylogenetic relationships of this fluke with other trematodes based on the mtDNA dataset., Findings: The complete F. magna mt genome sequence is 14,047 bp. The gene content and arrangement of the F. magna mt genome is similar to those of Fasciola spp., except that trnE is located between trnG and the only non-coding region in F. magna mt genome. Phylogenetic relationships of F. magna with selected trematodes using Bayesian inference (BI) was reconstructed based on the concatenated amino acid sequences for 12 protein-coding genes, which confirmed that the genus Fascioloides is closely related to the genus Fasciola; the intergeneric differences of amino acid composition between the genera Fascioloides and Fasciola ranged 17.97-18.24 %., Conclusions: The determination of F. magna mt genome sequence provides a valuable resource for further investigations of the phylogeny of the family Fasciolidae and other trematodes, and represents a useful platform for designing appropriate molecular markers.

Fasciolopsis buski is a food-borne zoonotic parasite which is transmitted by aquatic plants, with pigs and humans as the definitive hosts. The objective of the present study was to characterize the microRNA (miRNA) profiles of this parasite by Solexa deep sequencing and bioinformatic analysis. Approximately 12 million high-quality reads were obtained from adult F. buski. A total of 286 miRNA candidates were found and 24 miRNA candidates were conserved miRNAs in the miRBase database. Three novel miRNAs were identified and confirmed by stem-loop reverse transcriptase polymerase chain reaction (RT-PCR). The miRNAs found in the present study belong to 13 families whose members showed high bias. The guanine (G) was the dominant nucleotide at the beginning and middle of the conserved miRNAs, particularly at the positions of 2nd, 6th, and 13th. To our knowledge, this is the first report of the miRNA profiles of F. buski, which would lay a foundation for further functional studies of miRNAs of F. buski.

The giant liver fluke, Fascioloides magna, is a veterinary important liver parasite of free living and domestic ruminants. This originally North American parasite was introduced along with its cervid hosts to Europe where it has established three permanent natural foci - in northern Italy, central and southern parts of the Czech Republic and the Danube floodplain forests. The first record on fascioloidosis in Poland originated from the Lower Silesian Forest in south-western Poland and since then an occurrence of F. magna in this country has not been documented. Recently, the parasitological examination of red deer (Cervus elaphus elaphus) from the Lower Silesian Wilderness (south-western Poland) revealed the presence of F. magna eggs. In order to determine the genetic interrelationships of the Polish giant liver fluke individuals, they were molecularly analyzed by mitochondrial cytochrome c oxidase subunit I (cox1) and nicotinamide dehydrogenase subunit I (nad1) and compared with haplotypes of so far studied European populations of the parasite. The study revealed the genetic uniformity of F. magna specimens from Poland with part of individuals from the Czech natural focus. Note: Nucleotide sequence data reported in this paper are available in the GenBank, EMBL and DDBJ databases under the accession numbers KP635008-9.

The microsatellite markers were designed for the giant liver fluke, Fascioloides magna, veterinary important liver parasite of free-living and domestic ruminants. Due to its geographic distribution (five enzootic regions across USA and Canada, three permanent European foci) and invasive character, F. magna is an interesting model for population genetics. Out of 667 amplicon candidates generated after NGS, 118 provided the best resolution and were tested with PCR analysis. In total, 56 yielded PCR products of expected size and in 36 of them the declared repetitive motif was identified by Sanger sequencing. After fragment analysis, 12 loci were proved to be polymorphic in individuals from one tested European and four North American populations. These loci were selected for setup of multiplex STR assays and utilized in genotyping of larger sample cohort. The outputs of statistical analyses indicate further global application of 11 conclusive loci in population genetics of the parasite., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The study was conducted in 2012-2013 on 75 fecal samples of red deer from the Lower Silesian Wilderness which were examined to determine the prevalence of Fascioloides magna in the game population. Finding liver fluke eggs in a single sample which were larger in size than Fasciola hepatica eggs indicated that further molecular analysis was necessarily. The partial sequence (116 bp long) of ITS-2 of the investigated eggs was identical to the sequences of F. magna from red deer (Cervus elaphus) (GenBank, EF534993; GenBank, EF534992) and from wapiti deer (Cervus elaphus canadensis) (GenBank, EF534994) from Slovakia, as well as from fallow deer (Dama dama) from the USA (GenBank, EF051080). This is the first molecular confirmation of the occurrence of F. magna in Poland.

