Oxylipin responses to fasting and insulin infusion in a large mammalian model of fasting-induced insulin resistance, the northern elephant seal.

The prolonged, postweaning fast of northern elephant seal (Mirounga angustirostris) pups is characterized by a reliance on lipid metabolism and reversible, fasting-induced insulin resistance, providing a unique model to examine the effects of insulin on lipid metabolism. We have previously shown that acute insulin infusion induced a shift in fatty acid metabolism dependent on fasting duration. This study complements the previous study by examining the effects of fasting duration and insulin infusion on circulating levels of oxylipins, bioactive metabolites derived from the oxygenation of polyunsaturated fatty acids. Northern elephant seal pups were studied at two postweaning periods (n = 5/period): early fasting (1-2 wk postweaning; 127 ± 1 kg) and late fasting (6-7 wk postweaning; 93 ± 4 kg). Different cohorts of pups were weighed, sedated, and infused with 65 mU/kg of insulin. Plasma was collected prior to infusion (T0) and at 10, 30, 60, and 120 min postinfusion. A profile of ∼80 oxylipins was analyzed by UPLC-ESI-MS/MS. Nine oxylipins changed between early and late fasting and eight were altered in response to insulin infusion. Fasting decreased prostaglandin F2α (PGF2α) and increased 14,15-dihydroxyicosatrienoic acid (14,15-DiHETrE), 20-hydroxyeicosatetraenoic acid (20-HETE), and 4-hydroxy-docosahexaenoic acid (4-HDoHE) (P < 0.03) in T0 samples, whereas insulin infusion resulted in an inverse change in area-under-the-curve (AUC) levels in these same metabolites (P < 0.05). In addition, 12-12-hydroperoxyeicosatetraenoic acid (HpETE) and 12-HETE decreased with fasting and insulin infusion, respectively (P < 0.04). The oxylipins altered during fasting and in response to insulin infusion may contribute to the manifestation of insulin resistance and participate in the metabolic regulation of associated cellular processes.

Exogenous GLP-1 stimulates TCA cycle and suppresses gluconeogenesis and ketogenesis in late-fasted northern elephant seals pups.

The postweaning fast of northern elephant seal pups is characterized by a lipid-dependent metabolism and associated with a decrease in plasma glucagon-like peptide-1 (GLP-1), insulin, and glucose and increased gluconeogenesis (GNG) and ketogenesis. We have also demonstrated that exogenous GLP-1 infusion increased plasma insulin despite simultaneous increases in cortisol and glucagon, which collectively present contradictory regulatory stimuli of GNG, ketogenesis, and glycolysis. To assess the effects of GLP-1 on metabolism using primary carbon metabolite profiles in late-fasted seal pups, we dose-dependently infused late-fasted seals with low (LDG; 10 pM/kg; n = 3) or high (HDG; 100 pM/kg; n = 4) GLP-1 immediately following a glucose bolus (0.5 g/kg), using glucose without GLP-1 as control (n = 5). Infusions were performed in similarly aged animals 6-8 wk into their postweaning fast. The plasma metabolome was measured from samples collected at five time points just prior to and during the infusions, and network maps constructed to robustly evaluate the effects of GLP-1 on primary carbon metabolism. HDG increased key tricarboxylic acid (TCA) cycle metabolites, and decreased phosphoenolpyruvate and acetoacetate (P < 0.05) suggesting that elevated levels of GLP-1 promote glycolysis and suppress GNG and ketogenesis, which collectively increase glucose clearance. These GLP-1-mediated effects on cellular metabolism help to explain why plasma GLP-1 concentrations decrease naturally in fasting pups as an evolved mechanism to help conserve glucose during the late-fasting period.

Rapid determination of thyroid hormones in blood plasma from Glaucous gulls and Baikal seals by HybridSPE®-LC-MS/MS.

