A repetitive DNA probe for the sensitive detection of Fasciola hepatica infected snails

Epizootiologic studies on F. hepatica frequently use microscopic techniques for the detection of infected snails, however, the poor efficiency, sensitivity, and specificity associated with these techniques limit their usefulness. A DNA-based test for the identification of snails infected with larval stages of F. hepatica would solve these problems and enable a level of detection accuracy previously unavailable. We have cloned and sequenced a 124 bp fragment of repetitive DNA from F. hepatica which hybridizes specifically with DNA of F. hepatica but not with DNA of its snail intermediate hosts Fossaria cubensis and Pseudosuccinea columella, or with DNA of Fascioloides magna and Paramphistomum liorchis, ruminant trematodes which share the same intermediate host and same enzootic range as F. hepatica. Using this 124 bp fragment as a probe, infection in snails was detected immediately following miracidial penetration, thus a sensitivity equivalent to the minimum biologic unit of the parasite was achieved. This 124 bp repeated sequence belongs to a large family of 124 bp repeats that share a high level of sequence identity and constitute approximately 15% of the F. hepatica genome. We also report here the development of a quick and inexpensive DNA extraction protocol for use in field-collected snails. Thus, we have developed both a highly sensitive and specific DNA probe and a means to use the probe in a large epizootiologic study of F. hepatica where thousands of field-collected snails need to be assayed for infection.