The high-resolution melting (HRM) method, recently optimized as a reliable technique for population study of the European Fascioloides magna populations, was applied to determine an origin of F. magna individuals from Croatia. The structure and frequency of mitochondrial cytochrome c oxidase subunit I (439 bp; cox1) haplotypes of 200 Croatian flukes coming from 19 red deer (Cervus elaphus elaphus) livers were screened and compared with recently determined reference samples of F. magna from all European foci-Italy, Czech Republic, and Danube floodplain forests. While the reference haplotypes Ha1 and Ha2 were specific for flukes from the first European focus of fascioloidosis, the Natural Park La Mandria in Italy, the remaining three haplotypes (Ha3, Ha4, and Ha5) represented parasites from the second focus, Czech Republic. Besides, Ha3 and Ha4 were found also in the third, latest, and still expanding European focus, the Danube floodplain forests. The HRM screening of cox1 haplotypes of Croatian F. magna individuals resulted in classification of samples into the two mitochondrial haplogroups characterized by well-distinguished melting curves. They corresponded to Ha3 and Ha4 reference haplotypes that confirmed the Danube origin of F. magna from Croatia. The results support the theory that the Danube floodplain forests population of F. magna represents uniform genetic pool of the parasite. The spread of F. magna alongside the Danube River down to Croatia was possible due to suitable ecological conditions for definitive and intermediate hosts present in this unique biotope.

Parasitic liver flukes of the family Fasciolidae are responsible for major socioeconomic losses worldwide. However, at present, knowledge of the fundamental molecular biology of these organisms is scant. Here, we characterize, for the first time, the transcriptome and secreted proteome of the adult stage of the "giant liver fluke," Fascioloides magna, using Illumina sequencing technology and one-dimensional SDS-PAGE and OFFGEL protein electrophoresis, respectively. A total of ∼54,000,000 reads were generated and assembled into ∼39,000 contiguous sequences (contigs); ∼20,000 peptides were predicted and classified based on homology searches, protein motifs, gene ontology, and biological pathway mapping. From the predicted proteome, 48.1% of proteins could be assigned to 384 biological pathway terms, including "spliceosome," "RNA transport," and "endocytosis." Putative proteins involved in amino acid degradation were most abundant. Of the 835 secreted proteins predicted from the transcriptome of F. magna, 80 were identified in the excretory/secretory products from this parasite. Highly represented were antioxidant proteins, followed by peptidases (particularly cathepsins) and proteins involved in carbohydrate metabolism. The integration of transcriptomic and proteomic datasets generated herein sets the scene for future studies aimed at exploring the potential role(s) that molecules might play at the host-parasite interface and for establishing novel strategies for the treatment or control of parasitic fluke infections.

Chromosomal characteristics, i.e., number, size, morphology, and location of ribosomal DNA (rDNA) clusters were examined in two medically important liver flukes, Fasciola hepatica and Fascioloides magna (Fasciolidae), using conventional Giemsa staining and fluorescent in situ hybridization (FISH) with ribosomal 18S rDNA probe. A comparison of F. magna and F. hepatica karyotypes confirmed significant differences in all chromosomal features. Whilst the karyotype of F. hepatica comprised ten pairs of chromosomes (one metacentric and nine medium-sized subtelocentrics and submetacentrics; 2n = 20, n = 1 m + 5 sm + 4 st; TCL = 49.9 μm), the complement of F. magna was composed of 11 pairs of medium-sized subtelocentrics and submeta-metacentrics (2n = 22, n = 9 st + 1 sm + 1 sm-m; TCL = 35.2 μm). Noticeable differences were found mainly in length and morphology of first chromosome pair. It was metacentric and 9.0 μm long in F. hepatica while subtelocentric and 4.7 μm long in F. magna. Although FISH with rDNA probe revealed a single cluster of ribosomal genes in both species, conspicuous interspecific differences were displayed by chromosomal location of ribosomal loci (i.e., NORs). The signals were found on short arms of fifth homologous pair in F. hepatica; however, they were detected in pericentromeric regions of the long arms of tenth pair in F. magna. The observed cytogenetic differences were interpreted in terms of karyotype evolution of fasciolid flukes; F. hepatica may be regarded phylogenetically younger than F. magna. The present paper provides a pilot study on molecular cytogenetics within a group of hermaphroditic digenetic flukes.

The giant liver fluke, Fascioloides magna, a liver parasite of free-living and domestic ruminants of Europe and North America, was analysed in order to determine the origin of European populations and to reveal the biogeography of this originally North American parasite on the European continent. The variable fragments of the mitochondrial cytochrome c oxidase subunit I (cox1; 384bp) and nicotinamide dehydrogenase subunit I (nad1; 405bp) were used. Phylogenetic trees and haplotype networks were constructed and the level of genetic structuring was evaluated using population genetic tools. In F. magna individuals originating from all European foci of infection (Italy, Czech Republic and Danube floodplain forests involving the territories of Slovakia, Hungary and Croatia) and from four of five major North American enzootic areas, 16 cox1 and 18 nad1 haplotypes were determined. The concatenated sequence set produced 22 distinct haplotypes. The European fluke populations were less diverse than those from North America in that they contained proportionately fewer haplotypes (eight), while a more substantial level of genetic diversity and a greater number of haplotypes (15) were recorded in North America. Only one haplotype was shared between the European (Italy) and North American (USA/Oregon and Canada/Alberta) flukes, supporting a western North American origin of the Italian F. magna population. Haplotypes found in Italy were distinct from those determined in the remaining European localities which indicates that introduction of F. magna to the European continent occurred more than once. In the Czech focus of infection, a south-eastern USA origin was revealed. Identical haplotypes, common to parasites from the Czech Republic and from an expanding focus in Danube floodplain forests, implies that the introduction of F. magna to the Danube region came from an already established Czech focus of infection., (Copyright © 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

