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.

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.

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.

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.

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.

Clinicoimmunopathologic findings in Atlantic bottlenose dolphins Tursiops truncatus with positive Chlamydiaceae antibody titers.

Sera from free-ranging Atlantic bottlenose dolphins Tursiops truncatus inhabiting the Indian River Lagoon (IRL), Florida, and coastal waters of Charleston (CHS), South Carolina, USA, were tested for antibodies to Chlamydiaceae as part of a multidisciplinary study of individual and population health. A suite of clinicoimmunopathologic variables was evaluated in Chlamydiaceae-seropositive dolphins (n = 43) and seronegative healthy dolphins (n = 83). Fibrinogen, lactate dehydrogenase, amylase, and absolute numbers of neutrophils, lymphocytes, and basophils were significantly higher, and serum bicarbonate, total alpha globulin, and alpha-2 globulin were significantly lower in dolphins with positive Chlamydiaceae titers compared with seronegative healthy dolphins. Several differences in markers of innate and adaptive immunity were also found. Concanavalin A-induced T lymphocyte proliferation, lipopolysaccharide-induced B lymphocyte proliferation, and granulocytic phagocytosis were significantly lower, and absolute numbers of mature CD 21 B lymphocytes, natural killer cell activity and lysozyme concentration were significantly higher in dolphins with positive Chlamydiaceae antibody titers compared to seronegative healthy dolphins. Additionally, dolphins with positive Chlamydiaceae antibody titers had significant increases in ELISA antibody titers to Erysipelothrix rhusiopathiae. These data suggest that Chlamydiaceae infection may produce subclinical clinicoimmunopathologic perturbations that impact health. Any potential subclinical health impacts are important for the IRL and CHS dolphin populations, as past studies have indicated that both dolphin populations are affected by other complex infectious and neoplastic diseases, often associated with immunologic perturbations and anthropogenic contaminants.

Molt-associated changes in hematologic and plasma biochemical values and stress hormone levels in African penguins (Spheniscus demersus).

Handling, including blood collection, has often been discouraged in molting penguins because it is considered an additional stress imposed on birds already experiencing major physiologic stress associated with molting. To evaluate the degree of physiologic stress posed by molting, we compared the hematologic and plasma biochemical values and hormone levels of molting and nonmolting African penguins, Spheniscus demersus. Five male and 5 female penguins randomly chosen were given complete physical examinations, were weighed, and blood samples were taken at 7 time points before, during, and after the molt. Data were analyzed by linear mixed-model analysis of variance. Throughout the study, behavior and appetite remained normal. Catecholamine levels were highly variable within and among subjects, whereas mean corticosterone levels were significantly different between baseline, molt, and postmolt values. Significant differences from baseline values were observed in many of the hematologic analytes; however, only decreases in hematocrit and red blood cell count values were considered clinically significant. Anemia due to experimentally induced blood loss as a possible cause of the significant hematologic changes was ruled out based on results of a follow-up control study during the nonmolt season, which showed no significant changes in hematocrit level or total red blood cell counts when using similar sampling protocols, which indicates that these changes were associated with molt.

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.

Serologic response in bottlenose dolphins Tursiops truncatus infected with Brucella sp. using a dolphin-specific indirect ELISA.

Marine-origin Brucella infections and serologic evidence of exposure have been documented in multiple cetacean species. A dolphin-specific indirect enzyme-linked immunosorbent assay (ELISA) was developed to screen bottlenose dolphin sera for anti-Brucella antibodies. A total of 131 serum samples collected over a 2 to 18 yr period from 6 bottlenose dolphins Tursiops truncatus with confirmed Brucella infections were analyzed for the presence and magnitude of antibody titers against marine-origin Brucella to compare individual antibody responses to various disease manifestations. Additionally, an epidemiologic serologic survey of a managed population of 64 bottlenose dolphins was performed to evaluate for the presence of antibodies and to determine whether there were any clinical pathology predictors for exposure or infection. The serologic results revealed that the dolphins with Brucella-associated abortions were seronegative for 7 to 18 yr until after the abortion and maintained positive titers for several years, with 2 of 3 animals returning to seronegative status. In contrast, the dolphins with Brucella-associated pulmonary or bone lesions maintained persistent positive titers for 2 to 18 yr. The population serosurvey revealed no significant differences in antibody levels among males and females, and dolphins between the ages of 17 and 25 yr were 6.8 times more likely to be Brucella antibody positive compared to those that were younger or older. Seropositive dolphins did not have significant inflammation compared to seronegative dolphins but were more likely to have higher levels of aspartate aminotransferase and gamma-glutamyl transpeptidase. Among 16 dolphins that tested seropositive, 13 (81.3%) had previously been seropositive for at least 3 to 5 yr.

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.

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.

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.

Clinicoimmunopathologic findings in Atlantic bottlenose dolphins Tursiops truncatus with positive cetacean morbillivirus antibody titers.

