Rapid detection of Galba truncatula in water sources on pasture-land using loop-mediated isothermal amplification for control of trematode infections

Abstract

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.

eDNA-based detection of the invasive crayfish Pacifastacus leniusculus in streams with a LAMP assay using dependent replicates to gain higher sensitivity

“…Advances have been made in using LAMP assays to process environmental samples such as hive debris 24 and water samples 23 , 25 , 26 . Three of the LAMP studies based on eDNA detection in water samples were targeted at mussel species from the Dreissena genus 23 , 26 , and one was developed to detect the presence of a parasite’s intermediate host (a snail species) in drinking spots for livestock 25 . The coupling of eDNA sampling with a LAMP detection approach was thus proven operational.…”

Temporal dynamics of trematode intermediate snail host environmental DNA in small water body habitats

“…All filters, including blank controls, were homogenized using a sterile pipette tip with each whole filter subjected to DNA extraction via the PowerSoil® kit protocol. PCR targeting a 288 bp strand of the G. truncatula ITS2 gene and gel electrophoresis were used to identify G. truncatula eDNA in extracts as previously described by Jones et al (2018) with the modifications stated by Davis et al (2020). The limit of detection of this PCR assay is 0.5 pg μL −1 (Davis et al, 2020) and the assay has been shown to be capable of detecting G. truncatula eDNA in 100% of habitats where the snail was surveyed (Jones et al, 2018).…”

“…In recent years, eDNA capture and detection protocols have been developed to detect intermediate snail host eDNA, including that of Galba truncatula (Jones et al, 2018;Davis et al, 2020), Austropeplea tomentosa (Rathinasamy et al, 2018;Rathinasamy et al, 2021), Oncomelania hupensis (Fornillos et al, 2019) and Bulinus truncatus (Mulero et al, 2020). As these snails are often present at low densities and are difficult to find and differentiate from non-intermediate host snails, eDNA surveys can outperform traditional physical detection methods (Jones et al, 2018) as has been shown for other organisms (Wilcox et al, 2016).…”

“…Steps may be taken to limit DNA contamination (Goldberg et al, 2016) and to ensure assay specificity (Davis et al, 2020); however, the potential for eDNA to be transported to the sampling site or its persistence following previous inhabitation cannot be controlled. The calculation and incorporation of false-positive probability rates into site occupancy models has been suggested as a method for improving eDNA survey results (Ficetola et al, 2016) and may be vital when surveying across a wide range of habitat types that certain intermediate snail hosts can inhabit.…”

Towards the comprehension of fasciolosis (re-)emergence: an integrative overview

“…Zumaquero-Ríos et al ., 2013; Alba et al ., 2016). Furthermore, other types of samples beyond blood, stool (definitive host) and snails (intermediate host) have been added to the panel of possibilities for active screening; from meat juice and bulk milk for serological assessment in livestock (Charlier et al ., 2009; Villa-Mancera and Reynoso-Palomar, 2019) to water for environmental DNA detection of the parasite and its snail hosts (Jones et al ., 2018; Davis et al ., 2020).…”