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: CMax (obs) 70.6 ± 14.6 µg/mL, TMax (obs) 2.9 ± 1.5 h, elimination rate constant (kel ) 0.017 ± 0.002/h, elimination half-life (t1/2kel) 41.6 ± 4.7 h, area under the plasma concentration-vs.-time curve to last sample (AUClast) 3438.7 ± 437.7 h·µg/mL and AUC extrapolated to infinity (AUC0→∞ ) 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.

ANTIDOG IgG SECONDARY ANTIBODY SUCCESSFULLY DETECTS IgG IN A VARIETY OF AQUATIC MAMMALS.

Serological tests play an important role in the detection of wildlife diseases. However, while there are many commercial assays and reagents available for domestic species, there is a need to develop efficient serological assays for wildlife. In recent years, marine mammals have represented a wildlife group with emerging infectious diseases, such as influenza, brucellosis, and leptospirosis. However, with the exception of disease-agent-specific assays or functional assays, few reports describe the use of antibody detection assays in marine mammals. In an indirect enzyme-linked immunoassay (EIA) or an immunofluorescence assay, antibody is detected using an antitarget species secondary conjugated antibody. The sensitivity of the assay depends on the avidity of the binding reaction between the bound antibody and the detection antibody. A commercial polyclonal antidog IgG conjugated antibody was tested in an EIA for its ability to sensitively detect the IgG of seven marine mammals including sea otter ( Enhydra lutris ), polar bear ( Ursus maritimus ), grey seal ( Halichoerus grypus ), harbor seal ( Phoca vitulina ), northern elephant seal ( Mirounga angustirostris ), California sea lion ( Zalophus californianus ), Pacific walrus ( Odobenus rosmarus ) and one freshwater mammal: Asian small-clawed otter ( Aonyx cinerea ). With the exception of Asian small-clawed sea otters, the detection of IgG in these marine mammals either exceeded or was nearly equal to detection of dog IgG. The use of the tested commercial antidog IgG antibody may be a valid approach to the detection of antibody response to disease in sea mammals.

Accumulation of PBDEs in an urban river otter population and an unusual finding of BDE-209.

River otter scat samples (n = 77) and blood samples (n = 16) collected through non-invasive field collections and live-capture activities (November 2009 to October 2010) along the coastline of Southern Vancouver Island, near Victoria, British Columbia (BC) were analyzed for polybrominated diphenyl ethers (PBDEs). ∑PBDEs were highest in urbanized regions of Victoria Harbour for blood (1.12 µg/g lipid weight) and scat (0.35 µg/g lipid weight). A location effect between zones was confirmed statistically for blood but not for scat. Specific congeners with the highest concentrations overall were BDE-47 in blood samples (0.37 µg/g lipid weight) and BDE-206 (0.18 µg/g lipid weight) and BDE-47 (0.16 µg/g lipid weight) in scat samples. There was also an unusual finding of extremely high levels of BDE-209 in 2 scat samples (163 and 956 µg/g lipid weight). The patterns of select congeners (BDE 47, 99, 100, 153, 154) measured in blood and scat were found not to be significantly different (Chi-square Test, X2 = 21.08, DF = 4, p = 0003). The most prominent congeners within Victoria Harbour were BDE-47 for both blood (0.82 mg/kg lipid weight) and scat (0.26 mg/kg lipid weight) followed by BDE-206 (0.18 µg/g lipid weight) and BDE-207 (0.10 µg/g lipid weight) for scat only. Comparable levels of BDE-47 were reported across the study area whereas BDE 206 and 207 were only observed in Victoria Harbour (scat). Toxicological effects of PBDEs in rivers otters from Victoria, BC are still unknown however the predominance of BDE-47 could have negative implication as an endocrine disruptor.

Bartonella spp. exposure in northern and southern sea otters in Alaska and California.

