Isolation and characterization of microsatellite markers in the liver fluke (Fasciola hepatica)

The liver fluke (Fasciola hepatica) is a re-emerging food-borne Trematode that causes important veterinary and public health problems worldwide (see Hurtrez-Boussès et al. 2001). The highest human infection rates have been recorded in the Bolivian Altiplano (Esteban et al. 1999). In this area of hyperendemicity, the liver fluke has been recently introduced from European strains (see Mas-Coma et al. 2001), conjointly with its intermediate host, the freshwater snail Lymnaea truncatula (see Meunier et al. 2001). Here, we report on the first isolation of microsatellite markers in F. hepatica.

Identification and characterization of microsatellite markers were made according to Estoup & Martin (1996). A complete digestion of F. hepatica DNA was made with Sau3A enzyme. Fragments between 300 and 900 bp served to construct a genomic library (3185 clones). The clones were screened for (CA)₁₀ and (GA)₁₀ probes, marked with Digoxygenin. Positive clones were detected using an anti-Dig labelling kit (Roche Diagnostics). In total, 39 positive clones were sequenced in 4% polyacrylamide gels (8 m urea), using (α-³³P)-ATP (Amersham) with T7 sequencing kit (Pharmacia). We retained five of these sequences that presented microsatellite motifs and suitable flanking regions (see Table 1). Moreover, we screened 122 sequences already published in EMBL or GenBank and retained one of them (EMBL Accession no.: FH222CBP), which presented a suitable microsatellite. Therefore, we used a total of six sequences, for which we defined primers (see Table 1) using primer 3 software (Rozen & Skaletsky 2000; http://biotools.umassmed.edu/bioapps/primer3_www.cgi).

Adults of F. hepatica (N = 52) were collected in February 1996, in slaughterhouses of the Bolivian Altiplano. Liver flukes were sampled by dissecting the livers of definitive hosts (sheep, pig and cow) and were immediately stored in ethanol 80%.

DNA extraction was performed using 500 µL Chelex 10% (Estoup & Martin 1996). DNA extracts were diluted 1/10 and used as the template for microsatellite amplifications. Microsatellite loci were amplified from 6 µL of diluted DNA for the locus FH 26/1 and 4 µL DNA for all other loci, in a 20 µL reaction volume containing 2 µL buffer 10× (Promega), 1.5 mm Mg²⁺ (Promega), 2 mm dNTPs (Invitrogen/Life Technologies), 5–10 pmol of each primer (see Table 1 for exact amount) and 1.3 U of Taq DNA polymerase (Promega). Thermocycling, performed using a MJ-Research PTC 100 96-well, consisted of an initial denaturation at 94 °C for 4 min, 30 cycles at 94 °C for 30 s, annealing temperature (see Table 1 for specific annealing temperatures) for 30 s, 72 °C for 30 s and a final elongation step at 72 °C for 10 min. Fluorescently labelled primers for use on an ABI automated sequencer were purchased (Applera; Table 1). One microlitre of polymerase chain reaction (PCR) products amplified from each DNA sample was pooled in two groups of loci (FH1, FH25, FH26/1 and FH 222CBP group and FH15–FH23 group), together with 0.5 µL of internal size standards (GENESCAN 500 LIZ, Applera), and diluted in Hi-Di Formamide (20 µL QSP) for automatic electrophoresis. Measurements of allele length were automated with genescan software (Applied Biosystems). Each liver fluke was genotyped at the six studied loci.

Five of the six studied loci are polymorphic, with on average (± SD) 5.6 (± 3.8) different alleles (Table 1). There is no significant linkage disequilibrium between any pair of loci. Observed and expected heterozygosities are high (mean H_O = 0.374 ± 0.291; mean H_E = 0.524 ± 0.212). Deviations from Hardy–Weinberg equilibrium are nonsignificant, except at locus FH23 where there is a significant deficit in heterozygotes (Table 1). Such a divergence at one locus may be due to the presence of null alleles at this locus. Assuming the existence of a null allele at locus FH23, our results support a panmictic reproduction mode in liver fluke.

The demonstrated genetic variability of the liver fluke should be taken into account in future prospects of treatments and vaccines, because some variants may be able to escape the host immune system and/or treatments.