Campylobacter insulaenigrae Isolates from Northern Elephant Seals (Mirounga angustirostris) in California

There are only two reports in the literature demonstrating the presence of Campylobacter spp. in marine mammals. One report describes the isolation of a new species, Campylobacter insulaenigrae sp. nov., from three harbor seals (Phoca vitulina) and a harbor porpoise (Phocoena phocoena) in Scotland, and the other describes the isolation of Campylobacter jejuni, Campylobacter lari, and an unknown Campylobacter species from northern elephant seals (Mirounga angustirostris) in California. In this study, 72 presumptive C. lari and unknown Campylobacter species strains were characterized using standard phenotypic methods, 16S rRNA PCR, and multilocus sequence typing (MLST). Phenotypic characterization of these isolates showed them to be variable in their ability to grow either at 42°C or on agar containing 1% glycine and in their sensitivity to nalidixic acid and cephalothin. Based on both 16S rRNA PCR and MLST, all but 1 of the 72 isolates were C. insulaenigrae, with one isolate being similar to but distinct from both Campylobacter upsaliensis and Campylobacter helveticus. Phylogenetic analysis identified two C. insulaenigrae clades: the primary clade, containing exclusively California strains, and a secondary clade, containing some California strains and all of the original Scottish strains. This study demonstrates the inability of phenotypic characterization to correctly identify all Campylobacter species and emphasizes the importance of molecular characterization via 16S rRNA sequence analysis or MLST for the identification of Campylobacter isolates from marine mammals.     

Leptospirosis in northern elephant seals (Mirounga angustirostris) stranded along the California coast

Leptospirosis was identified in six northern elephant seals (Mirounga angustirostris) that were stranded in 1995 along the coast of California (USA). Histologic lesions in all seals included tubulointerstitial nephritis with tubular degeneration and necrosis. Infection was confirmed through identification of spirochetes using an immunohistochemical stain for Leptospira sp. antigens. One affected seal had an elevated titer to Leptospira interrogans serovar pomona. Four of the six seals developed leptospirosis during rehabilitation, and two seals had evidence of exposure in the wild. Potential sources of infection during rehabilitation include other elephant seals, California sea lions (Zalophus californianus), Pacific harbor seals (Phoca vitulina richardsii), or free-ranging wildlife. These results indicate that northern elephant seals are susceptible to leptospirosis and can develop disease both in the natural environment and in a rehabilitation setting.     

Infection with a novel gammaherpesvirus in northern elephant seals (Mirounga angustirostris)

Twenty juvenile northern elephant seals (Mirounga angustirostris) that died between 1998 and 2004 had ulcers on the tongue, palatine mucosa, and/or tonsils. Histologic examination of the lesions revealed cytoplasmic swelling, nuclear pyknosis, and eosinophilic to amphophilic intranuclear inclusions bodies suggestive of herpesviral infection. Electron microscopic examination and polymerase chain reaction analysis confirmed the presence of a herpesvirus. Subsequent DNA sequencing identified this to be a new gammaherpesvirus that was similar to Porcine lymphotropic virus 2, Alcephaline herpesvirus 1 (malignant catarrhal fever virus from wildebeest), and Chlorocebus rhadinovirus 1 from African green monkeys. Identical herpesviral DNA was also detected in blood and mucosal swabs collected from five healthy elephant seal pups.     

Early functional maturity of captive male northern elephant seals (Mirounga angustirostris)

A male northern elephant seal (Mirounga angustirostris) that was conceived in captivity was stillborn February 14, 1981, at Mystic Marinelife Aquarium in Mystic, Connecticut. Either of two males in the exhibit could have sired the pup. Both were only 3 yr at the time of conception.     

Physiology and behavior influence lactation efficiency in northern elephant seals (Mirounga angustirostris)

The efficiency with which mothers convert acquired energy into milk is a key determinant of the magnitude of parental investment in mammals; however, the mechanisms underlying lactation efficiency are poorly understood. Investigations on northern elephant seals have shown lactation efficiency, measured as the proportion of total energy expenditure that goes to the pup as milk, increases with age. In a cross-sectional study the physiological and behavioral determinants of lactation efficiency were investigated in eight young and seven prime (older) elephant seals by conducting behavioral observations and collecting milk, blood, and tissue on days 3 and 22 of lactation. Milk composition, circulating fatty acid and triglyceride concentrations, and mammary and blubber lipoprotein lipase activity were determined. Prime females had significantly greater percent milk fat and circulating fatty acids on day 3 than did young females, but these differences disappeared by day 22. The ability for prime females to produce higher-energy milk early in lactation may allow them to increase lactation efficiency by increasing the rate of energy transfer. In addition, prime females spent significantly more time resting. A combination of reduced activity and more rapid energy delivery likely explains the increase in lactation efficiency with age found in a previous study.     

