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.

Characterization of Current Husbandry and Veterinary Care Practices of the Giant Pacific Octopus (Enteroctopus dofleini) Using an Online Survey.

Giant Pacific octopuses (Enteroctopus dofleini) (GPOs) are commonly housed in zoos or aquaria, and sedation, anesthesia, and/or euthanasia may be indicated for a variety of reasons. Despite this need, evidence-based data on best practices is limited and focuses on smaller or more tropical species. The objectives of this study were to survey the aquatic community regarding the husbandry and veterinary care of GPOs, with a specific focus on anesthetic and euthanasia protocols. A two-part web-based survey was distributed to four aquatic and/or veterinary email listservs. Individuals from fifty-two institutions participated in phase one. Results documented that 40 (78 percent) participating institutions currently house GPOs, with most housing one and nine institutions housing two to three GPOs. The median (range) habitat volume is 5405 (1893-16,465) L, and 78 percent of systems are closed. Of the institutions surveyed, 23 have anesthetized or sedated a GPO for nonterminal procedures, including wound care, biopsies, and hemolymph collection. Reported methods of sedation or anesthesia include magnesium chloride, ethanol, isoflurane, tricaine methanesulfonate (MS-222), magnesium sulfate, benzocaine, and dexmedetomidine. Drugs or methods used for euthanasia include magnesium chloride, ethanol, mechanical decerebration, pentobarbital, isoflurane, MS-222, magnesium sulfate, benzocaine, potassium chloride, dexmedetomidine, and freezing. Reported observed side effects include ineffectiveness or inadequate sedation, inking, prolonged drug effects, and behavior changes. Survey data have the potential to guide the husbandry and veterinary care of GPOs and build the framework for future prospective studies on GPO sedation and anesthesia.

Serological Detection of Marine Origin Brucella Exposure in Two Alaska Beluga Stocks.

Among emerging threats to the Arctic is the introduction, spread, or resurgence of disease. Marine brucellosis is an emerging disease concern among free-ranging cetaceans and is less well-studied than terrestrial forms. To investigate marine-origin Brucella sp. exposure in two beluga stocks in Alaska, USA, this study used serological status as well as real-time polymerase chain reaction (rtPCR) and bacterial culture. In total, 55 live-captured-released belugas were tested for Brucella exposure in Bristol Bay (2008-2016) and 112 (8 live-captured; 104 subsistence-harvested) whales were tested in the eastern Chukchi Sea (2007-2017). In total, 73% percent of Bristol Bay live captures, 50% of Chukchi Sea live captures, and 66% of Chukchi Sea harvested belugas were positive on serology. Only 10 of 69 seropositive belugas were rtPCR positive in at least one tissue. Only one seropositive animal was PCR positive in both the spleen and mesenteric lymph node. All animals tested were culture negative. The high prevalence of seropositivity detected suggests widespread exposure in both stocks, however, the low level of rtPCR and culture positive results suggests clinical brucellosis was not prevalent in the belugas surveyed. Continued detection of Brucella exposure supports the need for long-term monitoring of these and other beluga populations.

Evaluating beluga (Delphinapterus leucas) blow samples as a potential diagnostic for immune function gene expression within the respiratory system.

