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The seal picornavirus 1, species Aquamavirus A, is currently the only recognized member of the genus Aquamavirus within the family Picornaviridae. The bear picornavirus 1 was recently proposed as the second species in the genus under the name aquamavirus B. Herein, we determined the complete genomes of two novel pinniped picornaviruses, the harbor seal picornavirus (HsPV) and the ribbon seal picornavirus (RsPV). The HsPV and the RsPV were isolated in Vero.DogSLAMtag cells from samples collected from stranded harbor (Phoca vitulina) and ribbon (Histriophoca fasciata) seals. RsPV-infected Vero.DogSLAMtag cells displaying extensive cytopathic effects were processed for transmission electron microscopy and revealed non-enveloped viral particles aggregated into paracrystalline arrays in the cytoplasm. A next-generation sequencing approach was used to recover the complete genomes of the HsPV and the RsPV (6,709 bp and 6,683 bp, respectively). Phylogenetic and genetic analyses supported the HsPV and the RsPV as members of the Aquamavirus genus. Based on these results, RsPV represents a novel strain of Aquamavirus A, while the HsPV is a novel strain of the proposed species aquamavirus B. These discoveries provide information on the evolutionary relationships and ultrastructure of aquamaviruses and expands the known host range of those viruses. Our results underscore the importance of the application of classical virology and pathology techniques coupled with high-throughput sequencing technologies for the discovery and characterization of pathogens in wild marine mammals.

Assessing the health of marine mammal sentinel species is crucial to understanding the impacts of environmental perturbations on marine ecosystems and human health. In Arctic regions, beluga whales, Delphinapterus leucas, are upper level predators that may serve as a sentinel species, potentially forecasting impacts on human health. While gene expression profiling from blood transcriptomes has widely been used to assess health status and environmental exposures in human and veterinary medicine, its use in wildlife has been limited due to the lack of available genomes and baseline data. To this end we constructed the first beluga whale blood transcriptome de novo from samples collected during annual health assessments of the healthy Bristol Bay, AK stock during 2012-2014 to establish baseline information on the content and variation of the beluga whale blood transcriptome. The Trinity transcriptome assembly from beluga was comprised of 91,325 transcripts that represented a wide array of cellular functions and processes and was extremely similar in content to the blood transcriptome of another cetacean, the bottlenose dolphin. Expression of hemoglobin transcripts was much lower in beluga (25.6% of TPM, transcripts per million) than has been observed in many other mammals. A T12A amino acid substitution in the HBB sequence of beluga whales, but not bottlenose dolphins, was identified and may play a role in low temperature adaptation. The beluga blood transcriptome was extremely stable between sex and year, with no apparent clustering of samples by principle components analysis and

An adult male harbour porpoise (Phocoena phocoena) stranded off the coast of Alaska displaying poor body condition, scattered mild ulcerative dermatitis, and necrotizing balanoposthitis. Necropsy findings included severe verminous panniculitis, pneumonia, hepatitis, and enteritis. Histopathological examination of skin lesions revealed a pustular epidermitis and dermatitis, with ballooning degeneration of keratinocytes and occasional amphophilic intracytoplasmic inclusion bodies. A swab sample collected from the ulcerative penile lesions was processed for virus isolation resulting in cytopathic effects observed in primary beluga whale kidney (BWK) cells. Transmission electron microscopy revealed bullet-shaped virions budding from the cell surface of infected BWK cells consistent with a rhabdovirus. A cDNA library was prepared using RNA extracted from infected cell culture supernatant and sequenced on an Illumina MiSeq sequencer. The near-complete genome of a novel rhabdovirus was recovered. Genetic and phylogenetic analyses based on the complete L gene supported the harbour porpoise rhabdovirus (HPRV) as a new species. HPRV clustered phylogenetically with dolphin rhabdovirus (DRV) and this cetacean rhabdovirus clade was found to be the sister group to members of the genus Perhabdovirus that infect fish. A specific nested RT-PCR assay detected HPRV RNA in the epaxial musculature of the harbour porpoise. Our results are consistent with a previous hypothesis that cetacean rhabdoviruses may have arisen following a host jump from fish and suggest that DRV and HPRV represent separate species belonging in a new genus within the family Rhabdoviridae. Further research is needed to determine the health impact of HPRV in harbour porpoise populations, its prevalence, and route of transmission.

