HISTOLOGIC ANALYSIS OF TESTICULAR DEVELOPMENT AND SEXUAL MATURATION IN REHABILITATED NORTHERN SEA OTTERS (ENHYDRA LUTRIS KENYONI).

As part of routine captive management of the species, sea otter (Enhydra lutris) males are often castrated to prevent reproduction, to preserve limited spaces for future nonreleasable stranded individuals, and to minimize potential aggression among cohorts. To determine the relative stage of testicular development, testicles from 14 castrated, rehabilitated northern sea otters (Enhydra lutris kenyoni) were submitted for histologic examination. Eight of the otters (aged 201, 304, 344, 352, 360, 373, 401, and 1,423 d old) had evidence of varying degrees of sexual maturity. Histologic grading of maturity of individual testicles showed inactive testes, spermatocytes with some maturation of spermatogenic precursors, and fully active spermatogenesis. Spermatozoa were seen in the otters that were 401 and 1,423 d old (1.1 and 3.9 yr old). Sexual maturity for wild male sea otters in Alaska has been previously reported to occur from 3 to 5-6 yr. Social maturity, or the ability to breed and reproduce, may occur a few years later than the onset of physiologic maturity; age, weight, territory quality, and the length of time holding a territory may influence a male otter's mating success. Early testicular development in rehabilitated sea otters may be related to abundant resources, lack of competition, and decreased environmental pressures. Additionally, these findings have implications for husbandry and management practices in short- and long-term care facilities.

Differential Progression of Lymphoma in Two Captive Steller's Eiders (Polysticta stelleri).

This report details 2 different presentations of lymphoma in captive Steller's eiders (Polysticta stelleri). A female Steller's eider, at least 10 years old, developed lameness and lethargy. A complete blood cell count (CBC) revealed a severely elevated total white blood cell (WBC) count with a lymphocytosis. A subsequent liver biopsy confirmed a diagnosis of lymphoma and lymphocytic leukemia. The female Steller's eider was euthanatized 11 days after presentation. On necropsy, neoplastic lymphoid infiltrates were present in multiple tissues, primarily in the skin and subcutis. The results of a CBC from an apparently healthy male Steller's eider, at least 14 years old, also indicated this bird had an increased WBC count with a lymphocytosis. Serum submitted for protein electrophoresis indicated that this bird had a monoclonal gammopathy. Lymphocytes isolated from the male Steller's eider had low levels of proliferation in response to mitogens. The bird survived nearly 2 years without treatment, and the WBC count continued to increase during this time. The male Steller's eider maintained good body condition and exhibited normal behavior throughout the course of the disease until a sudden decline just prior to death. Lymphoma was diagnosed based on histopathology results. Infiltrates of atypical lymphocytes were observed in the bone marrow, proventriculus, ventriculus, heart, and eyes.

Complete genome sequence of a novel sea otterpox virus.

Members of the Poxviridae family are large, double-stranded DNA viruses that replicate in the cytoplasm of their host cells. The subfamily Chordopoxvirinae contains viruses that infect a wide range of vertebrates including marine mammals within the Balaenidae, Delphinidae, Mustelidae, Odobenidae, Otariidae, Phocidae, and Phocoenidae families. Recently, a novel poxvirus was found in a northern sea otter pup (Enhydra lutris kenyoni) that stranded in Alaska in 2009. The phylogenetic relationships of marine mammal poxviruses are not well established because of the lack of complete genome sequences. The current study sequenced the entire sea otterpox virus Enhydra lutris kenyoni (SOPV-ELK) genome using an Illumina MiSeq sequencer. The SOPV-ELK genome is the smallest poxvirus genome known at 127,879 bp, is 68.7% A+T content, is predicted to encode 132 proteins, and has 2546 bp inverted terminal repeats at each end. Genetic and phylogenetic analyses based on the concatenated amino acid sequences of 7 chorodopoxvirus core genes revealed the SOPV-ELK is 52.5-74.1% divergent from other known chordopoxviruses and is most similar to pteropoxvirus from Australia (PTPV-Aus). SOPV-ELK represents a new chordopoxvirus species and may belong to a novel genus. SOPV-ELK encodes eight unique genes. While the function of six predicted genes remains unknown, two genes appear to function as novel immune-modulators. SOPV-ELK-003 appears to encode a novel interleukin-18 binding protein (IL-18 BP), based on limited sequence and structural similarity to other poxviral IL-18 BPs. SOPV-ELK-035 appears to encode a novel tumor necrosis factor receptor-like (TNFR) protein that may be associated with the depression of the host's antiviral response. Additionally, SOPV-ELK-036 encodes a tumor necrosis factor-like apoptosis-inducing ligand (TRAIL) protein that has previously only been found in PTPV-Aus. The SOPV-ELK genome is the first mustelid poxvirus and only the second poxvirus from a marine mammal to be fully sequenced. Sequencing of the SOPV-ELK genome is an important step in unraveling the position of marine mammal poxviruses within the larger Poxviridae phylogenetic tree and provides the necessary sequence to develop molecular tools for future diagnostics and epidemiological studies.