Evaluating respiratory health is important in the management of cetaceans, which are vulnerable to respiratory diseases. Quantifying the expression of genes related to immune function within the respiratory tract could be a valuable tool for directly assessing respiratory health. Blow (exhale) samples allow DNA analysis, and we hypothesized that RNA could also be isolated from blow samples for gene expression studies of immune function. We evaluated the potential to extract RNA from beluga blow samples and tested whether transcripts associated with immune function could be detected with endpoint polymerase chain reaction. A total of 54 blow samples were collected from clinically healthy aquarium belugas (n = 3), and 15 were collected from wild belugas temporarily restrained for health assessment in Bristol Bay, Alaska (n = 9). Although RNA yield varied widely (range, 0-265.2 ng; mean = 85.8; SD = 71.3), measurable RNA was extracted from 97% of the samples. Extracted RNA was assessed in 1-6 PCR reactions targeting housekeeping genes (Rpl8, Gapdh or ActB) or genes associated with immune function (TNFα, IL-12p40 or Cox-2). Fifty of the aquarium samples (93%) amplified at least one transcript; overall PCR success for housekeeping genes (96/110, 87%) and genes associated with immune function (90/104, 87%) were similarly high. Both RNA yield and overall PCR success (27%) were lower for wild beluga samples, which is most likely due to the reduced forcefulness of the exhale when compared with trained or free-swimming belugas. Overall, the high detection rate with PCR suggests measuring gene expression in blow samples could provide diagnostic information about immune responses within the respiratory tract. While further study is required to determine if quantitative gene expression data from blow samples is associated with disease states, the non-invasive nature of this approach may prove valuable for belugas, which face increasing anthropogenic disturbances.

INVESTIGATION OF THE USE OF SERUM BIOMARKERS FOR THE DETECTION OF CARDIAC DISEASE IN MARINE MAMMALS.

Cardiac disease has been extensively documented in marine mammals; however, it remains difficult to diagnose antemortem. Assays measuring cardiac troponin I (cTnI) and N-terminal pro-brain natriuretic peptide (NT-proBNP) are used as sensitive and specific biomarkers of cardiac disease in many species, but have not been widely investigated in marine mammals. This study aimed to provide a set of reference values for cTnI and NT-proBNP in belugas (BW) (Delphinapterus leucas), sea otters (SO) (Enhydra lutris), Steller sea lions (SSL) (Eumetopias jubatus), and California sea lions (CSL) (Zalophus californianus) with and without cardiac disease, and to determine if these biomarkers are useful indicators of cardiac disease in these species. First, existing immunoassays for cTnI and NT-proBNP were successfully validated utilizing species-specific heart lysate spiked serum. Cohorts were defined by histopathology as animals with no evidence of cardiac disease ("control"), with confirmed cardiac disease ("disease"), and with concurrent renal and cardiac disease ("renal") for which serum samples were then analyzed. Serum concentration ranges for cTnI (ng/ml) and NT-proBNP (pmol/L) were determined for control and disease cohorts. There was significantly higher cTnI (P= 0.003) and NT-proBNP (P= 0.004) concentrations in the CSL disease cohort, as well as positive trends in BW, SO, and SSL disease cohorts that did not reach statistical significance. NT-proBNP concentrations were significantly higher in the CSL renal cohort compared with the control (P < 0.001) and disease (P= 0.007) cohorts. These results suggest that cTnI and NT-proBNP may be clinically useful in the antemortem diagnosis of cardiac disease in CSL, and warrant further investigation in BW, SO, and SL.

Competency of common northern sea otter (Enhydra lutris kenyoni) prey items to harbor Streptococcus lutetiensis and S. phocae.

Streptococcus lutetiensis and S. phocae have been associated with significant morbidity and mortality in northern sea otters Enhydra lutris kenyoni in Alaska, USA, but the route and mechanism(s) of transmission remain unknown. The goal of this study was to determine the competence of common northern sea otter prey to harbor 2 species of pathogenic Streptococcus bacteria. Prey items (bay mussels Mytilus trossulus, butter clams Saxidomus giganteus, Dungeness crab Metacarcinus magister and black turban snails Tegula funebralis) were exposed to known concentrations of exponential phase cultures of S. lutetiensis and S. phocae in seawater for 24 h. A quantitative PCR assay was developed targeting the sodA gene of both S. lutetiensis and S. phocae to quantify DNA in the prey samples. Results (mean ± SD) revealed that butter clams had the highest concentration of bacteria (4.32 × 107 ± 8.20 × 106 CFU ml-1 of S. lutetiensis, 1.20 × 108 ± 2.08 × 107 CFU ml-1 of S. phocae), followed by mussels (4.26 × 107 ± 1.66 × 107 CFU ml-1, 1.16 × 108 ± 5.39 × 107 CFU ml-1), snails (1.90 × 107 ± 5.26 × 106 CFU ml-1, 5.97 × 107 ± 2.07 × 107 CFU ml-1) and crab (1.46 × 107 ± 0 CFU ml-1, 1.64 × 107 ± 0 CFU ml-1). All prey species harbored higher concentrations of S. phocae than S. lutetiensis.

