Infection with Brucella spp., long known as a cause of abortion, infertility, and reproductive loss in domestic livestock, has increasingly been documented in marine mammals over the past two decades. We report molecular evidence of Brucella infection in Asian sea otters (Enhydra lutris lutris). Brucella DNA was detected in 3 of 78 (4%) rectal swab samples collected between 2004 and 2006 on Bering Island, Russia. These 78 animals had previously been documented to have a Brucella seroprevalence of 28%, markedly higher than the prevalence documented in sea otters (Enhydra lutris) in North America. All of the DNA sequences amplified were identical to one or more previously isolated Brucella spp. including strains from both terrestrial and marine hosts. Phylogenetic analysis of this sequence suggested that one animal was shedding Brucella spp. DNA with a sequence matching a Brucella abortus strain, whereas two animals yielded a sequence matching a group of strains including isolates classified as Brucella pinnipedialis and Brucella melitensis. Our results highlight the diversity of Brucella spp. within a single sea otter population.
Bacteria of the genus Brucella are among the most common causes of zoonotic disease worldwide (Pappas et al. 2006), and they cause serious livestock losses (6–10% of income) in endemic areas (McDermott et al. 2013). Isolation of Brucella spp. from a marine mammal was first reported in 1994 (Ross et al 1994). Since then, serologic evidence of infection has been detected in many marine species. A previous review documented serologic evidence from 53 species (including 35 cetaceans and 14 pinnipeds), but active infection in only 11 cetacean and 6 pinniped species (Hernández-Mora et al. 2013), and the known host range continues to expand. Clinical illness has been observed in cetaceans, typically as reproductive and neurologic disease (Miller et al. 1999; González-Barrientos et al. 2010). Isolation of Brucella spp. from sea otters (Enhydra lutris) has not been reported, but one isolate was recovered from a European otter (Lutra lutra), although it is not known whether this animal had lesions (Foster et al. 1996). Serologic evidence of exposure has been reported previously in sea otters. Seroprevalence estimates using diagnostic tests developed for livestock have been low: Hanni et al. (2003) report values of 8% among wild northern sea otters (Enhydra lutris kenyoni) from Alaska in 1997 and 6% in Southern sea otters (Enhydra lutris nereis) from California between 1995 and 2000 by using the Rose Bengal test.
To address uncertain performance of serologic tests that were developed for livestock but used in marine mammals, a competitive enzyme-linked immunosorbent assay (cELISA; Meegan et al. 2010) was optimized specifically for detecting antibodies to marine Brucella spp. By using Brucella pinnipedialis antigen and was validated in bottlenose dolphins (Tursiops truncatus) and Hawaiian monk seals (Neomonachus schauinslandi). Using this test, a seroprevalence of 28% was estimated in Asian sea otters (Enhydra lutris lutris) on Bering Island, Russia, sampled between 2004 and 2006 (Goldstein et al. 2011). Northern sea otters from Kodiak Island, Alaska, sampled in summers 2004 and 2005 and tested with the cELISA yielded a much lower seroprevalence (2.7%; Goldstein et al. 2011). The reason for the comparatively high Brucella prevalence at Bering Island is unknown. Unlike other Brucella serologic tests, the cELISA does not seem to cross-react with Yersinia enterocolitica (Meegan et al. 2010). The cELISA test has not been validated in sea otters, but in cetaceans, a sensitivity and specificity of 100% and 73%, respectively, was determined, whereas in pinnipeds, it was 67% and 77%, respectively. Although this test will detect antibodies to any Brucella species, Meegan et al. (2010) concluded that it is more sensitive in marine species than the terrestrial antigen-based tests. Retesting Hawaiian monk seal samples previously analyzed with the terrestrial Brucella serologic tests yielded two additional positives using the cELISA test (Nielsen et al. 2005).
We examined 89 rectal swab samples from the same live-captured Asian sea otters from Bering Island (55°11′N, 166°8′E) collected by Goldstein et al. (2011) between 2004 and 2006 and stored at −70 C. We extracted DNA (DNeasy Blood and Tissue Kit, QIAGEN, Venlo, the Netherlands), and 78 of 89 samples contained amplifiable DNA based on mammalian ferritin PCR (Goldstein et al. 2004). Samples were tested by PCR, targeting a 150-base pair (bp) fragment of the gene encoding the insertion sequence IS711, by using primers IS711F (5′-TACCGCTGCGAATAAAGCCAAC-3′) and IS711R (5′-TGAGATTGCTGGCAATGAAGGC-3′) described by Wu et al. (2014). Samples testing negative with this primer set were retested with the same forward primer and an alternative reverse primer IS711R2 (5′-GCATCATAGGCTGCATCAGCAA-3′), which yields a shorter (118-bp) product. The PCR products of the expected size were purified (QIAquick Gel Extraction Kit, QIAGEN), cloned (pCR 2.1 TOPO TA Vector, One-Shot® TOP10 Escherichia coli; Invitrogen, Carlsbad, California, USA), and sequenced (BigDye® Terminator v3.1 chemistry, Invitrogen). Sequences were analyzed in Geneious Pro 5.3.6 (Biomatters, Auckland, New Zealand) and phylogenetic analysis was conducted using MRBAYES 2.03 (Huelsenbeck and Ronquist 2001).
