Diagnosis of fluke infective stages in the environment (PhD)

Determining species identity of any metacercariae isolated from pasture is important for the correct diagnosis of disease risk. Incorrect identification could lead to unnecessary and potentially damaging implementation of disease avoidance techniques and targeted treatment. Both PCR and LAMP were demonstrated to be capable of detecting very small amounts of metacercarial DNA from spiked grass. This is a proof-of-concept method for DNA-based detection of F. hepatica from the environment is achievable, the very nature of working with environmental samples is challenging. Farmers can avoid fasciolosis in the stock by avoiding high risk pasture.

Other methods of disease avoidance include the reduction of G. truncatula numbers through drainage of habitat. Molluscicides were previously implemented but are now banned because of the potential harm to the wider environment that they pose (Torgerson & Claxton, 1998). Reducing water from the surface and sub-surface of a field using drainage offers a less dangerous method for the removal of G. truncatula habitat.

Where the removal of snail habitat is not possible, livestock can be prevented from grazing such areas by use of fencing and or moving them to low risk pasture. In some west, upland areas of Scotland most of the grazing pasture could be deemed suitable habitat for G. truncatula and therefore identified as high risk for fasciolosis. Farmers in these areas may not have the option to graze their animals on low risk pasture and drainage may not be an economically viable option. In this situation farmers are advised to ‘protect’ pasture by reducing the egg output onto fields, thus reducing the risk of snails becoming infected. LAMP has the potential to be transformed into an on farm-based test.

For practicality and ease of use on farm, the current method of detecting the amplified target would need to be altered. Instead of using an intercalating fluorochrome dye (MAST), a fluorescence dye could be used to measure the amplification of the target from samples. The appearance of fluorescence within a sample tube can be detected by eye. This is beneficial for the development of a farm-based test as this does not require the use of RT-PCR thermo cyclers which are expensive and not suitable for easy determination of sample status by potential none lab based end point users.

As the origin of the amplified DNA cannot be known, any results should be considered in relation to any metacercarial recovery data, the fasciolosis status of stock on the farm, the presence and status of G. truncatula on the field. The epidemiological strength of any theoretical results gathered from pasture samples would be poor, especially considering the amount of processing these methods require. Without physical confirmation of metacercariae on the pasture by their physical recovery, any DNA positive results are effectively meaningless from a F. hepatica risk perspective. F. hepatica COX 1 amplification can offer a quick, but crude pin point determination of F. hepatica present in a field or on a farm but does not inform the user as to the number or viable metacercariae present. However, positive and or negative confirmation of F. hepatica presence on fields/parts of fields does benefit some farms where confirmation of any low risk grazing would be helpful. The amount of grass needed to determine the presence of F. hepatica on a field is considerable.

Indeed, the MAFF (1986) method of metacercarial recovery from pasture requires 100g of grass per individual sample. Recognising that many hundreds of grams of grass would be required to survey a complete field amplifies the workload considerably. Practical application of pasture DNA analysis to detect F. hepatica on this volume of grass is not realistic or appropriate for determining fasciolosis risk. Alternative methods of sample processing and DNA extraction that could handle larger samples sizes efficiently and at low cost would be the ideal solution. However, the more valuable solution would be focus on further development of efficient method for the recovery of wild metacercariae from pasture samples.

This project has highlighted that there is not a single, all-encompassing method or answer for the determination of liver fluke risk on farms. For an accurate assessment of fasciolosis risk on farms, all information must be considered, including: knowledge of the history of fasciolosis on farm, the current status of livestock, treatment plans, snail surveys and weather readings. All of this information, including the viability and quantity of metacercariae from pasture can be combined from farms across the country to build up a more comprehensive understanding of liver fluke epidemiology and more accurate predictions of disease outbreaks on individual farms in the U.K.

The Challenge

Liver fluke, Fasciola hepatica is a significant burden to the UK livestock industry causing production losses in the live animals as well as condemnations at slaughter as a result of fluke damage, in 2013 alone ~25% of cattle livers and ~11% of sheep livers were condemned. Liver fluke epidemiology is changing with the emergence of drug resistant fluke and an increase in reports of unseasonal fluke due to increased survival over winter. Rumen fluke has also emerged, very little is known about its epidemiology, clinical signs or effects upon production. Forecasting currently only occurs on a crude regional level using traditional seasonal patterns and diagnosis in the definitive host. However, the ultimate indicator of infection is the metacercarial challenge on pasture therefore this project will aim to quantify the metacercarial challenge by using imaging, biochemical and molecular methods as well as investigate factors influencing cyst availability and viability.

The Project

The project aims:

• to investigate factors affecting metacercarial availability and viability, and
• to develop methods to investigate viable liver fluke and rumen fluke metacercariae in the environment to increase understanding of fluke epidemiology and help to identify disease risk to inform avoidance strategies and allow targeted disease control.

Year 1 – Methods development in the lab

Student

Grace Cuthill