Many organohalogen compounds (OHCs) are persistent organic pollutants (POPs) found in appreciable concentrations in marine predators. While production of some POPs has declined or ceased in recent decades, their capacity for global transport and bioaccumulation results in observations of unchanging or increasing concentrations in marine systems. Sea otters ( Enhydra lutris ) have been advocated as an environmental sentinel for contaminants due to their longevity, site fidelity and prey species that often overlap with human consumption. Using archived (1992–2010) samples of livers from Northern sea otters (n = 50) from Alaska we examine concentrations of chlordanes (CHLs), polychlorinated biphenyls (PCBs) dichlorodiphenyltrichloroethane (DDTs), and polybrominated diphenyl ethers (PBDEs) and associated metabolites. We found some evidence for declining ΣPCBs over the two decades, however for most animals concentrations were low compared to toxicological thresholds. Six animals had relatively high concentrations of ΣPCBs (mean = 262,000 ng/g lipid weight), ΣDDTs (mean = 8800 ng/g lw), and ΣPBDEs (mean = 4600 ng/g lw), with four of these six animals experiencing hepatic parasitism or hepatitis. In order to assess whether differences in POPs concentrations are associated with feeding ecology, we examined stable isotopes of C and N in archived muscle and whisker samples. In general, there were no significant relationships between ΣPOP concentrations and stable isotope ratios. There were small differences in stable isotope profiles in animals with high POP concentrations, although it was unclear if these differences were due to feeding ecology or disease processes. This study highlights the importance of considering feeding ecology and necropsy (health and disease status) data while conducting contaminant surveys, and confirms some previous reports of trends in OHCs in Alaska marine mammals.

Beluga whale alphaherpesvirus 1 was isolated from a blowhole swab taken from a juvenile beluga whale. The genome is 144,144 bp in size and contains 86 putative genes. The virus groups phylogenetically with members of the genus Varicellovirus in subfamily Alphaherpesvirinae and is the first alphaherpesvirus sequenced from a marine mammal.

Oil spills of unknown origin were detected in three oil-fouled, ice-associated seals from the Alaska Bering Strait region collected by Alaska Native subsistence hunters during fall 2012. Bile analyses of two oiled seals indicated exposure to fluorescent polycyclic aromatic hydrocarbon (PAH) metabolites but levels of some metabolites were similar to or lower than biliary levels in harvested unoiled ice seals. Oiled seals had elevated tissue PAH concentrations compared to tissue levels of PAHs determined in unoiled ice seals. However, regardless of oiling status, tissue PAH levels were relatively low (

We collected blood from 18 beluga whales (Delphinapterus leucas), live-captured in Bristol Bay, Alaska, USA, in May and September 2008, to establish baseline hematologic and serum chemistry values and to determine whether there were significant differences in hematologic values by sex, season, size/age, or time during the capture period. Whole blood was collected within an average of 19 min (range=11-30 min) after the net was set for capture, and for eight animals, blood collection was repeated in a later season after between 80-100 min; all blood was processed within 12 hr. Mean hematocrit, chloride, creatinine, total protein, albumin, and alkaline phosphatase were significantly lower in May than they were in September, whereas mean corpuscular hemoglobin concentration, monocytes, phosphorous, magnesium, blood urea nitrogen, alanine aminotransferase, aspartate aminotransferase, γ-glutamyltranspeptidase, and creatinine kinase were significantly higher. Mean total protein, white blood cell count, neutrophils, and lymphocytes were significantly higher early in the capture period than they were later. No significant differences in blood analyte values were noted between males and females. Using overall body length as a proxy for age, larger (older) belugas had lower white blood cell, lymphocyte, and eosinophil counts as well as lower sodium, potassium, and calcium levels but higher creatinine levels than smaller belugas. These data provide values for hematology and serum chemistry for comparisons with other wild belugas.

