Serum vitamin A concentrations in captive sea otters (Enhydra lutris)

Individual dietary preferences and difficulty with animal training create challenges and nutritional concerns when evaluating a captive sea otter (Enhydra lutris) diet. The importance of vitamin A within the body reflects the necessity that it be ingested in adequate amounts to ensure optimal health. To compare levels of serum vitamin A concentrations from captive sea otters on daily oral vitamin A supplementation, serum samples from eight adult sea otters from three institutions were evaluated for serum vitamin A concentrations. The eight animals were fed a total of four different diets and received oral supplementation via three different methods. Multiple diet items were analyzed for vitamin A content and were found to have low to nondetectable levels of vitamin A. Oral vitamin A supplementation, as a slurry with dietary items, was shown to be effective and a mean serum concentration of approximately 170 +/- 51 microg/L was obtained for serum vitamin A concentrations in captive sea otters. Captive diets can be modified to increase vitamin A concentration and supplementation and, if accepted, can be used as a means to ensure adequate vitamin A intake.     

Age-related change and allometry of skull and canine of sea otters,Enhydra lutris

Skulls and canines of 460 sea otters from Lopatka Cape, Kamchatka, were examined to assess development patterns, individual variation and sexual differences. An allometric formula was applied to morphometrical data, and the relative growth of each character to total length of skull was analyzed. In both sexes, most morphometrical characters ceased growth at about 2 years of age. Canine root length increased rapidly during the first year of life, while crown length decreased due to remarkable wear. There was large individual variation in the feeding and breathing/sniffing apparatus, while there was little variation in braincase size. There were sexual differences in most characteristics, although males and females showed similar growth patterns. The coronoid process of the mandible showed positive allometry in both sexes, and we attributed this finding to feeding habits. The fact that only male mastoids showed positive allometry may be due to the need for male otters to maintain a passing territory.     

Chemical anesthesia of northern sea otters (Enhydra lutris): results of past field studies.

Between 1987 and 1997, we chemically immobilized 597 wild sea otters (Enhydra lutris) in Alaska for the collection of biological samples or for surgical instrumentation. One drug-related sea otter fatality occurred during this time. Fentanyl in combination with diazepam produced consistent, smooth inductions with minimal need for supplemental anesthetics during procedures lasting 30-40 min. Antagonism with naltrexone or naloxone was rapid and complete, although we observed narcotic recycling in sea otters treated with naloxone. For surgical procedures, we recommend a fentanyl target dose of 0.33 mg/kg of body mass and diazepam at 0.11 mg/kg. For nonsurgical biological sample collection procedures, we recommend fentanyl at 0.22 mg/kg and diazepam at 0.07 mg/kg. We advise the use of the opioid antagonist naltrexone at a ratio of 2:1 to the total fentanyl administered during processing.     

Pharmacokinetic indices for cefovecin after single‐dose administration to adult sea otters (Enhydra lutris)

Seven sea otters received a single subcutaneous dose of cefovecin at 8 mg/kg body weight. Plasma samples were collected at predetermined time points and assayed for total cefovecin concentrations using ultra‐performance liquid chromatography and tandem mass spectrometry. The mean (±SD) noncompartmental pharmacokinetic indices were as follows: C_Max (obs) 70.6 ± 14.6 μg/mL, T_{Max (obs)} 2.9 ± 1.5 h, elimination rate constant (k_el) 0.017 ± 0.002/h, elimination half‐life (t_1/2kel) 41.6 ± 4.7 h, area under the plasma concentration‐vs.‐time curve to last sample (AUC_last) 3438.7 ± 437.7 h·μg/mL and AUC extrapolated to infinity (AUC_0→∞) 3447.8 ± 439.0 h·μg/mL. The minimum inhibitory concentrations (MIC) for select isolates were determined and used to suggest possible dosing intervals of 10 days, 5 days, and 2.5 days for gram‐positive, gram‐negative, and Vibrio parahaemolyticus bacterial species, respectively. This study found a single subcutaneous dose of cefovecin sodium in sea otters to be clinically safe and a viable option for long‐acting antimicrobial therapy.     