Since 2002, an increased number of northern sea otters (Enhydra lutris kenyoni) from southcentral Alaska have been reported to be dying due to endocarditis and/or septicemia with infection by Streptococcus infantarius subsp. coli. Bartonella spp. DNA was also detected in northern sea otters as part of mortality investigations during this unusual mortality event (UME) in Kachemak Bay, Alaska. To evaluate the extent of exposure to Bartonella spp. in sea otters, sera collected from necropsied and live-captured northern sea otters, as well as necropsied southern sea otters (Enhydra lutris nereis) unaffected by the UME, were analyzed using an immunofluorescent antibody assay. Antibodies against Bartonella spp. were detected in sera from 50% of necropsied and 34% of presumed healthy, live-captured northern sea otters and in 16% of necropsied southern sea otters. The majority of sea otters with reactive sera were seropositive for B. washoensis, with antibody titers ranging from 1:64 to 1:256. Bartonella spp. antibodies were especially common in adult northern sea otters, both free-living (49%) and necropsied (62%). Adult stranded northern sea otters that died from infectious causes, such as opportunistic bacterial infections, were 27 times more likely to be Bartonella seropositive than adult stranded northern sea otters that died from noninfectious causes (p<0.001; 95% confidence interval 2.62-269.4). Because Bartonella spp. antibodies were detected in necropsied northern sea otters from southcentral (44%) and southwestern (86%) stocks of Alaska, as well as in necropsied southern sea otters (16%) in southcentral California, we concluded that Bartonella spp. exposure is widely distributed among sea otter populations in the Eastern Pacific, providing context for investigating future disease outbreaks and monitoring of Bartonella infections for sea otter management and conservation.

Efficacy of two canine distemper vaccines in wild Nearctic river otters (Lontra canadensis).

Canine distemper virus (CDV), a contagious morbillivirus, infects families in the order Carnivora, including Nearctic river otters (Lontra canadensis). As a preventative measure, vaccinations against CDV are frequently given to mustelids in captive environments. The Pennsylvania River Otter Reintroduction Project (PRORP) used wild-caught river otters to evaluate the efficacy and need for vaccinations against CDV as part of any reintroduction project. The objectives of this study were to: 1) evaluate the prevalence of exposure to CDV in wild river otters, 2) determine the immunologic response of river otters (i.e., seroconversion) after vaccination with a single (primary) vaccine dose compared to a second (booster) dose of Galaxy-D, a modified live-virus canine distemper (CD) vaccine (MLV CDV), and 3) determine the immunologic response after being vaccinated with a primary vaccination compared to a booster dose of Fervac-D, an MLV CDV. River otters were injected subcutaneously in the nape of the neck with their designated vaccine. Timeframes for collection of blood samples and/or injection of booster vaccines varied depending on the parameters of PRORP. Ten of the 22 river otters had positive prevaccination titer levels to CD. Both vaccines, Galaxy-D and Fervac-D, produced sufficient seroconversion or rise of titer levels (86% and 57%, respectively) to recommend the use of vaccines in wild river otters. Future studies are recommended to evaluate currently produced CD vaccines. Future research should also focus on the number of days required between administration of primary and booster vaccines to achieve sufficient immune response. If only a primary dose is required, then hard-release reintroduction projects for river otters could be recommended. If primary and booster vaccines are required then soft-release reintroduction projects should be recommended. Soft-release projects should include captive management periods that allow for appropriate vaccination intervals and boosters needed to maximize the probability of protection against CDV.

Pathogen exposure and blood chemistry in the Washington, USA population of northern sea otters (Enhydra lutris kenyoni).

