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.

Social Network and Behavioral Synchrony Influences On Maternal and Infant Cortisol Response.

This study examined the contributions of mothers' social network stability and mother-infant behavioral synchrony on cortisol response in infants and their mothers during separation. The quality and stability of mothers' social network system and mother-infant bond have both been shown to affect infant neuroendocrine response. Yet, no studies have directly addressed how these two forms of social relationships might differentially affect infants' and mothers' neuroendocrine responses during separation. First-time mothers (N = 133) and their 3-month-old infants participated in the study. Maternal social network stability, mother-infant behavioral synchrony, and mother and infant cortisol response during an infant challenge task were assessed. Behavioral synchrony accounted for significant variance in infants' cortisol response, and after adjusting for synchrony, mothers' network stability measures did not explain variance in infant cortisol. Social network stability, but not synchrony, accounted for significant variance in mothers' cortisol response. These results demonstrate that, when mothers and infants experience brief separation, the quality of their bond is associated with a lower stress response for infants; but for mothers, it is the longevity of her social relationships outside of the mother-infant relationship context that is associated with her lower stress response.

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.

"Does your baby watch TV?": The associations between at-home TV watching and laboratory challenge cortisol are different for young infants and their mothers.

This longitudinal study examined associations between at-home TV watching and the biological stress response (cortisol) during a laboratory infant cognitive challenge task in 240 3- and 5-month infants and their mothers. Cortisol levels were lower in mothers of 5-month-old infants whose infants were exposed to TV at home, compared to mothers of infants that were not TV-exposed. Cortisol patterns were different across three laboratory sampling intervals for 3-month-old infants as a function of TV watching, revealing a sharp increase in laboratory cortisol only for infants who were exposed to TV at home. In contrast, there was no effect of TV exposure in 5-month-old infants' cortisol. Infant temperament and demographic measures were included as control variables in regression models to predict maternal and infant cortisol. At 3 months, for the T3 cortisol sampling interval, and at 5 months across all three sampling intervals, maternal cortisol levels were significantly predicted by infant TV exposure after accounting for variance due to these control variables. Our findings show the strong influence of the infant TV-exposure factor in the biological stress response of mothers of young infants, and suggest that infant TV exposure may influence self-regulation in 3-month-old infants.

Variation in the hemostatic complement (C5a) responses to in vitro nitrogen bubbles in monodontids and phocids.

Immune responses to nitrogen gas bubbles, particularly activation of inflammation via the complement cascade, have been linked to the development of symptoms and damage associated with decompression sickness (DCS) in humans. Marine mammals were long thought not to be susceptible to such dive-related injury, yet evidence of DCS-like injury and new models of tissue nitrogen super-saturation suggest that bubbles may routinely form. As such, it is possible that marine mammals have protective adaptations that allow them to deal with a certain level of bubble formation during normal dives, without acute adverse effects. This work evaluated the complement response, indicative of inflammation, to in vitro nitrogen bubble exposures in several marine mammal species to assess whether a less-responsive immune system serves a protective role against DCS-like injury in these animals. Serum samples from beluga (Delphinapterus leucas), and harbor seals (Phoca vitulina) (relatively shallow divers) and deep diving narwhal (Monodon monoceros), and Weddell seals (Leptonychotes weddellii) were exposed to nitrogen bubbles in vitro. Complement activity was evaluated by measuring changes in the terminal protein C5a in serum, and results suggest marine mammal complement is less sensitive to gas bubbles than human complement, but the response varies between species. Species-specific differences may be related to dive ability, and suggest moderate or shallow divers may be more susceptible to DCS-like injury. This information is an important consideration in assessing the impact of changing dive behaviors in response to anthropogenic stressors, startle responses, or changing environmental conditions that affect prey depth distributions.

RADIOGRAPHIC DETERMINATION OF GASTRIC EMPTYING AND GASTROINTESTINAL TRANSIT TIME IN COWNOSE RAYS (RHINOPTERA BONASUS) AND WHITESPOTTED BAMBOO SHARKS (CHILOSCYLLIUM PLAGIOSUM) AND THE EFFECT OF METOCLOPRAMIDE ON GASTROINTESTINAL MOTILITY.

