title: Characterisation of a novel panel of polymorphic microsatellite loci for the liver fluke, Fasciola hepatica, using a next generation sequencing approach.
abstract: The liver fluke, Fasciola hepatica is an economically important pathogen of sheep and cattle and has been described by the WHO as a re-emerging zoonosis. Control is heavily reliant on the use of drugs, particularly triclabendazole and as a result resistance has now emerged. The population structure of F. hepatica is not well known, yet it can impact on host-parasite interactions and parasite control with drugs, particularly regarding the spread of triclabendazole resistance. We have identified 2448 potential microsatellites from 83 Mb of F. hepatica genome sequence using msatfinder. Thirty-five loci were developed and optimised for microsatellite PCR, resulting in a panel of 15 polymorphic loci, with a range of three to 15 alleles. This panel was validated on genomic DNA from 46 adult F. hepatica; 38 liver flukes sourced from a Northwest abattoir, UK and 8 liver flukes from an established isolate (Shrewsbury; Ridgeway Research). Evidence for null alleles was found at four loci (Fh_1, Fh_8, Fh_13 and Fh_14), which showed markedly higher levels of homozygosity than the remaining 11 loci. Of the 38 liver flukes isolated from cattle livers (n=10) at the abattoir, 37 genotypes were identified. Using a multiplex approach all 15 loci could be amplified from several life cycle stages that typically yield low amounts of DNA, including metacercariae, the infective life cycle stage present on pasture, highlighting the utility of this multiplex microsatellite panel. This study reports the largest panel of microsatellite markers available to date for population studies of F. hepatica and the first multiplex panel of microsatellite markers that can be used for several life cycle stages.
authors: Veterinary Parasitology, Department of Infection Biology, Institute of Infection and Global Health, University of Liverpool, Liverpool L3 5RF, UK. Electronic address: k.cwiklinski@qub.ac.uk.
authors: Krystyna
authors: K
authors: Cwiklinski
authors: Veterinary Parasitology, Department of Infection Biology, Institute of Infection and Global Health, University of Liverpool, Liverpool L3 5RF, UK.
authors: Katherine
authors: K
authors: Allen
authors: Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK.
authors: James
authors: J
authors: LaCourse
authors: Veterinary Parasitology, Department of Infection Biology, Institute of Infection and Global Health, University of Liverpool, Liverpool L3 5RF, UK.
authors: Diana J
authors: DJ
authors: Williams
authors: Institute of Integrative Biology, University of Liverpool, L69 7ZB, UK.
authors: Steve
authors: S
authors: Paterson
authors: Veterinary Parasitology, Department of Infection Biology, Institute of Infection and Global Health, University of Liverpool, Liverpool L3 5RF, UK.
authors: Jane E
authors: JE
authors: Hodgkinson
mesh_terms: D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology; D002417:Cattle; D002418:Cattle Diseases / Q000469:parasitology; D018509:DNA, Helminth / Q000235:genetics; D005210:Fasciola hepatica / Q000187:drug effects / Q000235:genetics*; D005211:Fascioliasis / Q000188:drug therapy / Q000469:parasitology / Q000662:veterinary; D056426:Genetic Loci; D049751:Genome, Helminth; D060005:Genotyping Techniques; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006790:Host-Parasite Interactions / Q000187:drug effects / Q000235:genetics; D018895:Microsatellite Repeats; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D012756:Sheep; D012757:Sheep Diseases / Q000469:parasitology; D000077682:Triclabendazole
chemical_list: D001562:Benzimidazoles; D018509:DNA, Helminth; D000077682:Triclabendazole
keywords: Fasciola; Microsatellite; Next generation sequencing; Population structure