title: New multiplex PCR method for the simultaneous diagnosis of the three known species of equine tapeworm.
abstract: Although several techniques exist for the detection of equine tapeworms in serum and feces, the differential diagnosis of tapeworm infection is usually based on postmortem findings and the morphological identification of eggs in feces. In this study, a multiplex polymerase chain reaction (PCR)-based method for the simultaneuos detection of Anoplocephala magna, Anoplocephala perfoliata and Anoplocephaloides mamillana has been developed and validated. The method simultaneously amplifies hypervariable SSUrRNA gene regions in the three tapeworm species in a single reaction using three pairs of primers, which exclusively amplify the internal transcribed spacer 2 (ITS-2) in each target gene. The method was tested on three types of sample: (a) 1/10, 1/100, 1/500, 1/1000, 1/2000 and 1/5000 dilutions of 70 ng of genomic DNA of the three tapeworm species, (b) DNA extracted from negative aliquots of sediments of negative control fecal samples spiked with 500, 200, 100, 50 and 10 eggs (only for A. magna and A. perfoliata; no A. mamillana eggs available) and (c) DNA extracted from 80, 50, 40, 30, 10 and 1 egg per 2 μl of PCR reaction mix (only for A. magna and A. perfoliata; no A. mamillana eggs available). No amplification was observed against the DNA of Gasterophilus intestinalis, Parascaris equorum and Strongylus vulgaris. The multiplex PCR method emerged as specific for the three tapeworms and was able to identify as few as 50 eggs per fecal sample and as little as 0.7 ng of control genomic DNA obtained from the three species. The method proposed is able to differentiate infections caused by the two most frequent species A. magna or A. perfoliata when the eggs are present in feces and is also able to detect mixed infections by the three cestode species.
authors.affiliation: Department of Animal Health, Faculty of Veterinary Medicine, Universidad Complutense de Madrid, 28040 Madrid, Spain.
authors.forename: G Alejandro
authors.initials: GA
authors.lastname: Bohórquez
authors.affiliation: Department of Animal Health, Faculty of Veterinary Medicine, Universidad Complutense de Madrid, 28040 Madrid, Spain.
authors.forename: Mónica
authors.initials: M
authors.lastname: Luzón
authors.affiliation: Regional Agricultural Center & Parque Científico de Albacete (INCRECYT, FEDER) Junta de Comunidades de Castilla La Mancha, 19180 Marchamalo, Spain.
authors.forename: Raquel
authors.initials: R
authors.lastname: Martín-Hernández
authors.affiliation: Department of Animal Health, Faculty of Veterinary Medicine, Universidad Complutense de Madrid, 28040 Madrid, Spain. Electronic address: ameana@ucm.es.
authors.forename: Aránzazu
authors.initials: A
authors.lastname: Meana
mesh_terms: D000818:Animals; D002589:Cestoda / Q000235:genetics / Q000302:isolation & purification*; D002590:Cestode Infections / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary*; D017931:DNA Primers / Q000235:genetics; D018509:DNA, Helminth / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D003937:Diagnosis, Differential; D005243:Feces / Q000469:parasitology; D006734:Horse Diseases / Q000175:diagnosis* / Q000469:parasitology; D006736:Horses; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D018509:DNA, Helminth; D021903:DNA, Ribosomal Spacer
keywords: Anoplocephala magna; Anoplocephala perfoliata; Anoplocephaloides mamillana; Diagnostic; Multiplex-PCR