Enteric bacterial pathogen detection in southern sea otters (Enhydra lutris nereis) is associated with coastal urbanization and freshwater runoff

Although protected for nearly a century, California’s sea otters have been slow to recover, in part due to exposure to fecally-associated protozoal pathogens like Toxoplasma gondii and Sarcocystis neurona. However, potential impacts from exposure to fecal bacteria have not been systematically explored. Using selective media, we examined feces from live and dead sea otters from California for specific enteric bacterial pathogens (Campylobacter, Salmonella, Clostridium perfringens, C. difficile and Escherichia coli O157:H7), and pathogens endemic to the marine environment (Vibrio cholerae, V. parahaemolyticus and Plesiomonas shigelloides). We evaluated statistical associations between detection of these pathogens in otter feces and demographic or environmental risk factors for otter exposure, and found that dead otters were more likely to test positive for C. perfringens, Campylobacter and V. parahaemolyticus than were live otters. Otters from more urbanized coastlines and areas with high freshwater runoff (near outflows of rivers or streams) were more likely to test positive for one or more of these bacterial pathogens. Other risk factors for bacterial detection in otters included male gender and fecal samples collected during the rainy season when surface runoff is maximal. Similar risk factors were reported in prior studies of pathogen exposure for California otters and their invertebrate prey, suggesting that land-sea transfer and/or facilitation of pathogen survival in degraded coastal marine habitat may be impacting sea otter recovery. Because otters and humans share many of the same foods, our findings may also have implications for human health.     

Evaluation of cardiac lesions and risk factors associated with myocarditis and dilated cardiomyopathy in southern sea otters (Enhydra lutris nereis)

OBJECTIVE: To describe cardiac lesions and identify risk factors associated with myocarditis and dilated cardiomyopathy (DCM) in beach-cast southern sea otters. ANIMALS: Free-ranging southern sea otters. PROCEDURE: Sea otters were necropsied at the Marine Wildlife Veterinary Care and Research Center from 1998 through 2001. Microscopic and gross necropsy findings were used to classify sea otters as myocarditis or DCM case otters or control otters. Univariate, multivariate, and spatial analytical techniques were used to evaluate associations among myocarditis; DCM; common sea otter pathogens; and potential infectious, toxic, and nutritional causes. RESULTS: Clusters of sea otters with myocarditis and DCM were identified in the southern aspect of the sea otter range from May to November 2000. Risk factors for myocarditis included age, good body condition, and exposure to domoic acid and Sarcocystis neurona. Myocarditis associated with domoic acid occurred predominantly in the southern part of the range, whereas myocarditis associated with S. neurona occurred in the northern part of the range. Age and suspected previous exposure to domoic acid were identified as major risk factors for DCM. A sample of otters with DCM had significantly lower concentrations of myocardial L-carnitine than control and myocarditis case otters. CONCLUSIONS AND CLINICAL RELEVANCE: Cardiac disease is an important cause of death in southern sea otters. Domoic acid toxicosis and infection with S. neurona are likely to be 2 important causes of myocarditis in sea otters. Domoic acid-induced myocarditis appears to progress to DCM, and depletion of myocardial L-carnitine may play a key role in this pathogenesis.     

Sarcocystis neurona retinochoroiditis in a sea otter (Enhydra lutris kenyoni)

Sarcocystis neurona is an important cause of fatal disease in sea otters in the USA. Encephalitis is the predominant lesion and parasites are confined to the central nervous system and muscles. Here we report retinochoroiditis in a sea otter (Enhydra lutris kenyoni) found dead on Copalis Beach, WA, USA. Salient lesions were confined to the brain and eye. Multifocal nonsuppurative meningoencephalitis was present in the cerebrum and cerebellum associated with S. neurona schizonts. The retina of one eye had a focus of inflammation that contained numerous S. neurona schizonts and merozoites. The focus extended from the retinal pigment epithelium inward through all layers of the retina, but inflammation was most concentrated at the inner surface of the tapetum and the outer retina. The inner and outer nuclear layers of the retina were disorganized and irregular at the site of inflammation. There was severe congestion and mild hemorrhage in the choroid, and mild hemorrhage into the vitreous body. Immunohistochemistry with S. neurona-specific polyclonal rabbit antibodies stained schizonts and merozoites. To our knowledge this is the first report of S. neurona-associated retinochoroiditis in any naturally infected animal.     