Gastrointestinal (GI) pathology is common in elasmobranchs; however, information regarding normal GI transit time and the effect of therapeutics on GI motility is lacking. The objective of this study was to determine baseline gastric emptying and GI transit times in cownose rays (Rhinoptera bonasus) and whitespotted bamboo sharks (Chiloscyllium plagiosum) via radiographic barium sulfate contrast studies. Additionally, a pilot study was undertaken to determine the effect of metoclopramide on GI transit time in whitespotted bamboo sharks. Eight cownose rays and eight whitespotted bamboo sharks were administered a 98% w/w barium sulfate suspension at 8 ml/kg via orogastric tube. Post-contrast radiographs were obtained at 2 min, 3, 6, 12, and 23 hr for rays; and 2 min, 3, 6, 9, 12, 16, 25, 30, 36, and every 12 hr until complete gastric emptying occurred for sharks. In cownose rays, the mean and standard error were established for time of initial spiral colon filling (3.4 ± 0.4 hr), complete spiral colon opacification (12 ± 0 hr), initial spiral colon emptying (21.6 ± 1.4 hr), and complete gastric emptying (23 ± 0 hr). In bamboo sharks, the mean and standard error were established for time of initial spiral colon filling (5.3 ± 0.5 hr), complete spiral colon opacification (12.4 ± 1.3 hr), initial spiral colon emptying (22.5 ± 2.7 hr), and complete gastric emptying (39.9 ± 3.3 hr). Cownose rays had a significantly shorter time to spiral colon filling and complete gastric emptying compared with bamboo sharks (P < 0.05). Whitespotted bamboo sharks (n = 8) were administered metoclopramide (0.4 mg/kg orally once daily for 10 days) and the barium series was repeated. Complete gastric emptying time was significantly shorter in treated sharks compared with control (P < 0.05), suggesting that metoclopramide may be a useful therapeutic for GI motility disorders in elasmobranchs.

Behavioral responses of beluga whales (Delphinapterus leucas) to environmental variation in an Arctic estuary.

Some Arctic estuaries serve as substrate rubbing sites for beluga whales (Delphinapterus leucas) in the summer, representing a specialized resource for the species. Understanding how environmental variation affects the species' behavior is essential to management of these habitats in coming years as the climate changes. Spatiotemporal and environmental variables were recorded for behavioral observations, during which focal groups of whales in an estuary were video-recorded for enumeration and behavioral analysis. Multiple polynomial linear regression models were optimized to identify the effects of spatiotemporal and environmental conditions on group size, composition, and the frequency of behaviors being performed. Results suggest that belugas take advantage of environmental variation to express behaviors that 1) protect young, e.g., bringing calves close to shore during cloudier days, obscuring visualization from terrestrial predators; 2) avoid predation, e.g., rubbing against substrates at higher Beaufort sea states to obscure visualization, and resting during low tides while swimming on outgoing tides to avoid stranding; and 3) optimize bioenergetic resources, e.g., swimming during lower Beaufort sea states and clearer days. Predictive models like the ones presented in this study can inform conservation management strategies as environmental conditions change in future years.

Prenatal maternal cortisol measures predict learning and short-term memory performance in 3- but not 5-month-old infants.

Little is known about relations between maternal prenatal stress and specific cognitive processes-learning and memory-in infants. A modified crib-mobile task was employed in a longitudinal design to test relations between maternal prenatal cortisol, prenatal subjective stress and anxiety, psychosocial variables, and learning and memory in 3- and 5-month-old infants. Results revealed that maternal prenatal cortisol was affected by particular psychosocial variables (e.g., maternal age, whether or not the infant's grandmother provided childcare, financial status), but was unrelated to measures of maternal depression, anxiety, and stress. Although maternal prenatal cortisol was not predictive of learning or memory performance in 5-month-old infants, higher levels of basal maternal cortisol and reduced prenatal cortisol response was predictive of some learning and short-term memory measures in 3-month-old infants. These results suggest an influence of maternal neuroendocrine functioning on fetal neurological development, and the importance of separate examination of subjective and biological measures of stress.

Pressure Induced Changes in Adaptive Immune Function in Belugas (Delphinapterus leucas); Implications for Dive Physiology and Health.