Sequences encoding the major and minor capsid proteins (VP1 and VP2) from two marine vesivirus isolates (Steller sea lion viruses V810 and V1415) were engineered for expression of virus-like particles (VLPs) in the baculovirus system. The resulting VLPs were morphologically similar to native vesivirus virions. Purified VLPs were probed in immunoblots with pooled antisera specific for nine San Miguel sea lion virus (SMSV) types, and a predominant protein of approximately 60 kDa was detected. An enzyme linked immunosorbent assay (ELISA) for the detection of antibodies was developed in which the VLPs served as antigen. The VLPs were adsorbed to the wells of a microplate, and the specificity of the ELISA was established with hyperimmune sera raised against 24 serotypes of the genus Vesivirus . The ELISA was used to screen for the presence of vesivirus specific antibodies in the sera of free-ranging Steller sea lions. The ELISA results demonstrated that Steller sea lions that inhabit the Pacific Ocean waters of southeast Alaska are widely exposed to antigenically related marine vesiviruses, while no previous exposure could be demonstrated using VLP antigens in 17 Steller sea lions from the Aleutian Islands. The broad reactivity of these VLPs and their non-infectious nature will facilitate global sero-epidemiological studies aimed at determining the incidence and prevalence of marine vesiviruses in mammals that inhabit the Pacific and Atlantic oceans as well as susceptible terrestrial animals.

Marine vesiviruses were isolated in cell culture from oral and rectal swabs and vesicular fluid from Alaskan Steller sea lions (SSL; Eumetopias jubatus). Further characterization by RT-PCR, complete genomic sequencing, and phylogenetic analyses indicated that these viruses are most closely related to the marine vesiviruses, but are distinct viruses and represent two novel genotypes. The complete genome of these two SSL isolates was sequenced after cloning their viral cDNA. The genomes were found to be 8302 and 8305 nucleotides in length, organized in three open reading frames and contained 5' and 3' untranslated regions (UTR) of 19 and 180 nucleotides, respectively. The complete genomes of both SSL viruses were most closely related to each other and shared 83.0% nucleotide identity. Using the very limited number of complete genomic vesivirus sequences available in the NCBI database, these novel SSL vesiviruses seem most closely related to vesicular exanthema of swine virus-A48 and least related to rabbit vesivirus and walrus calicivirus. Specific antiserum against some evolutionary closer marine vesiviruses did not neutralize these isolates supporting the novel nature of these SSL viruses.

The lack of integrated long-term data on health, diseases, and toxicant effects in Arctic marine mammals severely limits our ability to predict the effects of climate change on marine mammal health. The overall health of an individual animal is the result of complex interactions among immune status, body condition, pathogens and their pathogenicity, toxicant exposure, and the various environmental conditions that interact with these factors. Climate change could affect these interactions in several ways. There may be direct effects of loss of the sea ice habitat, elevations of water and air temperature, and increased occurrence of severe weather. Some of the indirect effects of climate change on animal health will likely include alterations in pathogen transmission due to a variety of factors, effects on body condition due to shifts in the prey base/food web, changes in toxicant exposures, and factors associated with increased human habitation in the Arctic (e.g., chemical and pathogen pollution in the runoff due to human and domestic-animal wastes and chemicals and increased ship traffic with the attendant increased risks of ship strike, oil spills, ballast pollution, and possibly acoustic injury). The extent to which climate change will impact marine mammal health will also vary among species, with some species more sensitive to these factors than others. Baseline data on marine mammal health parameters along with matched data on the population and climate change trends are needed to document these changes.

