Authors

Discussion

Giardia duodenalis(Giardia)is an intestinal protozoan parasite which causes disease in both animals and humans across the globe. It is becoming increasingly apparent that in developed countries there exist major knowledge gaps on Giardia ecology and epidemiology. For example, in Scotland, despite being considered an “exotic” disease, recent studies have suggested the presence of an endemic cycle of disease within the country which is not associated with travel-related infections. However, endemic transmission routes are poorly understood and, in particular, the level of zoonotic risk is unknown. Current molecular markers for parasite genotyping are low-resolution and are only effective in a minority of field samples. Moreover, only a low proportion of Giardia isolates can be adapted to culture, which presents a major obstacle to generating good-quality DNA preps for genomic sequencing. Therefore, the present study aims: (i) to establish a laboratory workflow to allow sequencing of Giardia directly from faecal samples collected from human and animal cases in Scotland; (ii) to generate high-quality exomes to establish a robust minimum set of genotyping markers for analysing relationships between parasite isolates and (iii) to compare the genomes of endemic and foreign travel-associated isolates to begin to investigate transmission routes in Scotland. The laboratory workflow fundamentally consists of four steps: library preparation performed on Giardia DNA extracted directly from faecal samples, validation of a hybridisation capture protocol targeting the parasite exome, enrichment amplification conducted on the captured libraries and, lastly, short read sequencing. For the genome capture hybridisation, we designed an array of overlapping biotinylated RNA capture probes evenly spaced across the Giardia exome. An additional set of capture probes was designed to specifically cover the loci most commonly used for typing, including the bg, gdh and tpi genes located on chromosomes 4 and 5 of the parasite. A process of iterative testing and refinement of the library preparation and target enrichment methodology has allowed us to develop an optimised protocol. We have successfully applied this to a Giardia-positive faecal sample from a Scottish wildcat, achieving a high level of parasite DNA enrichment. Reads mapped to the genome corresponding to the exome probe design. The loci which were over-represented on the probe design had a higher depth than the other regions, including the three key loci used for typing. The gene sequences at these loci identified the isolate as belonging to ‘assemblage F’ typically found in felines. Having demonstrated ‘proof of principle’ for this approach, we now intend to apply it to a number of human and animal samples. Ultimately, it is envisioned that the genome capture methodology may provide a rapid and accurate typing method for this parasite, including samples that do not readily PCR, allowing a better understanding of the epidemiology of Giardia in Scotland and further afield.