title: Non-invasive detection and quantification of the parasitic ciliate Ichthyophthirius multifiliis by real-time PCR.
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.
authors.affiliation: Dipartimento di Patologia Animale, Profilassi e Igiene degli Alimenti, Università di Pisa, Via delle Piagge 2, 56124 Pisa, Italy. olivier.jousson@vet.unipi.it
authors.forename: Olivier
authors.initials: O
authors.lastname: Jousson
authors.forename: Carlo
authors.initials: C
authors.lastname: Pretti
authors.forename: Domenica
authors.initials: D
authors.lastname: Di Bello
authors.forename: Anna M
authors.initials: AM
authors.lastname: Cognetti-Varriale
mesh_terms: D000818:Animals; D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D005456:Fluorescent Dyes; D005618:Fresh Water / Q000469:parasitology*; D016803:Hymenostomatida / Q000235:genetics*; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D013045:Species Specificity
chemical_list: D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D012337:RNA, Ribosomal, 18S; C098022:SYBR Green I