Isolation of a novel adenovirus from California sea lions Zalophus californianus

Viral hepatitis associated with adenoviral infection has been reported in California sea lions Zalophus californianus admitted to rehabilitation centers along the California coast since the 1970s. Canine adenovirus 1 (CAdV-1) causes viral hepatitis in dogs and infects a number of wildlife species. Attempts to isolate the virus from previous sea lion hepatitis cases were unsuccessful, but as the hepatitis had morphologic features resembling canine infectious hepatitis, and since the virus has a wide host range, it was thought that perhaps the etiologic agent was CAdV-1. Here, we identify a novel adenovirus in 2 stranded California sea lions and associate the infection with viral hepatitis and endothelial cell infection. Phylogenetic analysis confirmed the classification of the sea lion adenovirus in the Mastadenovirus genus with the most similarity to tree shrew adenovirus 1 (TSAdV-1, 77%). However, as the sea lion adenovirus appeared to be equally distant from the other Mastadenovirus species based on phylogenetic analysis, results indicate that it represents an independent lineage and species. Although sequences from this novel virus, otarine adenovirus 1 (OtAdV-1), show some similarity to CAdV-1 and 2, it is clearly distinct and likely the cause of the viral hepatitis in the stranded California sea lions.     

Antibodies to marine caliciviruses in the Steller sea lion (Eumetopias jubatus Schreber)

Sera from 145 Steller sea lions (76 adults, three subadults, 37 pups, and 29 fetuses) were tested for neutralizing antibodies to nine marine calicivirus serotypes. Antibodies were found to San Miguel sea lion virus (SMSV) types 1, 5, 6, 7, 8, 10 and 13, and to Tillamook (bovine) calicivirus, but no antibodies were found to the walrus calicivirus. Titers (microtiter neutralization assay) ranged from 1:20 to 1:320, with many positive reactions at the higher dilutions (greater than or equal to 1:80). Antibodies to SMSV's 5 and 10 were most common among animals sampled in Alaskan waters, while antibodies to SMSV-6 were most common among pups from the southern Oregon coast. These data provide evidence that Steller sea lions, like their California sea lion (Zalophus c. californianus Lesson) counterparts, have experienced widespread exposure to multiple serotypes of marine caliciviruses.     

Prevalence and characterization of Salmonella spp. among marine animals in the Channel Islands, California

Salmonella enterica is a zoonotic pathogen that has been isolated from free-ranging marine mammals throughout the world, with animals in the Channel Islands of California (USA) showing the highest prevalence. The goal of this study was to determine prevalence, antimicrobial sensitivity and genetic similarity using pulsed-field gel electrophoresis (PFGE) of Salmonella in several non-domestic animal species on San Miguel and San Nicolas Islands. Fecal samples were collected from 90 California sea lion Zalophus californianus pups, 30 northern elephant seal Mirounga angustirostris pups and 87 western gulls Larus occidentalis in the Channel Islands and 59 adult male sea lions in Puget Sound, WA (USA). Salmonella were isolated, identified and serotyped, followed by antimicrobial susceptibility testing and PFGE. Of the California sea lion pups that were sampled on the islands, 21% (n = 19) were positive for Salmonella, whereas no adults males in Puget Sound were positive. Of the northern elephant seal pups sampled, 87% (n = 26) were harboring Salmonella. Only 9% (n = 8) of western gulls were shedding Salmonella, with one of these gulls harboring the only antimicrobial resistant isolate. The serotypes found in these animals were Enteritidis, Montevideo, Newport, Reading, and Saint Paul. The only serotype that showed variation on PFGE was Newport. The pinnipeds of the Channel Islands harbor Salmonella at a higher prevalence than pinnipeds from other geographic areas observed in previous studies. Researchers and veterinarians should exercise increased caution when working with these animals due to the zoonotic potential of Salmonella.     

Molecular characterization of expressed DQA and DQB genes in the California sea lion ( Zalophus californianus)

