Comparative Evaluation of Molecular Diagnostic Tests for Nucleospora salmonis and Prevalence in Migrating Juvenile Salmonids from the Snake River, USA

Abstract

Nucleospora salmonis is an intranuclear microsporidian that primarily infects lymphoblast cells and contributes to chronic lymphoblastosis and a leukemia-like condition in a range of salmonid species. The primary goal of this study was to evaluate the prevalence of N. salmonis in out-migrating juvenile hatchery and wild Chinook salmon Oncorhynchus tshawytscha and steelhead O. mykiss from the Snake River in the U.S. Pacific Northwest. To achieve this goal, we first addressed the following concerns about current molecular diagnostic tests for N. salmonis: (1) nonspecific amplification patterns by the published nested polymerase chain reaction (nPCR) test, (2) incomplete validation of the published quantitative PCR (qPCR) test, and (3) whether N. salmonis can be detected reliably from nonlethal samples. Here, we present an optimized nPCR protocol that eliminates nonspecific amplification. During validation of the published qPCR test, our laboratory developed a second qPCR test that targeted a different gene sequence and used different probe chemistry for comparison purposes. We simultaneously evaluated the two different qPCR tests for N. salmonis and found that both assays were highly specific, sensitive, and repeatable. The nPCR and qPCR tests had good overall concordance when DNA samples derived from both apparently healthy and clinically diseased hatchery rainbow trout were tested. Finally, we demonstrated that gill snips were a suitable tissue for nonlethal detection of N. salmonis DNA in juvenile salmonids. Monitoring of juvenile salmonid fish in the Snake River over a 3-year period revealed low prevalence of N. salmonis in hatchery and wild Chinook salmon and wild steelhead but significantly higher prevalence in hatchery-derived steelhead. Routine monitoring of N. salmonis is not performed for all hatchery steelhead populations. At present, the possible contribution of this pathogen to delayed mortality of steelhead has not been determined.

Received September 14, 2010; accepted November 14, 2010

ACKNOWLEDGMENTS

This research was supported by the U.S. Army Corps of Engineers (contracts DACW57–00-0009, number 11, W68SBV60307671, and W68SBV6041861), the U.S. Geological Survey, the U.S. Fish and Wildlife Service, and the California Department of Fish and Game. The authors acknowledge the technical assistance of Constance McKibben, LynnMarie Applegate, Anthony Murray, Carla Conway, Lisa Wetzel, and Sacha Mosterd (Western Fisheries Research Center). Representative DNA from Ovipleistophora ovariae, Loma salmonae, and Ceratomyxa shasta were obtained from Andy Goodwin (University of Arkansas), Mike Kent (Oregon State University), and Sascha Hallet (Oregon State University), respectively. Positive N. salmonis material was obtained from Carla Hogge (Idaho Fish and Game). The mention of trade, firm, or corporation names in this publication is for the information and convenience of the reader and does not constitute an official endorsement or approval by the U.S. Fish and Wildlife Service, the U.S. Geological Survey, or the U.S. Department of Interior of any product or service to the exclusion of others that may be suitable.