INFECTIOUS DISEASE AND TOXICOLOGICAL MONITORING OF STRANDED PACIFIC HARBOR SEALS (PHOCA VITULINA RICHARDSI) IN COOK INLET AS SURROGATES FOR MONITORING ENDANGERED BELUGAS (DELPHINAPTERUS LEUCAS).

Pacific harbor seals ( Phoca vitulina richardsi) and belugas ( Delphinapterus leucas ) eat many of the same prey species, occupy the same geographic area, and demonstrate site fidelity in Cook Inlet, Alaska. Although most direct research involving the critically endangered belugas is currently prohibited, studying harbor seals may provide important information about this beluga population. In recent years, harbor seal populations in Alaska have declined for unknown reasons. As part of its stranding program, the Alaska SeaLife Center (ASLC) managed 59 cases of live and dead stranded harbor seals from Cook Inlet between 1997 and 2011. Animals were screened for a variety of diseases and contaminants of concern. Animals were negative by serology to the following diseases: avian influenza, canine distemper virus, dolphin morbillivirus, porpoise morbillivirus, Leptospira canicola, L. grippotyphosa, L. pomona, Neospora caninum , Sarcocystis neurona , and Toxoplasma gondii . Positive titers were found against Brucella spp., phocine distemper virus, seal herpesvirus-1, L. bratislava, L. hardjo, and L. icterohemorrhagiae. All titers were stable or declining except in one animal with an increasing titer for seal herpesvirus-1. Fecal pathogen screenings identified normal flora as well as stable or declining low levels of potentially pathogenic and opportunistic bacteria, though most were of little concern for seal health. In most animals, toxicology screening showed that the majority of tested contaminants were below detectable limits. The level of evidence of exposure to pathogens of concern was low in harbor seals. Although the infectious disease burden and contaminant levels in belugas in Cook Inlet cannot be definitively determined without direct testing, pathogen and contaminant exposure is expected to be similar to that found in harbor seals in this region, as the harbor seals and belugas share the habitat and food resources.

CIRCULATING CONCENTRATIONS OF THYROID HORMONE IN BELUGA WHALES (DELPHINAPTERUS LEUCAS): INFLUENCE OF AGE, SEX, AND SEASON.

Thyroid hormones play a critical physiologic role in regulating protein synthesis, growth, and metabolism. To date, because no published compilation of baseline values for thyroid hormones in beluga whales (Delphinapterus leucas) exists, assessment of thyroid hormone concentrations in this species has been underused in clinical settings. The purpose of this study was to document the concentrations of total thyroxine (tT4) and total triiodothyronine (tT3) in healthy aquarium-maintained and free-ranging beluga whales and to determine the influence of age, sex, and season on the thyroid hormone concentrations. Archived serum samples were collected from healthy aquarium-maintained (n=43) and free-ranging (n=39) belugas, and serum tT4 and tT3 were measured using chemiluminescence immunoassay. The mean tT4 concentration in aquarium-maintained belugas was 5.67±1.43 µg/dl and the mean tT3 concentration was 70.72±2.37 ng/dl. Sex comparisons showed that aquarium-maintained males had significantly greater tT4 and tT3 (9.70±4.48 µg/dl and 92.65±30.55 ng/dl, respectively) than females (7.18±2.82 µg/dl and 77.95±20.37 ng/dl) (P=0.004 and P=0.013). Age comparisons showed that aquarium-maintained whales aged 1-5 yr had the highest concentrations of tT4 and tT3 (8.17±0.17 µg/dl and 105.46±1.98 ng/dl, respectively) (P=0.002 and P<0.001). tT4 concentrations differed significantly between seasons, with concentrations in winter (4.59±1.09 µg/dl) being significantly decreased compared with spring (P=0.009), summer (P<0.0001), and fall (P<0.0001) concentrations. There was a significant difference in tT4 and tT3 concentrations between aquarium-maintained whales (5.67±1.43 µg/dl and 70.72±15.57 ng/dl, respectively) and free-ranging whales (11.71±3.36 µg/dl and 103.38±26.45 ng/dl) (P<0.0001 and P<0.001). Clinicians should consider biologic and environmental influences (age, sex, and season) for a more accurate interpretation of thyroid hormone concentrations in belugas. The findings of this study provide a baseline for thyroid health monitoring and comprehensive health assessments in both aquarium-maintained and free-ranging beluga whales.

