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.

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.

Systemic mycosis in a California sea lion (Zalophus californianus) with detection of cystofilobasidiales DNA.

A 6-yr-old, intact male California sea lion (Zalophus californianus) with a systemic mycosis died after 5 wk of antifungal drug therapy. Antemortem clinical findings included hind flipper swelling, ring-lesions on skin of the flippers, and dermal nodules that increased in size and number spreading from the hind flippers and ventral abdomen to the foreflippers and muzzle. Lesions were accompanied by severe lymphadenopathy and development of systemic clinical signs despite therapy using itraconazole and later voriconazole. Histopathologic evaluation of biopsies revealed granulomatous dermatitis due to infection by fungus-producing yeast cells in tissue. Isolation attempts, using biopsied skin and tissue samples collected at necropsy, failed to yield growth of a fungus producing yeast cells like those in histologic section. Consensus polymerase chain reaction (PCR) tests of biopsied skin for fungal DNA produced an amplicon having significant sequence identity with a Cystofilobasidiales, a fungus belonging to a subclade that includes several Cryptococcus spp. Histopathologic evaluation of necropsy tissues revealed a systemic mycosis with yeast cells disseminated throughout subcutis, lymph nodes, and viscera. Hepatic necrosis was identified associated with acute liver failure, possibly from the voriconazole administration. This is the first report documenting the clinical presentation, treatment, and pathologic findings of infection associated with Cystofilobasidiales in a marine mammal and serves to expand the understanding of mycoses in pinnipeds.

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.

Neuroimmunological response of beluga whales (Delphinapterus leucas) to translocation and a novel social environment.

This study assessed changes in phagocyte function and activation of the sympatho-adrenal medullary and hypothalamo-pituitary adrenal axes of beluga whales (Delphinapterus leucas) in response to translocation and introduction to a novel social environment. Transported belugas exhibited increases in epinephrine (E), norepinephrine (NE), and cortisol levels in response to the translocation process. In response to the introduction of the transported belugas, resident belugas exhibited an increase in E and NE but not cortisol. Moreover, the increase in E and NE shown by the transported belugas was significantly greater than the increase exhibited by the resident belugas. Resident belugas exhibited a concomitant decrease in neutrophil and monocyte phagocytosis associated with the introduction of the transported belugas. In contrast, transported belugas exhibited an attendant increase in phagocytosis and respiratory burst activity immediately following transport. Differences in phagocyte response may derive from differences in hormonal milieu, stressor modality and/or intensity, or phagocyte priming. Investigating the complex interactions between types of stressors, neuroendocrine response, and immunocompetence will lead to a better understanding of the impacts of environmental challenges, including anthropogenic perturbations, on the health of cetacean populations.

Development of microsatellite markers in Cordia bifurcata (Boraginaceae) and cross-species amplification in Cordia inermis and Cordia pringlei.

We developed 16 microsatellite markers in Cordia bifurcata, a Central and South American shrub. The markers show low polymorphism in C. bifurcata, a species suspected of self-fertilization or apomixis. Of four polymorphic loci, three had only two alleles. However, current research indicates that these markers hold value for interpopulational comparisons of C. bifurcata and for analyses of congeners. In Cordia inermis, a dioecious or subdioecious shrub, seven of the markers produced interpretable amplification products of which five showed polymorphism. In Cordia pringlei, a distylous shrub, nine of the markers produced interpretable amplification products of which six showed polymorphism.