Brucella Infection in Asian Sea Otters (Enhydra lutris lutris) on Bering Island, Russia.

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.

INFECTIOUS DISEASE AND TOXICOLOGICAL MONITORING OF STRANDED PACIFIC HARBOR SEALS (PHOCA VITULINA RICHARDSI) IN COOK INLET AS SURROGATES FOR MONITORING ENDANGERED BELUGAS (DELPHINAPTERUS LEUCAS).

Pacific harbor seals ( Phoca vitulina richardsi) and belugas ( Delphinapterus leucas ) eat many of the same prey species, occupy the same geographic area, and demonstrate site fidelity in Cook Inlet, Alaska. Although most direct research involving the critically endangered belugas is currently prohibited, studying harbor seals may provide important information about this beluga population. In recent years, harbor seal populations in Alaska have declined for unknown reasons. As part of its stranding program, the Alaska SeaLife Center (ASLC) managed 59 cases of live and dead stranded harbor seals from Cook Inlet between 1997 and 2011. Animals were screened for a variety of diseases and contaminants of concern. Animals were negative by serology to the following diseases: avian influenza, canine distemper virus, dolphin morbillivirus, porpoise morbillivirus, Leptospira canicola, L. grippotyphosa, L. pomona, Neospora caninum , Sarcocystis neurona , and Toxoplasma gondii . Positive titers were found against Brucella spp., phocine distemper virus, seal herpesvirus-1, L. bratislava, L. hardjo, and L. icterohemorrhagiae. All titers were stable or declining except in one animal with an increasing titer for seal herpesvirus-1. Fecal pathogen screenings identified normal flora as well as stable or declining low levels of potentially pathogenic and opportunistic bacteria, though most were of little concern for seal health. In most animals, toxicology screening showed that the majority of tested contaminants were below detectable limits. The level of evidence of exposure to pathogens of concern was low in harbor seals. Although the infectious disease burden and contaminant levels in belugas in Cook Inlet cannot be definitively determined without direct testing, pathogen and contaminant exposure is expected to be similar to that found in harbor seals in this region, as the harbor seals and belugas share the habitat and food resources.

Esophageal squamous cell carcinoma in six harbor seals (Phoca vitulina spp.).

Six cases of esophageal squamous cell carcinoma were identified in six captive adult Pacific (Phoca vitulina richardsii; n = 2) and Atlantic (Phoca vitulina concolor; n = 4) harbor seals. These seals presented with intermittent dysphagia, regurgitation, inappetence, and abnormal posturing. Common clinical pathology findings in these seals included azotemia, hyperproteinemia, hyperglobulinemia, and leukocytosis. Gastrointestinal endoscopy commonly revealed an ulcerated mass near the gastroesophageal junction. Each seal was euthanized (n = 3) due to poor prognosis, subsequently died while undergoing an anesthetic procedure (n = 2), or found dead (n = 1). The diagnosis of squamous cell carcinoma was confirmed via biopsy of esophageal mucosa during endoscopy or histopathologic examination of affected tissues after necropsy. On the basis of clinical and postmortem findings, esophageal squamous cell carcinoma should be considered as a differential diagnosis in aged harbor seals exhibiting clinical signs of regurgitation, decreased appetite or anorexia, vomiting, and/or abnormal posturing.