To date, there are no published MHC sequences from the California sea lion (Zalophus californianus), a thriving species that, by feeding high on the marine food web, could be a sentinel for disturbances in marine and coastal ecosystems. In this study, degenerate primers and RACE technology were used to amplify near-full-length (MhcZaca- DQB) and full-length (MhcZaca- DQA) expressed class II MHC gene products from the peripheral blood mononuclear cells of two California sea lions in rehabilitation. Five unique Zaca- DQA sequences and eight unique Zaca- DQB sequences, all encoding functional proteins, were identified in the two animals, indicating the presence of multiple DQ- loci in this species. An additional three Zaca- DQB sequences containing features compatible with pseudogenes or null alleles were also identified. Despite the identification of multiple DQA and DQB sequences, the degree of heterogeneity between them was extremely low. To confirm the limited degree of Zaca-DQ nucleotide variation between individuals, we used denaturing gradient gel electrophoresis to examine putative peptide binding region sequences from the peripheral blood leukocyte-derived RNAs of 19 wild-caught California sea lions from physically distinct populations. The pattern of Zaca-DQ sequence migration was identical between individuals and independent of geographical region. This apparent Zaca-DQ sequence identity between sea lions was confirmed by direct sequencing of individual bands. In combination, these findings raise important questions regarding immunogenetic diversity within this thriving species, and should prompt further research into the existence of a highly polymorphic sea lion class II MHC molecule with sequence features that support traditional peptide binding functions.     

Parasites of forage fishes in the vicinity of Steller sea lion (Eumetopias jubatus) habitat in Alaska

Fish serve as intermediate hosts for a number of larval parasites that have the potential of maturing in marine mammals such as Steller sea lions (Eumetopias jubatus). We examined the prevalence of parasites from 229 fish collected between March and July 2002 near two islands used by Steller sea lions in Southeast Alaska and island habitats in the Aleutian Islands. Sea lion populations have remained steady in Southeast Alaska but have been declining over the last 30 yr in the Aleutian Islands. Even though the fish samples near the Southeast Alaska haul-outs were composed of numerous small species of fish and the Aleutian Islands catch was dominated by juveniles of commercially harvested species, the parasite fauna was similar at all locations. Eleven of the 20 parasite taxa identified were in their larval stage in the fish hosts, several of which have been described from mammalian final hosts. Four species of parasite were more prevalent in Southeast Alaska fish samples, and seven parasite species, including several larval forms capable of infecting marine mammals, were more prevalent in fish from the Aleutian Islands. Nevertheless, parasites available to Steller sea lions from common fish prey are not likely to be a major factor in the decline of this marine mammal species.     

Foreflipper propulsion in the California sea lion, Zalophus californianus

The family Otariidae comprises the only group of marine mammals that habitually use their pectoral appendages to generate propulsive forces during swimming. This method of propulsion was examined in the California sea lion ( Zalophus californianus ), a representative member of the family. High-speed films were taken as a sea lion swam against a water current generated inside a large flow channel. Thrust production was determined by examining the body's movement at various stages of the propulsive cycle. Sea lions generate thrust continuously throughout the stroke. Over its initial three-quarters, foreflippers act as hydrofoils creating forward thrust and lift as they move vertically through the water. Thrust production is greatest, however, near the end of the stroke, when flippers are used as paddles and are oriented broad side to the oncoming flow. The force generated by this three-phased system of propulsion is likely to be greater than that attainable by either an exclusively lift-based hydrofoil or drag-based paddling style of swimming.
The kinematic changes that enable sea lions to change speed were also investigated. Film records revealed that stroke amplitude became greater with speed, although total stroke duration remained essentially constant. Sea lions increase stroke frequency with velocity but large variations in the measured values suggest that changes in amplitude and flipper angle of attack are also important parameters for modulating swimming speed.     

Common cancer in a wild animal: the California sea lion (Zalophus californianus) as an emerging model for carcinogenesis

Naturally occurring cancers in non-laboratory species have great potential in helping to decipher the often complex causes of neoplasia. Wild animal models could add substantially to our understanding of carcinogenesis, particularly of genetic and environmental interactions, but they are currently underutilized. Studying neoplasia in wild animals is difficult and especially challenging in marine mammals owing to their inaccessibility, lack of exposure history, and ethical, logistical and legal limits on experimentation. Despite this, California sea lions (Zalophus californianus) offer an opportunity to investigate risk factors for neoplasia development that have implications for terrestrial mammals and humans who share much of their environment and diet. A relatively accessible California sea lion population on the west coast of the USA has a high prevalence of urogenital carcinoma and is regularly sampled during veterinary care in wildlife rehabilitation centres. Collaborative studies have revealed that genotype, persistent organic pollutants and a herpesvirus are all associated with this cancer. This paper reviews research to date on the epidemiology and pathogenesis of urogenital carcinoma in this species, and presents the California sea lion as an important and currently underexploited wild animal model of carcinogenesis.     

