Using digital droplet PCR to improve the detection of helminth infections of ruminants in Northern Ireland

Fasciola hepatica, usually termed "the temperate liver fluke", is found worldwide and causes a disease known as fasciolosis. These helminth parasites is primarily a damaging infection of production ruminants. In Europe, the disease has been reported in UK, Ireland, France, Portugal, Spain, Switzerland, Italy, The Netherlands, Germany and Poland. As a food-borne disease, fasciolosis results from consumption of the infective larvae by ruminant animals. These helminths greatly reduce global agricultural capacity and outputs of both developing and developed communities, causing production inefficiency in millions of ruminants worldwide. Fasciolosis is also an important zoonosis, and is acknowledged by the World Health Organization (WHO).
All organisms shed DNA into their environment, this material is known as eDNA. The detection of this eDNA enables the classification of species diversity and abundance in an environment. When Fasciola parasites are present, during part of their lifecycle they are exposed in the environment in order to be infectious to the snail or animal host, they also leave eDNA behind. The improved detection of this eDNA is the basis of this project. While low levels of this materials is expected in water or soil, or on plants, new molecular methods will be used to overcome these difficulties.
These methods include the use of third generation technology droplet digital PCR (ddPCR) which greatly increases the sensitivity and reproducibility of DNA detection. ddPCR has the distinct value of being a fully quantitative method, and being highly sensitive and reproducible.
Our proposal will drive a step-change in the way eDNA analysis is performed for the identification of helminth parasite infection risk. This will be involve examining a range of sample types, including water, soil, grass and faeces. These challenging samples will be characterised by the highly sensitive ddPCR technology to provide comprehensive quantitative measurements of parasite/vector eDNA on collected samples. Sheep farms in Northern Ireland will sampled over one year and these new sample collected to find molecular indicators of disease. This represents a less invasive and potentially less labor intensive approach to disease monitoring.
In detecting parasitic infections earlier, the use of drugs (anthelmintics) can be reduced or avoided through intelligent pasture management - rotating animals around pastures according to their age, species and season, relative to the perception of infection risk in those pastures. Farmers would be able to make these decisions more effectively if they had access to relevant data from their own farm.

As a third generation technology droplet digital PCR (ddPCR) provides increased DNA detection sensitivity and reproducibility. The lifecycle of the important agricultural pathogen F. hepatica presents sources of environmental eDNA which we proposed to detect in plants, soil or water samples.
A multiplex ddPCR assay will use published primers for the snail vector G. truncatula and F. hepatica. The amplicons sizes (288bp Gt & 169bp Fh), will provide appropriate intensities to differentiate the species. In targeting ITS-2, thousands of copies within the genome are available, while allowing specific detection, since F. hepatica ITS-2 primers are reported to not amplify closely related species. Experiments to quantitate the degree of variation and reproducibility will be performed.
The School of Biological Sciences QUB, has a long history of collaboration with farmers in Northern Ireland. One hundred and fifty (150) sheep farms have been screened repeatedly for parasite infection across the province over a ten-year period, using traditional direct detection methods. These datasets have been used to identify two individual pilot sites based on their ecology and infection status, one with F. hepatica present, the other where it is absent. Calculations for field sampling: considering a single study group, with a dichotomous endpoint (presence of eDNA or not), since the known incidence of fascioliasis in Northern Ireland is ~50%, the use of a known infection sites (75%), assuming a beta at 95% (from the ddPCR power calculations of a 2 log10 improvement of sensitivity over qPCR), 100 samples will produce an alpha < 0.001. The 2 log10 improvement in the ddPCR lower detection limit will allow smaller water volumes sampling and the investigation of other previous not considered sources (soil and plant). All of these samples in containing lower levels of snail or parasite eDNA, ddPCR will be key to the viability of this novel and powerful assay.