SUMMARY: Reports ofCalicophoron daubneyiinfecting livestock in Europe have increased substantially over the past decade; however, there has not been an estimate of its farm level prevalence and associated risk factors in the UK. Here, the prevalence ofC. daubneyiacross 100 participating Welsh farms was recorded, with climate, environmental and management factors attained for each farm and used to create logistic regression models explaining its prevalence. Sixty-one per cent of farms studied were positive forC. daubneyi, with herd-level prevalence for cattle (59%) significantly higher compared with flock-level prevalence for sheep (42%,P= 0·029). Co-infection betweenC. daubneyiandFasciola hepaticawas observed on 46% of farms; however, a significant negative correlation was recorded in the intensity of infection between each parasite within cattle herds (rho = −0·358,P= 0·007). Final models showed sunshine hours, herd size, treatment regularity againstF. hepatica, the presence of streams and bog habitats, and Ollerenshaw index values as significant positive predictors forC. daubneyi(P< 0·05). The results raise intriguing questions regardingC. daubneyiepidemiology, potential competition withF. hepaticaand the role of climate change inC. daubneyiestablishment and its future within the UK.

Increasing trematode prevalence and disease occurrence in livestock is a major concern. With the global spread of anthelmintic resistant trematodes, future control strategies must incorporate approaches focusing on avoidance of infection. The reliance of trematodes on intermediate snail hosts to successfully complete their life-cycle means livestock infections are linked to the availability of respective snail populations. By identifying intermediate snail host habitats, infection risk models may be strengthened whilst farmers may confidently apply pasture management strategies to disrupt the trematode life-cycle. However, accurately identifying and mapping these risk areas is challenging. In this study, environmental DNA (eDNA) assays were designed to reveal Galba truncatula, Fasciola hepatica and Calicophoron daubneyi presence within water sources on pasture land. eDNA was captured using a filter-based protocol, with DNA extracted using the DNeasy® PowerSoil® kit and amplified via PCR. In total, 19 potential G. truncatula habitats were analysed on four farms grazed by livestock infected with both F. hepatica and C. daubneyi. Galba truncatula eDNA was identified in 10/10 habitats where the snail was detected by eye. Galba truncatula eDNA was also identified in four further habitats where the snail was not physically detected. Fasciola hepatica and C. daubneyi eDNA was also identified in 5/19 and 8/19 habitats, respectively. This study demonstrated that eDNA assays have the capabilities of detecting G. truncatula, F. hepatica and C. daubneyi DNA in the environment. Further assay development will be required for a field test capable of identifying and quantifying F. hepatica and C. daubneyi infection risk areas, to support future control strategies. An eDNA test would also be a powerful new tool for epidemiological investigations of parasite infections on farms.

Environmental DNA (eDNA) is a powerful tool for identifying the spatial and temporal presence and density of species in a range of aquatic habitats. The analysis of eDNA has a wide range of application, one of which may be to inform of Fasciola hepatica infection risk on pastures based on the detection of its eDNA as well as that of its intermediate snail host, Galba truncatula eDNA. Here, droplet digital PCR (ddPCR) and quantitative real‐time PCR (qPCR) assays were developed to detect the eDNA of F. hepatica, and its intermediate snail host, G. truncatula in water samples collected from pastures grazed by cattle and/or sheep. Environmental factors associated with species presence, as detected via an eDNA survey, were identified using zero‐inflated linear mixed models. Sixty‐four habitats were sampled across six farms in Ceredigion, Wales, UK, with ddPCR and qPCR identifying 42 and 33 habitats to be positive for G. truncatula eDNA, respectively. G. truncatula eDNA was significantly less likely to be detected in habitats fully shaded by trees, those that contained black or dark brown soils and habitats that contained deep water pools (p < 0.05). Significantly higher G. truncatula eDNA concentrations were observed in habitats that tend to dry up during Summer (i.e., temporary habitats) (p < 0.05). ddPCR also identified five habitats to be positive for F. hepatica eDNA; however, questions remain regarding the utility of F. hepatica eDNA detection due to a lack of specificity toward infective F. hepatica larval stages. The results of this study inform of factors which influences G. truncatula distribution and ecology on pastures and also provided practical information for farmers to aid F. hepatica control in their flocks and herds.

Background: Fascioliasis caused by the trematodes Fasciola hepatica and F. gigantica, is a global neglected zoonotic disease estimated to cost the livestock industry over €2.5 billion annually. Farm management measures and sustainable use of anthelmintics can, in principle, effectively control trematode infection in livestock and reduce the rate of developing anthelmintic resistance. Previously, we designed an environmental DNA (eDNA) assay to identify a common trematode intermediate host, the freshwater snail Galba truncatula, in water sources to measure specific trematode infection risk areas on pasture-land. To improve this procedure, we now report a loop-mediated isothermal amplification (LAMP) assay to identify G. truncatula eDNA. Methods: A LAMP assay was designed and optimised (e.g. temperature, time duration and primer concentration) to identify G. truncatula DNA. The ability of the LAMP assay to target G. truncatula DNA was identified, and LAMP assay limit of detection was investigated in comparison to conventional PCR. In the field, 48 water samples were collected from stream, ditch and water pool habitats in four locations at two Aberystwyth University farms over a seven week period to investigate the applicability of the LAMP assay for use on eDNA samples, in comparison to conventional PCR. Results: The LAMP assay delivered detectable results in 30 min at 63 °C. The assay discriminated between G. truncatula DNA and non-target DNA, presenting a level of DNA detection comparable to conventional PCR. No significant difference was found between the ability of the LAMP and PCR assay to identify G. truncatula eDNA in water samples. Kappa coefficient analysis revealed a moderate level of agreement between LAMP and PCR assays. Conclusions: This study demonstrated that the LAMP assay can detect G. truncatula eDNA in a simple and rapid manner. The LAMP assay may become a valuable tool to determine optimum pasture management for trematode parasite control.

Galba truncatula is one of the most distributed intermediate hosts of Fasciola hepatica across Europe, North Africa and South America. Therefore, understanding the environmental preferences of this species is vital for developing control strategies for fascioliasis and other trematodes such as Calicophoron daubneyi. This systematic literature review evaluates the current understanding of the snail's environmental preferences to identify factors which might aid control and areas where further research is needed. Searches were conducted using Google Scholar and PubMed and included papers published up to August 2023. After filtration, 198 papers with data from 64 countries were evaluated, and data regarding habitat type and habitat pH were noted, along with any other information pertaining to the snail's environmental preferences. The results show that G. truncatula can survive in a diverse range of climates and habitats, generally favours shallow slow-moving water or moist bare mud surfaces, temperatures between 10 and 25°C and was found in habitats with a water pH ranging from 5.0 to 9.4. However, there is limited understanding of the impact of several factors, such as the true optimum pH and temperature preferences within the respective tolerance limits or the reason for the snail's apparent aversion to peatland. Further research is needed to clarify the impact of biotic and abiotic factors on the snail to create robust risk assessments of fluke infection and assess opportunities for environmental control strategies, and for predicting how the snail and fluke transmission may be impacted by climate change.