Northern sea otters (Enhydra lutris kenyoni) from Washington State, United States were evaluated in 2011 to determine health status and pathogen exposure. Antibodies to Brucella spp. (10%) and influenza A virus (23%) were detected for the first time in this population in 2011. Changes in clinical pathology values (serum chemistries), exposure to pathogens, and overall health of the population over the last decade were assessed by comparing 2011 data to the data collected on this population in 2001-2002. Several serum chemistry parameters were different between study years and sexes but were not clinically significant. The odds of canine distemper virus exposure were higher for otters sampled in 2001-2002 (80%) compared to 2011 (10%); likelihood of exposure significantly increased with age. Prevalence of exposure to Sarcocystis neurona was also higher in 2001-2002 (29%) than in 2011 (0%), but because testing methods varied between study years the results were not directly comparable. Exposure to Leptospira spp. was only observed in 2001-2002. Odds of Toxoplasma gondii exposure were higher for otters sampled in 2011 (97%) than otters in 2001-2002 (58%). Substantial levels of domoic acid (n = 2) and saxitoxin (n = 2) were found in urine or fecal samples from animals sampled in 2011. No evidence of calicivirus or Coxiella burnetii exposure in the Washington population of northern sea otters was found in either 2001-2002 or 2011. Changes in exposure status from 2001-2002 to 2011 suggest that the Washington sea otter population may be dealing with new disease threats (e.g., influenza) while also increasing their susceptibility to diseases that may be highly pathogenic in naïve individuals (e.g., canine distemper).

Hydrocarbon concentrations and patterns in free-ranging sea otters (Enhydra lutris) from British Columbia, Canada.

With oil pollution recognized as a major threat to British Columbia's recovering sea otter (Enhydra lutris) population, it is important to distinguish acute from chronic exposures to oil constituent groups in this marine mammal. Concentrations and patterns of alkanes and polycyclic aromatic hydrocarbons (PAHs) were determined in blood samples from 29 live-captured sea otters in two coastal areas of British Columbia, as well as in representative samples of their invertebrate prey. Hydrocarbon concentrations in sea otters were similar between areas and among age and sex classes, suggesting that metabolism dominates the fate of these compounds in sea otters. Biomagnification factors derived from PAH ratios in otter:prey supported this notion. Although some higher alkylated three- and four-ring PAHs appeared to biomagnify, the majority of PAHs did not. The apparent retention of alkyl PAHs was reflected in the composition of estimated sea otter body burdens, which provided an alternative way of evaluating hydrocarbon exposure. Alkyl PAHs made up 86 ± 9% of estimated body burdens (4,340 ± 2,950 µg), with no differences between males and females (p = 0.18). The importance of measuring both parent and alkyl PAHs is underscored by their divergent dynamics in sea otters, with ready depuration of parent PAHs (metabolized or excreted) by sea otters on the one hand and biomagnification of alkyl PAHs on the other.

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.

Persistent organic pollutants in the blood of free-ranging sea otters (Enhydra lutris ssp.) in Alaska and California.

As part of tagging and ecologic research efforts in 1997 and 1998, apparently healthy sea otters of four age-sex classes in six locations in Alaska and three in California were sampled for persistent organic pollutants (POPs) and other chemicals of ecologic or environmental concern (COECs). Published techniques for the detection of POPs (specifically ∑polychlorinated biphenyls [PCBs], ∑DDTs, ∑hexachlorocyclohexanes [HCHs], ∑polycyclic aromatic hydrocarbons [PAHs], ∑chlordanes [CHLs], hexachlorobenzene [HCB], dieldrin, and mirex) in the tissue of dead otters were modified for use with serum from live sea otters. Toxic equivalencies (TEQs) were calculated for POPs with proven bioactivity. Strong location effects were seen for most POPs and COECs; sea otters in California generally showed higher mean concentrations than those in Alaska. Differences in contaminant concentrations were detected among age and sex classes, with high levels frequently observed in subadults. Very high levels of ∑DDT were detected in male sea otters in Elkhorn Slough, California, where strong freshwater outflow from agricultural areas occurs seasonally. All contaminants except mirex differed among Alaskan locations; only ∑DDT, HCB, and chlorpyrifos differed within California. High levels of ∑PCB (particularly larger, more persistent congeners) were detected at two locations in Alaska where associations between elevated PCBs and military activity have been established, while higher PCB levels were found at all three locations in California where no point source of PCBs has been identified. Although POP and COEC concentrations in blood may be less likely to reflect total body burden, concentrations in blood of healthy animals may be more biologically relevant and less influenced by state of nutrition or perimortem factors than other tissues routinely sampled.

