Response of metabolic hormones and blood metabolites to realimentation in rehabilitated harbor seal (Phoca vitulina) pups.

Mammals with increased requirements for adipose tissue stores, such as marine mammals, have altered nutrient allocation priorities compared to many terrestrial mammals and thus the physiological response to undernutrition (low nutritional status) and realimentation (refeeding) may differ. Key regulators of nutrient allocation and tissue specific growth include metabolic hormones of the somatotropic axis, growth hormone (GH) and insulin-like growth factor (IGF)-I, as well as satiety and adipose promoting ghrelin and the stress hormone cortisol. Longitudinal measurements of metabolic hormones, blood metabolites, and morphometrics were collected over a 10-week period in twelve (male n = 3, female n = 9) harbor seal pups (< 6 weeks of age). Blood metabolites were used to indicate metabolic response during realimentation while morphometrics estimated tissue specific growth priorities. Harbor seal pups undergoing refeeding after nutritional deprivation show a preference for protein sparing despite severe malnutrition. Both BUN and total protein were negatively associated with GH and positively associated with IGF-I and ghrelin highlighting the importance of these metabolic hormones in the regulation of protein metabolism. While the response of the somatotropic axis to realimentation was typical of the mammalian pattern, the surprising increase of ghrelin across the study period suggests the priority of adipose accretion in addition to a possible mechanism regulating compensatory growth of vital adipose stores in a species, which prioritizes adipose accretion for survival.

Determining pregnancy status in harbour seals using progesterone concentrations in blood and blubber.

Pregnancy status in harbour seals can be estimated from concentrations of progesterone in blubber as well as in blood samples, which are significantly higher in pregnant than non-pregnant animals. This study investigated the accuracy of estimating pregnancy rates using samples from live-captured and released harbour seals from three regions around Scotland, coupled with observed pregnancy outcomes. Concentrations of progesterone in blood (plasma) and blubber were obtained during the capture of animals early in the year (February to May). Individual animals were identified from the unique markings on their pelage, with a proportion (n = 51) of females re-sighted during the subsequent breeding season and the reproductive outcomes determined (pregnant or possibly non-pregnant) during observations from long-term photo-identification studies. Generalised linear models with a binomial link function were fitted to training (60% of the data) and test datasets (40% of the data) to estimate pregnancy status from progesterone concentrations in blubber, plasma or both, and a received operating curves (ROC) approach was used to evaluate the performance of each classifier. The accuracy for the plasma concentrations was 85% with a high classification performance (as estimated from an area under the curve (AUC) of 0.82). The Youden method to determine the cut-point (threshold) and bootstrapping the training dataset resulted in a cut-point of 58 ng ml-1 (95th percentiles, 25-102 ng ml-1). For blubber, the accuracy was 77% (AUC = 0.86) with an optimal cut-point of 56 ng g-1 (95th percentiles, 26-223 ng g-1). In the combined analysis (both blubber and plasma), the accuracy was 87.5% (AUC 0.81) with the cut-points of 72 ng ml-1 (95th percentiles, 25-103 ng ml-1) in plasma and 56 ng g-1 (95th percentiles, 26-223 ng g-1) in blubber. These thresholds were then used to estimate the pregnancy proportions among adult females at the three study sites, including those that were not included in the photo-id studies. Proportions were high at all sites, (63%-100%) regardless of which matrices were used and were not statistically significantly different from each other but suggested that analysing concentrations in both sample matrices would minimise the uncertainty.

Deiminated proteins and extracellular vesicles as novel biomarkers in pinnipeds: Grey seal (Halichoerus gryptus) and harbour seal (Phoca vitulina).