A rapid hybrid solid phase extraction (HybridSPE®) protocol tailored to liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) analysis, was developed for the determination of four thyroid hormones, L-Thyroxine (T4), 3,3',5-triiodo-L-thyronine (T3), 3,3',5'-triiodo-L-thyronine (rT3) and 3,3'-diiodo-L-thyronine (T2) in blood plasma from Glaucous gulls (Larus hyperboreus) and Baikal seals (Phoca sibirica). The use of target analyte specific 13C internal standards allowed quantification to be performed through the standard solvent calibration curves and alleviated the need to perform quantification with matrix match curves. The relative recoveries were 100.0-110.1 % for T4, 99.1-102.2 % for T3, 100.5-108.0 % for rT3, and 100.5-104.6 % for T2. The matrix effects ranged from -1.52 to -6.10 %, demonstrating minor signal suppression during analysis. The method intra-day precision (method repeatability, RSD %, N = 5, k = 1 day) and inter-day precision (method reproducibility, RSD %, N = 10, k = 2 days) at the 1 ng/mL concentration of fortification were 8.54-15.4 % and 15.4-24.8 %, respectively, indicating acceptable chromatographic peak stabilities for all target THs even at trace level concentrations. The method limit of detection (LOD) for T4, T3, rT3 and T2 was 0.17, 0.16, 0.30 and 0.17 ng/mL, respectively. The HybridSPE® protocol was simple and rapid (<1 min) upon application, while the HybridSPE® cartridge did not require (as in classical SPE cartridges) any additional equilibration nor conditioning step prior sample loading. A total of 46 blood plasma samples, 30 samples collected from Glaucous gulls and 16 samples collected from Baikal seals, were analyzed for thyroid hormones to demonstrate the applicability of the developed method in these wildlife species. The concentrations of T4 and T3 in blood plasma from the Glaucous gulls were 5.95-44.2 and 0.37-5.61 ng/mL, respectively, whereas those from Baikal seals were 3.57-46.5 and 0.45-2.07 ng/mL, respectively. In both species, rT3 demonstrated low detection rate, while T2 was not detected. Furthermore, cross-array comparison between the HybridSPE®-LC-MS/MS protocol and an established routine radioimmunoassay (RIA) kit-based method was performed for T4 and T3 concentrations from selected Baikal seal plasma samples.

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.

Decadal changes in blood δ13C values, at-sea distribution, and weaning mass of southern elephant seals from Kerguelen Islands.

Changes in the foraging environment and at-sea distribution of southern elephant seals from Kerguelen Islands were investigated over a decade (2004-2018) using tracking, weaning mass, and blood δ13C values. Females showed either a sub-Antarctic or an Antarctic foraging strategy, and no significant shift in their at-sea distribution was detected between 2004 and 2017. The proportion of females foraging in sub-Antarctic versus Antarctic habitats did not change over the 2006-2018 period. Pup weaning mass varied according to the foraging habitat of their mothers. The weaning mass of sub-Antarctic foraging mothers' pups decreased by 11.7 kg over the study period, but they were on average 5.8 kg heavier than pups from Antarctic foraging mothers. Pup blood δ13C values decreased by 1.1‰ over the study period regardless of their sex and the presumed foraging habitat of their mothers. Together, these results suggest an ecological change is occurring within the Indian sector of the Southern Ocean with possible consequences on the foraging performance of southern elephant seals. We hypothesize that this shift in δ13C is related to a change in primary production and/or in the composition of phytoplankton communities, but this requires further multidisciplinary investigations.

Sample preparation and liquid chromatography-tandem mass spectrometry for the analysis of selected Pacific ciguatoxins in blood samples.

Consumption of ciguatoxin-contaminated seafood can lead to ciguatera poisoning (CP). The diagnosis of CP in humans is based on the clinical symptoms after eating the fish from tropical or subtropical areas because no confirmatory clinical tests are available. One of the challenges for ciguatoxin analysis is their extremely low but toxicologically relevant concentration in biological samples. We previously reported a method using acetonitrile to precipitate proteins and extract the ciguatoxins simultaneously in whole blood samples from animals for toxin quantification by N2A cell-based assay. However, a test method for unambiguous confirmation of exposure of marine animals or humans to ciguatoxins is still needed. In the present study, we adopted the acetonitrile extraction method and added sample clean-up in the sample preparation for the determination of Pacific ciguatoxins CTX1B (aka P-CTX-1), 52-epi-54-deoxyCTX1B (aka P-CTX-2), and CTX3C (aka P-CTX-3C) in blood plasma by LC-MS/MS. We investigated sample clean-up, LC mobile phases, LC solvent programming, and settings of the two mass spectrometers (4000 Q TRAP and AB SCIEX Triple Quad 5500) in order to improve the ability to detect the Pacific ciguatoxins at ppt level. Rat blood plasma was used for the method development. Average recoveries of the three toxins in the rat plasma samples ranged from 90% to 116% with relative standard deviations of less than 15%. The method detection limits were still not low enough for the determination of the Pacific ciguatoxins in individual blood samples from Hawaiian monk seals with the two LC-MS systems. The methods were applied to a pooled sample of blood plasma collected from Hawaiian monk seals for confirmation of toxin exposure. This study will benefit monitoring of Pacific ciguatoxins in marine mammals and potentially humans by LC-MS/MS.

