Location: Infectious Bacterial Diseases ResearchTitle: Development of a real-time PCR for the detection of pathogenic Leptospira spp. in California sea lions) Author
Submitted to: Veterinary Microbiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 4/11/2014
Publication Date: 8/11/2014
Publication URL: http://handle.nal.usda.gov/10113/59506
Citation: Wu, Q., Prager, K.C., Goldstein, T., Alt, D.P., Galloway, R.L., Zuerner, R.L., Lloyd-Smith, J.O., Schwacke, L. 2014. Development of a real-time PCR for the detection of pathogenic Leptospira spp. in California sea lions. Diseases of Aquatic Organisms. 110(3):165-172. Interpretive Summary: Leptospira spp are pathogens that can cause clinical illness in livestock, humans and wildlife. A disease syndrome in sea lions has been linked to infection with Leptospira. Current methods for diagnosis of this infection suffer from several drawbacks. Sampling of tissues is too invasive, culture suffers from low sensitivity and is time consuming, and testing of serum cannot differentiate prior exposure from current infection. In this study, we report development of a rapid and specific real-time PCR assay for the detection of pathogenic Leptospira spp. in California sea lions (Zalophus californianus). Comparisons between the assay and culture for detection of pathogenic Leptospira spp. in urine and kidney tissue from California sea lions showed that the assay was more sensitive than culture. These studies indicated that real-time PCR is a sensitive and specific assay for the rapid detection of pathogenic Leptospira spp. in marine mammals. This work will be of value to veterinarians and marine biologists in guiding diagnosis and treatment options for sea lions which are suspected of being infected with Leptospira. Because Leptospira can also spread to livestock and humans, this work may also have veterinary and public health implications in that infection in sea lions may indicate a potential risk for both livestock and clinical disease in humans.
Technical Abstract: Rapid detection of pathogenic Leptospira spp. in marine mammals is challenging: microbiological culture can take 3-6 months and has low sensitivity, immunohistochemical staining of kidney to detect leptospires is invasive and time consuming, and serological methods, such as the microscopic agglutination test, detect antibodies indicative of prior exposure but are not diagnostic for current infection. This study reports a rapid and specific assay for the detection of pathogenic Leptospira spp. in California sea lions (Zalophus californianus) using real-time PCR using primers and a probe targeting the lipL32 gene, which is present only in pathogenic Leptospira spp. The limit of detection of this assay was determined to be three genome copies per PCR volume using L. interrogans serovar Pomona DNA. This PCR assay was found to be 100% sensitive for all pathogenic leptospiral serovars tested. Cross-reaction did not occur with strains of L. biflexa and L. inadai and any of the non-Leptospira pathogens tested. 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 real-time PCR was more sensitive than culture. Inclusion of an internal amplification control in the real-time PCR assay showed no inhibitory effects in PCR negative samples. These studies indicated that real-time PCR is a sensitive and specific assay for the rapid detection of pathogenic Leptospira spp. in marine mammals.