Mapping triclabendazole resistance in Fasciola hepatica using next generation sequencing technologies

Fasciolosis is a common and important disease of livestock. In the UK it is the most commonly reported infection associated with the digestive tract of ruminants. It is caused by Fasciola hepatica, a flatworm found in the liver and transmitted by a mud snail. Fasciolosis is controlled predominantly using drug treatment and the most commonly used drug is triclabendazole (TCBZ). This is the only drug that is effective against the immature stages of the parasite that are responsible for acute, often fatal disease. It is also the drug of choice for human fasciolosis, caused by the same parasite. Resistance to TCBZ was first reported in Australia in 1995 and is now thought to be widespread worldwide. Very little is known about mechanisms of drug resistance in F. hepatica, which genes are involved and how resistance is evolving. This proposal aims to exploit our ability to create clones of resistant and susceptible F. hepatica; to derive genetically identical infections of TCBZ-susceptible and TCBZ-resistant isolates in sheep, from which we will generate second generation populations. These populations will be used to identify markers, in this case single mutations at the DNA level that associate with resistance genes during the crossing of resistant and susceptible clones. We will use rapid, high throughput, next generation sequencing technologies, which allow us to produce a preliminary F. hepatica genome map. The resistance markers will be mapped onto this preliminary genome and allow us to identify regions of the genome where the gene or genes responsible for encoding resistance are found. At the end of the project we will have genetic markers for resistance that could be used to detect and track the emergence of resistance in populations of F. hepatica in naturally infected sheep and cattle in the UK, but, importantly they will also be used in future projects to start to home in and identify genes encoding resistance. There will be other important outputs for the wider scientific community from the project, such as a bank of genome data for the parasite and clones of the parasite whose resistance status is well characterised. Genome data will be made available through a publicly accessible website and the parasite clones will be made available to other groups by lodging them with our industrial partner, Ridgeways Research Ltd.

Fasciola hepatica causes serious disease in livestock worldwide. It is common in the UK, its prevalence is increasing and the costs of controlling infection have a significant impact on the UK agricultural sector. Resistance to triclabendazole (TCBZ), the most widely used drug for the control of fasciolosis, is reported in the UK, Europe and Australia. This project will take a new approach to identifying markers for resistance that will, in the long term, be useful in the identification of gene(s) encoding resistance and the development of assays to measure the prevalence and spread of resistance. Clonal lines will be generated from resistant and susceptible field isolates and F1 and F2 generations created based on crosses of these lines. A draft genome sequence for F. hepatica will be created using 454 fragment and mate pair libraries at 10x coverage producing a rough assembly (N50 of 30kb) complemented by Solexa reads and the MAKER pipeline used for basic annotation. Resistant and susceptible clonal lines will be resequenced at 20x coverage to identify single nucleotide polymorphisms (SNP) using SOLiD fragment libraries. Differences in SNP allele frequencies will be identified between parasites that have developed after exposure to TCBZ versus unexposed controls. Six pairs of treated vs control populations will be sequenced at 20x coverage using SOLiD fragment libraries. Allele frequencies will be analyzed using logistic regression plotted against the draft genome sequence to identify genomic regions associated with TCBZ resistance. Finally a sample of 150 SNPs linked to TCBZ resistance, plus 100 unlinked controls will be validated. Individual miracidia will be SNP genotyped to determine linkage between markers to indicate if a single locus is responsible for resistance and if TCBZ resistance is a dominant or recessive trait. Finally experimental infections using F2 individuals will be used to confirm linkage of SNPs to TCBZ resistance in vivo.

The work we are proposing uses the latest genomic technologies combined with knowledge and exploitation of the life cycle of the parasite to elucidate the molecular mechanisms of triclabendazole resistance. The innovative nature of the work is only possible because of the recent advances in genome sequencing, but the work is necessarily technically complex and long term. In the short term the outputs of the project will be the identification of markers for resistance genes. This will lead to further research aimed at identifying specific genes of interest using the parasite material generated in this project, which, combined with the likely advances in sequencing and annotation technologies, will lead to identification of genes encoding resistance. From this we can start to understand the biochemical pathways associated with resistance, leading to ways to prevent resistance occurring - such as through modifications to the drug itself. Thus the tangible outputs and impact of the project are necessarily long term, but will ultimately lead to improved application of drug, markers for resistance and circumventing resistance mechanisms developed by the parasite. Markers for resistance will form the basis of sensitive and specific diagnostic tests for resistance in the field and ultimately provide evidence based recommendations for anthelmintic use. The principle, immediate beneficiaries from this work is the scientific community in general and specifically groups working on anthelmintic resistance. The new approach we are taking will provide information to groups working on related systems. The majority of molecular studies on anthelmintic resistance have focused on possible associations of putative candidate genes with a resistance phenotype. What we are proposing represents a timely and novel molecular approach using a combination of genetic and genomic resources to address drug resistance at a genome-wide level. As such, the work will have important outputs, useful to other academics, both those working directly in fluke research but also those working on other parasites and in the drug resistance field as a whole. Those groups working on the development of vaccines, the immunology and pathogenesis of fluke and improved diagnosis will benefit from free access to the partly annotated fluke genome. Fluke research will be greatly advance by having access to new, fully validated parasite clones. These will be made available through our commercial collaborator, Ridgeways Research Ltd. In the long term, veterinarians, farmers and food retailers will benefit from diagnostics for triclabendazole resistance, improved, strategic use of triclabendazole leading to reduced use of drug, which ultimately will lead to reduced drug residues in food and improve the safety of meat and milk products for the whole community.