Otarine herpesvirus-1: a novel gammaherpesvirus associated with urogenital carcinoma in California sea lions (Zalophus californianus)

The incidence of neoplasia in California sea lions (CSLs) is considered to be unusually high. Electron microscopic examination of some of these urogenital tumours revealed the presence of virions with typical herpes-like structure. While current attempts to cultivate this virus have not been successful, molecular studies employing DNA extracted from tumour tissues allowed both the classification of the agent and its identification in tumours and archived tissue samples. Two genome fragments generated using degenerate primers in PCR demonstrated highest identities with other mammalian gammaherpesviruses. Phylogenetic analysis showed that this novel virus, tentatively designated Otarine herpesvirus-1 (OtHV-1), grouped with members of the gammaherpesvirus subfamily and was distinct from PHV-2, a previously described pinniped gammaherpesvirus. An OtHV-1 specific PCR was established and used to investigate the presence of this virus in CSL tissues. PCR of DNA isolated from animals with these tumours, demonstrated that this virus was present in 100% (16/16) of tumours. Furthermore, DNA extracted from archived brain and muscle tissues was also positive in 29% (4/14) and 50% (7/14) of cases examined. This preliminary study provides evidence to support the hypothesis that the presence of this novel gammaherpesvirus is a factor in the development of urogenital carcinoma in CSLs.     

Pathology of domoic acid toxicity in California sea lions (Zalophus californianus)

Over 100 free-ranging adult California sea lions (Zalophus californianus) and one Northern fur seal (Callorhinus ursinus), predominantly adult females, were intoxicated by domoic acid (DA) during three harmful algal blooms between 1998 and 2000 in central and northern California coastal waters. The vector prey item was Northern anchovy (Engraulis mordax) and the primary DA-producing algal diatom was Psuedonitzschia australis. Postmortem examination revealed gross and histologic findings that were distinctive and aided in diagnosis. A total of 109 sea lions were examined, dying between 1 day and 10 months after admission to a marine mammal rehabilitation center. Persistent seizures with obtundation were the main clinical findings. Frequent gross findings in animals dying acutely consisted of piriform lobe malacia, myocardial pallor, bronchopneumonia, and complications related to pregnancy. Gross findings in animals dying months after intoxication included bilateral hippocampal atrophy. Histologic observations implicated limbic system seizure injury consistent with excitotoxin exposure. Peracutely, there was microvesicular hydropic degeneration within the neuropil of the hippocampus, amygdala, pyriform lobe, and other limbic structures. Acutely, there was ischemic neuronal necrosis, particularly apparent in the granular cells of the dentate gyrus and the pyramidal cells within the hippocampus cornu ammonis (CA) sectors CA4, CA3, and CA1. Dentate granular cell necrosis has not been reported in human or experimental animal DA toxicity and may be unique to sea lions. Chronically, there was gliosis, mild nonsuppurative inflammation, and loss of laminar organization in affected areas.     

Disseminated blastomycosis in two California sea lions (Zalophus californianus).

Two captive California sea lions (Zalophus californianus) from different facilities were diagnosed with disseminated blastomycosis. The first, a 12-yr-old male, died after a 3-wk history of progressive anorexia and lethargy. Gross examination revealed acute jejunitis with focal perforation and associated peritonitis, along with severe purulent bronchopneumonia. The second, a 15-yr-old female, was euthanized after a 2-wk history of severe cutaneous ulceration and declining clinical condition. Gross examination revealed severe pyogranulomatous bronchopneumonia and ulcerative dermatitis. Histopathologic examination in both individuals revealed severe multifocal subacute to chronic pyogranulomatous pneumonia associated with massive numbers of fungal organisms morphologically compatible with Blastomyces sp. Fungal organisms were 8-20-microm-diameter broad-based budding yeasts with thick, refractile, double-contoured walls. The male sea lion had multifocal transmural Blastomyces-induced enteritis with subsequent rupture and peritonitis. The organism was also present in the liver, with minimal associated inflammation. The female had severe multifocal pyogranulomatous ulcerative dermatitis associated with large numbers of intralesional fungal organisms. Dissemination to the spleen had occurred in both animals. A serologic immunodiffusion test for Blastomyces dermatitidis was positive in the male. The presumptive primary pathogen in both cases was Blastomyces dermatitidis.     

