Serologic Detection of Subtype-specific Antibodies to Influenza A Viruses in Southern Sea Otters (Enhydra lutris nereis)

Southern sea otters (Enhydra lutris nereis) are a federally threatened marine mammal species that occupies nearshore marine habitats throughout central California, US (US Fish and Wildlife Service 2015). By inhabiting the coastal interface, sea otters serve as bioindicators of pathogens that can move between terrestrial and marine ecosystems (Jessup et al. 2004). Influenza A viruses (IAVs) are zoonotic pathogens that infect birds and mammals and can cause high morbidity and mortality in humans (Liu et al. 2009). Marine mammals (especially pinnipeds) are susceptible to IAVs, and widespread morbidity and mortality have been associated with IAV infection in Atlantic harbor seals (Phoca vitulina) in the eastern US (Yang et al. 2015) and the Netherlands (Bodewes et al. 2015). Exposure to IAV has been detected in a small number of northern sea otters (Enhydra lutris kenyoni; White et al. 2013; Li et al. 2014), but there is no information on exposure of southern sea otters to IAVs, or potential impacts of IAV infection on sea otter health. A related mustelid, the ferret (Mustela putorius furo), is highly susceptible and is widely used as a laboratory animal model for human influenza infection (Oh and Hurt 2016).

In this study, we used serologic assays to assess southern sea otter exposure to human- and avian-origin IAVs and to determine the specific subtypes of IAV that may have infected southern sea otters over a 19-yr period. From 1997 to 2015, sera were collected postmortem from southern sea otters during necropsy as part of the Sea Otter Research Program at the Marine Wildlife Veterinary Care and Research Center in Santa Cruz, California. We initially evaluated IAV exposure by screening sera for the presence of nonsubtype-specific serum antibodies by using a commercial enzyme-linked immunosorbent assay (ELISA) (Competitive ELISA, protocol for chicken, turkey, quail, guinea fowl and horse; ID Screen® Influenza A Antibody Competition Multi-species, Innovative Diagnostics, Montpellier, France) to the influenza virus nucleoprotein ([NP], Lebarbenchon et al. 2012). Serum was considered positive for antibodies to influenza when the ratio of the absorbance of the test sample to the negative control was less than 0.45 (Table 1). Virus microneutralization (VN) was then performed as described by Ramey et al. (2014) to identify subtype-specific antibodies to the IAV surface protein hemagglutinin (HA). Sera from ELISA-positive (n=114) and ELISA-negative (n=5) sea otters were tested against 12 avian-origin HA antigens and one human-origin HA antigen, pandemic H1N1 (pdmH1N1). Isolated antigens (A/California/04/2009 [pdmH1N1], A/mallard/NJ/AI10-4263/2010 [H1N1], A/mallard/MN/AI08-2755/2008 [H2N3], A/mallard/MN/AI10-2593/2010 [H3N8], A/mallard/MN/AI10-3208/2010 [H4N6], A/mallard/MN/AI11-3933/2011 [H5N1], A/mallard/MN/SG-01048/2008 [H6N1], A/mallard/MN/AI09-3770/2009 [H7N9], A/mallard/MN/AI08-2721/2008 [H8N4], A/RUTU/DE/AI11-809/2011 [H9N2], A/mallard/MN/SG-00999/2008 [H10N7], A/mallard/MN/SG-00930/2008 [H11N9], A/mallard/MN/SG-3285/2007 [H12N5]) for VN were propagated in Madin Darby canine kidney cells as described by Ramey et al. (2014). Sera were considered positive for antibodies to a given HA subtype when no cytopathic effects were observed in infected Madin Darby canine kidney epithelial cells at dilutions ≥1:40 (Table 2).

We detected NP antibodies by ELISA in 160 (24.2%) of 661 southern sea otters sampled from 1997 to 2015 (Table 1). The ELISA testing revealed two distinct peaks in seroprevalence: one peak from 1997 to 2002 and the other peak from 2010 to 2015, raising the possibility that the southern sea otter population experienced two periods of epizootic transmission involving two different viruses. However, subtype-specific antibody testing by VN indicated likely exposure to a variety of IAV subtypes from 2000 to 2007, including avian subtypes H1, H3, H4, H5, H6, H7, H9, and H11 (Table 2). As expected, sera from individual otters often cross-reacted with multiple viral subtypes, but VN antibody responses to a single subtype were observed for avian strains H1, H3, H4, H9, and H11. In contrast, although several different avian-origin viruses apparently infected sea otters from 2000 to 2007, a single virus, pdmH1N1, was responsible for the strong antibody responses detected in ELISA-positive otters from 2010 to 2015.

