Abstract
BackgroundFascioliasis 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.MethodsA 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.ResultsThe 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.ConclusionsThis 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.
Highlights
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
Comparison of loop-mediated isothermal amplification (LAMP) and conventional PCR limit of detection To determine the limit of detection of the LAMP assay, 10-fold dilutions (101–109) of a known concentration
Similar 10-fold dilutions were used to compare the limit of detection of the LAMP assay with conventional PCR
Summary
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. 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 report a loop-mediated isothermal amplification (LAMP) assay to identify G. truncatula eDNA. Fasciola hepatica is a trematode of worldwide economic importance [4] as the disease it causes is estimated to cost the global livestock production industry €2.5 billion annually [5]. In the absence of vaccines, trematode control in the definitive host is primarily via anthelmintic administration; anthelmintic resistance and the limited availability of novel anthelmintic compounds threatens sustainable control [9]
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