Fecal glucocorticoid analysis as a health monitoring tool for endangered African penguins (Spheniscus demersus).

African penguins (Spheniscus demersus) are an endangered species, with approximately 70,000 mature adults remaining in the wild. Population loss is linked to a combination of environmental and anthropogenic stressors. The aim of the study was to validate a commercially available enzyme immunoassay (EIA) to assess adrenal activity and measure the response to stressors in the feces of African penguins. Fecal samples (n = 609) were collected from 12 African penguins housed at Mystic Aquarium throughout their natural lifecycle, including breeding and molt, where measurable changes in fecal glucocorticoid metabolite (FGM) levels are predicted to occur. Fecal samples collected post-veterinary exam were used for biological validation. Longitudinal analysis shows a significant difference (p = <0.0001) between the average FGM levels during baseline and breeding season, 33.97 ± 1.30 ng/g and 50.21 ± 3.18 ng/g, respectively. Females displayed significantly higher FGM levels than males during both baseline (p = 0.0386; females = 38.80 ± 2.19 ng/g; males = 29.34 ± 1.37 ng/g) and breeding periods (p = 0.0175; females = 57.53 ± 4.84 ng/g; males = 42.69 ± 3.95 ng/g). Average FGM levels decreased significantly over the three-week molting period, from 85.40 ± 20.35 ng/g at week one to 20.23 ± 5.30 ng/g at week three. A seasonal difference in FGM levels was observed in both male and female fecal samples, with Fall having the highest average FGM levels, 54.38 ± 3.64 ng/g, and Summer the lowest, 30.87 ± 2.21 ng/g. General linear mixed model analysis determined that lifecycle (females) and visitor presence (males) were the two factors which best explained the variation in FGM levels observed, however neither factor was found to be significant. These results show FGM analysis can detect physiologically meaningful changes in endocrine activity in African penguins and can be used to monitor health for penguins in aquaria and in the wild, thus contributing to conservation efforts for the survival of the species.

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.

Longitudinal analysis of pinnipeds in the northwest Atlantic provides insights on endemic circulation of phocine distemper virus.

Phocine distemper virus (PDV) is a morbillivirus that circulates within pinnipeds in the North Atlantic. PDV has caused two known unusual mortality events (UMEs) in western Europe (1988, 2002), and two UMEs in the northwest Atlantic (2006, 2018). Infrequent cross-species transmission and waning immunity are believed to contribute to periodic outbreaks with high mortality in western Europe. The viral ecology of PDV in the northwest Atlantic is less well defined and outbreaks have exhibited lower mortality than those in western Europe. This study sought to understand the molecular and ecological processes underlying PDV infection in eastern North America. We provide phylogenetic evidence that PDV was introduced into northwest Atlantic pinnipeds by a single lineage and is now endemic in local populations. Serological and viral screening of pinniped surveillance samples from 2006 onward suggest there is continued circulation of PDV outside of UMEs among multiple species with and without clinical signs. We report six full genome sequences and nine partial sequences derived from harbour and grey seals in the northwest Atlantic from 2011 through 2018, including a possible regional variant. Work presented here provides a framework towards greater understanding of how recovering populations and shifting species may impact disease transmission.

Characterizing the culturable surface microbiomes of diverse marine animals.

Biofilm-forming bacteria have the potential to contribute to the health, physiology, behavior and ecology of the host and serve as its first line of defense against adverse conditions in the environment. While metabarcoding and metagenomic information furthers our understanding of microbiome composition, fewer studies use cultured samples to study the diverse interactions among the host and its microbiome, as cultured representatives are often lacking. This study examines the surface microbiomes cultured from three shallow-water coral species and two whale species. These unique marine animals place strong selective pressures on their microbial symbionts and contain members under similar environmental and anthropogenic stress. We developed an intense cultivation procedure, utilizing a suite of culture conditions targeting a rich assortment of biofilm-forming microorganisms. We identified 592 microbial isolates contained within 15 bacterial orders representing 50 bacterial genera, and two fungal species. Culturable bacteria from coral and whale samples paralleled taxonomic groups identified in culture-independent surveys, including 29% of all bacterial genera identified in the Megaptera novaeangliae skin microbiome through culture-independent methods. This microbial repository provides raw material and biological input for more nuanced studies which can explore how members of the microbiome both shape their micro-niche and impact host fitness.