Immunomodulatory effects upon in vitro exposure of California sea lion and southern sea otter peripheral blood leukocytes to domoic acid.

During red tide bloom events, the marine diatom Pseudo-nitzschia produces the toxin domoic acid (DA), which has been associated with stranding and mortality events involving California sea lions (Zalophus californianus) and southern sea otters (Enhydra lutris). In addition to these well-documented DA-induced neurotoxic events, there is increasing concern that DA may exert chronic effects, such as immunomodulation, which may potentially increase an individual's susceptibility to a number of opportunistic infections following nonlethal exposure. We investigated the effects of DA on innate (phagocytosis and respiratory burst) and adaptive (mitogen-induced lymphocyte proliferation) immune functions with the use of peripheral blood leukocytes collected from healthy California sea lions and southern sea otters upon in vitro exposure to 0 (unexposed control), 0.0001, 0.001, 0.01, 0.1, 1.0, 10, and 100 microM DA. Domoic acid did not significantly modulate phagocytosis or respiratory burst in either species. For California sea lions, DA significantly increased ConA-induced T-lymphocyte proliferation upon exposure to DA concentrations ranging from 0.0001 to 10 microM, resulting in a nonlinear dose-response curve. There was no effect on lymphocyte proliferation at the highest concentration of DA tested. No effects on lymphocyte proliferation were observed in southern sea otters. Importantly, the in vitro DA concentrations affecting T-cell proliferation were within or below the range of DA in serum measured in free-ranging California sea lions following natural exposure, suggesting a risk for immunomodulation in free-ranging animals. Understanding the risk for immunomodulation upon DA exposure will contribute in the health assessment and management of California sea lions and southern sea otters, as well as guide veterinarians and wildlife rehabilitators in caring for and treating afflicted animals.

PCB exposure in sea otters and harlequin ducks in relation to history of contamination by the Exxon Valdez oil spill.

Exposure to contaminants other than petroleum hydrocarbons could confound interpretation of Exxon Valdez oil spill effects on biota at Prince William Sound, Alaska. Hence, we investigated polychlorinated biphenyls (PCBs) in blood of sea otters and harlequin ducks sampled during 1998. PCB concentrations characterized by lower chlorinated congeners were highest in sea otters from the unoiled area, whereas concentrations were similar among harlequin ducks from the oiled and unoiled area. Blood enzymes often elevated by xenobiotics were not related to PCB concentrations in sea otters. Only sea otters from the unoiled area had estimated risk from PCBs, and PCB composition or concentrations did not correspond to reported lower measures of population performance in sea otters or harlequin ducks from the oiled area. PCBs probably did not influence limited sea otter or harlequin duck recovery in the oiled area a decade after the spill.

Characterization of basal seminal traits and reproductive endocrine profiles in North American river otters and Asian small-clawed otters.

In this study, fecal samples were collected from 24 North American river (NARO) and 17 Asian small-clawed otters (ASCO) for 6-36 months and semen collected seasonally from NARO males (n=4/season) via electroejaculation. Our main objectives were to: (1) characterize endocrine parameters by longitudinal monitoring of fecal hormone metabolites and (2) investigate semen collection and basal seminal traits in NARO. NARO demonstrated a distinct seasonality in the spring, with females having a monoestrual estrogen elevation lasting 15.33+/-1.98 (mean+/-SEM) days and males peaking in testosterone production for 25.50+/-7.51 days. Pregnancy was characterized by 7-9 months of basal fecal progesterone, presumably corresponding to embryonic diapause, followed by a rapid increase over the final 68-73 days to term. Pseudopregnancy exhibited a similar late winter progesterone peak of 68-72 days, which could not be differentiated from pregnancy. Geographic latitude possibly influenced the timing of increased testosterone in males and increased progesterone in pregnant/pseudopregnant females. In ASCO, monitoring of fecal estrogens did not allow consistent detection of peak values associated with behavioral estrus. Both pregnancy and pseudopregnancy were characterized by a moderate rise in fecal progesterone for 14-16 days postovulation followed by a marked increase. Total gestation length was 67-77 days compared with 62-84 days for pseudopregnancy. In NARO, optimal sperm recovery and quality occurred only in the spring, corresponding with seasonal increases in testicular volume and fecal testosterone. These findings represent the first comprehensive information on normative endocrine and seminal traits in freshwater otter species.