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.

Polychlorinated Biphenyls (PCBs) and Sex Hormone Concentrations in Ringed and Grey Seals: A Possible Link to Endocrine Disruption?

Polychlorinated biphenyls (PCBs) are recognised reproductive and immune system toxicants in marine mammals mediated by endocrine-disrupting mechanisms. As with other predators, seals are exposed to elevated bioaccumulated concentrations of PCBs and other persistent organic pollutants (POPs). Cryopreserved plasma samples from adult ringed (Phoca hispida; n = 39) and grey (Halichoerus grypus; n = 38) seals, sampled between 1998 and 2002 from Baltic Sea, Svalbard, and Sable Island (Canada) were used to investigate relationships between PCB exposure and sex hormone concentrations (progesterone; P4, 17α-hydroxy progesterone; 17α-OH-P4, testosterone; T4, 17ß-estradiol; E2, estrone; E3). Immunoassay methods were used for quantification of analytes due to the limited sample volumes available. PCB concentrations were found to be significantly higher in Baltic seals than other sampling locations and were classed as "Exposed" seals while Svalbard and Sable Is seal were classed "Reference" seals (sexes and species separate). Mean hormone concentrations in Exposed seal were lower than Reference seals, and this was statistically significantly for 17α-OH-P4 (both sexes and both species), E2 (ringed and grey seal females), and E3 (grey seal females). Regression analyses (PCB v hormone concentrations) for each sex and species revealed significant correlations for P4 (Sable Is. female grey seals and female ringed seals), 17α-OH-P4 (Sable Is. male grey seals and Svalbard male ringed seals), T4 (Svalbard male ringed seals), E2 (female ringed seals), and E3 (female ringed seals and Baltic female grey seals). Although significant correlations are not evidence of cause and effect, the potential impact of hormone changes on endocrine homeostasis and reproductive health for seal populations warrants further investigation given that PCB concentrations found here are in the same range as those currently reported in seals from these populations.

Effects of health status on pressure-induced changes in phocid immune function and implications for dive ability.

The ability of marine mammals to cope with environmental challenges is a key determining factor in strandings and successful release of rehabilitated animals. Dive behavior is related to foraging and thus survival. While dive adaptations have been well studied, it is unknown how the immune system responds to diving and whether health status impacts immune function during diving. This study investigated the functional response of ex situ immune cells from stranded phocids to in vitro increased pressure, over the course of rehabilitation. Blood samples were drawn from stranded harbor seals (Phoca vitulina), gray seals (Halichoerus grypus) and harp seals (Phoca groenlandica) at the time of admit to the Mystic Aquarium, Mystic, CT and again after rehabilitation (pre-release). Phagocytosis, lymphocyte proliferation and immune cell activation were measured in vitro, with and without exposure to 2000 psi (simulated dive depth of 1360 m). Plasma epinephrine and norepinephrine, and serum cortisol were measured in vivo. All hormone values decreased between admit and release conditions. Under admit or release conditions, pressure exposures resulted in significant changes in granulocyte and monocyte phagocytosis, granulocyte expression of CD11b and lymphocyte expression of the IL2 receptor (IL2R). Overall, pressure exposures resulted in decreased phagocytosis for admit conditions, but increased phagocytosis in release samples. Expression of leukocyte activation markers, CD11b and IL2R, increased and the response did not differ between admit and release samples. Specific hematological and serum chemistry values also changed significantly between admit and release and were significantly correlated with pressure-induced changes in immune function. Results suggest (1) dive duration affects the response of immune cells, (2) different white blood cell types respond differently to pressure and (3) response varies with animal health. This is the first study describing the relationship between diving, immune function and health status in phocids.

EVALUATION OF SERIAL BLOOD LACTATE AND THE USE OF A POINT-OF-CARE LACTATE METER IN LIVE-STRANDED PINNIPEDS.

