qPCR-based environmental monitoring of the whirling disease-causing parasite Myxobolus cerebralis and phylogenetic analysis of the tubificid hosts in Alberta, Canada

Myxobolus cerebralis is the causative agent of whirling disease in salmonid fishes. In 2016, this invasive parasite was detected in Alberta, Canada, for the first time, initiating a comprehensive 3 yr monitoring program to assess where the parasite had spread within the province. As part of this program, a qPCR-based test was developed to facilitate detection of the environmental stages of M. cerebralis and from the oligochaete host, Tubifex tubifex. During this program, ~1500 environmental samples were collected and tested over 3 yr. Fish were collected from the same watersheds over 2 yr and tested as part of the official provincial monitoring effort. Substrate testing identified sites positive for M. cerebralis in 3 of 6 watersheds that had been confirmed positive by fish-based testing and 3 novel detections where the parasite had not been detected previously. Testing of individually isolated Tubifex from each sample site was used to further confirm the presence of M. cerebralis. DNA barcoding of the cytochrome oxidase I (cox1) gene of 567 oligochaete specimens collected from 6 different watersheds yielded 158 unique sequences belonging to 21 genera and 37 putative species. Phylogenetic analyses of sequences assigned to the genus Tubifex predicted 5 species of Tubifex arising from this assessment. Based on our results, we propose that environmental and worm samples can be a valuable complement to the gold-standard fish testing and will be especially useful for monitoring in areas where fish collection is challenging or prohibitive because of site accessibility or vulnerability of the fish populations.