Identification of viable liver fluke metacercarial challenge to livestock
Cuthill, Grace Victoria
The parasitic liver fluke, Fasciola hepatica, presents a significant economic burden to the UK livestock industry. Liver fluke disease (fasciolosis) control is underpinned by forecasting, which is currently performed on a relatively crude regional scale, informed by traditional seasonal disease patterns and prevailing climatic conditions. The ultimate indicator of fluke infection risk is the infectious cyst (metacercarial) challenge on pasture; hence precise forecasting of the fasciolosis risk to grazing livestock requires the development of new methods to identify and quantify viable metacercariae on pasture. The work in this thesis focuses on the development of assays to evaluate the viability of metacercariae, methods to recover metacercariae from herbage, and DNA detection techniques to identify and quantify the numbers of viable metacercariae from pasture samples. Furthermore, there is investigation and discussion of the risk of fasciolosis to livestock grazing on wetland areas as part of agri-environmental schemes, and to housed livestock consuming grass silage. Determination of metacercarial viability is key to the prediction of metacercarial challenge on pasture. Methods which are quick and relatively easy to perform are appropriate for liver fluke forecasting as avoidance of infections is important to the livestock industry. Assessment of metacercarial morphology, using light microscopy, offers a quick and relatively easy method to determine cyst viability. Excystment assays are another method used to determine metacercarial viability in the lab, each assay varying in design and ease to perform. The reagents used and the design of ‘C. E. Bennett excystment assay’ show promise as a method which is both easy to perform and potentially applicable to the determination of metacercarial challenge from pasture on farm. The aim of Chapter 2 was to evaluate the reliability of both light microscopy and the ‘C. E. Bennett excystment assay’ as methods to determine metacercarial viability. This was demonstrated on metacercariae subjected to one well researched factor of viability, temperature, and another lesser known factor of viability, salinity. The ability of the ‘C. E. Bennett excystment assay’ and light microscopy were evaluated to determine metacercarial viability; both were found to be unreliable. For this thesis a robust and reliable method of determining metacercarial viability was required: this was provided by the excystment method published by Hernández- González et al. (2010). Despite the poor excystment percentages obtained using the ‘C. E. Bennett excystment assay’, it was straightforward to perform. Shop-bought carbonated water was used to modify the excystment assay published by Hernández-González et al. (2010). This modified assay gave excystment percentages of 97% using ovine bile and 90% using bovine bile with freshly encysted metacercariae. The use of carbonated water made this method more applicable to an ‘on-farm’ testing scenario. Metacercarial recovery from pasture is essential to determine metacercarial challenge on farms/fields. The physical removal and isolation of metacercariae from grass provides information as to the number of metacercariae on the pasture. Currently, a routine method of metacercarial recovery from pasture does not exist. One aim of Chapter 3 was to demonstrate the combined use of vinegar, as a detachment solution, and a salad spinner, to isolate metacercariae from their original encystment material. This method was evaluated on F. hepatica metacercariae encysted on cellophane; on metacercariae allowed to re-adhere to both grass and cellophane; and on grass exposed to snails shedding metacercariae of another species of parasitic fluke, Notocotylus. About 44% of metacercariae encysted on cellophane were estimated to be recovered. Similarly, 44% of re-adhered metacercariae were recovered from grass, compared to 29% of metacercariae re-adhered to cellophane. The limitations of using re-adhered metacercariae to represent freshly encysted metacercariae are discussed. In the absence of direct access to a laboratory Galba truncatula mud snail colony, Radix sp. snails shedding Notocotylus sp. metacercariae were placed on laboratory grown grass. Seven Notocotylus sp. metacercariae were recovered. The total number of Notocotylus sp. metacercariae encysted upon the grass sample was unknown and because of this the efficiency of recovery could not be measured. Another aim of Chapter 3 was to evaluate the specific risk of liver fluke to livestock grazing on wader scrapes over time. Another method of metacercarial recovery, cellophane rafts, was used to determine the risk of fasciolosis to livestock grazing around wader scrapes, established to promote biodiversity on farms. Wader scrapes can overlap with and create habitats suitable for the G. truncatula intermediate mud snail hosts for F. hepatica, causing concern to farmers who are required to graze their livestock around these areas in order to receive environmental subsidies. Two newly installed wader scrapes and a ditch located on Scottish hill farms were sampled over three years. Cellophane rafts were deployed in all three areas in an attempt to collect any available metacercariae. Snail numbers were monitored and specimens were collected. Faeces from both wildlife and livestock were collected opportunistically and fluke egg counts were performed. A total of 198 snails, either G. truncatula or Radix sp. were collected over the 3 years. 