Innervation patterns of mystacial vibrissae support active touch behaviors in California sea lions (Zalophus californianus)

Vibrissae or follicle-sinus complexes (F-SCs) are highly developed mammalian sensory structures. These blood-filled sinuses are richly innervated and possess novel mechanoreceptors. Although much is known regarding the function of F-SCs in terrestrial mammals, much less is known regarding marine carnivores such as pinnipeds. Pinnipeds possess the largest, most highly innervated vibrissae of any mammal. One such pinniped is the California sea lion, which are generalist marine predators that rely heavily upon tactile discrimination capabilities. Psychophysical studies demonstrate that haptic tactile discrimination using F-SCs is exceptionally sensitive. However, our knowledge of the structure and function of F-SCs in otariids is limited. Our objectives were to investigate the innervation and microstructure of F-SCs across the mystacial vibrissal field and infer function from haptic performance studies in California sea lions. Innervation and microstructure of vibrissae differed considerably compared to similar data available for phocids. Total innervation of mystacial vibrissae was estimated to be 86,042 axons. Investigations of innervation density and investment of microvibrissae versus macrovibrissae demonstrated a significantly increased axon density per F-SC in medial microvibrissal regions compared to lateral macrovibrissae, which supports psychophysical data and somatotopic organization of the central nervous system involved with tactile discrimination capability. Innervation increased from medial microvibrissae (705 ± 125 axons/F-SC) to lateral macrovibrissae (1,447 ± 154) as well as from dorsal (541 ± 60) to ventral (1,493 ± 327) vibrissal regions. These data provide a more complete picture of the sensory ecology of this important aquatic mammalian lineage; the specialization of peripheral sensory structures, central nervous structures with demonstrated enhanced haptic capabilities behaviorally has likely led to the ecological success of California sea lions.     

Molecular and morphometric evidence for separate species of Uncinaria (Nematoda: Ancylostomatidae) in California sea lions and northern fur seals: hypothesis testing supplants verification

California sea lions (Zalophus californianus) and northern fur seals (Callorhinus ursinus) are each believed to host distinct hookworm species (Uncinaria spp.). However, a recent morphometric analysis suggested that a single species parasitizes multiple pinniped hosts, and that the observed differences are host-induced. To explore the systematics of these hookworms and test these competing hypotheses, we obtained nucleotide sequences of nuclear ribosomal DNA (D2/D3 28S, D18/D19 28S, and internal transcribed spacer [ITS] regions) from 20 individual hookworms parasitizing California sea lion and northern fur seal pups where their breeding grounds are sympatric. Five individuals from an allopatric population of California sea lions were also sampled for ITS-1 and D18/D19 28S sequences. The 28S D2/D3 sequences showed no diagnostic differences among hookworms sampled from individual sea lions and fur seals, whereas the 28S D18/D19 sequences had one derived (apomorphic) character demarcating hookworms from northern fur seals. ITS sequences were variable for 7 characters, with 4 derived (apomorphic) states in ITS-1 demarcating hookworms from California sea lions. Multivariate analysis of morphometric data also revealed significant differences between nematodes representing these 2 host-associated lineages. These results indicate that these hookworms represent 2 species that are not distributed indiscriminately between these host species, but instead exhibit host fidelity, evolving independently with each respective host species. This evolutionary approach to analyzing sequence data for species delimitation is contrasted with similarity-based methods that have been applied to numerous diagnostic studies of nematode parasites.     

Novel symptomatology and changing epidemiology of domoic acid toxicosis in California sea lions (Zalophus californianus): an increasing risk to marine mammal health

Harmful algal blooms are increasing worldwide, including those of Pseudo-nitzschia spp. producing domoic acid off the California coast. This neurotoxin was first shown to cause mortality of marine mammals in 1998. A decade of monitoring California sea lion (Zalophus californianus) health since then has indicated that changes in the symptomatology and epidemiology of domoic acid toxicosis in this species are associated with the increase in toxigenic blooms. Two separate clinical syndromes now exist: acute domoic acid toxicosis as has been previously documented, and a second novel neurological syndrome characterized by epilepsy described here associated with chronic consequences of previous sub-lethal exposure to the toxin. This study indicates that domoic acid causes chronic damage to California sea lions and that these health effects are increasing.     