Valvular endocarditis has been well described in northern sea otters Enhydra lutris kenyoni of Alaska and in many cases no cause has been identified. It is also one of the most com- mon conditions observed in people with chronic Coxiella burnetii infection. Given the high levels of C. burnetii exposure in marine mammals distributed throughout the same geographic range as the northern sea otter, and the presence of valvular lesions seen in otters, the objective of this study was to determine the level of C. burnetii exposure in otters and investigate any association between exposure, infection and valvular disease in this species. Archived serum from 75 live cap- tured, apparently healthy otters (25 from each of 3 stocks) and 30 dead otters were tested for C. burnetii antibodies by indirect florescent antibody assay (IFA). Archived bone marrow and heart valves were tested for C. burnetii DNA by real-time PCR (qPCR). Overall, the seroprevalence in live otters was 17%, with significantly more exposed animals in the south central (40%) stock rel- ative to the southwest (8%) and southeast (4%). The seroprevalence of animals sampled post mortem was 27%, although none of the bone marrow or heart valve samples were positive by qPCR. Results of this study failed to demonstrate a significant association between C. burnetii infection and valvular endocarditis in sea otters; however, the differing seroprevalence suggests that exposure opportunities vary geographically.

Pacific sleeper sharks Somniosus pacificus were captured near Steller sea lion Eumetopias jubatus rookeries during the period when Steller sea lion pups are most vulnerable to Pacific sleeper shark predation (first water entrance and weaning). Analysis of stomach contents revealed that teleosts were the dominant prey in August and cephalopods were the dominant prey in May (n = 198). Marine mammals were found in 15% of stomachs regardless of season, but no Steller sea lion tissues were detected. Molecular genetic analysis identified grey whale Eschrichtius robustus and harbour seal Phoca vitulina remains in some Pacific sleeper shark stomachs. Most mammals were cetacean and at least 70% of the cetaceans were probably scavenged. Although Pacific sleeper shark and Steller sea lion ranges overlapped, so predation could potentially occur, the diet study suggested that predation on Steller sea lions is unlikely, at least when pups first enter the water or during weaning. Harbour seals were infrequent prey and may have been consumed alive. Pacific sleeper sharks consume fast-swimming prey like Pacific salmon Oncorhynchus sp., most likely live animals rather than scavenged animals. Pacific sleeper sharks appeared to be opportunistic consumers of the available prey and carrion, feeding both on the bottom and in the water column, and their diet shifted to teleosts and cetacean carrion as the fish grew larger.

Characterization of the polygenic and polymorphic features of the Steller sea lion major histo- compatibility complex (MHC) provides an ideal window for evaluating immunologic vigor of the population and identifying emergence of new genotypes that reflect ecosystem pressures. MHC genotyping can be used to measure the potential immunologic vigor of a population. However, since ecosystem-induced changes to MHC genotype can be slow to emerge, measurement of differential expression of these genes can potentially provide real-time evidence of immunologic perturbations. MHC DRB genes were cloned and sequenced using peripheral blood mononuclear leukocytes derived from 10 Steller sea lions from Southeast Alaska, Prince William Sound, and the Aleutian Islands. Nine unique DRB gene sequences were represented in each of 10 animals. MHC DRB gene expression was measured in a subset of six sea lions. Although DRB in genomic DNA was identical in all individuals, relative levels of expressed DRB mRNA was highly variable. Selective sup- pression of MHC DRB genes could be indicative of geographically disparate environmental pressures, thereby serving as an immediate and sensitive indicator of population and ecosystem health.

Phocine distemper virus (PDV) has caused 2 epidemics in harbor seals in the Atlantic Ocean but had never been identified in any Pacific Ocean species. We found that northern sea otters in Alaska are infected with PDV, which has created a disease threat to several sympatric and decreasing Pacific marine mammals.