Peptidylarginine deiminases (PADs) are phylogenetically conserved calcium-dependent enzymes which post-translationally convert arginine into citrulline in target proteins in an irreversible manner, leading to functional and structural changes in target proteins. Protein deimination can cause the generation of neo-epitopes, affect gene regulation and also allow for protein moonlighting and therefore facilitate multifaceted functions of the same protein. PADs are furthermore a key regulator of cellular release of extracellular vesicle (EVs), which are found in most body fluids and participate in cellular communication via transfer of cargo proteins and genetic material. In this study, post-translationally deiminated proteins and EVs were assessed in sera of two seal species, grey seal and harbour seal. We report a poly-dispersed population of serum-EVs, which were positive for phylogenetically conserved EV-specific markers and characterised by transmission electron microscopy. A number of deiminated proteins critical for immune and metabolic functions were identified in the seal sera and varied somewhat between the two species under study, while some targets were in common. EV profiles of the seal sera further revealed that key microRNAs for inflammation, immunity and hypoxia also vary between the two species. Protein deimination and EVs profiles may be useful biomarkers for assessing health status of sea mammals, which face environmental challenges, including opportunistic infection, pollution and shifting habitat due to global warming.

HEMATOLOGY AND SERUM BIOCHEMISTRY OF HARBOR SEAL (PHOCA VITULINA) PUPS AFTER REHABILITATION IN THE NETHERLANDS.

Hematology and serum biochemistry profiles are used to evaluate the health status of animals ongoing rehabilitation. The aim of this project was to develop blood and biochemistry ranges for harbor seal pups (Phoca vitulina) after rehabilitation; thus, 22 different blood parameters in 60 animals were tested before release. The second goal was to test for differences due to sex, stranding location, body condition at admission, and presence or absence of umbilical cord. The alanine aminotransferase, ALT (or glutamate pyruvate transaminase, GPT), (ALT-GPT) differed significantly (P bq = 0.00851) between sexes. Lower leukocyte counts and higher liver enzyme values were the most remarkable findings when comparing the results of this study to other published data. This is the first study to report blood reference ranges for harbor seal pups in the Dutch Wadden Sea after rehabilitation.

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.

T lymphocyte-proliferative responses of harbor seal (Phoca vitulina) peripheral blood mononuclear cells (PBMCs) exposed to pharmaceuticals in vitro.

The ubiquity of pharmaceuticals in the aquatic environment and the accumulation in organisms of lower trophic levels have been documented. The immunotoxicity of these xenobiotics has however been little investigated. This study assessed the effects of pharmaceuticals on the immune responses of harbor seal lymphocytes. Peripheral blood mononuclear cells isolated from harbor seal pups were exposed to varying concentrations of 17α-ethinyl estradiol (250-50,000µg/L), naproxen (500-100,000µg/L), carbamazepine (500-100,000µg/L), erythromycin (750-150,000µg/L) and binary mixtures thereof in vitro. All individual compounds and mixtures inhibited lymphocyte proliferation. Mixture effects were non-additive and predictive values overestimated the inhibition of proliferation. Male pups were more sensitive to erythromycin exposure. Comparison with the sensitivity of the 11B7501 cell line showed a higher sensitivity of pups to individual compounds and the inverse trend for mixtures. Based on our results, we hypothesize that pharmaceuticals may have the potential to interrupt immune functions in harbor seals.

Seroprevalence of Brucella antibodies in harbor seals in Alaska, USA, with age, regional, and reproductive comparisons.

Populations of harbor seal Phoca vitulina in the Gulf of Alaska have dramatically declined during the past 4 decades. Numbers of seals in Glacier Bay, in southeast Alaska, USA, have also declined despite extensive protection. Causes of the declines and slow recovery are poorly understood. Brucellosis is a zoonotic disease that adversely affects reproduction in many domestic species. We measured the seroprevalence of Brucella antibodies in 554 harbor seals in 3 Alaska locations: Prince William Sound (PWS), Glacier Bay (GB), and Tracy Arm Fords Terror (TAFT) Wilderness Area. Objectives included testing for regional, sex, age, and female reproductive state differences in Brucella antibody seroprevalence, persistence in titers in recaptured seals, and differences in titers between mother seals and their pups. Overall, 52% of adults (AD), 53% of subadults (SA), 77% of yearlings (YRL), and 26% of <5 mo old pups were seropositive. Matched mother-pup samples were consistent with dependent pups acquiring maternal passive immunity to Brucella. Results show higher seroprevalence (64%) for AD and SA seals in the depressed and declining populations in PWS and GB than in TAFT (29%). Lactating females were less likely to be seropositive than other AD females, including pregnant females. Further research is needed to seek evidence of Brucella infection in Alaskan harbor seals, identify effects on neonatal viability, and assess zoonotic implications for Alaska Natives who rely on harbor seals for food.

