In the present study, 25 samples representing Fasciola (Platyhelminthes: Trematoda: Digenea) from nine host species and 19 geographical locations in Spain were characterized genetically by sequences of the first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA). The ITS rDNA was amplified from individual liver flukes by polymerase chain reaction (PCR), and the amplicons were sequenced directly. The lengths of the ITS-1 and ITS-2 sequences were 422 and 362 bp, respectively, for all Spanish liver fluke samples sequenced. Comparison of the ITS sequences of the Spanish Fasciola samples examined in the present study with that of Fasciola hepatica, Fasciola gigantica and the "intermediate Fasciola" revealed that all Spanish Fasciola samples examined represent the single species of F. hepatica, with only slight sequence variation in the ITS-2 (1/362, 0.3%) among the sequenced samples, but the sequence variation was not related to particular host species and/or geographical origins of the samples. The Spanish F. hepatica examined differed from Fasciola from elsewhere by two nucleotides in the ITS-2, which provided genetic marker for the differentiation of Spanish F. hepatica from Fasciola from other geographical localities. These results have implications for studying the population genetic structure of the Spanish F. hepatica and for the diagnosis and control of the disease it causes.

A collection of483 samples representing Fasciola from six naturally infected host species and 16 localities in Spain, previously identified morphologically and genetically as Fasciola hepatica, was characterized by a novel genetic marker, namely sequence-related amplified polymorphism (SRAP), aiming to reveal genetic variability within F. hepatica in Spain. Visualization of amplification fragments was carried out on 6% denaturing polyacrylamide gels, followed by staining with 0.1% AgNO3 solution. Ten SRAP primer combinations were tested--six of them turned out to be polymorphic. Thirty-four representative F. hepatica samples from six host species and 16 geographical localities showed polymorphic banding patterns using SRAP primer combinations and were grouped into four major clusters using the unweighted pair-group method with arithmetic averages, indicating the existence of genetic variability within the examined F. hepatica samples. These four clusters were not related to particular host species and/or geographical origins of the samples. The results of the present study revealed that SRAP markers were useful in revealing sufficient polymorphism in F. hepatica samples from Spain and had implications for studying the population genetic structure of the Spanish F. hepatica. To our knowledge, this is the first application of SRAP marker to study genetic variation in parasites of human and animal health significance.