The endangered Western population of the Steller sea lion declined for three decades for uncertain reasons. We present baseline data of metal concentrations in pups as a first step towards investigating the potential threat of developmental exposures to contaminants. Seven metals were investigated: arsenic, cadmium, silver, aluminum, mercury, lead and vanadium. Vanadium was detected in only a single blubber sample. Mercury appears to be the most toxicologically significant metal with concentrations in the liver well above the current action level for mercury in fish. The concentrations of aluminum, arsenic, silver, cadmium and lead were present in one-fourth to two-thirds of all samples and were at either comparable or below concentrations previously reported. Neither gender nor region had a significant effect on metal burdens. Future work should consider metal concentrations in juveniles and adults and toxicological studies need to be performed to begin to assess the toxicity of these metals.

Lesions suggestive of poxvirus infection were observed in two Steller sea lions (Eu- metopias jubatus) in Alaska during live capture-and-release studies during 2000 and 2001. Both of these animals, female pups in poor body condition, were from Prince William Sound; this population is part of the declining western stock. Umbilicated, typically ulcerated dermal nodules were present, primarily on the fore flippers in one case, and over most of the body in the second case. Histologically, there were discrete masses in the superficial dermis composed of epithelial cells, some of which contained eosinophilic intracytoplasmic inclusion bodies. Negative staining of skin biopsy homogenates demonstrated the presence of orthopoxvirus-like particles. Total DNA extracted from skin biopsies were analyzed by polymerase chain reaction (PCR) using primers that targeted the DNA polymerase and DNA topoisomerase genes. These primers directed the amplification of fragments 543 base pairs (bp) and 344 bp, respectively, whose deduced amino acid sequences indicated the presence of a novel poxvirus within the Chordopoxvirinae subfamily. Comparison of these amino acid sequences with homologous sequences from members of the Chordopoxvirinae indicated highest identity with orthopoxviruses.

Serologic data were examined to determine whether infectious disease may have played a role in the decline of Steller sea lions (Eumetopias jubatus) in the Gulf of Alaska and Aleutian Islands, USA. Available published data, unpublished data, and recent collections (1997-2000) were compared and reviewed. Data were stratified by geography to compare the declining western Alaskan population in the Aleutian Islands through eastern Prince William Sound to the increasing population in southeastern Alaska. Prevalences of antibodies from the 1970s to the early 1990s were noted for Leptospira interrogans, Chlamydophila psittaci, Brucella spp., phocid herpesvirus-1, and calciviruses. Serum samples collected from 1997-2000 were tested for antibodies to these agents as well as to marine mammal morbilliviruses, canine parvovirus, and canine adenovirus-1 and -2. Conclusions could not be drawn about changes in antibody prevalence to these agents during the decline of Steller sea lions, however, because data were incomplete or not comparable as a result of inconsistencies in testing techniques. Despite these shortcomings, results provided no convincing evidence of significant exposure of Steller sea lions to morbilliviruses, Brucella spp., canine parvovirus, or L. interrogans. Steller sea lions have been exposed to phocid herpesviruses, caliciviruses, canine adenovirus, and C. psittaci or to cross-reactive organisms in regions of both increasing and decreasing sea lion abundance. Based on similar antibody prevalence estimates from the increasing and decreasing populations, these agents are unlikely to have been the primary cause of the population decline. They may have contributed to the decline or impeded population recovery, however, because of undetected mortality and morbidity or reductions of fecundity and body condition in animals under other stresses. Systematic monitoring for disease agents and their effects is needed to determine whether infectious disease currently plays a role in the decline and lack of recovery of Steller sea lions.