Characterization of a novel papillomavirus species (ZcPV1) from two California sea lions (Zalophus californianus)

A seven-year old California sea lion (Zalophus californianus) presented with focally extensive, bilaterally symmetric, proliferative axillary skin lesions and preputial lesions. A second California sea lion in the same population presented with similar proliferative lesions on the underside of the tail. Histopathology revealed epidermal hyperplasia with severe hyperkeratosis, with proliferating keratinocytes forming broad, branching pegs that extended into the dermis. Pan-papillomaviral consensus PCR was used to obtain initial E1 sequence template and the complete genome was determined using a combination of rolling circle amplification and specific-primer PCR. Analysis revealed a novel papillomavirus, Zalophus californianus papillomavirus 1 (ZcPV1), with seven open reading frames encoding five early proteins (E6, E7, E1, E2 and E4) and two late proteins (L1 and L2). Phylogenetic analysis revealed that (ZcPV1) is most closely related to Equine papillomavirus 1 (EcPV1) in the genus Zetapapillomavirus, and Canine papillomaviruses 3 and 4 (CPV3, CPV4) in the genus Chipapillomavirus. The lesions regressed without intervention over a period of several months.     

Immobilisation of free-ranging Galapagos sea lions (Zalophus californianus wollebaeki)

Nine free-ranging Galápagos sea lions were immobilised for marking, using a combination of ketamine (3 to 5 mg per kg) and xylazine (0.5 to 1.0 mg per kg). The degree of effect depended largely on the animals' behavioural and physiological state before immobilisation (after parturition, high arousal, subject to aggression, etc.) All nine animals survived. Overheating in the immobilised state may quickly result in heart and circulatory failures.     

Common metastatic carcinoma of California sea lions (Zalophus californianus): evidence of genital origin and association with novel gammaherpesvirus

Tissues from 10 adult California sea lions (Zalophus californianus, seven females and three males) that had metastatic carcinoma in sublumbar area lymph nodes were examined histologically. A distinctive epithelial proliferative lesion interpreted as intraepithelial neoplasia was found in genital tracts of all ten animals; in vagina (5/7), cervix (7/7), uterus (3/7), penis (3/3) and prepuce (3/3). Intraepithelial neoplasia closely resembled metastatic carcinomas and was directly contiguous with invasive carcinoma in one animal. Rare eosinophilic intranuclear inclusion bodies were found in penile and preputial intraepithelial neoplasia (one animal), cervical intraepithelial neoplasia (one animal), invasive cervical carcinoma (one animal) and metastatic carcinoma (two animals). Electron microscopic examination of tissues from two sea lions (one with intraepithelial neoplasia and one with metastatic carcinoma) demonstrated viral particles consistent with a herpesvirus. An immunohistochemical stain for the latent membrane protein of Epstein-Barr virus was positive in intraepithelial neoplasia in one sea lion. Herpesvirus DNA sequences were detected by consensus primer polymerase chain reaction (PCR) in metastatic carcinomas from all four sea lions from which unfixed tumor samples were available. Results of sequencing were consistent with a novel gammaherpesvirus in the genus Rhadinovirus. DNA extracted from the four metastatic carcinomas also was tested for papillomavirus by Southern blot and PCR with consensus papillomavirus primers; all samples were negative by both methods. These findings support the genital origin of the sea lion carcinoma and implicate a novel gammaherpesvirus as a possible cause.     

Isoflurane anaesthesia in four sea lions (Otaria byronia and Zalophus californianus)

OBJECTIVE: To (1) evaluate a purpose-built chamber for inducing isoflurane anaesthesia in sea lions and (2) assess isoflurane as an anaesthetic for dental surgery in these species. STUDY DESIGN: Prospective case study. ANIMALS: Four sea lions, aged between 5 and 12 years and weighing 74-110 kg, with dental disease. METHODS: Sea lions were restrained in a custom-built acrylic chamber into which 5% isoflurane (vapourizer setting) was delivered in O(2) (30 L minute(-1)) from three anaesthetic machines. When the animals were recumbent, the chamber was opened and induction completed using a face mask. Anaesthesia was maintained with 1.5-3.0% isoflurane (vapourizer setting) for 10-15 minutes and after tracheal intubation, was maintained with 1.0-3.0% isoflurane (end-tidal) for 41-255 minutes, using a partial rebreathing system with CO(2) absorption. RESULTS: During induction, the sea lions attempted to support their weight on their thoracic limbs before slipping into sternal or lateral recumbency. Sea lions underwent either root canal surgery, 'crowning' or canine tooth extraction. The animals were completely unresponsive during these operations and apnoea was never encountered at any point during anaesthesia. After surgery, the sea lions were placed into transport cages and their tracheae extubated once they began to move their heads. Thereafter, recovery from anaesthesia was smooth and no signs of distress were observed. Animals were able to raise their forequarters using their flippers within 1 hour of the operation and were returned to the aquarium about 2 hours after surgery. CONCLUSIONS AND CLINICAL RELEVANCE: The purpose-built induction chamber is safe for inducing isoflurane anaesthesia in spontaneously breathing sea lions and isoflurane is an effective anaesthetic in this species.     

