Non-invasive detection and quantification of the parasitic ciliate Ichthyophthirius multifiliis by real-time PCR

DAO Diseases of Aquatic Organisms Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials DAO 65:251-255 (2005) - doi:10.3354/dao065251 Non-invasive detection and quantification of the parasitic ciliate Ichthyophthirius multifiliis by real-time PCR Olivier Jousson*, Carlo Pretti, Domenica Di Bello, Anna M. Cognetti-Varriale Dipartimento di Patologia Animale, Profilassi e Igiene degli Alimenti, Università di Pisa, Via delle Piagge 2, 56124 Pisa, Italy *Email: olivier.jousson@vet.unipi.it ABSTRACT: The main parasitic threat to freshwater fish is the ciliate Ichthyophthirius multifiliis. We developed a real-time PCR assay using SYBR Green™ intercalating fluorescent dye for rapid detection and quantification of I. multifiliis. This non-invasive assay was based on the quantification of I. multifiliis free-swimming stages from filtered water samples, and thus made it possible to preserve host individuals. An alignment of 18S rDNA sequences of I. multifiliis and related species of the ciliate order Hymenostomatida was used to design amplification primers specifically targeting the I. multifiliis 18S rDNA gene. Different standard curves consisting of 2-fold serial dilutions of DNA extracted from 20, 60, 100 and 1000 I. multifiliis cells were constructed. The assay was able to detect less than 0.5 cell equivalent and showed a strong linearity (R2 = 0.984). Water samples were collected from 2 tanks containing heavily infected and apparently uninfected Carassius auratus specimens and were used to test this technique. Positive signals were obtained from water samples collected from both tanks, with a deduced concentration ranging from 3 to 58 I. multifiliis cells l–1. The assay can detect low concentrations of the parasite in water, presumably corresponding to an early phase of the disease. It may, thus, be a valuable tool in assisting in the monitoring and control of ichthyophthiriasis in aquaculture. KEY WORDS: Ichthyophthirius multifiliis · Real-time PCR · Non-invasive assay · Quantification Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in DAO Vol. 65, No. 3. Online publication date: July 18, 2005 Print ISSN: 0177-5103; Online ISSN: 1616-1580 Copyright © 2005 Inter-Research.