Molecular Markers, Mat and Modeling: New Evidence for Leptospirosis in California Sea Lions Being Endemic with Periodic Epizootics, Defying the Host-Adapted Strain Paradigm for Leptospirosis
IAAAM 2009
Frances M.D. Gulland1; Denise Greig1; Jamie Lloyd-Smith2; Richard Zuerner3
1The Marine Mammal Center, Sausalito, CA, USA; 2Center for Infectious Disease Dynamics, Pennsylvania State University, University Park, PA, USA; 3Bacterial Diseases of Livestock, National Animal Disease Center, Ames, IA, USA


Leptospirosis is a zoonotic disease infecting a broad range of mammalian hosts, and is re-emerging globally in humans and domestic dogs. Disease outbreaks have occurred periodically in California sea lions (Zalophus californianus) off the central and northern coasts of California, with hundreds of animals dying in each outbreak since 1970.2,3,7,12 Repeated epidemics have occurred at approximately three to five year intervals since 1984.2,3,4 Leptospira interrogans serovar Pomona has been isolated from infected sea lions, but it is unknown whether the pathogen persists in the sea lion population or is introduced repeatedly from external reservoirs. The potential for transmission to and from both domestic animals and humans, coupled with the large numbers of sick and dying sea lions lying on beaches during epidemics, makes the question of the source of the infection in these free-living marine mammals one of public as well as veterinary interest. Here we present serological surveillance and modeling results, combined with molecular data from ten sea lion isolates, to elucidate the epidemiology of leptospirosis in California sea lions, and investigate whether the disease is endemic in sea lions or results from spill-over from a terrestrial source.

Serum samples collected over an 11-year period from 1,344 California sea lions that stranded alive on the California coast were evaluated using the microscopic agglutination test (MAT) for antibodies to L. interrogans serovar Pomona.1 Seroprevalence among yearlings was evaluated as a measure of incidence in the population, and antibody persistence times based on temporal changes in the distribution of titer scores were characterized. Multivariate logistic regression was used to determine individual risk factors for seropositivity with high and low titers. The serosurvey revealed cyclical patterns in seroprevalence to L. interrogans serovar Pomona, with 4-5 year periodicity and peak seroprevalence above 50%.6 Seroprevalence in yearling sea lions was an accurate index of exposure among all age classes, and indicated on-going exposure to leptospires in non-outbreak years. Analysis of titer decay rates showed that some individuals probably maintain high titers for more than a year following exposure.

Strains of Leptospira interrogans (n = 63) from a variety of domestic animals and ten strains obtained from sea lion kidneys during leptospirosis epizootics since 1971 were propagated in liquid EMJH medium at 29 °C. All strains were identified as L. interrogans serovar Pomona using RFLP analysis.10 Genomic DNA samples were used as templates for PCR amplification at previously described VNTR loci. PCR products were separated by agarose gel electrophoresis and visualized, and product sizes were calculated by comparison to size standards using linear regression. The number of tandem repeats at nine loci (VNTR4, VNTR7, VNTR10, VNTR23, VNTR27, VNTR29, VNTR30, VNTR31, and VNTR36) was determined for all strains.8,9 Eight loci (VNTR4, VNTR7, VNTR10, VNTR23, VNTR29, VNTR30, VNTR31, and VNTR36) were used to differentiate serovar Pomona isolates from L. interrogans serovar Copenhageni, used as an out-group in this study. Three loci (VNTR29, VNTR30, and VNTR36) were invariant among serovar Pomona isolates but differentiated serovar Pomona from serovar Copenhageni, while variation among serovar Pomona isolates at five loci (VNTR4, VNTR7, VNTR10, VNTR23, and VNTR31) was used to differentiate isolates within this serovar.