Reversible anesthesia of captive California sea lions (Zalophus californianus) with medetomidine, midazolam, butorphanol, and isoflurane.

Two adult California sea lions (Zalophus californianus) were effectively anesthetized 13 times with medetomidine (0.010-0.013 mg/kg), midazolam (0.2-0.26 mg/kg), and butorphanol (0.2-0.4 mg/kg) by i.m. hand or pole syringe injection. For each anesthetic event, atropine (0.02 mg/kg, i.m.) was administered 6-20 min after initial injections, and oxygen administration via face mask or nasal insufflation began at the same time. Light anesthesia was induced in 8-22 min and lasted 13-78 min. During eight of the procedures, isoflurane (0.5-2.0%) was administered via face mask or endotracheal tube for an additional 30-120 min to facilitate longer procedures or surgery. Anesthesia was antagonized with atipamezole (0.05-0.06 mg/kg) and naltrexone (0.1 mg/kg) in seven events, with the addition of flumazenil (0.0002-0.002 mg/kg) in six events. The antagonists were administered by i.m. injection 42-149 min after administration of the induction agents. All sea lions recovered to mild sedation within 4-17 min after administration of the antagonists.     

Plasma concentrations of itraconazole following a single oral dose in juvenile California sea lions (Zalophus californianus)

The objective of this study was to establish a single-dose pharmacokinetic profile for orally administered itraconazole in California sea lions (Zalophus californianus). Twenty healthy rehabilitated juvenile California sea lions were included in this study. Itraconazole capsules were administered orally with food at a target dose of 5–10 mg/kg. Blood samples were collected from each animal at 0 hr and at two of the following timepoints: 0.5, 1, 2, 4, 6, 8, 12, 24, 48, and 72 hr. Quantitative analysis of itraconazole in plasma samples was performed by high-performance liquid chromatography. An average maximum concentration of 0.22 µg/ml ± 0.11 was detected 4 hr after administration. The average concentration fell to 0.12 µg/ml ± 0.11 by 6 hr and 0.02 µg/ml ± 0.02 at 12 hr. At no point did concentrations reach 0.5 µg/ml, the concentration commonly accepted for therapeutic efficacy. While this formulation was well tolerated by the sea lions, oral absorption was poor and highly variable among individuals. These data indicate that a single oral dose of itraconazole given as a capsule at 5–10 mg/kg, under the conditions used in this study, does not achieve therapeutic plasma concentrations in California sea lions.     

Domoic acid toxicity in Californian sea lions (Zalophus californianus): clinical signs, treatment and survival

Eighty-one Californian sea lions (Zalophus californianus) with signs of domoic acid toxicity stranded along the coast of California in 1998 when there were blooms of the domoic acid-producing alga Pseudonitzschia australis off-shore. In 2000, a further 184 sea lions stranded with similar clinical signs, but the strandings occurred both during detectable algal blooms and after the blooms had subsided. The clinical signs in these 265 Californian sea lions included seizures, ataxia, head weaving, decreased responsiveness to stimuli and scratching behaviour. Affected animals had high haematocrits, and eosinophil counts, and high activities of serum creatine kinase. They were treated supportively by using fluid therapy, diazepam, lorazepam and phenobarbitone. Fifty-five of the 81 sea lions (68 per cent) affected in 1998 and 81 of the 184 (44 per cent) affected in 2000 died despite the treatment. Three of the 23 sea lions which survived in 1998 were tracked with satellite and radiotransmitters; they travelled as far south as San Miguel Island, California, and survived for at least three months. Eleven of the 129 animals which were released stranded within four months of being released.     