Trapping and handling of North American river otters (Lontra canadensis) in a managed marsh.

North American river otters (Lontra canadensis) were trapped in a managed marsh in northern California between July and November of 1998. Five trap types using several set configurations were utilized in this study. Otters were successfully captured with minimal injury and a relatively high trap rate (1 capture per 48 trap nights), with the use of No. 1.5 and No. 1.75 double-coil spring traps, and No. 11 double long spring traps on short chains in blind land sets, or in bank sets on one-way cables leading to land-buried stakes. Only 3 of 14 captures incurred more than minimal injuries, and all 3 of these were exacerbated by complications from traps attached to long chains. Otters were captured at any time of day, although activity appeared lowered between 0900 and 1900 hours. Otters were anesthetized for handling. Monitoring results and anesthetic complications are reported for 14 captures. Ketamine (15 mg/kg) and midazolam (0.5 mg/kg) provided a wide margin of safety, rapid induction at low injection volume, good anesthetic quality, handling time of about 30 min, and few complications. White blood cell counts were taken and appeared high relative to reported values in other studies. This trend is likely attributable to blood draws immediately after the animal had been held in a trap for several hours, in contrast to most other studies, in which blood was drawn days to weeks after being held in captivity.

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.

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.

Evaluation of injury severity and hematologic and plasma biochemistry values for recently captured North American river otters (Lontra canadensis).

As part of a reintroduction program, blood samples from free-ranging, recently captured Nearctic river otters (Lontra canadensis) in eastern New York state were collected and analyzed to determine baseline hematology and plasma biochemistry values for the source population, and to determine whether these values were significant predictors of trap-injury status. Based on physical exam, each otter was classified as uninjured, moderately injured, or severely injured. Clinical pathology parameters were compared across sex, age class, and injury classification. The increase in likelihood of a change in each parameter in injured versus uninjured otters was determined using logistic regression. Baseline hematology and plasma biochemistry values did not differ significantly from published values for captive otters in zoos or other reintroduction programs. Plasma aspartate aminotransferase levels increased as time from capture to venipuncture decreased. Some otters in this study showed clinical signs consistent with exertional myopathy, possibly altering our calculation of baseline values. Our results suggest that the hematology and plasma biochemistry values obtained in this recently captured population of otters are generally not good predictors of capture-related injury. This could be due to disease processes that are not readily visible upon physical examination or because changes in these values may be associated with factors independent of capture-related injury.

Chronic fuel oil toxicity in American mink (Mustela vison): systemic and hematological effects of ingestion of a low-concentration of bunker C fuel oil.