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.

Molt-associated changes in hematologic and plasma biochemical values and stress hormone levels in African penguins (Spheniscus demersus).

Handling, including blood collection, has often been discouraged in molting penguins because it is considered an additional stress imposed on birds already experiencing major physiologic stress associated with molting. To evaluate the degree of physiologic stress posed by molting, we compared the hematologic and plasma biochemical values and hormone levels of molting and nonmolting African penguins, Spheniscus demersus. Five male and 5 female penguins randomly chosen were given complete physical examinations, were weighed, and blood samples were taken at 7 time points before, during, and after the molt. Data were analyzed by linear mixed-model analysis of variance. Throughout the study, behavior and appetite remained normal. Catecholamine levels were highly variable within and among subjects, whereas mean corticosterone levels were significantly different between baseline, molt, and postmolt values. Significant differences from baseline values were observed in many of the hematologic analytes; however, only decreases in hematocrit and red blood cell count values were considered clinically significant. Anemia due to experimentally induced blood loss as a possible cause of the significant hematologic changes was ruled out based on results of a follow-up control study during the nonmolt season, which showed no significant changes in hematocrit level or total red blood cell counts when using similar sampling protocols, which indicates that these changes were associated with molt.

Serologic response in bottlenose dolphins Tursiops truncatus infected with Brucella sp. using a dolphin-specific indirect ELISA.

Marine-origin Brucella infections and serologic evidence of exposure have been documented in multiple cetacean species. A dolphin-specific indirect enzyme-linked immunosorbent assay (ELISA) was developed to screen bottlenose dolphin sera for anti-Brucella antibodies. A total of 131 serum samples collected over a 2 to 18 yr period from 6 bottlenose dolphins Tursiops truncatus with confirmed Brucella infections were analyzed for the presence and magnitude of antibody titers against marine-origin Brucella to compare individual antibody responses to various disease manifestations. Additionally, an epidemiologic serologic survey of a managed population of 64 bottlenose dolphins was performed to evaluate for the presence of antibodies and to determine whether there were any clinical pathology predictors for exposure or infection. The serologic results revealed that the dolphins with Brucella-associated abortions were seronegative for 7 to 18 yr until after the abortion and maintained positive titers for several years, with 2 of 3 animals returning to seronegative status. In contrast, the dolphins with Brucella-associated pulmonary or bone lesions maintained persistent positive titers for 2 to 18 yr. The population serosurvey revealed no significant differences in antibody levels among males and females, and dolphins between the ages of 17 and 25 yr were 6.8 times more likely to be Brucella antibody positive compared to those that were younger or older. Seropositive dolphins did not have significant inflammation compared to seronegative dolphins but were more likely to have higher levels of aspartate aminotransferase and gamma-glutamyl transpeptidase. Among 16 dolphins that tested seropositive, 13 (81.3%) had previously been seropositive for at least 3 to 5 yr.

Endoscopic evaluation and biopsy collection of the gastrointestinal tract in the green moray eel (Gymnothorax funebris): application in a case of chronic regurgitation with gastric mucus gland hyperplasia.

A green moray eel (Gymnothorax funebris) was evaluated for chronic regurgitation. By using flexible endoscopy, the gastrointestinal tract was evaluated and revealed multifocal proliferative gastric masses and an intestinal ulcer. Biopsy specimens revealed gastric mucus gland hyperplasia, intestinal nematodiasis, and mild enteritis. Esophagoscopy and gastroscopy were performed by using a larger endoscope (length, 200 cm). A smaller endoscope (length, 100 cm) facilitated entering the intestinal tract in normograde or retrograde directions. A control eel was also evaluated, and no gross or histologic abnormalities were detected. The case eel was treated with metoclopramide and fenbendazole, responded well to therapy, and regurgitation decreased. A year later, the animal died of unrelated causes. Necropsy revealed coelomic gastric adhesions. The gastric proliferative lesions were associated with degeneration and necrosis of gastric pit mucosa without significant inflammation; etiology was unknown. Gastrointestinal endoscopy proved a useful diagnostic tool for evaluation and biopsy collection in this eel species.