Novel poxviruses were identified in skin lesions of several species of cetaceans and pinnipeds using polymerase chain reaction targeting DNA polymerase and DNA topoisomerase I genes of members of the subfamily Chordopoxvirinae. With the exception of parapoxviruses, no molecular data of marine mammal poxviruses were available to infer genetic and evolutionary relatedness to terrestrial vertebrate poxviruses. Viruses were assigned to a cetacean poxvirus 1 (CPV-1) group based on nucleotide and amino acid identities of gene fragments amplified from skin lesions of Asian bottlenose (Tursiops aduncus), Atlantic bottlenose (Tursiops truncatus), rough-toothed (Steno bredanensis), and striped (Stenella coeruleoalba) dolphins. A different poxvirus was detected in skin lesions of a bowhead whale (Balaena mysticetus) and provisionally assigned to a CPV-2 group. These viruses showed highest identity to terrestrial poxviruses of the genera Orthopoxvirus and Suipoxvirus. A novel species-specific poxvirus was also identified in skin lesions of Steller sea lions (Eumetopias jubatus). None of these poxviruses were found to have amplifiable hemagglutinin gene sequences. Novel parapoxviruses were also identified in skin lesions of Steller sea lions and spotted seals (Phoca largha). A significant degree of divergence was observed in sequences of Steller sea lion parapoxviruses, while those of spotted seals and harbor seals (Phoca vitulina) were highly conserved.

We identified Echinococcus multilocularis for the first time in brown lemmings (Lemmus trimucronatus) from Barrow, Alaska, USA. Of 467 brown lemmings trapped between 1995 and 2000, two males and two females (0.9%; 95% confidence interval = 0.9±0.9%) were found to be infected with metacestodes of E. multilocularis. No metacestodes were found in 17 collared lemmings (Dicrostonyx rubricatus) also trapped at Barrow. In humans, E. multilocularis causes alveolar echinococcosis, which is potentially fatal. Knowledge of the distribution of this parasite is important to protect the public health.

We measured element concentrations (As, Cd, Cu, Pb, Zn, Fe) and body condition (gross and histologic endpoints) of animals from a caribou (Rangifer tarandus) mortality event that occurred in Alaska, in the area of Point Hope and Cape Thompson (including the Chariot site), in 1995. These were compared to results from hunter-killed caribou from reference sites (Barrow and Teshekpuk Lake, Alaska) and from the area of a mine (Red Dog Mine) to determine whether heavy metals had played a role in the mortality event or whether any elements were at concentrations of concern for human consumers. Starvation and malnutrition were major factors leading to death or severe weakness, as very little or no fat (very low body condition scores) and serous atrophy of fat (observed as watery contents of the marrow cavity, with no apparent fat, and histologically) were more prevalent in caribou associated with the mortality event than in hunter-killed animals from reference sites. Accumulation of hepatic (liver) hemosiderin in Kupffer cells (macrophages) was noted as an indicator of cachexia. Concentrations of lead in fec es and liver, copper in the rumen contents, and arsenic in muscle were higher in caribou harvested near Red Dog Mine, as might be expected in that mineral-rich area, but were not at levels of concern for toxicoses. Kidney concentrations of cadmium, which increased significantly with increasing age, present a potential concern for human consumers, and this is an expected finding. We concluded that caribou had starved and that heavy metals had played no role in the mortality event. Further investigation of re gional mineral differences is required to understand the sources and transport mechanisms that explain these findings and to properly address mining activity. Mortality events on the north slope of Alaska are common and likely involve starvation as described he re, but in most cases they are not investigated, even though recent industrial activities have heightened concern among some local residents and wildlife managers.

Seven Pacific harbor seals with meningoencephalitis associated with Sarcocystis neurona-like protozoa are described. Six of the 7 seals were free-ranging and were found stranded over an 80-km stretch of central California coastline; the other was captive. All had marked to severe nonsuppurative meningoencephalitis, most severe in the cerebellar cortex. Immunohistochemistry for S. neurona antigens was positive on brain tissue in all cases, revealing numerous merozoites as well as developing and mature schizonts, including rosette forms. Electron microscopy performed on 3 animals revealed merozoites and schizonts consistent with Sarcocystis sp., with the absence of rhoptries in merozoites, lack of a parasitophorous vacuole around schizonts, and division by endopolygeny. Serology using western blotting revealed the presence of anti-S. neurona immunoglobulins in the sera of 4 of 5 seals tested. Four animals also had a concurrent mild to moderate nonsuppurative myocarditis; in 1 seal, rare sarcocysts of undetermined species were present within cardiomyocytes.