Immunomodulatory effects of organochlorine mixtures upon in vitro exposure of peripheral blood leukocytes differ between free-ranging and captive southern sea otters (Enhydra lutris)

Organochlorines (OCs), notably polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are ubiquitous environmental contaminants. Contaminant-induced immunosuppression by OCs has been implicated as a co-factor in the deaths of thousands of marine mammals in infectious disease epizootics over the last two decades, and limited studies support the hypothesis that PCBs are immunomodulatory. This study represented a unique opportunity to assess the potential differences in susceptibility to OCs between captive and free-ranging sea otters originating from the same genetic population. In vitro immune assays were utilized to evaluate both innate (phagocytosis and respiratory burst) and acquired (mitogen-induced B and T lymphocyte proliferation) immune functions. Individual PCBs (138, 153, 169 and 180) as well as TCDD and all 26 possible combinations were tested. Mixtures were tested as they represent 'real life' exposure. Our results suggest that (1) different immune functions were sensitive to different OC mixtures in both magnitude and direction (enhancement/suppression) and (2) differences in sensitivities upon in vitro exposure to OCs occurred between free-ranging and captive otters. Differences in susceptibility could be explained by the acute stress of capture, the chronic stress of captivity or nutritional differences. Understanding differences in toxicity to different populations of sea otters will have important implications for risk assessment as well as conservation and management strategies.     

Transplacental toxoplasmosis in a wild southern sea otter (Enhydra lutris nereis)

In September 2004, a neonatal sea otter pup was found alive on the beach in northern Monterey Bay, CA. Efforts to locate the mother were unsuccessful. Due to a poor prognosis for successful rehabilitation, the pup was euthanized. Postmortem examination revealed emaciation, systemic lymphadenopathy and a malformation of the left cerebral temporal lobe. On histopathology, free tachyzoites and tissue cysts compatible with Toxoplasma gondii were observed in the brain, heart, thymus, liver, lymph nodes and peri-umbilical adipose. The presence of T. gondii within host tissues was associated with lymphoplasmacytic inflammation and tissue necrosis. Immunofluorescent antibody tests using postmortem serum were positive for anti-T. gondii IgM and IgG (at 1:320 and 1:1280 serum dilution, respectively), but were negative for IgG directed against Sarcocystis neurona and Neospora caninum (<1:40 each). Brain immunohistochemistry revealed positive staining for tachyzoites and tissue cysts using antiserum raised to T. gondii, but not S. neurona or N. caninum. T. gondii parasite DNA was obtained from extracts of brain and muscle by PCR amplification using the diagnostic B1 locus. Restriction enzyme digestion followed by gel electrophoresis and DNA sequencing confirmed the presence of Type X T. gondii, the strain identified in the majority of southern sea otter infections.     

Validation of a cell culture bioassay for detection of petroleum exposure in mink (Mustela vison) as a model for detection in sea otters (Enhydra lutris)

OBJECTIVE: To validate a luciferase bioassay, which is based on a recombinant mouse hepatoma cell line, for the detection of exposure to petroleum in mustelid species. ANIMALS: 122 American mink (Mustela vison) and 15 sea otters (Enhydra lutris). PROCEDURES: Mink were exposed to Bunker C fuel oil or Alaska North Slope crude oil externally as a single exposure or internally via low dose concentrations in their ration for 6 months. Serum samples were analyzed for cytochrome P450 1A1 induction by quantification of luciferase activity in the bioassay. Mink liver specimens were also evaluated for cytochrome P450 1A1 induction by quantification of ethoxyresorufin-o-deethylase activity. Serum collected from exposed and unexposed sea otters was also analyzed using the luciferase bioassay. RESULTS: Serum samples from mink externally exposed to petroleum had significantly increased luciferase activities at 1 week after exposure. Serum samples taken at later time points or from mink exposed to either product in the ration did not cause significant luciferase induction. Samples from otters exposed to petroleum had significantly higher luciferase induction as compared with samples from otters not exposed to petroleum at 2 and 8 years after the spill. Cytochrome P450 1A1 activity in liver specimens collected from mink that were internally exposed through diet was significantly increased at the conclusion of our study. CONCLUSION AND CLINICAL RELEVANCE: The luciferase bioassay is a sensitive and specific method for determining recent exposure to petroleum in mink. The lack of luciferase activity in serum samples collected from mink greater than 1 week after experimental exposure was likely attributable to lower overall petroleum exposure in our trial, compared with natural exposures.     

