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Diseases of Aquatic Organisms

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DAO 110:165-172 (2014)  -  DOI:

Development of a real-time PCR for the detection of pathogenic Leptospira spp. in California sea lions

Qingzhong Wu1,*, Katherine C. Prager2,3,4, Tracey Goldstein5, David P. Alt6, Renee L. Galloway7, Richard L. Zuerner6,**, James O. Lloyd-Smith2,3, Lori Schwacke1

1Hollings Marine Laboratory, National Centers for Coastal Ocean Science, National Ocean Service, National Oceanic Atmospheric Administration, Charleston, South Carolina 29412, USA
2Department of Ecology and Evolutionary Biology, University of California, Los Angeles, California 90095, USA
3Fogarty International Center, National Institutes of Health, Bethesda, Maryland 20892, USA
4The Marine Mammal Center, Sausalito, California 94965, USA
5One Health Institute, School of Veterinary Medicine, University of California, Davis, California 95616, USA
6Infectious Bacterial Diseases Research Unit, National Animal Disease Center, Ames, Iowa 50010, USA
7Centers for Disease Control and Prevention, Atlanta, Georgia 30333, USA
*Corresponding author:
**Retired from the US Department of Agriculture Agricultural Research Service (ARS)

ABSTRACT: Several real-time PCR assays are currently used for detection of pathogenic Leptospira spp.; however, few methods have been described for the successful evaluation of clinical urine samples. This study reports a rapid assay for the detection of pathogenic Leptospira spp. in California sea lions Zalophus californianus using real-time PCR with primers and a probe targeting the lipL32 gene. The PCR assay had high analytic sensitivity—the limit of detection was 3 genome copies per PCR volume using L. interrogans serovar Pomona DNA and 100% analytic specificity; it detected all pathogenic leptospiral serovars tested and none of the non-pathogenic Leptospira species (L. biflexa and L. meyeri serovar Semaranga), the intermediate species L. inadai, or the non-Leptospira pathogens tested. Our assay had an amplification efficiency of 1.00. Comparisons between the real-time PCR assay and culture isolation for detection of pathogenic Leptospira spp. in urine and kidney tissue samples from California sea lions showed that samples were more often positive by real-time PCR than by culture methods. Inclusion of an internal amplification control in the real-time PCR assay showed no inhibitory effects in PCR negative samples. These studies indicated that our real-time PCR assay has high analytic sensitivity and specificity for the rapid detection of pathogenic Leptospira species in urine and kidney tissue samples.

KEY WORDS: Sea lions · Pathogenic Leptospira spp. · lipL32 gene · Real-time PCR · Urine · Kidney

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Cite this article as: Wu Q, Prager KC, Goldstein T, Alt DP and others (2014) Development of a real-time PCR for the detection of pathogenic Leptospira spp. in California sea lions. Dis Aquat Org 110:165-172.

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