Novel poxvirus infection in northern and southern sea otters (Enhydra lutris kenyoni and Enhydra lutris neiris), Alaska and California, USA.

Small superficially ulcerated skin lesions were observed between October 2009 and September 2011 during captive care of two orphaned sea otter pups: one northern (Enhydra lutris kenyoni) in Alaska and one southern (Enhydra lutris nereis) in California. Inclusions consistent with poxviral infection were diagnosed by histopathology in both cases. Virions consistent with poxvirus virions were seen on electron microscopy in the northern sea otter, and the virus was successfully propagated in cell culture. DNA extraction, pan-chordopoxviral PCR amplification, and sequencing of the DNA-dependent DNA polymerase gene revealed that both cases were caused by a novel AT-rich poxvirus. Bayesian and maximum likelihood phylogenetic analyses found that the virus is divergent from other known poxviruses at a level consistent with a novel genus. These cases were self-limiting and did not appear to be associated with systemic illness. To our knowledge, this is the first report of a poxvirus in a mustelid species. The source of this virus, mode of transmission, zoonotic potential, and biological significance are undetermined.

Genotypic characterization of Streptococcus infantarius subsp. coli isolates from sea otters with infective endocarditis and/or septicemia and from environmental mussel samples.

Pulsed-field gel electrophoresis (PFGE) was used to type 128 Streptococcus infantarius subsp. coli isolates from sea otters and mussels. Six SmaI PFGE groups were detected, with one predominant group representing 57% of the isolates collected over a wide geographic region. Several sea otter and mussel isolates were highly related, suggesting that an environmental infection source is possible.

Vitamin A deficiency and hepatic retinol levels in sea otters, Enhydra lutris.

Vitamin A deficiency has rarely been reported in captive or free-ranging wildlife species. Necropsy findings in two captively housed southern sea otters (Enhydra lutris nereis) included irregular thickening of the calvaria characterized by diffuse hyperostoses on the internal surface. One animal also had moderate squamous metaplasia of the seromucinous glands of the nose. There was no measurable retinol in the liver of either sea otter. For comparison, hepatic retinol concentration was determined for 23 deceased free-ranging southern and northern (Enhydra lutris kenyoni) sea otters from California and Alaska. Free-ranging otters were found to have similar hepatic retinol concentrations (316 +/- 245 mg/kg wet weight) regardless of their location and subspecies. All of these values were significantly higher than the levels in the affected animals. Consumption of a diet with very low vitamin A concentrations and noncompliance in daily supplementation are hypothesized as the causes of vitamin A deficiency in these two sea otters.

Chemical immobilization of Weddell seals (Leptonychotes weddellii) by ketamine/midazolam combination.

OBJECTIVE: To provide reliable, effective immobilization for Weddell seals under extreme field conditions using an injectable ketamine/midazolam combination. STUDY DESIGN: Observational study. ANIMALS: Thirty adult Weddell seals (12 male, 18 female) in Erebus Bay, Antarctica, body mass (mean +/- SD) 412 +/- 47 kg, aged 9-27 years. METHODS: Seals were immobilized with a target dose of 2 mg kg(-1) ketamine hydrochloride and 0.1 mg kg(-1) midazolam hydrochloride (IM), based on visually estimated body mass. When required, maintenance doses were administered at a target of 0.5 mg kg(-1) ketamine hydrochloride and 0.025 mg kg(-1) midazolam hydrochloride (IV). RESULTS: Complete immobilization was achieved in 33 of 40 injections (14 of which were repeat events on the same individual). Time to immobilization averaged 12 +/- 4 minutes, with a duration of initial immobility of 38 +/- 19 minutes. Total immobilization time varied by handling protocol, including condition assessment and muscle biopsy (Protocol 1, 60 +/- 13 minutes), condition assessment and instrument attachment (Protocol 2, 154 +/- 13 minutes), and condition assessment, muscle biopsy and instrument retrieval (Protocol 3, 48 +/- 8 minutes). Overall, a total immobilization time of 114 +/- 60 minutes was accomplished with 4 +/- 4 maintenance doses, and an average recovery time of 36 +/- 17 minutes. Most effects of the anesthetic combination were unrelated to mass, age, sex or total body fat. However, leaner seals had longer duration of initial immobility (% and kg total body fat) and recovery times (kg fat). Apnea events were uncommon and treated effectively with doxapram. No animals died. CONCLUSIONS AND CLINICAL RELEVANCE: Reliable and effective field immobilization of Weddell seals was accomplished with a low dose of ketamine hydrochloride and midazolam hydrochloride, utilizing IM injection initially and IV maintenance methods.