Plasma melatonin concentration in neonatal northern elephant seals,Mirounga angustirostris

The development of pineal function in northern elephant seals was examined in an attempt to understand the physiological basis for previously observed high daytime levels of melatonin in neonatal southern elephant seals. Pineal glands from four northern elephant seal pups, estimated age less than 1 week, weighed 3.0 ± 0.80 g, which was significantly less than that previously found in southern elephant seals (4.6 ± 0.35 g). Midday concentrations of plasma melatonin in pups averaged more than 3000 pmol/l in the first 5 days post-partum, but declined rapidly to less than 400pmol/l after day 9. Daytime melatonin levels in northern elephant seals tended to be lower than in southern elephant seals, although they were very high compared with other species. A circadian cycle of plasma melatonin concentration was observed in newborn northern elephant seals, with levels of 3000–5000 pmol/1 during the day, rising to more than 10,000 pmol/1 late in the dark phase. Soon after weaning at 4 weeks of age, daytime and night-time levels were in the range 60–100 pmol/1 and 100–400 pmol/1, respectively. When approximately 10 weeks old, most samples were in the range 100–400 pmol/1 with no discernible difference between day and night levels. The results do not support the hypothesis that the pineal gland is involved in thermogenesis in new-born southern elephant seals. Instead, the very active pineal gland may contribute to energy conservation, by lowering body temperature, particularly at night. As physical insulation is acquired by the deposition of blubber, the mechanism is not required and melatonin falls to adult levels.     

Lactate flux and gluconeogenesis in fasting, weaned northern elephant seals (Mirounga angustirostris)

Elephant seals maintain rates of endogenous glucose production (EGP) typical of post-absorptive mammals despite enduring prolonged periods of food deprivation concurrent with low rates of glucose oxidation. These high rates of EGP suggest extensive glucose recycling during fasting. We investigated lactate metabolism in fasting elephant seals to assess its role in glucose recycling. Whole-animal glucose and lactate fluxes were measured as the rates of appearance of glucose and lactate (Ra _gluc and Ra _lac, respectively) using a primed constant infusion of [U-¹⁴C] lactate and [6-³H] glucose, and we calculated the minimum contribution of lactate to gluconeogenesis (GNG _lac). Ra _lac was high compared to resting values in other species (3.21 ± 0.71 mmol min^?1* kg^?1), did not change between 14 ± 1 and 31 ± 8 days of fasting and varied directly with Ra _glu. The minimum GNG _lac was 44.6 ± 6.0 % of EGP, varied directly with plasma lactate levels, and did not change over the fast. Ra _lac and Ra _glu both varied directly with plasma insulin concentrations. These data suggest that lactate is the predominant gluconeogenic precursor in fasting elephant seals and that high rates of glucose recycling through Cori cycle activity contribute to the maintenance of EGP during fasting. High levels of Cori cycle activity and EGP may be important components of metabolic adaptations that maintain glucose production while avoiding ketosis during extended fasting or are related to sustained metabolic alterations associated with extended breath-holds in elephant seals.     

Developmental aspects of sleep apnoea in northern elephant seals, Mirounga angustirostris

Northern elephant seals, Miroungu angustirostris , breathe irregularly while sleeping on land, alternating bouts of breath-holding (apnoea) that can last up to 25 min with periods of breathing (eupnoea). Our aims were to quantify changes in this behaviour during development and to determine the correspondence between these ontogenetic changes and those independently recorded in the dive durations of free-ranging seals. We observed 163 seals during periods of apparent sleep, ranging in age from new-born to adult. at Año Nuevo, California. Mean length of apnoea and percentage time spent in apnoea were 3·1 min and 59%, in neonates (0–4 days old). These values decreased to 1·8 min and 37% in suckling pups (5–28 days old), then increased with age thereafter, reaching about 8·0 min and 60% in adults of both sexes. Sleep apnoea duration and percentage time spent in sleep apnoea increased most markedly after weaning, when the animals were learning to swim and dive. Mean sleep apnoea duration and mean dive duration increased in a similar way during the first year of life; thereafter. mean sleep apnoea duration reached an asymptote while mean dive duration continued to increase. We conclude that the elephant seal's ability to sustain long apnoeas is not only an adaptation for foraging underwater but also a means for conserving water and energy while fasting on land.     

