Development of a qPCR Duplex Assay for simultaneous detection of Fascioloides magna and Galba truncatula in eDNA samples: Monitoring beyond boundaries.

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.

A new insight into genetic structure of Danube and Italian foci of fascioloidosis.

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.

Fasciolopsis buski Detected in Humans in Bihar and Pigs in Assam, India.

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.

FATAL FASCIOLOIDES MAGNA IN A LESSER SPOT-NOSED GUENON (CERCOPITHECUS PETAURISTA).

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.

Genetic Characterization of the Giant Liver Fluke, Fascioloides magna (Platyhelminthes: Fasciolidae) From the Adirondack Region of Northern New York.

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 (ФST) 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 (ФST = 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.

Description and phylogenetic analyses of ribosomal transcription units from species of Fasciolidae (Platyhelminthes: Digenea).

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.

Characterization and phylogenetic properties of the complete mitochondrial genome of Fascioloides jacksoni (syn. Fasciola jacksoni) support the suggested intergeneric change from Fasciola to Fascioloides (Platyhelminthes: Trematoda: Plagiorchiida).

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 tRNAGlu (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, tRNAS1(AGN) and tRNAS2(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.

Adaptive Radiation of the Flukes of the Family Fasciolidae Inferred from Genome-Wide Comparisons of Key Species.

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.

An Autochthonous Human Case of Fasciolopsiasis in Nepal.

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.

De novo genome and transcriptome analyses provide insights into the biology of the trematode human parasite Fasciolopsis buski.

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.

Mitochondrial genotyping of Fascioloides magna from Bavaria, Germany.

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.

Genetic homogeneity of Fascioloides magna in Austria.

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.

Fasciolopsis buski (Digenea: Fasciolidae) from China and India may represent distinct taxa based on mitochondrial and nuclear ribosomal DNA sequences.

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.

Population structure and dispersal routes of an invasive parasite, Fascioloides magna, in North America and Europe.

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.

A genetic structure of novel population of Fascioloides magna from Poland, Podkarpackie Province, indicates an expanding second European natural focus of fascioloidosis.

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.

Complete mitochondrial genome of the giant liver fluke Fascioloides magna (Digenea: Fasciolidae) and its comparison with selected trematodes.

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.

Identification and characterization of microRNAs in the zoonotic fluke Fasciolopsis buski.

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.

Molecular characterization of Fascioloides magna (Trematoda: Fasciolidae) from south-western Poland based on mitochondrial markers.

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.

Molecular identification of Fascioloides magna (Bassi, 1875) from red deer from South-Western Poland (Lower Silesian wilderness) on the basis of internal transcribed spacer 2 (ITS-2).

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.

Development and characterization of multiplex panels of polymorphic microsatellite loci in giant liver fluke Fascioloides magna (Trematoda: Fasciolidae), using next-generation sequencing approach.

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.