Caroline E. C. Goertz

Particulate and soluble hexavalent chromium are cytotoxic and genotoxic to Steller sea lion lung cells

Hexavalent chromium is an environmental contaminant. Within the environment, marine waters are a common site for hexavalent chromium deposition. We have recently reported significantly high levels of chromium in skin tissue from North Atlantic right whales. These findings demonstrate that marine species are being exposed to chromium. It is possible that such exposures may be playing a role in population declines evident among certain marine mammals, such as the Steller sea lion. We developed a Steller sea lion lung cell line from Steller sea lion lung tissue. Hexavalent chromium was cytotoxic to these primary lung fibroblasts as 1, 2.5, 5, 10 and 25microM sodium chromate induced 104, 99, 92, 58 and 11% relative survival, respectively. It was also genotoxic as 0, 1, 2.5, 5 and 10microM sodium chromate damaged chromosomes in 6, 11, 21, 36, and 39% of metaphases and damaged 6, 12, 27, 49 and 57 total aberrations in 100 metaphases, respectively. We also considered the toxicity of particulate hexavalent chromium, as it is the more potent carcinogen in humans. We found that 0.1, 0.5, 1, 5 and 10microg/cm(2) particulate chromate induced 95, 88, 91, 70, and 52% relative cell survival, respectively. These concentrations were genotoxic and damaged chromosomes in 9, 13, 18, and 23% of metaphases and induced 9, 15, 20 and 30 total aberrations per 100 metaphases, respectively. These data indicate that if sufficiently exposed, chromium may adversely affect the struggling Steller sea lion population. It would be prudent to investigate the effects chromium has in other Steller sea lion organs in order to derive a better understanding of how chromium in the marine environment may be affecting the declining Steller sea lion population.

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.

Baseline hearing abilities and variability in wild beluga whales (Delphinapterus leucas)

While hearing is the primary sensory modality for odontocetes, there are few data addressing variation within a natural population. This work describes the hearing ranges (4–150 kHz) and sensitivities of seven apparently healthy, wild beluga whales (Delphinapterus leucas) during a population health assessment project that captured and released belugas in Bristol Bay, Alaska. The baseline hearing abilities and subsequent variations were addressed. Hearing was measured using auditory evoked potentials (AEPs). All audiograms showed a typical cetacean U-shape; substantial variation (>30 dB) was found between most and least sensitive thresholds. All animals heard well, up to at least 128 kHz. Two heard up to 150 kHz. Lowest auditory thresholds (35–45 dB) were identified in the range 45–80 kHz. Greatest differences in hearing abilities occurred at both the high end of the auditory range and at frequencies of maximum sensitivity. In general, wild beluga hearing was quite sensitive. Hearing abilities were similar to those of belugas measured in zoological settings, reinforcing the comparative importance of both settings. The relative degree of variability across the wild belugas suggests that audiograms from multiple individuals are needed to properly describe the maximum sensitivity and population variance for odontocetes. Hearing measures were easily incorporated into field-based settings. This detailed examination of hearing abilities in wild Bristol Bay belugas provides a basis for a better understanding of the potential impact of anthropogenic noise on a noise-sensitive species. Such information may help design noise-limiting mitigation measures that could be applied to areas heavily influenced and inhabited by endangered belugas.

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.

Mycobacterium Marinum Dermatitis and Panniculitis with Chronic Pleuritis in a Captive White Whale (Delphinapterus Leucas) with Aortic Rupture

A 16-year-old female white whale, Delphinapterus leucas, died after nearly 18 months of chronic lymphopenia and pyogranulomatous dermatitis. Necropsy revealed rupture of the aorta with hemorrhage into the cranial mediastinum and between fascial planes of the ventral neck musculature. Multiple foci of ulcerative dermatitis and panniculitis were present across the thorax and abdomen and surrounded the genital folds. In addition, there was a chronic proliferative pleuritis with over 20 liters of histiocytic exudate in the thoracic cavity. Acid-fast bacteria consistent with Mycobacterium sp. were identified in sections of skin lesions and in cytospins of pleural exudate. Cultures of pleura and 1 skin lesion collected at necropsy yielded sparse growth of an acid-fast bacillus with colony characteristics and morphology consistent with Mycobacterium marinum. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis confirmed the presence of M. marinum DNA in samples of skin. This is the first documented occurrence of mycobacteriosis in a white whale and is a unique presentation of mycobacterial dermatitis and panniculitis with chronic pleuritis in a cetacean. The improved PCR-RFLP protocol utilized in this case unifies techniques from several protocols to differentiate between species of Nocardia and rapidly growing mycobacteria clinically relevant to aquatic animals.

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

ABSTRACT 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.

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.

NOVEL POXVIRUS INFECTION IN NORTHERN AND SOUTHERN SEA OTTERS (ENHYDRA LUTRIS KENYONI AND ENHYDRA LUTRIS NEIRIS), ALASKA AND CALIFORNIA, USA

Abstract 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.

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

Abstract 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.

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

Abstract:  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.