Systemic mycosis in a California sea lion (Zalophus californianus) with detection of cystofilobasidiales DNA.

A 6-yr-old, intact male California sea lion (Zalophus californianus) with a systemic mycosis died after 5 wk of antifungal drug therapy. Antemortem clinical findings included hind flipper swelling, ring-lesions on skin of the flippers, and dermal nodules that increased in size and number spreading from the hind flippers and ventral abdomen to the foreflippers and muzzle. Lesions were accompanied by severe lymphadenopathy and development of systemic clinical signs despite therapy using itraconazole and later voriconazole. Histopathologic evaluation of biopsies revealed granulomatous dermatitis due to infection by fungus-producing yeast cells in tissue. Isolation attempts, using biopsied skin and tissue samples collected at necropsy, failed to yield growth of a fungus producing yeast cells like those in histologic section. Consensus polymerase chain reaction (PCR) tests of biopsied skin for fungal DNA produced an amplicon having significant sequence identity with a Cystofilobasidiales, a fungus belonging to a subclade that includes several Cryptococcus spp. Histopathologic evaluation of necropsy tissues revealed a systemic mycosis with yeast cells disseminated throughout subcutis, lymph nodes, and viscera. Hepatic necrosis was identified associated with acute liver failure, possibly from the voriconazole administration. This is the first report documenting the clinical presentation, treatment, and pathologic findings of infection associated with Cystofilobasidiales in a marine mammal and serves to expand the understanding of mycoses in pinnipeds.

Evaluation of potential variables contributing to the development and duration of plantar lesions in a population of aquarium-maintained African penguins (Spheniscus demersus).

Bumblefoot (pododermatitis), often described as the most significant environmental disease of captive penguins, is commonly due to excessive pressure or trauma on the plantar surface of the avian foot, resulting in inflammation or necrosis and causing severe swelling, abrasions, or cracks in the skin. Although not formally evaluated in penguins, contributing factors for bumblefoot are thought to be similar to those initiating the condition in raptors and poultry. These factors include substrate, body weight, and lack of exercise. The primary purpose of this retrospective study was to evaluate variables potentially contributing to the development and duration of plantar lesions in aquarium-maintained African penguins (Spheniscus demersus), including sex, weight, age, season, exhibit activity, and territory substrate. Results indicate that males develop significantly more plantar lesions than females. Penguins weighing between 3.51 and 4.0 kg develop plantar lesions significantly more often than penguins weighing between 2.5 and 3.5 kg, and because male African penguins ordinarily weigh significantly more than females, weight is likely a contributing factor in the development of lesions in males compared with females. Significantly more plantar lesions were observed in penguins standing for greater than 50% of their time on exhibit than swimming. Penguins occupying smooth concrete territories developed more plantar lesions compared with penguins occupying grate territories. Recommendations for minimizing bumblefoot in African penguins include training penguins for monthly foot examinations for early detection of plantar lesions predisposing for the disease, encouraging swimming activity, and replacing smooth surfaces on exhibit with surfaces providing variable degrees of pressure and texture on the feet.

Brucella sp. vertebral osteomyelitis with intercurrent fatal Staphylococcus aureus toxigenic enteritis in a bottlenose dolphin (Tursiops truncatus).