Increased pressure, associated with diving, can alter cell function through several mechanisms and has been shown to impact immune functions performed by peripheral blood mononuclear cells (PBMC) in humans. While marine mammals possess specific adaptations which protect them from dive related injury, it is unknown how their immune system is adapted to the challenges associated with diving. The purpose of this study was to measure PBMC activation (IL2R expression) and Concanavalin A induced lymphocyte proliferation (BrdU incorporation) in belugas following in vitro pressure exposures during baseline, Out of Water Examination (OWE) and capture/release conditions. Beluga blood samples (n = 4) were obtained from animals at the Mystic Aquarium and from free ranging animals in Alaska (n = 9). Human blood samples (n = 4) (Biological Specialty Corporation) were run for comparison. In vivo catecholamines and cortisol were measured in belugas to characterize the neuroendocrine response. Comparison of cellular responses between controls and pressure exposed cells, between conditions in belugas, between belugas and humans as well as between dive profiles, were run using mixed generalized linear models (α = 0.05). Cortisol was significantly higher in Bristol Bay belugas and OWE samples as compared with baseline for aquarium animals. Both IL2R expression and proliferation displayed significant pressure induced changes, and these responses varied between conditions in belugas. Both belugas and humans displayed increased IL2R expression, while lymphocyte proliferation decreased for aquarium animals and increased for humans and Bristol Bay belugas. Results suggest beluga PBMC function is altered during diving and changes may represent dive adaptation as the response differs from humans, a non-dive adapted mammal. In addition, characteristics of a dive (i.e., duration, depth) as well as neuroendocrine activity can alter the response of beluga cells, potentially impacting the ability of animals to fight infection or avoid dive related pathologies.

Recurrence, retreatment, and rebleed rates of coiled aneurysms with respect to the Raymond-Roy scale: a meta-analysis.

BACKGROUND AND PURPOSE: The Raymond-Roy grading scale is used for aneurysm coiling with only limited data on its validity. The scale was developed based on the extent of initial aneurysm occlusion from 1 to 3. However, the model usefulness in evaluating recurrence, retreatment, and rebleeding is unknown. Our goal was to perform a meta-analysis to evaluate the predictiveness of the Raymond scale. METHODS: We performed a systematic review of the English literature for aneurysm coiling which reported the initial embolization results, based on the Raymond-Roy grading scale, and the respective recurrence rates, retreatment rates, and rebleed rates. This yielded data for 4587 aneurysms. We conducted a Bayesian random effects meta-analysis to evaluate the outcomes with respect to the reported initial embolization results. RESULTS: We found the Raymond scale to be predictive of retreatment, with statistically higher rates of retreatment with higher initial Raymond grade. Furthermore, we found a higher probability of rebleeding for initial grades 2 or 3 versus grade 1, which approached significance. The rebleed rates were probably affected by monitoring and treatment of recurrence. However, although there was a trend towards higher recurrence rates with initial grade, this was not statistically significant. CONCLUSIONS: The modified Raymond-Roy scale appears to provide reasonable predictive value for treated aneurysm, especially for the clinically more important aspects of retreatment and rebleed rates.

Self-regulation and working memory in musical performers.

Performing music in front of others can be stressful, even for experienced performers. The physiological effects of stress, namely, increases in cortisol and sympathetic nervous system activity, have been shown to have detrimental effects on cognition, particularly working memory. This study used an audition-like performance scenario to elicit a stress response in performers who differed in their degree of musical experience. We expected that participants with more musical experience would be better able to regulate their stress response, would report lower levels of anxiety, insecurity, and nervousness, and would show better working memory following the stressor, compared to participants with less musical experience. Although we did not find differences between more and less experienced performers in their sympathetic nervous system activity or their self-reported feelings of anxiety and nervousness, we did find some important differences: following the stressor, more experienced performers were less insecure, they showed better regulation of their cortisol response, and they demonstrated better working memory.

Beluga (Delphinapterus leucas) granulocytes and monocytes display variable responses to in vitro pressure exposures.

While it is widely known that marine mammals possess adaptations which allow them to make repetitive and extended dives to great depths without suffering ill effects seen in humans, the response of marine mammal immune cells to diving is unknown. Renewed interest in marine mammal dive physiology has arisen due to reports of decompression sickness-like symptoms and embolic damage in stranded and by-caught animals, and there is concern over whether anthropogenic activities can impact marine mammal health by disrupting adaptive dive responses and behavior. This work addresses the need for information concerning marine mammal immune function during diving by evaluating granulocyte and monocyte phagocytosis, and granulocyte activation in belugas (n = 4) in comparison with humans (n = 4), with and without in vitro pressure exposures. In addition, the potential for additional stressors to impact immune function was investigated by comparing the response of beluga cells to pressure between baseline and stressor conditions. Granulocyte and monocyte phagocytosis, as well as granulocyte activation, were compared between pressure exposed and non-exposed cells for each condition, between different pressure profiles and between conditions using mixed generalized linear models (α = 0.05). The effects of pressure varied between species as well by depth, compression/decompression rates, and length of exposures, and condition for belugas. Pressure induced changes in granulocyte and monocyte function in belugas could serve a protective function against dive-related pathologies and differences in the response between humans and belugas could reflect degrees of dive adaptation. The alteration of these responses during physiologically challenging conditions may increase the potential for dive-related in jury and disease in marine mammals.

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.