Live-stranded pinnipeds often present to rehabilitation centers systemically debilitated with dehydration and poor perfusion. In many terrestrial mammals, blood lactate elevation has been correlated with global tissue hypoxia and decreased circulating blood volume. Serial blood lactate measurements in companion animals and humans have been used to guide fluid resuscitation therapy and evaluate prognosis. The primary objective of this study was to evaluate the prognostic value of serial blood lactate levels in live-stranded pinnipeds in a rehabilitation setting. The secondary objectives were to evaluate the use of a point-of-care lactate meter, and potassium oxalate-sodium fluoride (gray-top) tubes for storing samples for lactate analysis in pinniped patients. Fifty-five live-stranded pinnipeds (30 northern elephant seals [Mirounga angustirostris], 21 Pacific harbor seals [Phoca vitulina richardsi], and four California sea lions [Zalophus californianus]) that presented to a rehabilitation center were manually restrained for blood collection and lactate measurement using a point-of-care analyzer (Lactate Plus™) prior to fluid or other medical therapy. Lactate measurements were repeated 72 hr post admission and prior to euthanasia or release. Plasma samples from gray-top tubes were frozen for 30-100 days at -80°C, then thawed and evaluated using both the LactatePlus and bench-top (ABL 815) analyzers. The point-of-care analyzer was successful in measuring lactate in pinnipeds. Gray-top tubes were effective at preserving lactate levels in frozen plasma samples for up to 100 days. Released animals had significantly greater lactate clearance at 72 hr (P = 0.039) than animals that died or were euthanized. Therefore, lactate clearance, determined by serial blood lactate measurements, may be useful for evaluating prognosis in live-stranded pinnipeds. Initial lactate and lactate values prior to euthanasia or release were not significantly associated with outcome. Given these findings, clinicians should interpret isolated lactate values in pinnipeds with caution.

Changes in total mercury, methylmercury, and selenium blood levels during different life history stages of the Baltic grey seal (Halichoerus grypus grypus).

Using the blood of grey seal pups, the blood and milk of female grey seals inhabiting the Hel Marine Station of Gdansk University's Institute of Oceanography (HMS), we monitored the transfer of total mercury (THg), methylmercury (MeHg), and selenium (Se) with blood during foetal life and nursing. Changes in the concentration of mercury and selenium were characterised in the pups' blood during their first three months of life when they transition from suckling, to a post-weaning fast, to eating fish. In the blood of pregnant females, there was a significant decrease in THg and MeHg concentrations throughout the gestation, indicating the transfer of these toxins through the placenta into the foetus. At no other stage of the pup's development was there such a high level of THg and MeHg as on the day of birth, despite the incorporation of mercury into the lanugo during foetal growth. This suggests that the maternal transfer of mercury during gestation may be the time of greatest mercury exposure for a young seal pup. The consumption of milk caused a rapid increase in weight and a lowering of the mercury level in the blood in the subsequent days of the pups' life. The postweaning fast was the period of the lowest mercury concentration. The switch to a diet consisting of fish caused a systematic increase in the concentration of mercury in the blood of the pups. Milk was the significant source of selenium for pups and the selenium concentration in females' blood was reduced during lactation. The nursing period seemed to have the greatest impact on the mercury and selenium blood levels in examined seals. Natural development of the grey seal pup created an opportunity to decrease the levels of toxic substances obtained through the maternal transfer during foetal growth.

ESTABLISHING HEMATOLOGY AND SERUM CHEMISTRY REFERENCE INTERVALS FOR WILD HAWAIIAN MONK SEALS ( NEOMONACHUS SCHAUINSLANDI).

Hematology and serum chemistry reference intervals have been previously established for the endangered Hawaiian monk seal ( Neomonachus schauinslandi) as an imperative measure for health assessments. Monitoring the health of the wild population depends upon reference intervals that are context specific; hence we developed reference intervals from fresh samples, as opposed to frozen, from wild monk seals. This study builds on the number of parameters from previous efforts by using samples collected between 2004 and 2015 from wild monk seals. Blood samples were analyzed by a single veterinary diagnostic laboratory within 24 hr of collection from apparently healthy, wild seals during research activities. Reference intervals were determined based on the analytical steps outlined by the American Society for Veterinary Clinical Pathology. These comprehensive hematology and serum chemistry reference intervals enable more consistent and systematic interpretation of results, which will guide individual and population-level health assessment and decision-making research and recovery activities.