Eleven distinct groups of L. interrogans serovar Pomona isolates were detected using VNTR analysis. Although most cattle isolates shared a common VNTR profile, isolates from pigs and most wildlife species exhibited greater diversity. Remarkably, all California sea lion isolates shared common VNTR profiles, despite separation by time and location of isolation, and were distinct from all other serovar pomona isolates.12 Isolate Po048, obtained during the first known leptospirosis outbreak among California sea lions,11 differs slightly from all isolates obtained from subsequent sea lion outbreaks and may represent the founder genotype for strains infecting this mammalian host. Isolates obtained during subsequent California sea lions outbreaks were identical to each other. These data strongly suggest that California sea lions are maintenance hosts for serovar Pomona, as it is unlikely that intermittent exposure of California sea lions to the same L. interrogans serovar Pomona clonal population has occurred over a 37-year period.

These results suggest that leptospirosis is endemic in California sea lions, but also causes periodic epidemics of acute disease. The findings call into question the classical dichotomy5 between maintenance hosts (infected with "host adapted" strains) of leptospirosis, which experience chronic but largely asymptomatic infections, and accidental hosts (infected with "non-host adapted" strains), which suffer acute illness or death as a result of disease spillover from reservoir species.


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2.  Dierauf L.A., D.J. Vandenbroek, J. Roletto, M. Koski, L. Amaya, and L.J. Gage. 1985. An epizootic of leptospirosis in California sea lions. J Am Vet Med Assoc 187: 1145-1148.

3.  Greig D.J, F.M.D. Gulland and C. Kreuder. 2005. A decade of live California sea lion (Zalophus californianus) strandings along the central California coast: causes and trends, 1991-2000. Aquat Mamm 31: 11-22.

4.  Gulland F.M.D, M. Koski, L.J. Lowenstine, A. Colagross, L. Morgan, and T. Spraker. 1996. Leptospirosis in California sea lions (Zalophus californianus) stranded along the central California coast, 1981-1994. J Wildl Dis 32: 572-580.

5.  Levett P.N. 2001. Leptospirosis. Clin Microbiol Rev 14: 296-326.

6.  Lloyd-Smith J.O., D.J. Greig, S. Hietala, G.S. Ghneim, L. Palmer, J. St. Leger, B.T. Grenfell and F.M. Gulland. 2007. Cyclical changes in seroprevalence of leptospirosis in California sea lions: endemic and epidemic disease in one host species? BMC Infec Dis 7: 125.

7.  McIlhattan T.J., J.W. Martin, R.J. Wagner and J.O. Iverson. 1971. Isolation of Leptospira pomona from a naturally infected California sea lion, Sonoma County, California. J Wildl Dis 7: 195-197.

8.  Salaun L., F. Merien, S. Gurianova, G. Baranton, and M. Picardeau. 2006. Application of multilocus variable-number tandem-repeat analysis for molecular typing of the agent of leptospirosis. J Clin Microbiol 44: 3954-3962.

9.  Slack A., M. Symonds, M. Dohnt, and L. Smythe. 2006. An improved multiple-locus variable number of tandem repeats analysis for Leptospira interrogans serovar Australis: a comparison with fluorescent amplified fragment length polymorphism analysis and its use to redefine the molecular epidemiology of this serovar in Queensland, Australia. J Med Microbiol 55: 1549-1557.

10. Thiermann A.B., A.L. Handsaker, S.L. Moseley, and B. Kingscote. 1985. New method for classification of leptospiral isolates belonging to serogroup Pomona by restriction endonuclease analysis: serovar Kennewicki. J Microbiol 21: 585-587.

11. Vedros N.A., A.W. Smith, J. Schonewa, G. Migaki, and R.C. Hubbard. 1971. Leptospirosis epizootic among California sea lions. Science 172: 1250-1251.

12. Zuerner, R.L., and D.P. Alt. 2009. Variable nucleotide tandem repeat analysis reveals a unique group of Leptospira interrogans serovar Pomona isolates associated with California sea lions. J Clin Microbiol (in press).


Speaker Information
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Frances M.D. Gulland
The Marine Mammal Center
Sausalito, CA, USA

MAIN : Epidemiology : Endemic Leptospirosis
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