This study is an extension of a previous assessment of exposure of infectious agents in this declining harbor seal population of GBNP , our work on brucellosis in polar bears  and moose (Alces alces) [23, 24], and of other investigators working on Alaska mammals [12, 25]. In the previous GBNP study that used the same serum samples as the present study, antibodies to Leptospira ssp. were detected while anti-distemper antibodies were not detected, potentially leaving the population in Glacier Bay vulnerable to this severe viral disease . To assess if exposure to Brucella bacteria was evident and could be involved in the past and current population decline in the Glacier Bay harbor seal population, we tested serum from harbor seals from this area for the presence of anti–Brucella antibodies. Based on the uncertainty of Brucella species present in pinnipeds in the North Pacific as well as assay dependent, cross reactivity with other bacterial pathogens  we tested the sera using six different assays. Serological prevalence rates differed between assays detecting antibodies over a range of 16 to 74 percent of samples. Two assays failed to detect antibody, which was expected as B. ovis and B. canis have not been described in harbor seals previously and would likely not cross react with Brucella organism(s) known to be circulating in harbor seal populations as they have rough lipopolysaccharides (LPS), indicating that the harbor seal most likely have been exposed to Brucella species exhibiting a smooth phenotype. These results indicate that Brucella bacteria are present in the harbor seal population of Glacier Bay; however the actual serological prevalence rate cannot be determined as different assays gave widely varying results. Prevalence of anti–Brucella antibodies ranged from 0 to 81 percent in other studies [20, 27–31] with variation attributed to annual differences  and assay type . Together, these findings highlight the need for cautious interpretation of serological results of exposure to Brucella bacteria.
Comparing the results from diagnostic tests without knowing the true prevalence of positive samples is difficult. No validated gold standard exists for the detection of antibodies against Brucella in pinnipeds, and developing such a standard for all marine mammal species and Brucella species is logistically difficult and cost prohibitive . Using total predictive agreement as a measure, the plate test shows less than 55% agreement with the other tests. The card test and the cELISA agreed reasonably well, with over 70% total predictive agreement (Table 1). While this agreement is useful at a population level, it is less useful for assessing the status of individual animals. The apparent higher serological prevalence using the plate test could be explained by the conversion of fibrinogen to fibrin, which can lead to false positive results ; cross reactivity with antibodies to other gram negative bacteria could be an alternative explanation. However, the card test employed the same antigen and did not result in such high prevalence rates, indicating that cross reactivity of antibodies against other pathogens with the antigen used in this assay is not the main reason for the higher rate of positive samples when using the plate test.
Due to the assay dependence of serological prevalence and the uncertain pathology of brucellosis in pinnipeds, the influence of brucellosis on the harbor seal population in GBNP cannot be established. Because the serological tests used in this study are designed to detect past exposure, no conclusions can be drawn on current infection status or if Brucella bacteria are causing adverse health effects. However, a potential role of Brucellosis in the possible multifactorial population decline cannot be excluded. In addition our results show the presence of a zoonotic agent in this population, which should be taken into consideration in any management and animal handling decisions.
The assay dependent differences in apparent exposure rates to Brucella bacteria reinforce the need for a careful approach to comparing literature on exposure to Brucella bacteria in pinnipeds using common diagnostic approaches, which are often not developed for or validated in marine mammals. While biological factors, such as age of animals sampled  are important in comparing different studies on Brucella exposure prevalence in pinnipeds, the serological test used should be considered an additional and possibly the main source of variation. Studies can only be compared and used to determine trends if similar assays were used in previous studies employing similar methods (including criteria to deem a sample positive). As most commonly used tests for antibodies against Brucella are based on methodology developed for livestock, companion animals, or humans, the marine cELISA used in this study is likely the most appropriate among the tests used here. In addition this test has been validated in detecting antibodies in harbor seals with brucellosis. It’s also a competitive ELISA and thus increases the usefulness across different host taxa as reactivity to antibodies of the host by secondary antibodies is not necessary. However the possibility that different biovars exist between B. pinnipedia and uncertainty about Brucella spp. distribution in marine mammals should lead to careful interpretation of even this assay, which is based on a marine derived Brucella isolate. We strongly suggest that if results from a study are intended to be compared to previously conducted serosurveys in pinnipeds that the same test from the historic study be included alongside the marine based cELISA.