Usefulness of serum cardiac troponin I concentration as a marker of survival of harbor seal (Phoca vitulina) pups during rehabilitation.

OBJECTIVE To measure serum cardiac troponin I (cTnI) concentrations in orphaned harbor seal (Phoca vitulina) pups at various points during rehabilitation in a seal rescue center and determine whether cTnI concentration was associated with survival during rehabilitation and duration of rehabilitation. DESIGN Serial cross-sectional study. ANIMALS Fifty-five 2- to 9-day-old harbor seal pups. PROCEDURES Blood samples for serum cTnI concentration measurement, CBC, and serum biochemical analysis were obtained from seal pups at admission into a seal rescue center, after 2 weeks of rehabilitation at the center, and prior to release. Serum cTnI concentrations were compared between seals that did or did not survive rehabilitation. RESULTS Median serum cTnI concentration was highest at admission (0.03 ng/mL). After 2 weeks, the median value was 0.01 ng/mL; prior to release, it was 0.01 ng/mL. Seal pups that were found to have died during or after rehabilitation (n = 7) had a significantly higher median serum cTnI concentration at admission (0.06 ng/mL) than did seal pups that survived rehabilitation (and for which the postrelease fate was unknown; 48; 0.03 ng/mL). No correlation was identified between serum cTnI concentration and duration of rehabilitation. CONCLUSIONS AND CLINICAL RELEVANCE The results of this study suggested some degree of myocardial injury was present in most of the orphaned seal pups admitted for rehabilitation. Measurement of serum cTnI concentration in seal pups at admission might provide prognostic information about their likelihood of survival during or after rehabilitation.

Essential and Toxic Elements in Blood Samples of Harbor Seals (Phoca vitulina) from the Islands Helgoland (North Sea) and Anholt (Baltic Sea): A Comparison Study with Urbanized Areas.

The harbor seals (Phoca vitulina) from Helgoland (North Sea) and Anholt (Kattegat, Baltic Sea) are top predators within the marine food web and an indicator species of the environmental contamination. Furthermore, they are a main tourist attraction. Despite these important roles, little is known about the health and pollutant contamination of these seals. The objective of this study was therefore to investigate 18 essential and nonessential/toxic elements (Al, As, Be, Ca, Cr, Cu, Fe, K, Mn, Mo, Ni, P, Pb, Rb, S, Se, Sr, and Zn) in blood samples using inductively coupled plasma mass spectrometry and total X-ray-fluorescence spectrometry. Blood concentrations of mineral nutrients, such as Ca, K, P, and S, were within the reference ranges described for harbor seals. Likewise, for the trace elements, As, Be, Rb, Se, and Sr, no significant differences were observed compared with previous studies. Interestingly, blood concentrations of nine nonessential as well as essential trace metals (Al, Cr, Cu, Fe, Mn, Mo, Ni, Pb, Zn) measured significantly lower in the offshore living seals from Helgoland and Anholt compared with results obtained from animals living close to urbanized areas, such as the Wadden Sea and Elbe estuary. This suggests that industrial emissions, sewage deposition, shipping traffic and dredging tasks might be the cause of increased metal concentrations of inshore harbor seals.

Seroprevalence of Antibodies against Seal Influenza A(H10N7) Virus in Harbor Seals and Gray Seals from the Netherlands.

In the spring and summer 2014, an outbreak of seal influenza A(H10N7) virus infection occurred among harbor seals (Phoca vitulina) off the coasts of Sweden and Denmark. This virus subsequently spread to harbor seals off the coasts of Germany and the Netherlands. While thousands of seals were reported dead in Sweden, Denmark and Germany, only a limited number of seals were found dead in the Netherlands. To determine the extent of exposure of seals in the Netherlands to influenza A/H10N7 virus, we measured specific antibody titers in serum samples from live-captured seals and seals admitted for rehabilitation in the Netherlands by use of a hemagglutination inhibition assay and an ELISA. In harbor seals in 2015, antibodies against seal influenza A(H10N7) virus were detected in 41% (32 out of 78) pups, 10% (5 out of 52) weaners, and 58% (7 out of 12) subadults or adults. In gray seals (Halichoerus grypus) in 2015, specific antibodies were not found in the pups (n = 26), but in 26% (5 out of 19) of the older animals. These findings indicate that, despite apparent low mortality, infection with seal influenza A(H10N7) virus was geographically widespread and also occurred in grey seals.