Audiogram of a Cook Inlet beluga whale (Delphinapterus leucas).

Noise is a stressor to wildlife, yet the precise sound sensitivity of individuals and populations is often unknown or unmeasured. Cook Inlet, Alaska belugas (CIBs) are a critically endangered and declining marine mammal population. Anthropogenic noise is a primary threat to these animals. Auditory evoked potentials were used to measure the hearing of a wild, stranded CIB as part of its rehabilitation assessment. The beluga showed broadband (4-128 kHz) and sensitive hearing (<80 dB) for a wide-range of frequencies (16-80 kHz), reflective of a healthy odontocete auditory system. Data were similar to healthy, adult belugas from the comparative Bristol Bay population (the only other published data set of healthy, wild marine mammal hearing). Repeated October and December 2017 measurements were similar, showing continued auditory health of the animal throughout the rehabilitation period. Hearing data were compared to pile-driving and container-ship noise measurements made in Cook Inlet, two sources of concern, suggesting masking is likely at ecologically relevant distances. These data provide the first empirical hearing data for a CIB allowing for estimations of sound-sensitivity in this population. The beluga's sensitive hearing and likelihood of masking show noise is a clear concern for this population struggling to recover.

Skin microbiome of beluga whales: spatial, temporal, and health-related dynamics.

BACKGROUND: Host-specific microbiomes play an important role in individual health and ecology; in marine mammals, epidermal microbiomes may be a protective barrier between the host and its aqueous environment. Understanding these epidermal-associated microbial communities, and their ecological- or health-driven variability, is the first step toward developing health indices for rapid assessment of individual or population health. In Cook Inlet, Alaska, an endangered population of beluga whales (Delphinapterus leucas) numbers fewer than 300 animals and continues to decline, despite more than a decade of conservation effort. Characterizing the epidermal microbiome of this species could provide insight into the ecology and health of this endangered population and allow the development of minimally invasive health indicators based on tissue samples. RESULTS: We sequenced the hypervariable IV region of bacterial and archaeal SSU rRNA genes from epidermal tissue samples collected from endangered Cook Inlet beluga whales (n = 33) and the nearest neighboring population in Bristol Bay (n = 39) between 2012 and 2018. We examined the sequences using amplicon sequence variant (ASV)-based analyses, and no ASVs were associated with all individuals, indicating a greater degree of epidermal microbiome variability among beluga whales than in previously studied cetacean species and suggesting the absence of a species-specific core microbiome. Epidermal microbiome composition differed significantly between populations and across sampling years. Comparing the microbiomes of Bristol Bay individuals of known health status revealed 11 ASVs associated with potential pathogens that differed in abundance between healthy individuals and those with skin lesions or dermatitis. Molting and non-molting individuals also differed significantly in microbial diversity and the abundance of potential pathogen-associated ASVs, indicating the importance of molting in maintaining skin health. CONCLUSIONS: We provide novel insights into the dynamics of Alaskan beluga whale epidermal microbial communities. A core epidermal microbiome was not identified across all animals. We characterize microbial dynamics related to population, sampling year and health state including level of skin molting. The results of this study provide a basis for future work to understand the role of the skin microbiome in beluga whale health and to develop health indices for management of the endangered Cook Inlet beluga whales, and cetaceans more broadly.

Comparing progesterone in blubber and serum to assess pregnancy in wild beluga whales (Delphinapterus leucas).