Petroleum oil enters the coastal marine environment through various sources; marine mammals such as sea otters that inhabit this environment may be exposed to low concentrations of petroleum hydrocarbons through ingestion of contaminated prey. The inability to perform controlled studies in free-ranging animals hinders investigations of the effects of chronic petroleum oil exposure on sea otter morbidity and mortality, necessitating the development of a reliable laboratory model. We examined the effects of oral exposure to 500 ppm bunker C fuel oil over 113-118 days on American mink, a species phylogenetically related to the sea otter. Hematological parameters and organs were examined for fuel oil-associated changes. Hepatic cytochrome P4501A1 mRNA expression and fecal cortisol concentrations were also measured. Ingestion of fuel oil was associated with a decrease in erythrocyte count, hemoglobin concentration (Hgb), hematocrit (HCT), and an increase in mean corpuscular volume (MCV). Total leukocytes were elevated in the fuel oil group from increases in neutrophils, lymphocytes, and monocytes. Significant interactions between fuel oil and antigen challenge were found for erythrocyte parameters, monocyte and lymphocyte counts. Liver and adrenal weights were increased although mesenteric lymph node weights were decreased in the fuel oil group. Hepatic cytochrome P4501A1 mRNA was elevated in the fuel oil group. Fecal cortisol concentration did not vary between the two groups. Our findings show that fuel oil exposure alters circulating leukocyte numbers, erythrocyte homeostasis, hepatic metabolism and adrenal physiology and establish a framework to use mink as a model for sea otters in studying the systemic effects of marine contaminants.

Clinical pathology and assessment of pathogen exposure in southern and Alaskan sea otters.

The southern sea otter (Enhydra lutris nereis) population in California (USA) and the Alaskan sea otter (E. lutris kenyoni) population in the Aleutian Islands (USA) chain have recently declined. In order to evaluate disease as a contributing factor to the declines, health assessments of these two sea otter populations were conducted by evaluating hematologic and/or serum biochemical values and exposure to six marine and terrestrial pathogens using blood collected during ongoing studies from 1995 through 2000. Samples from 72 free-ranging Alaskan, 78 free-ranging southern, and (for pathogen exposure only) 41 debilitated southern sea otters in rehabilitation facilities were evaluated and compared to investigate regional differences. Serum chemistry and hematology values did not indicate a specific disease process as a cause for the declines. Statistically significant differences were found between free-ranging adult southern and Alaskan population mean serum levels of creatinine kinase, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, calcium, cholesterol, creatinine, glucose, phosphorous, total bilirubin, blood urea nitrogen, and sodium. These were likely due to varying parasite loads, contaminant exposures, and physiologic or nutrition statuses. No free-ranging sea otters had signs of disease at capture, and prevalences of exposure to calicivirus, Brucella spp., and Leptospira spp. were low. The high prevalence (35%) of antibodies to Toxoplasma gondii in free-ranging southern sea otters, lack of antibodies to this parasite in Alaskan sea otters, and the pathogen's propensity to cause mortality in southern sea otters suggests that this parasite may be important to sea otter population dynamics in California but not in Alaska. The evidence for exposure to pathogens of public health importance (e.g., Leptospira spp., T. gondii) in the southern sea otter population, and the naïveté of both populations to other pathogens (e.g., morbillivirus and Coccidiodes immitis) may have important implications for their management and recovery.

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.

Evaluation of an indirect fluorescent antibody test (IFAT) for demonstration of antibodies to Toxoplasma gondii in the sea otter (Enhydra lutris).

An indirect fluorescent antibody test (IFAT) for detection of Toxoplasma gondii infection was validated using serum from 77 necropsied southern sea otters (Enhydra lutris nereis) whose T. gondii infection status was determined through immunohistochemistry and parasite isolation in cell culture. Twenty-eight otters (36%) were positive for T. gondii by immunohistochemistry or parasite isolation or both, whereas 49 (64%) were negative by both tests. At a cutoff of 1:320, combined values for IFAT sensitivity and specificity were maximized at 96.4 and 67.3%, respectively. The area under the receiver-operating characteristic curve for the IFAT was 0.84. A titer of 1:320 was used as cutoff when screening serum collected from live-sampled sea otters from California (n = 80), Washington (n = 21), and Alaska (n = 65) for T. gondii infection. Thirty-six percent (29 out of 80) of California sea otters (E. lutris nereis) and 38% (8 out of 21) of Washington sea otters (E. lutris kenyoni) were seropositive for T. gondii, compared with 0% (0 out of 65) of Alaskan sea otters (E. lutris kenyoni).