Diving Related Changes in the Blood Oxygen Stores of Rehabilitating Harbor Seal Pups (Phoca vitulina).

Harbor seal (Phoca vitulina) pups begin diving within hours of birth, stimulating the development of the blood oxygen (O2) stores necessary to sustain underwater aerobic metabolism. Since harbor seals experience a brief nursing period, the early-life development of these blood O2 stores is necessary for successful post-weaning foraging. If mothers and pups become prematurely separated, the pup may be transported to a wildlife rehabilitation center for care. Previous studies suggest that the shallow pools and lack of diving in rehabilitation facilities may lead to under-developed blood O2 stores, but diving behavior during rehabilitation has not been investigated. This study aimed to simultaneously study the diving behaviors and blood O2 store development of rehabilitating harbor seal pups. Standard hematology measurements (Hct, Hb, RBC, MCV, MCH, MCHC) were taken to investigate O2 storage capacity and pups were equipped with time-depth recorders to investigate natural diving behavior while in rehabilitation. Linear mixed models of the data indicate that all measured blood parameters changed with age; however, when compared to literature values for wild harbor seal pups, rehabilitating pups have smaller red blood cells (RBCs) that can store less hemoglobin (Hb) and subsequently, less O2, potentially limiting their diving capabilities. Wild pups completed longer dives at younger ages (maximum reported <25 days of age: 9 min) in previous studies than the captive pups in this study (maximum <25 days of age: 2.86 min). However, captivity may only affect the rate of development, as long duration dives were observed (maximum during rehabilitation: 13.6 min at 89 days of age). Further, this study suggests that there may be a positive relationship between RBC size and the frequency of long duration dives. Thus, rehabilitating harbor seal pups should be encouraged to make frequent, long duration dives to prepare themselves for post-release foraging.

Serum testosterone, progesterone, and estradiol concentrations and sexual maturation in spotted seals (Phoca largha).

Spotted seals (Phoca largha) are ice-breeding phocid found in eight different breeding colonies all over the world. They exhibit a seasonal breeding pattern, with annual and synchronous cycles; however, little is known about their reproductive endocrinology. In this study, we measured serum testosterone, progesterone, and 17ß-estradiol concentrations in captive spotted seals (simple number: female n = 68; male n = 89) throughout a full reproductive cycle. Males that were older than 4 years had significant testosterone fluctuations and were, therefore, classified as sexually mature. These animals show significant seasonal changes in testosterone levels, with average peak concentrations of 10.81 ± 9.57 nmol/L (±SD) from November to February, compared with mean concentrations of 1.42 ± 3.09 nmol/L throughout the remainder of the year. Females that reported a significant variation in progesterone concentrations and were older than 4 years were considered to be sexually mature. In these females, progesterone levels increased in February, remained elevated for 7 months with a mean value of 37.39 ± 17.03 nmol/L, and then dropped to 0.74 ± 0.54 nmol/L. Serum 17ß-estradiol levels were also found to be significantly increased in January, remained so for 8 months (15.80 ± 14.15 ng/L), and then declined after August (7.77 ± 6.78 ng/L). In seals, mating typically occurs in February and March, 1 month after the observed peaks in testosterone and estradiol concentrations and corresponding to the increase in progesterone. A moderate positive correlation between testosterone and progesterone concentrations in sexually mature males was also observed (Spearman rho, r = 0.63, P < 0.01). In sexually immature females, progesterone and estradiol concentrations were found to be significantly lower than those in mature females. Finally, the observed patterns of estradiol and progesterone in sexually mature females suggest that embryonic diapause or successful implantation occurs in August.

Selenium and mercury concentrations in harbor seals (Phoca vitulina) from central California: health implications in an urbanized estuary.