Biliary adenocarcinoma in a stranded northern elephant seal (Mirounga angustirostris)

A stranded adult female northern elephant seal (Mirounga angustirostris) was admitted to a rehabilitation hospital grossly underweight and lethargic in April 2001. The animal was icteric, had severe bilirubinemia, and elevated serum gamma glutamyl transferase concentrations. Laparoscopy under anesthesia revealed multiple masses up to 3 cm diameter throughout the liver and spleen and the animal was euthanized. Abnormal gross postmortem findings included green serous fluid in the abdominal cavity and 0.5 to 3 cm diameter yellow nodules scattered throughout the liver, spleen, and lymph nodes. Histopathology revealed a biliary adenocarcinoma with metastases to the liver, spleen, lymph nodes, adrenal gland, and pancreas. This is believed to be the first reported case of neoplasia in a northern elephant seal.     

Moult energetics of the northern elephant seal (Mirounga angustirostris)

Northern elephant seals, Mirounga angustirostris , undergo an annual moult during which they shed all of their pelage and underlying epidermis. Moulting takes place on land and lasts a mean of 32.0±6.6 days. During this time the mean mass loss of adult females was 24.7±6.1%. Mean body composition at arrival (25.6±4.8%fat) did not differ significantly from that at departure (24.9±3.2%fat). Fat catabolism accounted for 93.6%of derived energy and 41%of mass lost. Approximately 3.5%of total mass loss was associated with the shedding of the pelage and epidermis. Moulting female northern elephant seals express an average daily metabolic rate of 2.0±0.6 times that predicted for adult terrestrial mammals. This energy demand was met by losing 3.0 kg d^-1 of total body mass. These energy expenditures suggest that, similar to data for harbour seals, the moult period is a time of relatively low energy expenditure.     

Angiotensin II and aldosterone increase with fasting in breeding adult male northern elephant seals (Mirounga angustirostris)

The renin-angiotensin-aldosterone system (RAAS) appears to contribute significantly to osmoregulation of fasting northern elephant seal (Mirounga angustirostris) pups; however, RAAS has not been characterized in fasting adult seals. Therefore, this study examined the contribution of RAAS to water turnover rates in fasting adult male northern elephant seals. Blood samples were obtained twice during their breeding fast at an interval of 6.5 wk, and water efflux rate was estimated by isotopic dilution during the same period. Serum electrolytes (Na+, K+, Cl-) and osmolality were unaltered between the two sampling periods, indicating ionic and osmotic homeostasis during the fast. Despite the lack of an increase in vasopressin, serum angiotensin II and aldosterone were increased and were significantly and positively correlated. Changes in aldosterone concentration and water efflux rate were significantly and negatively correlated, suggesting that the greater the increase in aldosterone, the smaller the loss of water. Adult male seals maintain ionic and osmotic homeostasis similar to that of fasting weaned pups, and this homeostasis appears to be mediated, at least in part, by RAAS, which probably contributes to increased water retention as well. The hormonal mechanisms by which northern elephant seals maintain water and electrolyte balance during fasting conditions appear to be similar regardless of age.     