NEW APPROACH TO STRESS RESEARCH IN PHOCIDS-POTENTIAL OF DEHYDROEPIANDROSTERONE AND CORTISOL/DEHYDROEPIANDROSTERONE RATIO AS MARKERS FOR STRESS IN HARBOR SEALS ( PHOCA VITULINA) AND GRAY SEALS ( HALICHOERUS GRYPUS).

Cortisol is known to reflect the level of the stress response. However, measuring the cortisol concentration only once fails to provide sufficient information about the duration of the stress exposure. Moreover, handling is an acute stressor and increases cortisol secretion especially in wildlife species. Yet, in phocids reliable indicators are missing that reflect potential chronic effects of stress. The adrenal-derived steroid hormone dehydroepiandrosterone (DHEA) has been described as biomarker for the assessment of the stress status but has not been applied to marine mammal stress research. Therefore, DHEA, the sulfated precursor DHEAS and the cortisol/DHEA ratio were determined in serum of different seals. One group consisted of harbor ( Phoca vitulina) and gray seals ( Halichoerus grypus) that were habituated to human handling. The other two groups included healthy and free-ranging seals suffering from a disease. Blood samples were taken from 11 habituated (six males, five females), 17 wild_healthy (13 males, four females), and nine wild_diseased seals (four males, five females). No differences in serum cortisol levels could be detected between wild_healthy and wild_diseased seals. On the contrary, wild_diseased seals showed the lowest DHEA concentration compared to the other two groups. Wild_diseased seals also revealed the highest cortisol/DHEA ratio compared to the habituated_zoo (2,074.7 ± 351.4 vs. 121.5 ± 26.8, P < 0.001) and wild_healthy seals (827.9 ± 214.3, P < 0.01). DHEA and the cortisol/DHEA ratio may reflect differences in the functionality of the hypothalamic-pituitary-adrenal axis and therefore represent valuable tools for the assessment of stress-related effects in seals.

Maternal transfer of phenol derivatives in the Baltic grey seal Halichoerus grypus grypus.

Studies of circulating levels in difference sex and age classes, and maternal transfer of bisphenol A, 4-tert-octylphenol and 4- nonylphenol in the Baltic grey seal were performed from 2014-2017. Blood was collected from long-term captive adult males, pregnant females and pups. Milk was collected from nursing females. The aim of this study was not only to determine the concentrations of phenol derivatives, i.e. bisphenol A (BPA), 4-tert-octylphenol (OP) and 4-nonylphenol (NP), but also to try to evaluate the transfer of these compounds to the next generation in the final stage of foetal life and in the first few weeks of life in juvenile marine mammals. The measurements were carried out using high performance liquid chromatography. The obtained data show that all phenol derivatives are present in the blood of males, females and pups (range <0.07-101 ng·cm-3) and in female milk (range <0.1-406.3 ng·cm-3). The main source of phenol derivatives in organisms is food exposure. Gender, age, or number of births were not observed to have a significant effect on changes in phenol derivative levels in seal blood within the breeding group. In the prenatal stage of life, a small amount of BPA and alkylphenols was passed on to the offspring through the placenta. In the blood of the offspring the concentration of these compounds exceeded the concentration in the mother's blood 1.5-fold. During nursing, females detoxified their systems. Level of phenol derivatives in the pups blood increased linearly with its increasing concentrations in the mother's milk. On the other hand, the seafood diet which started after the physiological fasting stage of the pup, stabilised the levels of phenol derivatives below 10 ng ∙ cm-3.

Intrinsic anti-inflammatory properties in the serum of two species of deep-diving seal.