A previously beach-stranded, juvenile, male, bottlenose dolphin (Tursiops truncatus) was diagnosed with vertebral osteomyelitis of unknown etiology. Antemortem serological testing suggested past or current Brucella sp. infection; however, this could not be confirmed prior to death despite multiple isolation attempts from aspirates, blood, and biopsies. Systemic antibiotics were administered for over a year to control the suspected infection; however, the animal succumbed peracutely to infection by a highly pathogenic, enterotoxin-secreting Staphylococcus sp. Gross necropsy findings included a fistulous tract leading to locally extensive osteomyelitis of a coccygeal vertebra with sequestra and osteophytes from which a Brucella species was isolated. Histopathological examination of intestine revealed pseudomembranous enteritis with a uniform population of intraluminal Gram-positive cocci. Staphylococcus aureus was isolated in pure culture from the intestine and tested positive for the staphylococcal enterotoxin A gene by polymerase chain reaction analysis. Serum taken shortly before death had endotoxin and elevated antibody titers to staphylococcal enterotoxin A when compared to samples collected during a period of apparent good health 18 months earlier. The isolation of a pyrogenic toxin superantigen-producing staphylococcal isolate, clinical signs, and diagnostic findings in this animal resembled some of those noted in human toxic shock syndrome. The present case highlights the clinical challenges of treating chronic illnesses, complications of long-term antibiotic use, and promotion of pathogenic strains in cases of prolonged rehabilitation of marine mammals.

Assessment of clinical pathology and pathogen exposure in sea otters (Enhydra lutris) bordering the threatened population in Alaska.

Northern sea otter (Enhydra lutris kenyoni) abundance has decreased dramatically over portions of southwest Alaska, USA, since the mid-1980s, and this stock is currently listed as threatened under the Endangered Species Act. In contrast, adjacent populations in south central Alaska, USA, and Russia have been stable to increasing during the same period. Sea otters bordering the area classified in the recent decline were live-captured during 2004-2006 at Bering Island, Russia, and the Kodiak Archipelago, Alaska, USA, to evaluate differences in general health and current exposure status to marine and terrestrial pathogens. Although body condition was lower in animals captured at Bering Island, Russia, than it was at Kodiak, USA, clinical pathology values did not reveal differences in general health between the two regions. Low prevalences of antibodies (<5%) were found in Kodiak, USA, and on Bering Island, Russia, to Toxoplasma gondii, Sarcocystis neurona, and Leptospira interrogans. Exposure to phocine herpesvirus-1 was found in both Kodiak, USA (15.2%), and Bering Island, Russia (2.3%). Antibodies to Brucella spp. were found in 28% of the otters tested on Bering Island, Russia, compared with only 2.7% of the samples from Kodiak, USA. Prevalence of exposure to Phocine distemper virus (PDV) was 41% in Kodiak, USA, but 0% on Bering Island, Russia. Archived sera from southwest and south-central Alaska dating back to 1989 were negative for PDV, indicating exposure occurred in sea otters in Kodiak, USA, in recent years. Because PDV can be highly pathogenic in naïve and susceptible marine mammal populations, tissues should be examined to explore the contribution of this virus to otter deaths. Our results reveal an increase in exposure to pathogens in sea otters in Kodiak, Alaska, USA, since the 1990 s.

Evaluation of an alternative to feeding whole frozen fish in belugas (Delphinapterus leucas).

Feeding fish to captive piscivores can be challenging owing to cost, availability, variability in nutrient, and caloric composition, as well as handling and storage concerns. This trial evaluated the response of three belugas to being fed Fish Analog, an alternative to frozen fish. Body condition, gut transit time, serum chemistry and metabolic hormone analytes, immune function, and behavioral motivation were the dependent variables. Belugas (n=3) were fed various levels of Fish Analog (0-50%) over a 6-month period, and follow-up studies were conducted to further examine several dependent variables. When provided in gradually increasing amounts, belugas consumed the Fish Analog, with only minor fecal consistency changes and without behavioral responses indicative of gastric discomfort. Axillary girth and blubber thickness were positively correlated, and did not differ significantly with changes in the percentage of Fish Analog fed. Individual animal variation in initial passage time, some serum chemistry analytes, and immune function differences were noted following feeding of Fish Analog. Feeding Fish Analog reduced blood n9 fatty acids compared with captive belugas fed no Fish Analog. Feeding a DHA-enriched Fish Analog increased several n3 fatty acids, including eicosapentaenoic acid, but not DHA, compared with whales fed no Fish Analog or non-DHA-enriched Fish Analog. Fish Analog was shown to be a viable alternative to feeding fish at up to 50% of the dietary caloric density.

