Amplification of ribosomal DNA of Anoplocephalidae: Anoplocephala perfoliata diagnosis by PCR as a possible alternative to coprological methods.

The diagnosis of tapeworm infections in horses relies on copro-diagnostic methods, which are time-consuming and of limited sensitivity for determination of the exact prevalence. The development of serological tests has slightly improved the detection of tapeworm infections, but more sensitive methods are still required. A polymerase chain reaction (PCR)-based approach may constitute a valuable tool to improve tapeworm diagnosis. Nuclear ribosomal DNA (rDNA) is a useful target for species and/or strain markers. Partial 18S, the internal transcribed spacer 1 (ITS-1), the 5.8S, the internal transcribed spacer 2 (ITS-2), and partial 28S rDNA of the equine tapeworms Anoplocephala perfoliata and Anoplocephaloides mamillana were amplified and sequenced. The lengths and GC contents of the regions sequenced were 2087-2091bp and 49.35-49.69% for A. perfoliata, and 2110-2119bp and 49.15-49.32% for A. mamillana, respectively. Sequence alignment and comparison of both taxa showed 79.3-80.2% identity. The lowest identities were found in the ITS regions with 39.9-43.5% for the ITS-1 and 59.5-61.2% for the ITS-2. No matches of the ITS-2 of A. perfoliata and A. mamillana were found with other species by BLAST search. For this reason, ITS-2 sequences seemed appropriate as accurate species markers and A. perfoliata ITS-2 primers were developed. The ITS-2 PCR enabled the detection of genomic DNA as low as 0.5 pgs. First efforts on the practical application of the PCR-based approach were made. A 6-mg fragment of a tapeworm proglottid was detected in 0.5 and 1g of faeces.