Weddell and elephant seals are deep-diving mammals, which rely on lung collapse to limit nitrogen absorption and prevent decompression injury. Repeated collapse and re-expansion exposes the lungs to multiple stressors, including ischemia-reperfusion, alveolar shear stress and inflammation. There is no evidence, however, that diving damages pulmonary function in these species. To investigate potential protective strategies in deep-diving seals, we examined the inflammatory response of seal whole blood exposed to lipopolysaccharide (LPS), a potent endotoxin. Interleukin-6 (IL6) cytokine production elicited by LPS exposure was 50 to 500 times lower in blood of healthy northern elephant seals and Weddell seals compared with that of healthy human blood. In contrast to the ∼6× increased production of IL6 protein from LPS-exposed Weddell seal whole blood, isolated Weddell seal peripheral blood mononuclear cells, under standard cell culture conditions using medium supplemented with fetal bovine serum (FBS), produced a robust LPS response (∼300×). Induction of Il6 mRNA expression as well as production of IL6, IL8, IL10, KC-like and TNFα were reduced by substituting FBS with an equivalent amount of autologous seal serum. Weddell seal serum also attenuated the inflammatory response of RAW 267.4 mouse macrophage cells exposed to LPS. Cortisol level and the addition of serum lipids did not impact the cytokine response in cultured cells. These data suggest that seal serum possesses anti-inflammatory properties, which may protect deep divers from naturally occurring inflammatory challenges such as dive-induced hypoxia-reoxygenation and lung collapse.

Effects of a complex contaminant mixture on thyroid hormones in breeding hooded seal mothers and their pups.

There is a general lack of information on the possible effects of perfluoroalkyl substances (PFASs) on thyroid hormones (THs) in wildlife species. The effects of PFASs, which are known endocrine disruptors, on the TH homeostasis in hooded seals (Cystophora cristata) have yet to be investigated. Previously, correlations were found between plasma thyroid hormone (TH) concentrations in hooded seals, and organohalogen contaminants (OHCs) and hydroxyl (OH)-metabolites. Because animals are exposed to multiple contaminants simultaneously in nature, the effects of the complex contaminant mixtures that they accumulate should be assessed. Herein, we analyse relationships between plasma concentrations of multiple contaminants including protein-associated PFASs, hydroxylated metabolites of polychlorinated biphenyls (OH-PCBs) and lipid soluble OHCs and plasma concentrations of free and total THs, i.e. triiodothyronine (FT3, TT3) and thyroxine (FT4, TT4) in hooded seal mothers and their pups. The perfluoroalkyl carboxylates (PFCAs) were the most important predictors for FT3 concentrations and TT3:FT3 ratios in the mothers. The FT3 levels decreased with increasing PFCA levels, while the TT3:FT3 ratios increased. In the pups, hexachlorocyclohexanes (HCHs) were the most important predictors for TT3:FT3 ratios, increasing with increasing HCHs levels. Additionally, perfluoroalkyl sulfonates (PFSAs) and PFCAs were important predictors for FT4:FT3 ratios in hooded seal pups, and the ratio increased with increasing concentrations. The study suggests that PFASs contribute to thyroid disruption in hooded seals exposed to complex contaminant mixtures that include chlorinated and fluorinated organic compounds.

First isolation of Brucella pinnipedialis and detection of Brucella antibodies from bearded seals Erignathus barbatus.

Brucella species infecting marine mammals was first reported in 1994 and in the years since has been documented in various species of pinnipeds and cetaceans. While these reports have included species that inhabit Arctic waters, the few available studies on bearded seals Erignathus barbatus have failed to detect Brucella infection to date. We report the first isolation of Brucella pinnipedialis from a bearded seal. The isolate was recovered from the mesenteric lymph node of a bearded seal that stranded in Scotland and typed as ST24, a sequence type associated typically with pinnipeds. Furthermore, serological studies of free-ranging bearded seals in their native waters detected antibodies to Brucella in seals from the Chukchi Sea (1990-2011; 19%) and Svalbard (1995-2007; 8%), whereas no antibodies were detected in bearded seals from the Bering Sea or Bering Strait or from captive bearded seals.

ACUTE-PHASE RESPONSES IN HEALTHY, MALNOURISHED, AND OTOSTRONGYLUS-INFECTED JUVENILE NORTHERN ELEPHANT SEALS (MIROUNGA ANGUSTIROSTRIS).