Risk factors associated with perianesthetic mortality of stranded free-ranging California sea lions (Zalophus californianus) undergoing rehabilitation

The objective of this study was to identify risk factors associated with perianesthetic mortality of stranded free-ranging California sea lions (Zalophus californianus) undergoing rehabilitation. Hospital records of California sea lions that underwent heavy sedation or general anesthesia from 2004 through 2008 were reviewed, including records from 419 anesthetic events. Procedures that resulted in death during or in the subsequent 72 hr of anesthesia were classified as cases (n = 15). Procedures in which the animal survived were classified as controls (n = 334). Procedures that resulted in euthanasia (n = 70) were removed from subsequent analysis. The following risk factors were reviewed: gender, age class, health status, duration of anesthetic period, atropine premedication, induction protocols, maintenance protocols, and history of prior anesthesia. The prevalence of fatalities during anesthesia was 3.4% (n = 12) over the 5-yr period. With the inclusion of animals that died within 72 hr after anesthesia, the total mortality prevalence rose to 4.3% (n = 15). The most common time of death was during anesthetic maintenance. Health status was the single best predictor of anesthetic outcome, and sea lions premedicated with atropine had increased odds of anesthetic-related death.     

Multicentric neurofibromatosis with rectal prolapse in a California sea lion (Zalophus californianus)

An approximately 31-yr-old California sea lion (Zalophus californianus) with a history of chronic visual impairment and corneal disease presented with slow onset, progressive neurologic deficits. Treatment for rear flipper paresis was not effective and the animal was euthanatized. Histopathologic findings included hepatocellular and biliary neoplasia, ocular amyloidosis, adrenal adenoma and pheochromocytoma, and spinal cord changes consistent with multicentric neurofibromatosis. This is the first documentation of these conditions in a California sea lion.     

Secondary amyloidosis and renal failure in a captive California sea lion (Zalophus californianus)

A 16-yr-old, captive-born, female California sea lion (Zalophus californianus) was evaluated for intermittent lethargy, partial anorexia, and polydipsia of 2 wk duration. The animal was immobilized for physical examination. It was in thin body condition, with multifocal mucosal ulcerations over the caudal and ventral tongue. Blood was collected for hematology, serum chemistry, and leptospirosis serology. Serum chemistry revealed severe azotemia, mild hyperglycemia, and severe hyperphosphatemia. The animal went into cardiac arrest during recovery from anesthesia and died. On histopathology, abundant amorphous, finely fibrillar, eosinophilic material was deposited in the kidneys, and smaller amounts of the same material were found in the splenic and pancreatic vessels; these findings are consistent with systemic secondary amyloidosis. The animal also had chronic nephritis, which, coupled with renal amyloidosis, resulted in renal failure and death. Systemic amyloidosis should be considered as an additional differential diagnosis for renal failure in California sea lions.     

Aerobic bacterial flora of the vagina and prepuce of California sea lions (Zalophus californianus) and investigation of associations with urogenital carcinoma

To investigate the association between genital bacterial infection and urogenital carcinoma in California sea lions (Zalophus californianus), vaginal and preputial swabs for bacterial isolation were taken from 148 free-ranging and 51 stranded California sea lions including 16 animals with urogenital carcinoma. Cytological examination of vaginal or preputial smears showed a majority (65.5%, 57/87) of animals examined had mild or no inflammation. Aerobic bacteria were isolated from 116 (78.4%) wild sea lions and 100% of stranded animals. A total of 403 isolates were identified representing 51 unique bacterial species. The median number of isolates per animal increased with age in the wild group, but there was no difference in the number of isolates per animal between wild and stranded adults. The most common bacteria isolated from the wild sea lions were Psychrobacter phenylpyruvicus (39 isolates), non-hemolytic Streptococcus (35 isolates), Corynebacterium spp. (30 isolates), and Escherichia coli (20 isolates). More bacterial species were isolated from stranded animals than wild animals (33 versus 26) and there was significantly less growth of P. phenylpyruvicus, Corynebacterium spp., and Moraxella-like spp. in the stranded animals. Beta-hemolytic Streptococcus was the only bacterium significantly associated with urogenital carcinomas in California sea lions, but only in females.     

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.     

Lingual squamous cell carcinoma in a California sea lion (Zalophus californianus).

A 28-yr-old female California sea lion (Zalophus californianus) in a commercial aquarium developed an ulcerated lingual tumor and died. Necropsy revealed a moderately differentiated squamous cell carcinoma. Immunohistochemical staining and electron microscopic studies revealed that the tumor cells were strongly positive with anti-keratin-cytokeratin antibody and had abundant tonofibrils and desmosomes. The neoplasm had metastasized to a mandibular lymph node.