Development, validation, and utilization of a competitive enzyme-linked immunosorbent assay for the detection of antibodies against Brucella species in marine mammals.

A competitive enzyme-linked immunosorbent assay (cELISA) was developed by using a whole-cell antigen from a marine Brucella sp. isolated from a harbor seal (Phoca vitulina). The assay was designed to screen sera from multiple marine mammal species for the presence of antibodies against marine-origin Brucella. Based on comparisons with culture-confirmed cases, specificity and sensitivity for cetacean samples tested were 73% and 100%, respectively. For pinniped samples, specificity and sensitivity values were 77% and 67%, respectively. Hawaiian monk seal (Monachus schauinslandi; n  =  28) and bottlenose dolphin (Tursiops truncatus; n  =  48) serum samples were tested, and the results were compared with several other assays designed to detect Brucella abortus antibodies. The comparison testing revealed the marine-origin cELISA to be more sensitive than the B. abortus tests by the detection of additional positive serum samples. The newly developed cELISA is an effective serologic method for detection of the presence of antibodies against marine-origin Brucella sp. in marine mammals.

Validation of a homologous canine relaxin radioimmunoassay and application with pregnant and non-pregnant Northern fur seals (Callorhinus ursinus).

The primary objectives of this study were to validate a canine relaxin RIA for use in otariids and phocids and consider practical applications. For 6 captive Northern fur seal females, serum samples were grouped and examined according to pregnancy (n=13), post-partum (n=8) and non-pregnancy (n=6), and, for 2 captive Northern fur seal males, serum samples were grouped and examined together regardless of age (2 mo-15 yrs, n=6). Placental tissue was available for examination from one Northern fur seal, Steller sea lion and harbor seal. The validation process involved several steps using an acid-acetone extraction process to isolate a relaxin-containing fraction in pools of serum from each group of fur seals and placental tissue from each seal species. A relaxin-like substance was detected in extracts of pregnant, non-pregnant and male serum and placental tissue in a dose-responsive manner as increasing volumes of respective extracts or amounts of canine relaxin were introduced into the assay. In raw serum samples, mean immuno-reactive relaxin concentrations were higher (P<0.05) during pregnancy than post-partum and non-pregnancy, and lower (P<0.05) in male than female fur seals. During pregnancy, mean serum concentrations of relaxin progressively increased (P<0.05) over Months 4-10 and, in serial samples collected from the same fur seals before and after parturition, mean concentrations were higher (P<0.06) pre-partum than post-partum. In conclusion, validation of a homologous canine relaxin RIA for use in otariids and phocids resulted in the discovery of a relaxin-like substance in extracted and raw serum and placental tissue from Northern fur seals, a Steller sea lion and harbor seal. Distinctly higher immuno-reactive concentrations during pregnancy indicated the potential for relaxin to serve as a hormonal marker to differentiate between pregnant and non-pregnant or pseudopregnant pinnipeds.

The role of domoic acid in abortion and premature parturition of California sea lions (Zalophus californianus) on San Miguel Island, California.

Domoic acid is a glutaminergic neurotoxin produced by marine algae such as Pseudo-nitzschia australis. California sea lions (Zalophus californianus) ingest the toxin when foraging on planktivorous fish. Adult females comprise 60% of stranded animals admitted for rehabilitation due to acute domoic acid toxicosis and commonly suffer from reproductive failure, including abortions and premature live births. Domoic acid has been shown to cross the placenta exposing the fetus to the toxin. To determine whether domoic acid was playing a role in reproductive failure in sea lion rookeries, 67 aborted and live-born premature pups were sampled on San Miguel Island in 2005 and 2006 to investigate the causes for reproductive failure. Analyses included domoic acid, contaminant and infectious disease testing, and histologic examination. Pseudo-nitzschia spp. were present both in the environment and in sea lion feces, and domoic acid was detected in the sea lion feces and in 17% of pup samples tested. Histopathologic findings included systemic and localized inflammation and bacterial infections of amniotic origin, placental abruption, and brain edema. The primary lesion in five animals with measurable domoic acid concentrations was brain edema, a common finding and, in some cases, the only lesion observed in aborted premature pups born to domoic acid-intoxicated females in rehabilitation. Blubber organochlorine concentrations were lower than those measured previously in premature sea lion pups collected in the 1970s. While the etiology of abortion and premature parturition was varied in this study, these results suggest that domoic acid contributes to reproductive failure on California sea lion rookeries.

