Development and validation of a mtDNA multiplex PCR for identification and discrimination of Calicophoron daubneyi and Fasciola hepatica in the Galba truncatula snail

Paramphistomosis and Fasciolosis caused by Calicophoron daubneyi and Fasciola hepatica, respectively, are frequent and important trematodoses in ruminant livestock worldwide. Both parasites use the same snail, Galba truncatula, as intermediate host. The aim of this study was to develop and validate an analytical method based on a mitochondrial DNA (mtDNA) multiplex PCR technique which would allow the early and specific identification, in one step, of C. daubneyi and F. hepatica infection in G. truncatula. First of all, a 1035bp fragment of mtDNA from adult C. daubneyi worms was obtained. Then two pairs of specific mtDNA primers, which amplified a DNA fragment of 885pb in the case of C. daubneyi, and of 425pb in that of F. hepatica, were designed. By means of the multiplex PCR technique developed, there was always a specific amplification in samples from adult F. hepatica and C. daubneyi, but not from Calicophoron calicophorum, Cotylophoron cotylophorum, Cotylophoron batycotyle or Dicrocoelium dendriticum. Likewise, specific amplifications of the expected DNA fragments happened in all samples from snails harbouring larval stages of C. daubneyi or F. hepatica, previously detected by microscopy. However, amplifications were not seen when DNA from snails harbouring other Digenea (Plagiorchiidae, Notocotylidae and furcocercous cercariae) was analysed. Moreover, DNA from G. truncatula molluscs free from infection was not amplified. The multiplex PCR assay permitted infection in the snails experimentally infected with 4 miracidia to be detected as early as day 1 p.i. in the case of F. hepatica and with only 2 miracidia from day 2 p.i. in both, C. daubneyi and F. hepatica. Nevertheless it was necessary to wait until days 29 and 33 p.i. to see C. daubneyi and F. hepatica immature redia, respectively, using microscope techniques. The detection limit of the PCR technique was very low: 0.1ng of DNA from C. daubneyi and 0.001ng of DNA from F. hepatica. This allowed infection by either F. hepatica or C. daubneyi to be detected even when pools made up with only 1μl (60ng of DNA) from infected snail plus 99μl from non-infected ones were analyzed. Moreover, simultaneous detection of both parasites was experimentally possible in pools made up with uninfected (98μl), C. daubneyi infected (1μl) and F. hepatica infected (1μl) snails. The most precise and early diagnosis of the infections using the multiplex PCR technique designed will allow more realistic epidemiological model