title: Using real-time PCR and Bayesian analysis to distinguish susceptible tubificid taxa important in the transmission of Myxobolus cerebralis, the cause of salmonid whirling disease.
abstract: Aquatic oligochaetes have long been appreciated for their value in assessing habitat quality because they are ubiquitous sediment-dwelling filter feeders. Many oligochaete taxa are also important in the transmission of fish diseases. Distinguishing resistant and susceptible taxa is important for managing fish disease, yet challenging in practice. Tubifex tubifex (Oligochaeta: Tubificidae) is the definitive host for the complex life-cycle parasite, Myxobolus cerebralis, the causative agent of salmonid whirling disease. We developed two hydrolysis probe-based qualitative real-time PCR (qPCR) multiplex assays that distinguish among tubificid taxa collected from the Madison River, Montana, USA. The first assay distinguishes T. tubifex from Rhyacodrilus spp.; while the second classifies T. tubifex identified by the first assay into two genetic lineages (I and III). Specificity and sensitivity were optimized for each assay; the two assays showed specificity of 94.3% and 98.6% for the target oligochaetes, respectively. DNA sequencing verified the results. The development of these assays allowed us to more fully describe tubificid community composition (the taxa and their abundance at a site) and estimate the relative abundances of host taxa. To relate tubificid relative abundance to fish disease risk, we determined M. cerebralis infection prevalence in samples identified as T. tubifex using similar molecular techniques. Given prior information (i.e., morphological identification of sexually mature worms), Bayesian analysis inferred that the first qPCR assay improved taxonomic identification. Bayesian inference of the relative abundance of T. tubifex, combined with infection assay results, identified sites with a high prevalence of infected T. tubifex. To our knowledge, this study represents both the first assessment of oligochaete community composition using a qPCR assay based on fluorescent probes and the first use of Bayesian analysis to fully characterize the dominant infected taxa in streams where whirling disease is observed.
authors.affiliation: Civil and Environmental Engineering, University of Vermont, Burlington, VT, USA. Nikolaos.Fitilis@uvm.edu
authors.forename: Nikolaos
authors.initials: N
authors.lastname: Fytilis
authors.forename: Donna M
authors.initials: DM
authors.lastname: Rizzo
authors.forename: Ryan D
authors.initials: RD
authors.lastname: Lamb
authors.forename: Billie L
authors.initials: BL
authors.lastname: Kerans
authors.forename: Lori
authors.initials: L
authors.lastname: Stevens
mesh_terms: D000818:Animals; D001499:Bayes Theorem; D004199:Disease Vectors; D005618:Fresh Water / Q000469:parasitology; D009009:Montana; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D055766:Myxobolus / Q000302:isolation & purification*; D009835:Oligochaeta / Q000145:classification* / Q000469:parasitology*; D010274:Parasitology / Q000379:methods; D015995:Prevalence; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D045483:Rivers; D012680:Sensitivity and Specificity