Sera from free-ranging Atlantic bottlenose dolphins Tursiops truncatus inhabiting the Indian River Lagoon (IRL), Florida were tested for antibodies to cetacean morbilliviruses from 2003 to 2007 as part of a multidisciplinary study of individual and population health. A suite of clinicoimmunopathologic variables were evaluated in morbillivirus-seropositive dolphins (n = 14) and seronegative healthy dolphins (n = 49). Several important differences were found. Serum alkaline phosphatase, creatine phosphokinase, chloride, albumin and albumin/globulin ratios were significantly lower in seropositive dolphins. Innate immunity appeared to be upregulated with significant increases in lysozyme concentration and marginally significant increases in monocytic phagocytosis. Adaptive immunity was also impacted in dolphins with positive morbillivirus antibody titers. Mitogen-induced T lymphocyte proliferation responses were significantly reduced in dolphins with positive morbillivirus antibody titers, and marginally significant decreases were found for absolute numbers of CD4+ lymphocytes. The findings suggest impairment of cell-mediated adaptive immunity, similar to the immunologic pattern reported with acute morbillivirus infection in other species. In contrast, dolphins with positive morbillivirus antibody titers appeared to have at least a partially upregulated humoral immune response with significantly higher levels of gamma globulins than healthy dolphins, which may represent an antibody response to morbillivirus infection or other pathogens. These data suggest that subclinical dolphin morbillivirus infection in IRL dolphins may produce clinicoimmunopathologic perturbations that impact overall health.

Evaluation of an alternative to feeding whole frozen fish in belugas (Delphinapterus leucas).

Feeding fish to captive piscivores can be challenging owing to cost, availability, variability in nutrient, and caloric composition, as well as handling and storage concerns. This trial evaluated the response of three belugas to being fed Fish Analog, an alternative to frozen fish. Body condition, gut transit time, serum chemistry and metabolic hormone analytes, immune function, and behavioral motivation were the dependent variables. Belugas (n=3) were fed various levels of Fish Analog (0-50%) over a 6-month period, and follow-up studies were conducted to further examine several dependent variables. When provided in gradually increasing amounts, belugas consumed the Fish Analog, with only minor fecal consistency changes and without behavioral responses indicative of gastric discomfort. Axillary girth and blubber thickness were positively correlated, and did not differ significantly with changes in the percentage of Fish Analog fed. Individual animal variation in initial passage time, some serum chemistry analytes, and immune function differences were noted following feeding of Fish Analog. Feeding Fish Analog reduced blood n9 fatty acids compared with captive belugas fed no Fish Analog. Feeding a DHA-enriched Fish Analog increased several n3 fatty acids, including eicosapentaenoic acid, but not DHA, compared with whales fed no Fish Analog or non-DHA-enriched Fish Analog. Fish Analog was shown to be a viable alternative to feeding fish at up to 50% of the dietary caloric density.

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.

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.

A dolphin peripheral blood leukocyte cDNA microarray for studies of immune function and stress reactions.

A microarray focused on stress response and immune function genes of the bottlenosed dolphin has been developed. Random expressed sequence tags (ESTs) were isolated and sequenced from two dolphin peripheral blood leukocyte (PBL) cDNA libraries biased towards T- and B-cell gene expression by stimulation with IL-2 and LPS, respectively. A total of 2784 clones were sequenced and contig analysis yielded 1343 unigenes (archived and annotated at ). In addition, 52 dolphin genes known to be important in innate and adaptive immune function and stress responses of terrestrial mammals were specifically targeted, cloned and added to the unigene collection. The set of dolphin sequences printed on a cDNA microarray comprised the 1343 unigenes, the 52 targeted genes and 2305 randomly selected (but unsequenced) EST clones. This set was printed in duplicate spots, side by side, and in two replicates per slide, such that the total number of features per microarray slide was 19,200, including controls. The dolphin arrays were validated and transcriptomic profiles were generated using PBL from a wild dolphin, a captive dolphin and dolphin skin cells. The results demonstrate that the array is a reproducible and informative tool for assessing differential gene expression in dolphin PBL and in other tissues.

Characterization of the immunoglobulin A heavy chain gene of the Atlantic bottlenose dolphin (Tursiops truncatus).

Immunoglobulin constant region heavy chain genes of the dolphin (Tursiops truncatus) have been described for IgM and IgG but not for IgA. Here, the heavy chain sequence of dolphin IgA has been cloned and sequenced as cDNA. RT-PCR amplification from blood peripheral lymphocytes was carried out using degenerate primers and a single sequence was detected. The inferred heavy chain structure shows conserved features typical of mammalian IgA heavy chains, including three constant (C) regions, a hinge region between constant region domain 1 (C1) and constant region domain 2 (C2), and conserved residues for interaction with the Fc alpha R1 and N-glycosylation sites. Comparisons of the deduced amino acid sequences of the IgA heavy chain for the dolphin and the evolutionarily related artiodactyl species showed high similarity. In cattle and sheep, as in dolphins, a single IgA subclass has been identified. Southern blot analysis as well as genomic PCR confirmed the presence of multiple IGHA sequences suggesting that IGHA pseudogenes may be present in the dolphin genome.

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.