Isolation by distance among California sea lion populations in Mexico: redefining management stocks

Understanding the spatial structure of a population is critical for effective assessment and management. However, direct observation of spatial dynamics is generally difficult, particularly for marine mammals. California sea lions ( Zalophus californianus ) are polygynous pinnipeds distributed along the Pacific coast of North America. The species' range has been subdivided into three management stocks based on differences in mitochondrial DNA, but to date no studies have considered nuclear genetic variation, and thus we lack a comprehensive understanding of gene flow patterns among sea lion colonies. In light of recent population declines in the Gulf of California, Mexico, it is important to understand spatial structure to determine if declining sea lion colonies are genetically isolated from others. To define population subdivision and identify sex biases in gene flow, we analysed a 355-bp sequence of the mitochondrial DNA control region and 10 polymorphic microsatellite loci from 355 tissue samples collected from six colonies distributed along Mexican waters. Using a novel approach to estimate sex biases in gene flow, we found that male sea lions disperse on average 6.75 times more frequently than females. Analyses of population subdivision strongly suggest a pattern of isolation by distance among colonies and challenge current stock definitions. Based on these results, we propose an alternative classification that identifies three Mexican management units: Upper Gulf of California, Southern Baja Peninsula, and Upper Pacific Coast of Baja. This revised classification should be considered in future assessment and management of California sea lion populations in Mexican waters.     

Ancient DNA analysis of the Japanese sea lion (Zalophus californianus japonicus Peters, 1866): preliminary results using mitochondrial control-region sequences

In this study, we successfully extracted ancient DNA from skeletal remains of the Japanese sea lion-a species that is practically extinct-from archaeological sites and determined a partial sequence of its mitochondrial DNA control region. A molecular phylogenetic tree constructed by the neighbor-joining (NJ) method showed that the sequences from Japanese sea lions clustered together, with a high bootstrap value, and that this cluster was closest to the California sea lion cluster. The distinctly divergent cluster of Japanese sea lions reflected the morphological classification of these animals as a distinct species of the genus Zalophus; however, proximity to the California sea lion cluster simultaneously implied conformation with the traditional classification of these animals as a subspecies of Zalophus californianus. The average amount of nucleotide substitution between the Japanese and California sea lions was 7.02%. The Japanese and California sea lions were estimated to have diverged 2.2 million years ago, i.e., in the late Pliocene Epoch. This is the first report on a genetic analysis of the Japanese sea lion.     

Observations of the effect of a New Year’s fireworks display on the behavior of the South American sea lion (Otaria flavescens) in a colony of central-south Chile

The use of fireworks has increased worldwide in recent years. Despite this, their effects on sea lions and similar species are almost undocumented. In this study, we documented some effects of the 2015 New Year’s fireworks on the behavior of South American sea lions (Otaria flavescens), a marine mammal species that utilizes coastal rookeries for breeding, resting, and care and nursing of pups. An immediate cessation of vocalizations followed the start of the fireworks and a significant decrease in the number of sea lions in the colony was registered. This provided evidence that fireworks disrupted the ongoing behavior of the South American sea lion. Further studies will be necessary to determine whether this disruption has negative consequences for these animals or whether they have evolved behavioral mechanisms for dealing with these kinds of short-term disruptive events.     

Pathology and preliminary characterization of a parapoxvirus isolated from a California sea lion (Zalophus californianus)

Cutaneous pox-like lesions are a common complication in the rehabilitation of pinnipeds. However, the exact identity, taxonomy, and host range of pinniped parapoxviruses remain unknown. During a poxvirus outbreak in May 2003 in California sea lions (Zalophus californianus) at a marine mammal rehabilitation facility, multiple raised, firm, 1-3-cm skin nodules from the head, neck, and thorax of one sea lion weanling pup that spontaneously died were collected. Histologically, the nodules were characterized by inflammation and necrosis of the dermis and epidermis, acanthosis, and ballooning degeneration of the stratum spinosum. Large, coalescing eosinophilic cytoplasmic inclusions were observed in the ballooned cells. A parapoxvirus (sea lion poxvirus 1, SLPV-1) was isolated on early passage California sea lion kidney cells inoculated with a tissue homogenate of a skin nodule. The morphology of the virions on electron microscopy was consistent with that of parapoxviruses. Partial sequencing of the genomic region encoding the putative major virion envelope antigen p42K confirmed the assignment of the sea lion poxvirus to the genus Parapoxvirus. Although SLPV-1 is most closely related to the poxvirus of harbor seals of the European North Sea, it is significantly different from orf virus, bovine papular stomatitis virus, pseudocowpox virus and the parapoxvirus of New Zealand red deer.     