Evaluation of changes in hematologic and clinical biochemical values after exposure to petroleum products in mink (Mustela vison) as a model for assessment of sea otters (Enhydra lutris)

OBJECTIVE: To determine the effects of petroleum exposure on hematologic and clinical biochemical results of mink and to identify variables that may be useful for making management decisions involving sea otters (Enhydra lutris) that have been exposed to oil in their environment. ANIMALS: 122 American mink (Mustela vison). PROCEDURES: Mink were exposed once to a slick of oil (Alaskan North Slope crude oil or bunker C fuel oil) on seawater or via low-level contamination of their daily rations. RESULTS: In the acute phase of exposure, petroleum directly affected RBC, WBC, neutrophil, and lymphocyte counts, fibrinogen, sodium, calcium, creatinine, total protein, and cholesterol concentrations, and alanine transaminase, creatine kinase, alkaline phosphatase, and gamma-glutamyltransferase activities. Aspartate transaminase, alkaline phosphatase, gamma-glutamyltransferase, and lactate dehydrogenase activities and cholesterol concentration also varied as a result of chronic low-level contamination of feed. CONCLUSIONS AND CLINICAL RELEVANCE: Our results are in agreement with reports that attribute increased alanine transaminase and alkaline phosphatase activities and decreased total protein concentration to petroleum exposure in sea otters during an oil spill. Sodium, calcium, creatinine, cholesterol, and lactate dehydrogenase may be valuable variables to assess for guidance during initial treatment of sea otters exposed to oil spills as well as for predicting which petroleum-exposed sea otters will reproduce following an oil spill. Measurement of these variables should aid wildlife professionals in making decisions regarding treatment of sea otters after oil spills.     

The development of methods for immunophenotypic and lymphocyte function analyzes for assessment of Southern sea otter (Enhydra lutris nereis) health

The Southern sea otter (Enhydra lutris nereis) is listed as threatened under the Endangered Species Act. The population began a pattern of slow decline in 1995. The decline was attributed to high adult mortality rates with infectious disease being the major cause of death. Multiple pathogens were implicated in these deaths including opportunistic pathogens such as Coccidiodes immitis and Toxoplasma sp. These findings suggested that the immunological health of mature animals in this population might be compromised. The primary goal of this study was to establish techniques for assessing phenotypic and functional baseline data for peripheral blood mononuclear cells (PBMC) in free-ranging sea otters. Standard total and differential white blood cell counts were augmented by emumeration of T and B lymphocyte subsets. Lymphocyte function was determined by both mitogen-induced proliferation and expression of IL-2 receptors. In addition to establishing normal ranges for adult animals, age-related changes were identified in B lymphocyte numbers and cell-surface density of major histocompatability complex class II (MHC II) proteins. The predominant lymphocyte subpopulation in Southern sea otters is the T lymphocyte. Substantial variation among individual animals was observed within the B lymphocyte population both in cell number and density of MHC II expression. Pups had greater numbers of T and B lymphocyte, as well as, greater MHC II expression on B lymphocytes than adults. Mitogen-induced proliferation of peripheral blood mononuclear cells (PBMC) was variable among individual animals with no significant difference in cell response between age class and gender. Concanavalin (ConA) was a more effective mitogen in stimulating proliferation and interleukin (IL)-2 receptor expression than pokeweed. This data can be used to augment routine hematology profiles and aid in the identification of animals with immunologic perturbations.     

Molecular and antigenic characterization of Bordetella bronchiseptica isolated from a wild southern sea otter (Enhydra lutris nereis) with severe suppurative bronchopneumonia.

Bordetella bronchiseptica was isolated in pure culture from the lung, abdomen, and intestine of a wild free-ranging southern sea otter (Enhydra lutris nereis) with severe, suppurative bronchopneumonia. Immunohistochemistry, using antiserum raised to B. bronchiseptica, revealed strong positive staining of bacteria attached to bronchial ciliated epithelia as well as scattered positive staining in affected alveoli. Western blot analysis demonstrated that virulence factors, filamentous hemagglutinin, pertactin, and adenylate cyclase toxin are produced by the sea otter B. bronchiseptica isolate. Ribotype analysis using Pvu II restriction digests indicated that this isolate is most similar to strains commonly obtained in domestic dogs and cats.     

Vitamin A deficiency in turkey poults.