Particulate and soluble hexavalent chromium are cytotoxic and genotoxic to Steller sea lion lung cells.

Hexavalent chromium is an environmental contaminant. Within the environment, marine waters are a common site for hexavalent chromium deposition. We have recently reported significantly high levels of chromium in skin tissue from North Atlantic right whales. These findings demonstrate that marine species are being exposed to chromium. It is possible that such exposures may be playing a role in population declines evident among certain marine mammals, such as the Steller sea lion. We developed a Steller sea lion lung cell line from Steller sea lion lung tissue. Hexavalent chromium was cytotoxic to these primary lung fibroblasts as 1, 2.5, 5, 10 and 25microM sodium chromate induced 104, 99, 92, 58 and 11% relative survival, respectively. It was also genotoxic as 0, 1, 2.5, 5 and 10microM sodium chromate damaged chromosomes in 6, 11, 21, 36, and 39% of metaphases and damaged 6, 12, 27, 49 and 57 total aberrations in 100 metaphases, respectively. We also considered the toxicity of particulate hexavalent chromium, as it is the more potent carcinogen in humans. We found that 0.1, 0.5, 1, 5 and 10microg/cm(2) particulate chromate induced 95, 88, 91, 70, and 52% relative cell survival, respectively. These concentrations were genotoxic and damaged chromosomes in 9, 13, 18, and 23% of metaphases and induced 9, 15, 20 and 30 total aberrations per 100 metaphases, respectively. These data indicate that if sufficiently exposed, chromium may adversely affect the struggling Steller sea lion population. It would be prudent to investigate the effects chromium has in other Steller sea lion organs in order to derive a better understanding of how chromium in the marine environment may be affecting the declining Steller sea lion population.

Metal tissue levels in Steller sea lion (Eumetopias jubatus) pups.

The endangered Western population of the Steller sea lion declined for three decades for uncertain reasons. We present baseline data of metal concentrations in pups as a first step towards investigating the potential threat of developmental exposures to contaminants. Seven metals were investigated: arsenic, cadmium, silver, aluminum, mercury, lead and vanadium. Vanadium was detected in only a single blubber sample. Mercury appears to be the most toxicologically significant metal with concentrations in the liver well above the current action level for mercury in fish. The concentrations of aluminum, arsenic, silver, cadmium and lead were present in one-fourth to two-thirds of all samples and were at either comparable or below concentrations previously reported. Neither gender nor region had a significant effect on metal burdens. Future work should consider metal concentrations in juveniles and adults and toxicological studies need to be performed to begin to assess the toxicity of these metals.

Chronic pancreatitis with secondary diabetes mellitus treated by use of insulin in an adult California sea lion.

CASE DESCRIPTION: A 21-year-old neutered male captive California sea lion developed chronic polyuria; polydipsia; polyphagia; accelerated development of existing cataracts; and frequent episodes of gastrointestinal upset including anorexia, signs of abdominal discomfort, diarrhea, and vomiting. CLINICAL FINDINGS: Chronic hypercholesterolemia, hypertriglyceridemia, hyperglycemia, and glucosuria were identified. During episodes of gastrointestinal abnormalities, transient hyperbilirubinemia and increased serum J-glutamyltransferase activities developed. Clinical findings strongly suggested chronic pancreatitis with secondary diabetes mellitus and intermittent cholestasis. Multiple diagnostic tests, including abdominal ultrasonography, serial hematologic and serum biochemical analyses, fecal examinations, urinalyses and bacteriologic culture of urine, measurement of serum fructosamine and insulin concentrations, and evaluation of thyroid and adrenal function, did not reveal any specific parasitic, endocrine, hepatic, or neoplastic etiologies. TREATMENT AND OUTCOME: For 1.5 years, the sea lion received once-daily administration of glargine insulin, gastrointestinal protectants, and a strict high-protein, low-fat diet. Daily monitoring of glucose regulation was achieved by training the sea lion to submit to blood and urine sampling. Glucose regulation ranged from fair to good, and clinical signs of diabetes mellitus lessened. Episodes of gastrointestinal upset still occurred, although the frequency and severity decreased. Ultimately, a severe episode developed, associated with diabetic ketoacidosis and sepsis, and the sea lion died. Severe fibrosing pancreatitis with exocrine and endocrine atrophy and abscesses arising from ectatic pancreatic ducts were found. Peripancreatic fibrosis caused stricture of the common bile duct, resulting in gallbladder distension without cholecystitis. CLINICAL RELEVANCE: Diabetes mellitus can occur secondary to chronic pancreatitis in California sea lions and insulin therapy should be considered.

