A new multiplex PCR assay to distinguish among three cryptic Galba species, intermediate hosts of Fasciola hepatica

Abstract

A molecular tool described here allows in one step for specific discrimination among three cryptic freshwater snail species (genus Galba) involved in fasciolosis transmission, a worldwide infectious disease of humans and livestock. The multiplex PCR approach taken targets for each species a distinctive, known microsatellite locus which is amplified using specific primers designed to generate an amplicon of a distinctive size that can be readily separated from the amplicons of the other two species on an agarose gel. In this way, the three Galba species (G. cubensis, G. schirazensis, and G. truncatula) can be differentiated from one another, including even if DNA from all three were present in the same reaction. The accuracy of this new molecular tool was tested and validated by comparing multiplex PCR results with species identification based on sequences at mitochondrial and nuclear markers. This new method is accurate, inexpensive, simple, rapid, and can be adapted to handle large sample sizes. It will be helpful for monitoring invasion of Galba species and for developing strategies to limit the snail species involved in the emergence or re-emergence of fasciolosis.

Reviewing Fasciola hepatica transmission in the West Indies and novel perceptions from experimental infections of sympatric vs. allopatric snail/fluke combinations

“…A highly significant hyper pathological/physiological reaction, the Americas has been largely overlooked and is only relatively recently acknowledged (Correa et al, 2010;Bargues et al, 2011). This species may co-occur with other Galba species (in the present study we found G. schirazensis cooccurring with G. cubensis in Auyamas, Dominican Republic) and since they cannot be accurately differentiated at the morphological level, assuming an intermediate host role by either of the cryptic Galba, and particularly by G. schirazensis could be difficult without molecular discrimination (Alda et al, 2018).…”

“…In order to identify these three Galba species, up to 30 individuals from each Galba field population were tested using a molecular tool designed to this end by Alda et al (2018). Previously obtained egg clutches of field-collected snails were used for founding colonies after confirmation of the species using multiplex PCR.…”

“…DNA was extracted using a Chelex protocol following Estoup and Martin (1996) adapted to 96-well plates. The multiplex PCR (Alda et al 2018) was based on species-specific primers amplifying microsatellite loci targeting cryptic species and producing band sizes that are specific to the three targeted species (179-200 pb in G. cubensis, 227-232 pb in G. schirazensis, and 111-129 pb in G. truncatula).…”

Dinámica de la infestación por Fasciola hepatica en el sur de la provincia de Entre Ríos

“…Dentro de esta familia se encuentran un gran número de especies de caracoles de agua dulce cuya sistemática, aun, continúa sujeta a controversias (Kleiman et al, 2004;Prepelitchi, 2009;Correa et al, 2010;Lounnas et al, 2016;Alda et al, 2018). Antes de la masificación de las técnicas de biología molecular, la identificación de las especies se basaba en la morfología de la conchilla y del aparato reproductor masculino (Pontier et al, 2008).…”

“…gracias a un mayor polimorfismo que los anteriores, una clara diferenciación en especies (Lounnas et al, 2016;Alda et al, 2018).…”