Intraperitoneal implantation of life-long telemetry transmitters in otariids.

BACKGROUND: Pinnipeds, including many endangered and declining species, are inaccessible and difficult to monitor for extended periods using externally attached telemetry devices that are shed during the annual molt. Archival satellite transmitters were implanted intraperitoneally into four rehabilitated California sea lions (Zalophus californianus) and 15 wild juvenile Steller sea lions (Eumetopias jubatus) to determine the viability of this surgical technique for the deployment of long-term telemetry devices in otariids. The life history transmitters record information throughout the life of the host and transmit data to orbiting satellites after extrusion following death of the host. RESULTS: Surgeries were performed under isoflurane anesthesia and single (n = 4) or dual (n = 15) transmitters were inserted into the ventrocaudal abdominal cavity via an 8.5 to 12 cm incision along the ventral midline between the umbilicus and pubic symphysis or preputial opening. Surgeries lasted 90 minutes (SD = 8) for the 19 sea lions. All animals recovered well and were released into the wild after extended monitoring periods from 27 to 69 days at two captive animal facilities. Minimum post-implant survival was determined via post-release tracking using externally attached satellite transmitters or via opportunistic re-sighting for mean durations of 73.7 days (SE = 9.0, Z. californianus) and 223.6 days (SE = 71.5, E. jubatus). CONCLUSION: The low morbidity and zero mortality encountered during captive observation and post-release tracking periods confirm the viability of this surgical technique for the implantation of long-term telemetry devices in otariids.

Effects of ACTH, capture, and short term confinement on glucocorticoid concentrations in harlequin ducks (Histrionicus histrionicus).

Little is known about baseline concentrations of adrenal hormones and hormonal responses to stress in sea ducks, although significant population declines documented in several species suggest that sea ducks are exposed to increased levels of environmental stress. Such declines have been observed in geographically distinct harlequin duck populations. We performed an adrenocorticotropic hormone (ACTH) challenge to evaluate adrenal function and characterize corticosterone concentrations in captive harlequin ducks and investigated the effects of capture, surgery, and short term confinement on corticosterone concentrations in wild harlequin ducks. Harlequin ducks responded to the ACTH challenge with an average three-fold increase in serum corticosterone concentration approximately 90 min post injection, and a four- to five-fold increase in fecal glucocorticoid concentration 2 to 4 h post injection. Serum corticosterone concentrations in wild harlequin ducks increased within min of capture and elevated levels were found for several hours post capture, indicating that surgery and confinement maintain elevated corticosterone concentrations in this species. Mean corticosterone concentrations in wild harlequin ducks held in temporary captivity were similar to the maximum response levels during the ACTH challenge in captive birds. However, large variation among individuals was observed in responses of wild birds, and we found additional evidence suggesting that corticosterone responses varied between hatch year and after hatch year birds.

Assessment of ultrasound imaging as a noninvasive measure of blubber thickness in pinnipeds.

Portable ultrasound imaging was examined as a noninvasive measure of skin including blubber thickness in captive subadult Steller sea lions (Eumetopias jubatus) and adult harbor seals (Phoca vitulina). This method was validated through comparison with blubber biopsy. Ultrasound images provided depth measurements that were accurate to 99.8% of the actual. This method allowed clear images of the epidermis, dermis, and blubber layer to be rapidly obtained, with minimal animal restraint, and allowed differentiation of phocid and otariid blubber structure.