Development of a qPCR Duplex Assay for simultaneous detection of Fascioloides magna and Galba truncatula in eDNA samples: Monitoring beyond boundaries.

Parasites constitute a significant economic burden and highly impact environmental, public, and animal health. The emergence of many parasitic diseases is environmentally mediated and they share the same biogeography with humans and both domestic and wild animals. American liver fluke, Fascioloides magna - a trematode parasite of domestic and wild ungulates - is an example of the anthropogenic introduction of an "invasive alien species" in Italy and Europe. Multiple introductions to Europe have led to the biogeographical expansion of the parasite across the Danube region mainly provided by the presence of suitable habitats for all hosts involved in the parasite's life cycle, human-assisted transport, and drastic environmental events such as flooding. In Italy, it was introduced and established in La Mandria Regional Park (LMRP) near Turin in 1865 along with imported wapitis (Cervus elaphus canadensis) from North America (Bassi, 1875), but with no reported expansion to the surrounding areas. LMRP isolated F. magna focus, poses an important threat of possible expansion since the enclosed area is vulnerable to occasional bidirectional passage of roe deer. Additionally, tributary rivers to the Po river system, traversing the enclosed area, could further bolster the possibility of such spread. In this study, we developed a duplex qPCR assay for F. magna and its principal intermediate host Galba truncatula optimized for testing eDNA samples to meet the needs for surveillance of the parasite. Moreover, we validated the developed assay in natura by testing samples derived from filtered water and sediments collected inside and outside LMRP's fenced-off area. Our findings for the first time demonstrate the presence of F. magna's eDNA outside the park's internal fenced-off area.