Abstract - Hallett & Bartholomew 2006

Hallett, S. L. and Bartholomew, J. L. (2006)
Application of a real-time PCR assay to detect and quantify the myxozoan parasite Ceratomyxa shasta in water samples.
Diseases of Aquatic Organisms.71:109-118
Ceratomyxa shasta is a virulent pathogen of salmonid fish endemic in the Pacific Northwest of North America. Current detection methods involve sentinel fish exposures that are laborious and time-consuming. As a substitute, a filtering protocol and a quantitative real-time TaqMan polymerase chain reaction (QPCR) assay were developed to both detect and enumerate parasite spores in river water. Fluorescence was detected from both the myxospore and actinospore stages of the parasite but from neither the fish or polychaete hosts nor from 9 other myxozoans tested. Less than 1/1000th of a spore was detected, indicating each had greater than 1000 copies of the target 18S rDNA gene. The assay detected 1 spore in 1 L river water. Inhibition of the assay by some river samples was overcome by reducing the template volume and including bovine serum albumin in the reaction; occasionally a second purification step was required. The QPCR methodology was utilised to investigate the temporal and spatial distribution of C. shasta in the Klamath River, Oregon/California. The parasite was detected throughout the river and 2 of 5 tributaries tested contributed parasites to the mainstem. Correlation of QPCR Ct values with a standard curve for known starting numbers of whole spores revealed several sites where parasite abundance was in excess of 20 spores l-1. While QPCR data corroborated results of sentinel fish exposures, spore numbers did not correlate consistently with mortality in the exposure groups. The water sampling and filtering protocol combined with the QPCR assay was a simple and relatively rapid method for detection and quantification of parasite levels in environmental water samples.
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