The Cook Inlet population of beluga whales (Delphinapterus leucas) is listed as endangered and continues to decline for largely unknown reasons; however, there is some evidence that poor reproductive success is a contributing factor. Pregnancy is difficult to detect through observation, and, there is reluctance to capture endangered beluga whales for reproductive tract imaging via ultrasound or to obtain suitable samples for pregnancy assessments. An endocrine analysis of blubber biopsies collected by remote darting could represent a minimally invasive way to identify pregnant females and compare pregnancy rates among years or populations. Studies have validated the use of blubber biopsies to identify pregnant females in other cetacean species, but not beluga whales; therefore, validation of blubber progesterone levels to proven tests that reliably detect pregnancy was needed for this species. As part of a larger study, we sampled blood and blubber from live-captured beluga whales (21 females, 26 males) in Bristol Bay, Alaska. Progesterone levels were determined in serum samples obtained from all animals and in blubber samples from a subset (14 females, 13 males) to determine pregnancy status, estimate the stage of pregnancy, and evaluate the suitability of using blubber alone for these assessments. In general, there was distinct separation of high levels of progesterone in serum and blubber for presumed pregnant females and low levels for males and presumed non-pregnant females. Blubber progesterone levels in two females (14% of females tested) were intermediate (i.e. ambiguous); their corresponding serum levels were consistent with being pregnant in one case and not being pregnant in the other. Except for these two intermediate values, pregnancy status of beluga whales could be determined from blubber alone, thereby providing a valuable tool to better understand reproduction dynamics from populations that cannot be captured for examination.

Comparative Health Assessments of Alaskan Ice Seals.

Bearded (Erignathus barbatus), ringed (Pusa hispida), spotted (Phoca largha), and ribbon (Histriophoca fasciata) seals rely on seasonal sea-ice in Arctic and sub-Arctic regions. Many aspects of the biology and physiology of these seals are poorly known, and species-typical health parameters are not available for all species. Such information has proven difficult to obtain due to the challenges of studying Arctic seals in the wild and their minimal historic representation in aquaria. Here, we combine diagnostic information gathered between 2000 and 2017 from free-ranging seals, seals in short-term rehabilitation, and seals living in long-term human care to evaluate and compare key health parameters. For individuals in apparent good health, hematology, and blood chemistry values are reported by the source group for 10 bearded, 13 ringed, 73 spotted, and 81 ribbon seals from Alaskan waters. For a smaller set of individuals handled during veterinary or necropsy procedures, the presence of parasites and pathogens is described, as well as exposure to a variety of infectious diseases known to affect marine mammals and/or humans, with positive titers observed for Brucella, Leptospira, avian influenza, herpesvirus PhHV-1, and morbillivirus. These data provide initial baseline parameters for hematology, serum chemistries, and other species-level indicators of health that can be used to assess the condition of individual seals, inform monitoring and management efforts, and guide directed research efforts for Alaskan populations of ice-associated seals.

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.

Seasonal and developmental differences in blubber stores of beluga whales in Bristol Bay, Alaska using high-resolution ultrasound.

Diving mammals use blubber for a variety of structural and physiological functions, including buoyancy, streamlining, thermoregulation, and energy storage. Estimating blubber stores provides proxies for body condition, nutritional status, and health. Blubber stores may vary topographically within individuals, across seasons, and with age, sex, and reproductive status; therefore, a single full-depth blubber biopsy does not provide an accurate measure of blubber depth, and additional biopsies are limited because they result in open wounds. We examined high-resolution ultrasound as a noninvasive method for assessing blubber stores by sampling blubber depth at 11 locations on beluga whales in Alaska. Blubber mass was estimated as a proportion of body mass (40% from the literature) and compared to a function of volume calculated using ultrasound blubber depth measurements in a truncated cone. Blubber volume was converted to total and mass-specific blubber mass estimates based on the density of beluga blubber. There was no significant difference in mean total blubber mass between the 2 estimates (R2 = 0.88); however, body mass alone predicted only 68% of the variation in mass-specific blubber stores in juveniles, 7% for adults in the fall, and 33% for adults in the spring. Mass-specific blubber stores calculated from ultrasound measurements were highly variable. Adults had significantly greater blubber stores in the fall (0.48±0.02kg/kgMB) than in the spring (0.33±0.02kg/kgMB). There was no seasonal effect in juveniles. High-resolution ultrasound is a more powerful, noninvasive method for assessing blubber stores in wild belugas, allowing for precise measurements at multiple locations.