Characterization of blubber fatty acid signatures in northern elephant seals (Mirounga angustirostris) over the postweaning fast

Phocids routinely fast for extended periods. During these fasts, energetic requirements are met primarily through the catabolism of blubber lipid. To assess whether fatty acid (FA) composition changes during the postweaning fast in northern elephant seals, blubber biopsies were acquired longitudinally from 43 pups at 2.3 ± 1.5 and 55.2 ± 3.7 days postweaning in 1999 and 2000. At weaning, short-chain monounsaturated FA (SC-MUFA, ≤18 carbons) dominated the blubber while saturated FA (SFA) were found in the next highest proportion. The major FA (all ≥1 % by mass) comprised approximately 91 % of total blubber FA. In both years, 18:1n-9 and 16:0 were the most prevalent FA. Major FA mobilized during the fast consisted of polyunsaturated FA (PUFA), SFA, and SC-MUFA. Long-chain MUFA (>18 carbons) tended to be conserved. The fractional mobilization value of 20:5n-3 was the highest, resulting in significant reductions of this PUFA. Although concentrations of some blubber FA changed significantly during the postweaning fast, the general FA signature of blubber was similar at weaning and near the end of the fast. Changes in some FA differed across years. For example, the concentration of 20:4n-6, a minor PUFA, was significantly reduced in 1999 but not in 2000. FA mobilization patterns in northern elephant seal pups are somewhat similar to those reported previously for other fasting phocids and terrestrial mammals, though there are some notable differences. Differences in FA mobilization patterns across mammalian species may be related to differences in diets, geographical distribution, environmental factors, physiological adaptations, and life history stage.     

Hormone and metabolite changes associated with extended breeding fasts in male northern elephant seals (Mirounga angustirostris)

We measured metabolic hormones and several key metabolites in breeding adult male northern elephant seals to examine the regulation of fuel metabolism during extended natural fasts of over 3 months associated with high levels of energy expenditure. Males were sampled twice, early and late in the fast, losing an average of 23% of body mass and 47% of adipose stores between measurements. Males exhibited metabolic homeostasis over the breeding fast with no changes in glucose, non-esterified fatty acids, or blood urea nitrogen. Ketoacids increased over the fast but were very low when compared to other fasting species. Changes within individuals in total triiodothyronine (tT₃) were positively related to daily energy expenditure (DEE) and protein catabolism. Differences in levels of thyroid hormones relative to that observed in weaned pups and females suggest a greater deiodination of T₄ to support the high DEE of breeding males. Relative levels of leptin and ghrelin were consistent with the suppression of appetite but a significant reduction in growth hormone across the fast was contrary to expectation in fasting mammals. The lack of the increase in cortisol during fasting found in conspecific weaned pups and lactating females may contribute to the ability of breeding males to spare protein despite high levels of energy expenditure. Together these findings reveal significant differences with conspecifics under varying nutrient demands, suggesting metabolic adaptation to extended high energy fasts.     

Toxoplasmosis in an elephant seal (Mirounga angustirostris)

Toxoplasma gondii infections in fish-eating marine mammals is intriguing and indicative of contamination of the sea environment with oocysts. Toxoplasma gondii was identified in an elephant seal (Mirounga angustirostris) that had encephalitis. Tissue cysts were found in sections of cerebrum, and the diagnosis was confirmed by immunohistochemical staining with T. gondii-specific polyclonal rabbit serum. This is the first report of T. gondii infection in an elephant seal.     

Assessment of gestation,lactation and fasting on stable isotope ratios in northern elephant seals (Mirounga angustirostris)

Effects of physiological processes such as gestation, lactation and nutritional stress on stable isotope ratios remain poorly understood. To determine their impact, we investigated these processes in simultaneously fasting and lactating northern elephant seals (Mirounga angustirostris). Stable carbon and nitrogen isotope values were measured in blood and milk of 10 mother-pup pairs on days 5 and 22 of lactation. As long- and short-term integrators of diet, blood cells and serum may reflect foraging data or energy reserves from late gestation and lactation, respectively. Limited changes in isotopic signatures of maternal blood over the lactating period were highlighted. Nitrogen isotope fractionation associated with mother-to-offspring transfer of nutrients was generated between mother and offspring during gestation and lactation. This fractionation was tissue and time-specific, it varied between early and late lactation from +0.6‰ to +1.3‰ in blood cells and from +1.1‰ to nonsignificant value in serum. Therefore, if pups appear to be good proxies to investigate the female trophic ecology especially for C sources, much more caution is required in using δ¹⁵N values. Further studies are also needed to better define the relative impact of fasting and lactation on the enrichment or depletion of isotopes in different tissues.     