Acute-phase proteins (APPs) are utilized to detect early inflammation in many domestic and nondomestic species, but variability exists between species and inflammatory diseases as to which APPs are most useful. Stranded juvenile northern elephant seals (NESs; Mirounga angustirostris) undergoing rehabilitation at the Marine Mammal Center experience high mortality rates due to severe arteritis caused by the lungworm, Otostrongylus circumlitis (OC), and there are currently no effective antemortem diagnostic tools for this disease. To characterize patterns of the acute-phase response in the NES, two APPs-serum amyloid A (SAA) and C-reactive protein (CRP)-were measured, and serum protein electrophoresis was performed to measure albumin and globulin fractions in 81 serum samples from 58 NESs in four different health states: healthy, malnourished, preclinical for OC infection, or clinical for OC infection. Compared to healthy NESs (median, 11.2 mg/L), SAA concentrations were significantly increased in malnourished (33.9 mg/L), preclinical (247 mg/L), and clinical OC-infected NESs (328 mg/L) (P < 0.05). CRP concentrations were increased only in clinical OC-infected NESs (median, 53.9 mg/L) and were below detectable limits in the other three groups (<0.01 mg/L). These results show that SAA and CRP are positive APPs in NESs with OC infection, and that SAA may serve as the major APP for this species. Albumin : globulin ratios were significantly increased in malnourished NESs (median, 1.26) and decreased in clinical OC-infected NESs (0.53). As a result, albumin is a negative APP in the NES, similar to other mammalian species. APP monitoring can be helpful in detecting and monitoring inflammation in rehabilitating juvenile NESs.

Plasmatic coagulation and fibrinolysis in healthy and Otostrongylus-affected Northern elephant seals (Mirounga angustirostris).

BACKGROUND: Prepatent Otostrongylus arteritis results in hemorrhagic diathesis in free-ranging Northern elephant seals (Mirounga angustirostris) attributed to aberrant larval migration of the lungworm, Otostrongylus circumlitus. Clinical signs are often nonspecific, including lethargy, anorexia, and blepharospasm, but can progress to spontaneous frank hemorrhage and death within 72 hours of onset. Previously published case reports describe coagulopathy with prolonged PT and APTT, normal to elevated platelet counts, normal antithrombin concentrations, and low concentrations of fibrinogen degradation products. Disseminated intravascular coagulation was proposed as the cause of hemorrhage, but is inconsistent with some of the reported clinicopathologic changes. OBJECTIVE: The purpose of this study was to compare plasmatic coagulation and fibrinolysis in healthy and Otostrongylus-affected elephant seals, in order to identify potential therapy. We hypothesized that hyperfibrinolysis contributed to hemorrhage in these cases. METHODS: Citrated plasma samples were collected from 3- to 4-month-old Northern elephant seals in a wildlife rehabilitation hospital. The sampled population included 25 healthy, prerelease seals and 32 clinically ill seals diagnosed with presumptive Otostrongylus arteritis. Twenty-one of the included seals had Otostrongylus infestation confirmed at necropsy. Standard coagulation tests and plasma thromboelastography were performed for a complete assessment of coagulation and fibrinolysis. RESULTS: Northern elephant seals with definitive Otostrongylus infestation were hypocoagulable and hypofibrinolytic compared to healthy controls. CONCLUSIONS: Results were most consistent with disseminated intravascular coagulation. Treatment with antifibrinolytic drugs to control hemorrhage may be unrewarding; alternative therapies such as plasma transfusions or coagulation factor concentrates should be investigated.

Variation in adrenal and thyroid hormones with life-history stage in juvenile northern elephant seals (Mirounga angustirostris).

The classical approach to quantifying the impact of stressors on wildlife is through characterization of hormones associated with the generalized stress response. However, interpretation of hormone data can be difficult due to the range of natural variation within a species and potential confounds of individual and life-history variables. Blood adrenal and thyroid hormones were measured in 144 chemically immobilized yearling northern elephant seals (Mirounga angustirostris) to characterize variation between sexes and across semiannual haul-outs. There was no relationship between hormone concentrations and time needed for collecting blood nor evidence of diel patterns, suggesting that collection of samples for baseline values can be accomplished without bias due to handling artifacts or time of day. Serum cortisol concentrations did not vary with gender or across haul-out fasts but increased dramatically during molting. Cortisol was correlated with aldosterone across all measured life-history stages. Thyroid hormone levels were lower in females and decreased with fasting in both sexes during the fall haul-out. Cortisol concentrations were inversely associated with total triiodothyronine (T3) and positively associated with reverse T3 concentrations across all measured life-history stages suggesting an important impact of cortisol on deiodinase enzymes and thyroid function. Epinephrine concentrations increased across fasts and norepinephrine concentrations were higher in males than in females. Significant variation in stress hormone concentrations with gender and life-history stage emphasizes the importance of contextual variables when interpreting serum hormone concentrations.