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

Abstract

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 1035 bp fragment of mtDNA from adult C. daubneyi worms was obtained. Then two pairs of specific mtDNA primers, which amplified a DNA fragment of 885 pb in the case of C. daubneyi, and of 425 pb 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: 0.1 ng of DNA from C. daubneyi and 0.001 ng 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 1 μl (60 ng of DNA) from infected snail plus 99 μl from non-infected ones were analyzed. Moreover, simultaneous detection of both parasites was experimentally possible in pools made up with uninfected (98 μl), C. daubneyi infected (1 μl) and F. hepatica infected (1 μl) snails. The most precise and early diagnosis of the infections using the multiplex PCR technique designed will allow more realistic epidemiological models of both infections to be established and consequently a better strategic control.

Transmission of Calicophoron daubneyi and Fasciola hepatica in Galicia (Spain): Temporal follow-up in the intermediate and definitive hosts

“…The susceptibility has been shown to be associated with expression of certain stress genes, in the case of the snail Biomphalaria glabrata and the parasite trematode Schistosoma mansoni [34, 35]. Finally, the findings of the present study confirm the rarity of natural co-infections with different digenean species [20, 21, 36], probably as a result of competition, which would play an important role in the configuration of trematode infection status of snail populations in each habitat. The mechanisms underlying competitive processes remain largely unknown; however, such mechanisms may constitute a basis for developing strategies to interrupt the life-cycle of trematodes.…”

“…h Metacercaria of F. hepatica . Larval stages in a and b were confirmed by molecular techniques according to Martínez-Ibeas et al [20]. Larval stages in c - h were classified morphologically by microscopic techniques.…”

“…When sporocyst-like larval stages were observed by microscopy, samples were conserved in liquid nitrogen for subsequent molecular determination of the species using the technique developed by Martínez-Ibeas et al [20]. …”

“…Although the snail G. truncatula can act as an IH for C. daubneyi [18–20], the involvement of other gastropod species in the life-cycle of this parasite has scarcely been studied [21]. In view of the key role of lymnaeid snails in the life-cycle of trematodes of medical and veterinary importance, present knowledge of the dynamics of snail populations and parasite-host interactions is insufficient.…”

Estimating trematode prevalence in snail hosts using a single-step duplex PCR: how badly does cercarial shedding underestimate infection rates?

“…Martínez-Ibeas et al [18] used specific primers for the detection of Dicrocoelium dendriticum single infections in snail tissues, and documented a higher accuracy of PCR over cercarial release. Later, Martínez-Ibeas et al [54] designed a mtDNA multiplex PCR for identification and discrimination of Caliophoron daubneyi and Fasciola hepatica in the snail Galba truncatula , but they did not find natural double infections, neither by microscopy nor by PCR. As far as we know, our is the first study where species-specific primers have been designed in a duplex PCR for the accurate assessment of single and double infections with a blind sample of snail tissue.…”