CIRCULATING CONCENTRATIONS OF THYROID HORMONE IN BELUGA WHALES (DELPHINAPTERUS LEUCAS): INFLUENCE OF AGE, SEX, AND SEASON.

Thyroid hormones play a critical physiologic role in regulating protein synthesis, growth, and metabolism. To date, because no published compilation of baseline values for thyroid hormones in beluga whales (Delphinapterus leucas) exists, assessment of thyroid hormone concentrations in this species has been underused in clinical settings. The purpose of this study was to document the concentrations of total thyroxine (tT4) and total triiodothyronine (tT3) in healthy aquarium-maintained and free-ranging beluga whales and to determine the influence of age, sex, and season on the thyroid hormone concentrations. Archived serum samples were collected from healthy aquarium-maintained (n=43) and free-ranging (n=39) belugas, and serum tT4 and tT3 were measured using chemiluminescence immunoassay. The mean tT4 concentration in aquarium-maintained belugas was 5.67±1.43 µg/dl and the mean tT3 concentration was 70.72±2.37 ng/dl. Sex comparisons showed that aquarium-maintained males had significantly greater tT4 and tT3 (9.70±4.48 µg/dl and 92.65±30.55 ng/dl, respectively) than females (7.18±2.82 µg/dl and 77.95±20.37 ng/dl) (P=0.004 and P=0.013). Age comparisons showed that aquarium-maintained whales aged 1-5 yr had the highest concentrations of tT4 and tT3 (8.17±0.17 µg/dl and 105.46±1.98 ng/dl, respectively) (P=0.002 and P<0.001). tT4 concentrations differed significantly between seasons, with concentrations in winter (4.59±1.09 µg/dl) being significantly decreased compared with spring (P=0.009), summer (P<0.0001), and fall (P<0.0001) concentrations. There was a significant difference in tT4 and tT3 concentrations between aquarium-maintained whales (5.67±1.43 µg/dl and 70.72±15.57 ng/dl, respectively) and free-ranging whales (11.71±3.36 µg/dl and 103.38±26.45 ng/dl) (P<0.0001 and P<0.001). Clinicians should consider biologic and environmental influences (age, sex, and season) for a more accurate interpretation of thyroid hormone concentrations in belugas. The findings of this study provide a baseline for thyroid health monitoring and comprehensive health assessments in both aquarium-maintained and free-ranging beluga whales.

Morbidity and mortality in stranded Cook Inlet beluga whales Delphinapterus leucas.

The endangered Cook Inlet (Alaska, USA) stock of beluga whales Delphinapterus leucas declined 47% between 1994 and 1998, from an estimated 653 whales to 347 whales, with a continued decline to approximately 312 in 2012. Between 1998 and 2013, 164 known dead strandings were reported by the National Marine Fisheries Service. Only 38 of these animals, or 23% of the known stranded carcasses, were necropsied. Carcasses were found between April and October. The majority of animals necropsied were adults (n=25), followed by juveniles (n=6), calves (n=3), and aborted fetuses (n=4). Eight of the 11 mature females were pregnant, post-partum, or lactating. Many (82%) of these belugas were in moderate to advanced autolysis, which hampered determination of a cause of death (COD). Each animal had a single primary COD assigned within a broad set of categories. The CODs were unknown (29%), trauma (18%), perinatal mortality (13%), mass stranding (13%), single stranding (11%), malnutrition (8%), or disease (8%). Other disease processes were coded as contributory or incidental to COD. Multiple animals had mild to moderate verminous pneumonia due to Stenurus arctomarinus, renal granulomas due to Crassicauda giliakiana, and ulcerative gastritis due to Anisakis sp. Each stranding affords a unique opportunity to obtain natural history data and evidence of human interactions, and, by long-term monitoring, to characterize pathologies of importance to individual and population health.