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.

SERUM BIOCHEMICAL AND HEMATOLOGIC REFERENCE INTERVALS FOR WEANLING NORTHWEST ATLANTIC GRAY SEALS (HALICHOERUS GRYPUS).

Baseline health parameters are limited in the primary literature for gray seals (Halichoerus grypus) in the northwest Atlantic. Accurate normal physiologic reference ranges for both species and specific geographic populations are vital tools for assessing the health of individuals and understanding the health of the entire population. This study developed comprehensive reference intervals for biochemical and hematologic parameters of recently weaned gray seal pups on Cape Cod, Massachusetts from samples collected in 2013, 2016, and 2017. Reference ranges were developed using methodology outlined by the American Society of Clinical Veterinary Pathology. By establishing more comprehensive biochemical and hematologic reference ranges for this population based on a robust sample size, this study provides a new tool for clinicians, researchers, and rehabilitation organizations to improve individual patient care and population research.

Comparative Innate and Adaptive Immune Responses in Atlantic Bottlenose Dolphins (Tursiops truncatus) With Viral, Bacterial, and Fungal Infections.

Free-ranging Atlantic bottlenose dolphins (n = 360) from two southeastern U.S. estuarine sites were given comprehensive health examinations between 2003 and 2015 as part of a multi-disciplinary research project focused on individual and population health. The study sites (and sample sizes) included the Indian River Lagoon (IRL), Florida, USA (n = 246) and Charleston harbor and associated rivers (CHS), South Carolina, USA (n = 114). Results of a suite of clinicoimmunopathologic tests revealed that both populations have a high prevalence of infectious and neoplastic disease and a variety of abnormalities of their innate and adaptive immune systems. Subclinical infections with cetacean morbillivirus and Chlamydiaceae were detected serologically. Clinical evidence of orogenital papillomatosis was supported by the detection of a new strain of dolphin papillomavirus and herpesvirus by molecular pathology. Dolphins with cutaneous lobomycosis/lacaziasis were subsequently shown to be infected with a novel, uncultivated strain of Paracoccidioides brasiliensis, now established as the etiologic agent of this enigmatic disease in dolphins. In this review, innate and adaptive immunologic responses are compared between healthy dolphins and those with clinical and/or immunopathologic evidence of infection with these specific viral, bacterial, and fungal pathogens. A wide range of immunologic host responses was associated with each pathogen, reflecting the dynamic and complex interplay between the innate, humoral, and cell-mediated immune systems in the dolphin. Collectively, these studies document the comparative innate and adaptive immune responses to various types of infectious diseases in free-ranging Atlantic bottlenose dolphins. Evaluation of the type, pattern, and degree of immunologic response to these pathogens provides novel insight on disease immunopathogenesis in this species and as a comparative model. Importantly, the data suggest that in some cases infection may be associated with subclinical immunopathologic perturbations that could impact overall individual and population health.

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.

The environment as a driver of immune and endocrine responses in dolphins (Tursiops truncatus).

Immune and endocrine responses play a critical role in allowing animals to adjust to environmental perturbations. We measured immune and endocrine related markers in multiple samples from individuals from two managed-care care dolphin groups (n = 82 samples from 17 dolphins and single samples collected from two wild dolphin populations: Indian River Lagoon, (IRL) FL (n = 26); and Charleston, (CHS) SC (n = 19). The immune systems of wild dolphins were more upregulated than those of managed-care-dolphins as shown by higher concentrations of IgG and increases in lysozyme, NK cell function, pathogen antibody titers and leukocyte cytokine transcript levels. Collectively, managed-care care dolphins had significantly lower levels of transcripts encoding pro-inflammatory cytokine TNF, anti-viral MX1 and INFα and regulatory IL-10. IL-2Rα and CD69, markers of lymphocyte activation, were both lower in managed-care care dolphins. IL-4, a cytokine associated with TH2 activity, was lower in managed-care care dolphins compared to the free-ranging dolphins. Differences in immune parameters appear to reflect the environmental conditions under which these four dolphin populations live which vary widely in temperature, nutrition, veterinary care, pathogen/contaminant exposures, etc. Many of the differences found were consistent with reduced pathogenic antigenic stimulation in managed-care care dolphins compared to wild dolphins. Managed-care care dolphins had relatively low TH2 lymphocyte activity and fewer circulating eosinophils compared to wild dolphins. Both of these immunologic parameters are associated with exposure to helminth parasites which is uncommon in managed-care care dolphins. Less consistent trends were observed in a suite of hormones but significant differences were found for cortisol, ACTH, total T4, free T3, and epinephrine. While the underlying mechanisms are likely multiple and complex, the marked differences observed in the immune and endocrine systems of wild and managed-care care dolphins appear to be shaped by their environment.