This study provided convincing evidence that southern sea otters are susceptible to IAV infection; however, it is unknown whether IAV infection caused disease in any of these animals. Although the rise and fall in seroprevalence of NP antibodies (ELISA) suggested that sea otters may have undergone two periods of epizootic transmission (Table 1), the VN data showed that multiple avian-origin IAVs were likely introduced into the population over time and that only pdmH1N1 spread in an epizootic pattern (Table 2). The detection of high antibody titers against pdmH1N1 from 2010 to 2015 is consistent with the emergence of pdmH1N1 in humans in 2009, as well as the detection of pdmH1N1 in northern elephant seals (Mirounga angustirostris; Boyce et al. 2013; Goldstein et al. 2013) and northern sea otters (White et al. 2013; Li et al. 2014). The implications of IAV exposure on sea otter health have yet to be determined. In a recent study by Puryear et al. (2016), pdmH1N1 exposure was among the most frequently detected IAV subtypes in Northwest Atlantic gray seals (Halichoerus grypus), but exposure did not correlate with disease or mortality patterns. In contrast, H10N7 infection in Atlantic harbor seals in the Netherlands was associated with morbidity and mortality (Bodewes et al. 2015).

Although all ELISA-negative control sera were negative when tested by VN, some ELISA-positive samples (1997–99) were also negative by VN (Table 2). We also unexpectedly detected antibodies to pdmH1N1 in four animals, with VN titers of 640 (n=3) and 80 (n=1), before 2009. These unusual results may be due to cross-reactivity among viral subtypes; the viral strains chosen for VN testing; sample toxicity due to hemolysis; or other, unknown factors.

We presumed that the highest titer indicated the viral subtype that likely infected the otter, whereas lower titer reactions to other subtypes using the same sera represented cross-reactivity or multiple exposures to different viral subtypes. Although some otters sampled during this period only reacted to avian H1N1, the strongest antibody titers occurred against pdmH1N1, suggesting that the latter is the subtype responsible for the observed epizootic pattern of transmission. Avian H1, H5, and H6 viruses used in this study all share the same neuraminidase gene (N1), which may have also contributed to cross-reactive antibody responses. High titers and the lack of cross-reactivity with other subtypes (Table 2) provided the strongest evidence that southern sea otters were potentially infected with avian-origin influenza subtypes H1, H3, H4, H9, and H11.

Key questions for future research include the following: How do sea otters acquire IAVs of human and avian origin? What is the effect of IAV infection on individual sea otter health, and are there population-level effects? It was striking that only five of the 100 immature (<1-yr-old) sea otters sampled from 1998 to 2012 were seropositive for NP antibodies and that these samples were only from immature animals sampled from 2002 to 2007, when overall seroprevalence was in decline. Maternal antibodies to pdmH1N1 were documented among northern elephant seal pups sampled from 2010 to 2015 (Boyce et al. 2013), but we were unable to draw any conclusions about maternal antibodies to pdmH1N1 in sea otters because only two animals (both seronegative) were sampled after 2010 and the infection status of their dams was unknown. Future efforts should include a study directed at determining whether sea otter pups acquire maternal antibodies from their IAV-exposed mothers and whether such antibodies provide protection. In addition, research should evaluate whether sea otters serve as mixing vessels for IAVs and whether they play a role in generating viruses of potential public or animal health significance.

This study was supported in part by funding from National Institutes of Health Centers of Excellence for Influenza Research and Surveillance HHSN272201400008C, HHSN266200700010C, and HHSN272201400006C and the Students Training in Advanced Research program at the School of Veterinary Medicine, University of California, Davis. This work was also supported in part by the California Sea Otter Tax Checkoff, administered by the California Department of Fish and Wildlife and the State Coastal Conservancy. We thank the scientists at California Department of Fish and Wildlife; US Geological Survey; Monterey Bay Aquarium; University of California, Davis; and The Marine Mammal Center for carcass collection and staff at the Southeastern Cooperative Wildlife Disease Study at the University of Georgia for expertise and assistance with the virus microneutralization assay.