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During the 2001-02 and 2002-03 breeding seasons, epizootics of Klebsiella pneumoniae resulted in a dramatic increase of pup mortality in New Zealand sea lions (Phocarctos hookeri; NZSLs) on Enderby Island (Auckland Islands). To estimate the prevalence of infection in the NZSL population, a serologic test was developed using a Western blot and a polysaccharide antigen derived from a K. pneumoniae isolate from a NZSL pup. All archived serum samples collected between 1997 and 1998 and 2004 and 2005 at Sandy Bay Beach rookery, Enderby Island, were tested (314 pups and 302 adult females). Anti-Klebsiella antibodies were detected throughout this period, but overall, only 16% of NZSL pups between birth and 5 mo of age were seropositive compared with 95.7% of adults. There was no apparent change in antibody prevalence as a result of the two epizootics. A method to determine total immunoglobulin G (IgG) levels in sea lion serum also was developed to investigate passive immunoglobulin transfer to neonates and development of an acquired immune response. The IgG concentration was significantly lower in pups (median 2.1 mg/ml) than in adult females (median 80 mg/ml). Based on serologic results, it was not possible to determine whether K. pneumoniae was an endemic or a novel pathogen to the NZSL population because the test was not able to discriminate between Klebsiella species. However, this study suggested that the transfer of passive immunity to neonates was very low in the NZSL, especially for anti-Klebsiella antibodies.