Testosterone and progesterone concentrations in blow samples are biologically relevant in belugas (Delphinapterus leucas).

Steroid hormone analysis in blow (respiratory vapor) may provide a minimally invasive way to assess the reproductive status of wild cetaceans. Biological validation of the method is needed to allow for the interpretation of hormone measurements in blow samples. Utilizing samples collected from trained belugas (Delphinapterus leucas, n=20), enzyme immunoassays for testosterone and progesterone were validated for use with beluga blow samples. Testosterone concentrations in 40 matched blood and blow samples collected from 4 male belugas demonstrated a positive correlation (R2=0.52, p<0.0001). Progesterone concentrations in 64 matching blood and blow samples from 11 females were also positively correlated (R2=0.60, p<0.0001). Testosterone concentrations (mean±SD) in blow samples collected from adult males (119.3±14.2pg/ml) were higher (p<0.01) than that of a juvenile male (<8years) (59.4±6.5pg/ml) or female belugas (54.1±25.7pg/ml). Among adult males, testosterone concentrations in blow demonstrated a seasonal pattern of secretion, with peak secretion occurring during the breeding season (February-April, 136.95±33.8pg/ml). Progesterone concentrations in blow varied by reproductive status; pregnant females (410.6±87.8pg/ml) and females in the luteal phase of the estrous cycle (339.5±51.0pg/ml) had higher (p<0.0001) blow progesterone concentrations than non-pregnant females without a corpus luteum (242.5±27.3pg/ml). Results indicate that blow sample analysis can be used to detect variation in reproductive states associated with large differences in circulating testosterone or progesterone in belugas.

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.

Prevalence of influenza A virus in live-captured North Atlantic gray seals: a possible wild reservoir.

Influenza A virus (IAV) has been associated with multiple unusual mortality events (UMEs) in North Atlantic pinnipeds, frequently attributed to spillover of virus from wild-bird reservoirs. To determine if endemic infection persists outside of UMEs, we undertook a multiyear investigation of IAV in healthy, live-captured Northwest Atlantic gray seals (Halichoerus grypus). From 2013 to 2015, we sampled 345 pups and 57 adults from Cape Cod, MA, USA and Nova Scotia, Canada consistently detecting IAV infection across all groups. There was an overall viral prevalence of 9.0% (95% confidence interval (CI): 6.4%-12.5%) in weaned pups and 5.3% (CI: 1.2%-14.6%) in adults, with seroprevalences of 19.3% (CI: 15.0%-24.5%) and 50% (CI: 33.7%-66.4%), respectively. Positive sera showed a broad reactivity to diverse influenza subtypes. IAV status did not correlate with measures of animal health nor impact animal movement or foraging. This study demonstrated that Northwest Atlantic gray seals are both permissive to and tolerant of diverse IAV, possibly representing an endemically infected wild reservoir population.

Natural Variation in Stress Hormones, Comparisons Across Matrices, and Impacts Resulting from Induced Stress in the Bottlenose Dolphin.

Knowledge regarding stress hormones and how they vary in response to seasonality, gender, age, and reproductive status for any marine mammal is limited. Furthermore, stress hormones may be measured in more than one matrix (e.g., feces, blood, blubber), but the relationships between levels of a given hormone across these matrices are unknown, further complicating the interpretations of hormones measured in samples collected from wild animals. A study is underway to address these issues in a population of bottlenose dolphins trained for voluntary participation in sample collections from different matrices and across season and time of day.