We measured total selenium and total mercury concentrations ([TSe] and [THg]) in hair (n=138) and blood (n=73) of harbor seals (Phoca vitulina) from California to assess variation by geography and sex, and inferred feeding relationships based on carbon, nitrogen, and sulfur stable isotopes. Harbor seals from Hg-contaminated sites had significantly greater [THg], and lesser [TSe] and TSe:THg molar ratios than seals from a relatively uncontaminated site. Males had significantly greater [THg] than females at all locations. Sulfur stable isotope values explained approximately 25% of the variability in [THg], indicating increased Hg exposure for seals with a greater use of estuarine prey species. Decreased [TSe] in harbor seals from Hg-contaminated regions may indicate a relative Se deficiency to mitigate the toxic effects of Hg. Further investigation into the Se status and the potential negative impact of Hg on harbor seals from Hg-contaminated sites is warranted.

Validation of an enzyme-linked immunoassay (ELISA) for plasma oxytocin in a novel mammal species reveals potential errors induced by sampling procedure.

BACKGROUND: The neuropeptide oxytocin is increasingly the focus of many studies investigating human and animal social behaviours and diseases. However, interpretation and comparison of results is made difficult by a lack of consistent methodological approaches towards analysing this hormone. NEW METHOD: This study determined the sample collection and analysis protocols that cause the least amounts of protocol dependant variation in plasma oxytocin concentrations detected by ELISA. The effect of vacutainer type, sample extraction prior to analysis and capture and restraint protocol were investigated while validating an assay protocol for two novel species, grey seals (Halichoerus grypus) and harbour seals (Phoca vitulina). RESULTS: Where samples are extracted prior to analysis, vacutainer type (EDTA mean: 8.25±0.56 pg/ml, heparin mean: 8.25±0.62 pg/ml, p=0.82), time taken to obtain a sample and restraint protocol did not affect the concentration of oxytocin detected. However, concentrations of oxytocin detected in raw plasma samples were significantly higher than those in extracted samples, and varied significantly with vacutainer type (EDTA mean: 534.4±43.7 pg/ml, heparin mean: 300.9±19.6 pg/ml, p<0.001) and capture and restraint methodology. There was no relationship between oxytocin concentrations detected in raw and extracted plasma (p=0.25). COMPARISON WITH EXISTING METHOD(S): Over half the reviewed published studies analysing plasma oxytocin use raw plasma and different vacutainer types are used without consistency or justification through-out the literature. CONCLUSIONS: We caution that studies using raw plasma are likely to over estimate oxytocin concentrations, cannot be used to accurately infer true values via correlations and are susceptible to variation according vacutainer type.

Use of cellulose filter paper to quantify whole-blood mercury in two marine mammals: validation study.

Whole blood (WB) is commonly used to assess mercury (Hg) exposure in mammals, but handling and shipping samples collected in remote areas can be difficult. We describe and validate use of cellulose filter paper (FP) for quantifying WB total Hg concentration. Advantec Nobuto® FP was soaked with bottlenose dolphin (Tursiops truncatus) or harbor seal (Phoca vitulina) WB (collected between March and July 2012), then air dried. Untreated blood-soaked FPs were analyzed or were eluted with phosphate-buffered saline (PBS) and the eluate and PBS-treated FP Hg concentrations were determined. Total Hg from dried blood-soaked FPs, postelution FPs, and PBS-based eluate were compared with total Hg concentrations from WB. Recovery (on a concentration basis) for soaked FP relative to WB was 0.89 ± 0.15, for postelution FP was 0.86 ± 0.13, and for eluate (with a correction factor applied) was 0.96 ± 0.23. Least-squares linear regressions were fit for soaked papers (y = 1.15x, R(2) = 0.97), postelution FPs (y = 1.22x, R(2) = 0.95), and for eluate with a correction factor applied (y = 0.91x+0.03, R(2) = 0.97) as compared with WB. These data show that FP technology can have a valuable role in monitoring blood Hg concentrations in wildlife populations and FPs have the advantage of being easy to use, store, and transport as compared with WB.

Relationships between in vitro lymphoproliferative responses and levels of contaminants in blood of free-ranging adult harbour seals (Phoca vitulina) from the North Sea.