Eastern equine encephalitis in a flock of African penguins maintained at an aquarium.

Eastern equine encephalitis (EEE) was diagnosed in a flock of African penguins. Diagnosis was based on history and clinical signs and confirmed via serologic testing, virus isolation, reverse transcriptase-polymerase chain reaction (RT-PCR) assay, and histologic examination. Clinical signs in penguins included anorexia, behavior changes, depression, regurgitation, ataxia, recumbency, and seizures, and some penguins did not have any clinical signs. Mean +/- SD number of days that affected penguins had clinical signs was 12 +/- 5 days. Abnormalities initially detected on CBC included heterophilic leukocytosis and anemia; lymphocytosis and monocytosis were detected later. Plasma biochemical abnormalities included high activities of aspartate amino-transferase and creatine kinase, hyponatremia, hypochloremia, hyperglycemia, and high concentrations of globulin, triglycerides, and cholesterol. Mean +/- SD number of days required for resolution of CBC and plasma biochemical abnormalities was 67 +/- 24 days after the onset of clinical signs. Treatment consisted of supportive therapy. All penguins survived with the exception of one that was euthanatized; histopathologic findings were consistent with encephalitis. Results of RT-PCR assays performed on tissue from the right cerebrum of the penguin that was euthanatized were positive for EEE viral RNA. An inability to isolate virus several weeks after illness suggested successful viral clearance in recovered penguins. To the authors' knowledge, EEE infection in any penguin species has not been reported.

Fibrosarcoma of the brood pouch in an aquarium-reared lined seahorse (Hippocampus erectus).

An adult, male, captive lined seahorse (Hippocampus erectus) was presented with weight loss, anorexia, and brood pouch distention. Initial treatments included manual expression of the brood pouch followed by saline flushes and enrofloxacin infusions. Radiographs and physical examination identified a nodular swelling in the brood pouch wall. The mass was excised, but the seahorse died. Necropsy and histopathology revealed a malignant mesenchymal tumor with a collagenous stroma, and a brood pouch fibrosarcoma was diagnosed.

Evidence of Brucella sp. infection in marine mammals stranded along the coast of southern New England.

After recent isolations of Brucella sp. from pinnipeds and cetaceans, a survey was initiated to investigate the prevalence of Brucella sp. infections and serologic evidence of exposure in marine mammals stranded along the coasts of Connecticut and Rhode Island. One hundred and nineteen serum samples from four species of cetaceans and four species of pinnipeds were collected from 1985 to 2000 and tested for antibodies to Brucella sp. using the brucellosis card test, buffered acidified plate antigen test, and rivanol test. In addition, 20 of these were necropsied between 1998 and 2000, with lymphoid and visceral tissues cultured for Brucella sp. Three of 21 (14%) harbor seals (Phoca vitulina) and four of 53 (8%) harp seals (Phoca groenlandica) were seropositive. Brucella sp. was isolated from two of four (50%) harbor seals and three of nine (33%) harp seals. Of the five animals with positive cultures, two were seropositive and three seronegative. Brucella sp. was most frequently cultured from the lung and axillary, inguinal, and prescapular lymph nodes. Tissues from which Brucella sp. was isolated showed no gross or histopathologic changes. These results indicate that marine mammals stranded along the coast of southern New England can be exposed to and infected with Brucella sp.