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.

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.

Stress response of wild bottlenose dolphins (Tursiops truncatus) during capture-release health assessment studies.

There is a growing concern about the impacts of stress in marine mammals as they face a greater array of threats. The stress response of free-ranging dolphins (Tursiops truncatus) was examined by measuring their physiologic response to capture and handling. Samples were collected from 168 dolphins during capture-release health assessments 2003-2007 at two study sites: Charleston, SC (CHS) and the Indian River Lagoon, FL (IRL). Adrenocorticotropic hormone (ACTH), cortisol, aldosterone (ALD) and catecholamines (epinephrine (EPI), norepinephrine (NOR), dopamine (DA)), were measured in blood and cortisol in urine. Mean time to collect pre-examination samples after netting the animals was 22min; post-examination samples were taken prior to release (mean 1h 37min). EPI and DA concentrations decreased significantly with increased time to blood sampling. ACTH and cortisol levels increased from the initial capture event to the post-examination sample. EPI concentrations increased significantly with increasing time to the pre-examination sample and decreased significantly with time between the pre- and post-examination sample. Cortisol concentrations increased between the pre- and post-examination in CHS dolphins. Age- and sex-adjusted mean pre-examination values of catecholamines were significantly higher in CHS dolphins; ALD was higher in IRL dolphins. Significant differences related to age or sex included higher NOR concentrations in males; higher ALD and urine cortisol levels in juveniles than adults. Wild dolphins exhibited a typical mammalian response to acute stress of capture and restraint. Further studies that relate hormone levels to biological and health endpoints are warranted.

Validation of a commercial canine assay kit to measure pinniped cytokines.

The present study was conducted to assess and validate the cross-reactivity of commercially available multiplex human and canine cytokine kits coupled with the Bio-Plex 200 platform to measure cytokines in three pinniped species, harbor seals (Phoca vitulina), gray seals (Halichoerus grypus), and harp seals (Pagophilus groenlandicus). Cytokines are important small proteins that help direct a proper immune response to pathogens. The human cytokine kit allowed the detection of cytokines in the supernatant of mitogen-stimulated human peripheral blood mononuclear cells, but not in the three pinniped species studied, with the exception of TNFα and GM-CSF. In contrast, the canine cytokine kit appeared to cross-react with the majority of cytokines in the three pinniped species tested, including the pro-inflammatory cytokine TNFα, the Th1 cytokine INFγ, and the Th2 cytokine IL-10, an anti-inflammatory cytokine. In addition, the pro-inflammatory cytokines IL-6 and IL-8 were also measured in all pinniped species. Overall, the Bio-Plex 200 platform and the canine multiplex cytokine kit allowed the successful measurement of potentially clinically important pinniped cytokines. This additional tool may provide veterinarians with additional information to detect sub-clinical signs of inflammation or evidence for immune response, which may not be revealed during regular medical evaluation, e.g. physical examination, hematology, and serum chemistry.

Episodic and semantic components of autobiographical memories and imagined future events in post-traumatic stress disorder.

Individuals with post-traumatic stress disorder (PTSD) tend to retrieve autobiographical memories with less episodic specificity, referred to as overgeneralised autobiographical memory. In line with evidence that autobiographical memory overlaps with one's capacity to imagine the future, recent work has also shown that individuals with PTSD also imagine themselves in the future with less episodic specificity. To date most studies quantify episodic specificity by the presence of a distinct event. However, this method does not distinguish between the numbers of internal (episodic) and external (semantic) details, which can provide additional insights into remembering the past and imagining the future. This study employed the Autobiographical Interview (AI) coding scheme to the autobiographical memory and imagined future event narratives generated by combat veterans with and without PTSD. Responses were coded for the number of internal and external details. Compared to combat veterans without PTSD, those with PTSD generated more external than internal details when recalling past or imagining future events, and fewer internal details were associated with greater symptom severity. The potential mechanisms underlying these bidirectional deficits and clinical implications are discussed.