Three Asian sea otter rectal swab samples (from one adult male, one adult female, and one juvenile male) tested positive by PCR. Of these animals, only the adult female was seropositive to Brucella on the cELISA assay (Goldstein et al. 2011). Such imperfect agreement is expected, as infected animals may not yet have seroconverted, whereas many seropositive animals may no longer be shedding Brucella DNA. Sequencing produced four sequences of 115 bp (KU666447–KU666450; see Supplementary Material Table S1), each 100% identical to IS711 gene sequences from one or more previously characterized Brucella spp. (Fig. 1). The sample SO-024 yielded two IS711 sequences, SO-024 1/2 (GenBank accession no. KU666447) matching only Brucella abortus strain 86/8/59 and SO-024 2/2 (GenBank accession no. KU666448) that was identical to a different IS711 gene copy from B. abortus strain 86/8/59, as well as several Brucella suis, Brucella ovis, and Brucella melitensis sequences. The sequence from SO-025 (GenBank accession no. KU666450) was identical to Brucella melitensis (bv. 2 strain 63/9) only. The sequence from SO-123 (GenBank accession no. KU666449) was identical to two B. melitensis strains (bv. 2 63/9 and 16M); many B. pinnipedialis strains from pinnipeds; and the cetacean-origin strains B1/94, B14/94, and JM13/00.
The PCR test used in this study was modified from the method of Wu et al. (2014), who reported a 100% sensitivity at 0.27 fg per reaction volume, due in part to the presence of multiple copies of the IS711 gene in the Brucella genome. Unfortunately, the amplicon is a short fragment that is conserved and does not allow for speciation of Brucella. Furthermore, not all copies of the IS711 gene in a Brucella genome are identical, complicating phylogenetic inference. We attempted to characterize the sequences detected, targeting the bp26 gene (i.e., omp28), with several assays (Cloeckaert et al. 2000) and two multilocus sequence typing assays for omp25 and glucokinase genes (Whatmore et al. 2007), but all were unsuccessful. The additional protocols were developed by others to characterize cultured isolates with abundant genetic material, rather than the low DNA concentrations found in swab samples from apparently healthy animals. Thus, we were unable to confirm that the Brucella spp. detected was of marine or terrestrial origin, and further work is required to determine the species of Brucella resulting in seroconversion of almost 30% of Bering Island sea otters tested. Culture was not attempted on these samples as only DNA was available, but isolates in pure culture would allow more thorough characterization.
Exposure to Brucella in Asian sea otters on Bering Island may occur via land-sea spillover of the infectious agent, either from introduced reindeer (Rangifer tarandus) or domestic cattle (Bos taurus) grazing along the beaches in coastal areas of Bering Island. Information on the infection status of ungulates on Bering Island was not available, but B. abortus and B. suis biovar 4 are endemic (in cattle and reindeer, respectively) in mainland Russia (Meyer and Morgan 1973). Sea otters are reported to spend long periods hauled out on land Bering Island, potentially providing opportunities for interspecies transmission of Brucella spp. from terrestrial species or other marine mammal species sharing haulout sites, including harbor seals (Phoca vitulina stejnegeri), northern fur seals (Callorhinus ursinus), and Steller sea lions (Eumatopius jubatus jubatus). Alternatively, it has been proposed that fish are involved in Brucella transmission in marine environments. Brucella infection has been detected in lung worms that use a fish intermediate host (Garner et al. 1997) and in some fish species (El-Tras et al. 2010). Sea otters from Bering Island prey on benthic fish (Kornev and Korneva 2006), unlike southern sea otters that eat almost exclusively invertebrates. However, fish foraging is also observed in Northern sea otters in Alaska (Estes 1990), which had a much lower Brucella seroprevalence (Goldstein et al. 2011).
Our report of Brucella in sea otters in Russia adds to the already broad recognized host range of the genus Brucella. Our data provide evidence that Brucella spp. infect Asian sea otters, but information on the pathogenicity and transmission pathway is still limited. Evidence that Brucella can infect sea otters emphasizes that this pathogen should be included on the list of potential zoonotic risks to those handling sea otters.
This project was supported by the Karen C. Drayer Wildlife Health Center, School of Veterinary Medicine, University of California, Davis. Field sampling for this work was funded by the Alaska SeaLife Center, the US Fish and Wildlife Service, and the US Geological Survey (Alaska Science Center and Western Ecological Research Center). Thanks to personnel from the US Fish and Wildlife Service, US Geological Survey, Monterey Bay Aquarium, Alaska SeaLife Center, Marine Mammal Protection Division, Sevvostrybvod, ‘Komandorsky' Reserve, and the University of California at Davis, in particular J. Estes, T. Tinker, M. Murray, G. Bental, V. Nikulin, S. Zagrebelny, V. Fomin, D. Utkin, and B. Smith. Sea otter samples were collected under the Division of Management Authority permit MA041309-2 issued to the US Fish and Wildlife Service and approved by the University of Alaska, Anchorage, Institutional Animal Care and Use Committee.
Supplementary material for this article is online at http://dx.doi.org/10.7589/2016-09-220.