Water-soluble vitamin homeostasis in fasting northern elephant seals (Mirounga angustirostris) measured by metabolomics analysis and standard methods

Despite the importance of water-soluble vitamins to metabolism, there is limited knowledge of their serum availability in fasting wildlife. We evaluated changes in water-soluble vitamins in northern elephant seals, a species with an exceptional ability to withstand nutrient deprivation. We used a metabolomics approach to measure vitamins and associated metabolites under extended natural fasts for up to 7 weeks in free-ranging lactating or developing seals. Water-soluble vitamins were not detected with this metabolomics platform, but could be measured with standard assays. Concentrations of measured vitamins varied independently, but all were maintained at detectable levels over extended fasts, suggesting that defense of vitamin levels is a component of fasting adaptation in the seals. Metabolomics was not ideal for generating complete vitamin profiles in this species, but gave novel insights into vitamin metabolism by detecting key related metabolites. For example, niacin level reductions in lactating females were associated with significant reductions in precursors suggesting downregulation of the niacin synthetic pathway. The ability to detect individual vitamins using metabolomics may be impacted by the large number of novel compounds detected. Modifications to the analysis platforms and compound detection algorithms used in this study may be required for improving water-soluble vitamin detection in this and other novel wildlife systems.     

Protein catabolism in suckling and fasting northern elephant seal pups (Mirounga angustirostris)

Nursing elephant seal pups are hypothesized to be preadapted to the postweaning fast, yet no comparison of lipid or protein use for meeting metabolic costs has been made between these contrasting nutritional periods. To address this, protein catabolism was estimated in five elephant seal pups from measurements of urea turnover made twice during nursing and twice during the postweaning fast. Changes in body composition were measured in ten separate weaned pups via tritiated water dilution and matched to fasting urea turnover measurements in order to assess errors in protein catabolism derived from urea turnover rates. Estimates of lean mass loss based upon urea turnover and tritiated water dilution were in general agreement, supporting estimates of protein catabolism derived from urea turnover measurements. Protein catabolism was estimated to contribute less than 4% to the average metabolic rate of suckling and fasting pups implying strict protein conservation during both periods and supporting the shypothesis that suckling pups are pre-adapted to fasting. It is proposed that strict protein conservation across suckling and fasting compensates for relative reductions in maternal investment associated with the abbreviated lactation period of the elephant seal.     

Pattern and depth of dives in Northern elephant seals, Mirounga angustirostris

A time-depth recorder was attached to a female Northern elephant seal at the end of her lactation fast before she entered the sea to feed. The animal dived continuously during its first 11 days at sea, the period recorded for a total of 653 dives. Mean dive time was 21 min, with the longest submersion lasting 32 min. Mean surface interval between dives was 3 min, resulting in a total surface time of 11 %. Mean dive depth was 333 m and the deepest dive was 630 m, the deepest ever recorded for a pinniped. A epth histogram recorder attached to another female yielded a similar frequency distribution of dive depths.     

Thermoregulation of weaned northern elephant seal (Mirounga angustirostris) pups in air and water

Fasting weaned northern elephant seal pups (Mirounga angustirostris) experience diverse environmental conditions on land and in water on a daily basis. Each environment undoubtedly induces distinct energetic costs that may vary for pups of differing body condition. To determine the energetic costs associated with different environmental conditions and whether costs vary between individuals, body mass, surface area, volume, body composition, resting metabolic rate, and core body temperature were determined for 17 weaned northern elephant seal pups from A?o Nuevo, California. Metabolic rate and body temperature were measured for pups resting in air (20.9 degrees +/-0.8 degrees C), cold water (3.8 degrees+/-0.4 degrees ;C), and warm water (14.5 degrees+/-0.2 degrees C). Resting metabolic rate increased with body mass (range: 62.0-108.0 kg) and was also correlated with lean mass and lipid mass. Metabolic rates ranged from 293.6 to 512.7 mL O(2) min(-1) and were lowest for pups resting in cold water. Thermal conductance, calculated from metabolic rate and core body temperature, ranged from 3.1 to 15.2 W degrees C(-1), with the highest values in air and the lowest values in cold water. Metabolic responses to the three environmental conditions did not differ with individual variation in body condition. For all elephant seal pups, a consequence of high lipid content is that thermoregulatory costs are greatest on land and lowest in cold water, a pattern that contrasts markedly with terrestrial mammals.