In vitro culture of peripheral blood leucocytes (PBLs) is currently used in toxicological studies of marine mammals. However, blood cells of wild individuals are exposed in vivo to environmental contaminants before being isolated and exposed to contaminants in vitro. The aim of this study was to highlight potential relationships between blood contaminant levels and in vitro peripheral blood lymphocyte proliferation in free-ranging adult harbour seals (Phoca vitulina) from the North Sea. Blood samples of 18 individuals were analyzed for trace elements (Fe, Zn, Se, Cu, Hg, Pb, Cd) and persistent organic contaminants and metabolites (ΣPCBs, ΣHO-PCBs, ΣPBDEs, 2-MeO-BDE68 and 6-MeO-BDE47, ΣDDXs, hexachlorobenzene, oxychlordane, trans-nonachlor, pentachlorophenol and tribromoanisole). The same samples were used to determine the haematology profiles, cell numbers and viability, as well as the in vitro ConA-induced lymphocyte proliferation expressed as a stimulation index (SI). Correlation tests (Bravais-Pearson) and Principal Component Analysis with multiple regression revealed no statistically significant relationship between the lymphocyte SI and the contaminants studied. However, the number of lymphocytes per millilitre of whole blood appeared to be negatively correlated to pentachlorophenol (r=-0.63, p=0.005). In adult harbour seals, the interindividual variations of in vitro lymphocyte proliferation did not appear to be directly linked to pollutant levels present in the blood, and it is likely that other factors such as age, life history, or physiological parameters have an influence. In a general manner, experiments with in vitro immune cell cultures of wild marine mammals should be designed so as to minimize confounding factors in which case they remain a valuable tool to study pollutant effects in vitro.

Immune-relevant and new xenobiotic molecular biomarkers to assess anthropogenic stress in seals.

Harbour seals as top predators and indicators for ecosystem health are exposed to increasing pressure caused by anthropogenic activities in their marine environment. After their lactation period of about 24 days pups are weaned and left to hunt on their own. Little is known about the development of their immune system and a better understanding of anthropogenic impacts on the general health and immune system of harbour seal pups is needed. mRNA transcription of six immuno-relevant biomarkers was analysed in 13 abandoned harbour seal pups from the North Sea, fostered at the Seal Centre Friedrichskoog, Germany. RNAlater blood samples were taken at admission, day 22 and before release and analysed using RT-qPCR. Significant differences in HSP70, cytokine IL-2 and xenobiotic biomarkers AHR, ARNT and PPARα transcription were found between admission, during rehabilitation and before release. Highest levels at admission may result from dehydration, handling, transport and contaminant exposure via lactation. The significant decrease is linked to health improvement, feeding and adaptation. The increase before release is suspected to be due to infection pressure and contaminant exposure from feeding on fish. Molecular biomarkers are a sensitive tool to evaluate health and pollutant exposure and useful to serve as early warning indicators, monitoring and case-by-case tool for marine mammals in human care and the wild.

Immune status and function in harbor seal pups during the course of rehabilitation.

Routine hematological and serum chemistry parameters are important tools for the evaluation of health and the treatment of marine mammals admitted to rehabilitation centers. The evaluation of phagocytosis, oxidative burst and immunoglobulin G (IgG), as markers of immune system function, and haptoglobin (Hp), as a stress marker, were evaluated alongside routine hematology and chemistry as potentially informative diagnostic tools for marine mammal health assessments. Blood samples from harbor seal pups (Phoca vitulina) admitted to (n=46), and released from (n=28), the Vancouver Aquarium's Marine Mammal Rescue Center (VAMMRC) were collected (1) to perform routine and novel functional approaches to evaluate the health of pups at admission; (2) to determine how these parameters changed during the rehabilitation process; and (3) to generate baseline values for reference purposes. Sodium was the only blood parameter which differed between seal pups that survived and those that died, with the surviving pups exhibiting higher levels on admission diagnostics. Positive correlations between total protein concentrations, IgG and Hp levels were observed with globulin concentrations of seal pups. Changes in serum chemistry values between admission and release included a decrease in red blood cells (RBCs), glucose, bicarbonate, total bilirubin, γ-glutamyltransferase (GGT) levels, and an increase in mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), lymphocytes, eosinophils, urea, potassium, anion gap, calcium, phosphorus, total protein, albumin, globulin and osmolality levels. During the rehabilitation process, phagocytosis decreased, while Hp levels increased. Age and improved health appeared to underlie changes in these parameters during the rehabilitation period.