Aerial hearing thresholds and detection of hearing loss in male California sea lions (Zalophus californianus) using auditory evoked potentials

Auditory evoked potential (AEP) measurements are useful for describing the variability of hearing among individuals in marine mammal populations, an important consideration in terms of basic biology and the design of noise mitigation criteria. In this study, hearing thresholds were measured for 16 male California sea lions at frequencies ranging from 0.5 to 32 kHz using the auditory steady state‐response (ASSR), a frequency‐specific AEP. Audiograms for most sea lions were grossly similar to previously reported psychophysical data in that hearing sensitivity increased with increasing frequency up to a steep reduction in sensitivity between 16 and 32 kHz. Average thresholds were not different from AEP thresholds previously reported for male and female California sea lions. Two sea lions from the current study exhibited abnormal audiograms: a 26‐yr‐old sea lion had impaired hearing with a high‐frequency hearing limit (HFHL) between 8 and 16 kHz, and an 8‐yr‐old sea lion displayed elevated thresholds across most tested frequencies. The auditory brainstem responses (ABRs) for these two individuals and an additional 26‐yr‐old sea lion were aberrant compared to those of other sea lions. Hearing loss may have fitness implications for sea lions that rely on sound during foraging and reproductive activities.     

Salmonellae in feral pinnipeds off the Southern California coast

Rectal swabs were collected from 90 Northern fur seal (Callorhinus ursinus) and 50 California sea lion (Zalophus californianus) pups on San Miguel Island for Salmonella screening. Three serotypes (newport, heidelberg, and oranienburg) were recovered from 33% of the fur seals and 40% of the sea lions.     

Changes in the feeding ecology of South American sea lions on the southern Brazilian coast over the last two decades of excessive fishing exploration

In the last decades, an increasing fishing effort and a decreasing trend in fish catches have been observed in southern Brazil. Considering that marine mammals and fisheries usually compete for the same resources, it is reasonable to presume that the feeding ecology of these predators is affected by the current scenario. To evaluate this hypothesis, long-term variation in the diet of the South American sea lion (Otaria flavescens) relative to fisheries exploitation was analyzed for two periods (1993–2003 versus 2004–2014). The degree of overlap between the relative biomass of the sea lions’ diet and the target species of six types of local fishery was analyzed. An increase in prey overlap between sea lions and fisheries was observed in the more recent sampling period, along with an increase in prey diversity, richness, and niche breadth of the sea lions’ diet. These results suggest that the overfishing scenario could partly explain the modified feeding ecology of the sea lions. In this context, we recommend a review and better regulation of the current fishing effort in the region, which we believe will be an important step to maintain the fish stocks and minimize the impact of fishing on marine top predators.     

Trace Metal Concentrations in the Hair of Zalophus californianus Pups and their Relation to Feeding Habits

The levels of six trace metals (Hg, Se, Pb, Cu, Fe, and Zn) were measured in the hair of California sea lion pups from eight rookeries in the Gulf of California, with the aim of relating these levels with the animals feeding habits, trophic level, and delta(15)N signatures at each locality. Trace metal levels were seen to vary significantly between sample sites as were tendencies associated with feeding habits. Mercury concentrations showed a clear positive correlation with trophic level (r = 0.73, p = 0.06) as well as the delta(15)N values of the rookeries (r = 0.79, p = 0.033). This was explainable via the process of biomagnification, although other elements did not follow the same tendency. High Cu concentrations in the hair (18 and 47 microg g(-1); ANOVA, p < 0.05) were observed at San Pedro Mártir and San Esteban, where the consumption of squid is higher than at other localities (approximately 17% and approximately 27% of the diet, respectively). Se and Hg concentrations correlated positively (r = 0.73, p = 0.038), probably the result of detoxification processes, a phenomenon previously reported in marine mammals. In light of the high degree of philopatry among sea lions in the Gulf and their pups' dependence exclusively upon their mother's milk during the first months of life, the analysis of trace metals in the easily sampled hair of the pups, offers an indirect way of monitoring the toxicology of the sea lion populations.     

First isolation of a calicivirus from the Steller sea lion (Eumetopias jubatus)

A calicivirus was isolated from the rectum of a Steller sea lion (Eumetopias jubatus) pup on Rogue Reef, off the southern Oregon coast. Based on the results of neutralization tests with specific typing antisera, the isolate was identified as San Miguel sea lion virus serotype 6 (SMSV-6). Blood obtained from nine of 37 pups (24%) during virus sample collection procedures had specific neutralizing antibodies to SMSV-6. The isolation of SMSV-6 from a Steller sea lion represents, to our knowledge, the first isolation of any virus from this widely distributed marine mammal species, and serves to reconfirm the host-nonspecificity of yet another calicivirus of marine origin.