The high-resolution melting (HRM) technique was successfully optimized as fast and effective method for population study of digenetic fluke, Fascioloides magna (Trematoda: Fasciolidae), originally North American liver parasite of free-living and domestic ruminants. Previously selected variable region (439 bp) of mitochondrial cytochrome c oxidase subunit I (cox1) of 249 fluke individuals from enzootic European and North American regions were sequenced and mutually compared. The sequence analysis of partial cox1 revealed presence of seven structurally different haplotypes. Based on the sequence structure and alignments of six of them (Ha1-Ha6), three internal probes were designed and applied in HRM-based haplotype determination of all F. magna specimens. HRM analysis, performed with three designed probes, resulted in classification of samples into the seven haplogroups, equally with their assortment according to the sequence analysis. The representative of the haplotype, which was not involved in probe design (Ha7), was characterized by a unique melting curve shape as well. This provided an evidence of optimally settled conditions in HRM assay and indicated a probability of successful discrimination of novel haplotypes in future population studies on F. magna. The successful optimization of HRM method stands for an opportunity of detection of genetically unknown North American variants of F. magna and promises its application as fast and cheap screening technique for phylogeography studies of the giant liver fluke on its original continent.

Fasciolid flukes are among the largest and best known digenetic trematodes and have considerable historical and veterinary significance. Fasciola hepatica is commonly implicated in causing disease in humans. The origins, patterns of diversification, and biogeography of fasciolids are all poorly known. We have undertaken a molecular phylogenetic study using 28S, internal transcribed spacer 1 and 2 (ITS-1 and ITS-2) of nuclear ribosomal DNA, and mitochondrial nicotinamide dehydrogenase subunit 1 (nad1) that included seven of the nine recognized species in the family. The fasciolids examined comprise a monophyletic group with the most basal species recovered from African elephants. We hypothesize fasciolids migrated from Africa to Eurasia, with secondary colonization of Africa. Fasciolids have been conservative in maintaining relatively large adult body size, but anatomical features of their digestive and reproductive systems are available. These flukes have been opportunistic, with respect to switching to new snail (planorbid to lymnaeid) and mammalian hosts and from intestinal to hepatic habitats within mammals.

Complete sequences of ribosomal and mitochondrial genes of the giant liver fluke Fascioloides magna are presented. In particular, small subunit (18S) and internal transcribed spacers (ITS1 and ITS2) of the ribosomal gene (rDNA), as well as cytochrome c oxidase subunit I (cox1) and nicotinamide dehydrogenase subunit I (nad1) of the mitochondrial DNA (mtDNA), were analyzed. The 18S and ITS sequences were compared with previously published sequences of the liver fluke Fasciola hepatica. Fixed interspecific genetic differences were determined that allow molecular differentiation of F. magna and F. hepatica using either the PCR-RFLP method or PCR amplification of species-specific DNA regions. Additionally, intraspecific sequence polymorphism of the complete cox1 and nad1 mitochondrial genes in geographically distinct F. magna populations was determined. Based on the sequence divergences, short (< 500 bp) variable regions suitable for broader biogeographical studies of giant liver fluke were designed.

Fasciolopsis buski, the zoonotic intestinal flukes of pigs in South and Southeast Asia, is commonly prevalent in regions across Northeast India. Populations of the fluke collected from different parts of the region exhibit variations in morphology. The main objective of our study was to provide molecular characterization of the parasite so as to supplement morphological criteria, using ribosomal DNA cluster (rDNA), which is flanked by more conserved internal transcribed spacer (ITS) regions. We describe herein the ITS sequences of the parasite collected from swine hosts of Assam region. The ITS sequences of both egg and adult origins were found to be identical in length and composition. Phylogenetically, F. buski resembles closely the other members of family Fasciolidae, showing significant expectation value in the alignment. The results corroborate that the ITS sequences are not stage specific and are conserved through different stages of development of the fluke, and thus could be used as species markers.

The nucleotide sequence of the 3' end of the second internal transcribed spacer region (ITS2) was determined for four isolates of Fasciola hepatica, two isolates of Fasciola gigantica, one isolate of Fascioloides magna and one isolate of Fasciola sp. from Japan. Intraspecific sequence divergence was negligible. Sequence divergence between F. hepatica and F. gigantica was 2.8% and between Fasciola hepatica and Fascioloides magna was 13.2%. The sequence of Fasciola sp. from Japan matched closely that of F. gigantica. This study demonstrates variability in nucleotide sequence within the ITS2 region which allows discrimination between species of Fasciolidae.

Molecular variation, both intra- and inter-specific, in fasciolids and Paragonimus has been detected in isozymes and other proteins, antigens and nucleic acid sequences. The use of such data is reviewed for studies on population genetics, taxonomy, phylogeny, immunodiagnosis, ontogeny and host-induced variation.