A multiplex real-time polymerase chain reaction assay with two internal controls for the detection of Brucella species in tissues, blood, and feces from marine mammals.

Brucellosis has emerged as a disease of concern in marine mammals in the last 2 decades. Molecular detection techniques have the potential to address limitations of other methods for detecting infection with Brucella in these species. Presented herein is a real-time polymerase chain reaction (PCR) method targeting the Brucella genus-specific bcsp31 gene. The method also includes a target to a conserved region of the eukaryotic mitochondrial 16S ribosomal RNA gene to assess suitability of extracted DNA and a plasmid-based internal control to detect failure of PCR due to inhibition. This method was optimized and validated to detect Brucella spp. in multiple sample matrices, including fresh or frozen tissue, blood, and feces. The analytical limit of detection was low, with 95% amplification at 24 fg, or an estimated 7 bacterial genomic copies. When Brucella spp. were experimentally added to tissue or fecal homogenates, the assay detected an estimated 1-5 bacteria/µl. An experiment simulating tissue autolysis showed relative persistence of bacterial DNA compared to host mitochondrial DNA. When used to screen 1,658 field-collected marine mammal tissues in comparison to microbial culture, diagnostic sensitivity and specificity were 70.4% and 98.3%, respectively. In addition to amplification in fresh and frozen tissues, Brucella spp. were detected in feces and formalin-fixed, paraffin-embedded tissues from culture-positive animals. Results indicate the utility of this real-time PCR for the detection of Brucella spp. in marine species, which may have applications in surveillance or epidemiologic investigations.

Absolute quantification of transferrin in blood samples of harbour seals using HPLC-ICP-MS.

Harbour seals (Phoca vitulina) are bio-indicators for the assessment of their habitat and environmental changes. Besides population parameters and trends (survival, age structure, sex ratio), the individual health status represents a further important parameter for this assessment. The health status of seals is a complex and vague term, determined by a wide range of diagnostic parameters. Quantities of important blood proteins such as transferrin (Tf), as well as altered distribution patterns of its glycoforms, are frequently used as biomarkers in clinical diagnosis. Within this context Tf quantities and a varying pattern of its glycoforms are used as indicator for e.g. certain liver diseases, which also represents one of the most frequently observed pathological indication in harbour seals of the North Sea. Currently, most assay based quantification methods for Tf are limited since they often provide only information regarding the total Tf concentration rather than information of its different glycoforms. Due to a lack of suitable seal Tf antibodies also the application of more specific antibody based approaches is not possible. Within this background a new approach for the absolute quantification of the iron-transport protein Tf in the blood of harbour seals using its characteristic iron content and HPLC-ICP-MS detection is described. Method validation was performed using a certified human serum reference material (ERM-DA470K/IFCC). A Tf concentration of 2.33 ± 0.03 g L(-1) (sum of all quantified glycoforms) has been calculated, which is in good agreement with the certified total Tf concentration of 2.35 ± 0.08 g L(-1), confirming the accuracy of the proposed analytical method. Finally, different seal samples were analysed to demonstrate the suitability of the procedure for the quantification of Tf in real samples as well as to observe modified glycoform patterns. Compared to our previous studies for the first time it was possible to quantify the serum Tf baseline reference range for male (1.42-2.35 g L(-1)) and female German North Sea seals (1.93-2.74 g L(-1)) as well as a CDT level of 0.00-0.10 g L(-1), respectively, which provides valuable further diagnostic information regarding the health status of these specific marine mammals. Compared to assay based quantification approaches the proposed technique indicates great potential to obtain comparable and traceable absolute quantitative results, which are in particular important for long term investigations. This absolute quantification is based on an accurate, traceable element standard, while assay based approaches often show variations depending on the kit quality or changing activities of the used antibodies.