2.5% of these snails were positive for F. hepatica as determined by PCR. 10% of sheep faecal samples collected were positive for F. hepatica, as determined by faecal egg count. Of the 7 deer samples collected during the three year study period, one had a F. hepatica egg count of 1.67 eggs per gram. Putative metacercariae were observed on the cellophane rafts, but their identity could not be determined. It was concluded that wader scrapes could offer suitable habitats for G. truncatula and that there is a potential risk of fasciolosis to livestock grazing on these areas. It is important that farmers appreciate the risk relative to where livestock are grazing and the health status of their stock. The risk of fasciolosis to housed livestock fed grass silage is unknown. Silage fermentation is a many factorial process, Tarczynski & Podkowka (1964) suggested that pH impacts the survival of metacercariae when ensiled. Chapter 4 aimed to determine the longevity of viable F. hepatica metacercariae at different pH associated with silage fermentation, and to determine the longevity of F. hepatica metacercariae in silages of different qualities, produced under laboratory conditions. The modified Hernández-González et al. (2010) excystment method was used to determine metacercarial viability after incubation at different pH associated with silage fermentation. Metacercariae were incubated for 4 weeks in lactic acid solutions of pH 4, 5 and 6: viability remained high, being 88%, 82% and 91%, respectively. A longer exposure to lactic acid, pH 4, resulted in relatively high viability over a period of 12 weeks, but after 16 weeks, the percentage viability was significantly lower (p=0.008) than in the comparative control group; 38% vs. 72%, respectively. Small aliquots of pre-made grass silage were used to replicate ensiling conditions in the lab and to test metacercarial viability. Silages of ‘high’, ‘medium’ and ‘low’ quality were sampled from farms in Wales. These samples were split and spiked with F. hepatica metacercariae. Time intervals of 4, 8, 12 and 16 weeks were employed for the recovery of metacercariae from the ‘high’ quality silage sample. Recovery of metacercariae from the ‘medium’ and ‘low’ quality silages was determined at one time point of 14 weeks after spiking with fluke cysts. Metacercarial recovery was inconsistent regardless of time point or silage quality. Viability was determined using the modified Hernández- González et al. (2010) excystment method. The only group to contain viable metacercariae was the ‘high’ quality silage after 4 weeks of incubation. 16% of the metacercariae present in this sample of ‘high’ quality silage were recovered; 58% of the recovered metacercariae were viable. The multifactorial nature of grass silage fermentation means that fully understanding metacercarial survival in silage is not straightforward. pH alone does not appear to be the main determinant of cyst viability. Metacercariae could survive well in pH 4 lactic acid solution, but did not survive well in equivalent pH silage. It was concluded that it is possible for metacercariae to survive in silage, but the risk of fasciolosis when well-made silage is fed to livestock is very low. DNA methods are used routinely in applied research to identify F. hepatica in snail DNA and faecal DNA samples. These methods are highly specific and highly sensitive, highlighting the benefit that they could have for the detection of F. hepatica metacercariae from pasture samples. Polymerase chain reaction (PCR) and loop mediated isothermal amplification (LAMP) were the methods selected for the development of DNA-based detection of F. hepatica metacercariae from pasture samples. The work of Chapter 5 aimed to demonstrate the use of these methods and their effectiveness using DNA extracted from different prepared DNA samples. The mitochondrial cytochrome c oxidase subunit 1 (COX1) target was amplified from both DNA extracted from grass spiked with metacercarial DNA, and from grass spiked with viable metacercariae. The sensitivity levels of each assay were determined using DNA dilution series in water and in DNA extracted from grass. LAMP had a 100 times greater sensitivity than the equivalent PCR (0.001ng/µl vs 0.1ng/µl) when metacercarial DNA was diluted in grass DNA. LAMP was able to detect the target at a concentration of 0.001 ng/µl in 30 minutes. PCR and LAMP both showed capability of detecting metacercariae from grass samples. However, the translation of these tests for use with farm samples may not yet be practically feasible. DNA testing does not give any indication of the source of DNA being amplified: it cannot be known if the DNA amplified from a pasture sample has originated from a F. hepatica eggs, miracidia, cercariae, or metacercariae. Similarly, the results of a DNA test performed on pasture samples would be of little help to farmers without knowledge of the viability status of metacercariae amplified. The main findings of this thesis are: (1) metacercarial viability cannot be reliably determined using light microscopy or the ‘C. E. Bennett excystment assay’; (2) the modified Hernández- González et al. (2010) method is an efficient excystment method; (3) in the absence of a mud snail colony, demonstrating methods of metacercarial recovery and isolation is challenging; (4) there is a potential risk of fasciolosis to animals grazing on wader scrapes; (5) F. hepatica metacercariae are able to survive in grass silage for a short time; and (6) LAMP is x100 more sensitive than PCR and it is the most applicable method for DNA detection of metacercariae from grass. Overall, this work highlights that with future development of cyst recovery and viability techniques, the metacercarial challenge on pasture could be effectively quantified on farms. This would allow for more precise and accurate determination of liver fluke risk in time and space.