Development and application of a duplex QPCR for river water samples to monitor the myxozoan parasite Parvicapsula minibicornis

Abstract

A duplex quantitative polymerase chain reaction (QPCR) assay was developed to simultaneously quantify the myxozoan parasite Parvicapsula minibicornis in river water samples and detect inhibition, which may compromise recognition of the target organism. The assay combines a TaqMan MGB probe specific to the nuclear small subunit ribosomal RNA gene of P. minibicornis and a commercial TaqMan Exogenous Internal Positive Control. P. minibicornis is endemic to freshwaters of the Pacific Northwest of North America and contributes to reduced fish health in Klamath River (Oregon/California) salmonids. The prevalence of P. minibicornis in these fish can reach 100%, and infection can result in glomerulonephritis and impaired kidney function. To better understand the temporal and spatial occurrence of this parasite in the Klamath River basin, water samples were taken from 7 mainstem sites and 5 tributaries along the 400 km river from March through September 2006. The samples were filtered, and the captured DNA was extracted and tested for the presence of P. minibicornis with the duplex QPCR assay. The parasite was present throughout the river over the entire sampling period, but its distribution and abundance varied spatially and temporally by over 2 orders of magnitude. Spore densities were lowest in March (spring) and peaked in June/July (summer) when site variance was also greatest. Inhibition levels also varied. The assay is able to detect 1 actinospore (the life cycle stage infective to fish) in 1 l of water and offers an alternative to sampling fish to monitor this pathogen and develop management options.