Vitamin A deficiency was diagnosed in a commercial flock of 13,000 4-6-week-old turkey poults in the summer of 2004. The birds were initially submitted for examination because of a 3% increase in the reported daily mortality of the flock. Clinically, affected birds had stunted growth and ruffled feathers, showed signs of incoordination, and were depressed. At necropsy, pale white pseudomembranous to mucoid material was observed on the mucosal surface of the tongue, oral cavity, portions of the esophagus, and the crop of some birds. Histologically, there was squamous metaplasia of the mucosal epithelium of the oral mucosa, esophagus, sinuses, nasal glands, bronchi, proventriculus, and the bursa of Fabricius. Vitamin A was not detected in the feed sample at a detection limit of 0.5 mg/kg. Serum vitamin A concentrations in 7 birds were very low and ranged from 0.05 to 0.1 mg/L. Vitamin A concentrations in livers were extremely low (0.1 mg/kg wet weight, 1/7 poults) or undetectable (< 0.1 mg/kg wet weight, 6/7 poults). A diagnosis of vitamin A deficiency was made based on gross and microscopic lesions and vitamin A concentrations in serum, liver, and feed. To the authors' knowledge, this is the first documented case of vitamin A deficiency in poults submitted from a commercial meat turkey producer comparatively depicting the gross and microscopic lesions with those found in other species of birds and mammals.     

Measurement of serum immunoglobulin concentration in killer whales and sea otters by radial immunodiffusion

Killer whales and sea otters maintained in captivity are the subjects of routine health monitoring programs, and interest in immunologic studies in sea otters has been rising recently in response to potential impacts from infectious disease and environmental pollution on the threatened southern sea otter population. Development of species-specific reagents for immunologic studies in these two marine mammals is currently in its infancy. In this study, killer whale and sea otter immunoglobulin-specific polyclonal antibodies were generated, and used to develop tests for serum Ig concentration in the killer whale (Orcinus orca) and the southern (Enhydra lutris nereis) and northern sea otter (Enhydra lutris lutris). Killer whale serum IgG was purified using caprylic acid/ammonium sulfate precipitation. Sea otter plasma IgG was purified using protein-A-agarose. Polyclonal anti-Ig antisera were produced in rabbits, and specificity confirmed by immunoelectrophoresis. Radial immunodiffusion was used to measure Ig concentration in serum or plasma samples derived from 21 captive killer whales, 18 wild and 4 captive southern sea otters and 15 wild and 4 captive northern sea otters grouped by age. Mean killer whale serum Ig concentration (+/-95% confidence interval) ranged from 15.04 +/- 3.97 g/l for animals aged 0-5 years to 26.65 +/- 9.8 g/l for animals aged >10 years. Mean sea otter serum Ig concentration (+/-95% confidence interval) ranged from 28.39 +/- 11.00 g/l for southern sub-adults to 32.76 +/- 11.58 g/l for southern adults. No significant difference in serum Ig concentration was found between southern and northern sea otters. Serum Ig concentrations in two northern sea otter pups were low compared to those of adult sea otters. The two serum Ig quantitation assays produced were highly specific and reproducible and will be useful additions to the limited number of tests available for immune function in these marine mammal species.     

Vitamin A deficiency: serum cortisol and humoral immunity in lambs

Serum cortisol and antigen-specific and polyclonal immunoglobulin G (IgG) concentrations were measured to investigate the relationship between vitamin A status and immune function in lambs. Twenty-four 3-mo-old crossbred ewe lambs weighing approximately 10 kg were each fed 900 g/d of a carotene-deficient diet. The 12 control lambs also received a 100,000 IU oral dose of vitamin A palmitate every 2 wk. All lambs were given primary and secondary antigenic challenges. Lambs were slaughtered at the end of the secondary challenge period. Liver vitamin A concentrations were greater (P less than .001) in the control animals (69.5 vs 1.3 micrograms/g wet tissue). Both groups of lambs exhibited a similar growth response until d 105, after which daily gain of the control lambs exceeded (P less than .03) that of the A-deficient lambs. Polyclonal serum IgG concentrations were greater (P less than .05) in the A-deficient lambs on d 49 to 124 and on d 151 (P less than .10). Ovalbumin-specific serum IgG concentrations tended to be greater in the control lambs throughout the primary and secondary challenge periods. Control lambs had greater titers on d 164 (P less than .07) and d 190 (P less than .03). Vitamin A status appeared to have no consistent effects on serum cortisol concentrations. Spleen weights were greater (P less than .002) in the A-deficient lambs. Lungs from 11 of 12 A-deficient lambs contained abscesses, as opposed to 1 of 12 for the control lambs. Both polyclonal and antigen-specific IgG concentrations were affected by vitamin A status. Serum cortisol concentrations did not appear to mediate this effect.