Blow collection as a non-invasive method for measuring cortisol in the beluga (Delphinapterus leucas).

Non-invasive sampling techniques are increasingly being used to monitor glucocorticoids, such as cortisol, as indicators of stressor load and fitness in zoo and wildlife conservation, research and medicine. For cetaceans, exhaled breath condensate (blow) provides a unique sampling matrix for such purposes. The purpose of this work was to develop an appropriate collection methodology and validate the use of a commercially available EIA for measuring cortisol in blow samples collected from belugas (Delphinapterus leucas). Nitex membrane stretched over a petri dish provided the optimal method for collecting blow. A commercially available cortisol EIA for measuring human cortisol (detection limit 35 pg ml-1) was adapted and validated for beluga cortisol using tests of parallelism, accuracy and recovery. Blow samples were collected from aquarium belugas during monthly health checks and during out of water examination, as well as from wild belugas. Two aquarium belugas showed increased blow cortisol between baseline samples and 30 minutes out of water (Baseline, 0.21 and 0.04 µg dl-1; 30 minutes, 0.95 and 0.14 µg dl-1). Six wild belugas also showed increases in blow cortisol between pre and post 1.5 hour examination (Pre 0.03, 0.23, 0.13, 0.19, 0.13, 0.04 µg dl-1, Post 0.60, 0.31, 0.36, 0.24, 0.14, 0.16 µg dl-1). Though this methodology needs further investigation, this study suggests that blow sampling is a good candidate for non-invasive monitoring of cortisol in belugas. It can be collected from both wild and aquarium animals efficiently for the purposes of health monitoring and research, and may ultimately be useful in obtaining data on wild populations, including endangered species, which are difficult to handle directly.

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.

Seasonal hematology and serum chemistry of wild beluga whales (Delphinapterus leucas) in Bristol Bay, Alaska, USA.

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.

Brucella sp. vertebral osteomyelitis with intercurrent fatal Staphylococcus aureus toxigenic enteritis in a bottlenose dolphin (Tursiops truncatus).

A previously beach-stranded, juvenile, male, bottlenose dolphin (Tursiops truncatus) was diagnosed with vertebral osteomyelitis of unknown etiology. Antemortem serological testing suggested past or current Brucella sp. infection; however, this could not be confirmed prior to death despite multiple isolation attempts from aspirates, blood, and biopsies. Systemic antibiotics were administered for over a year to control the suspected infection; however, the animal succumbed peracutely to infection by a highly pathogenic, enterotoxin-secreting Staphylococcus sp. Gross necropsy findings included a fistulous tract leading to locally extensive osteomyelitis of a coccygeal vertebra with sequestra and osteophytes from which a Brucella species was isolated. Histopathological examination of intestine revealed pseudomembranous enteritis with a uniform population of intraluminal Gram-positive cocci. Staphylococcus aureus was isolated in pure culture from the intestine and tested positive for the staphylococcal enterotoxin A gene by polymerase chain reaction analysis. Serum taken shortly before death had endotoxin and elevated antibody titers to staphylococcal enterotoxin A when compared to samples collected during a period of apparent good health 18 months earlier. The isolation of a pyrogenic toxin superantigen-producing staphylococcal isolate, clinical signs, and diagnostic findings in this animal resembled some of those noted in human toxic shock syndrome. The present case highlights the clinical challenges of treating chronic illnesses, complications of long-term antibiotic use, and promotion of pathogenic strains in cases of prolonged rehabilitation of marine mammals.

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.