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.     

Viral hepatitis (adenovirus) in a California sea lion

A juvenile California sea lion (Zalophus californianus) died 28 days after admission to the California Marine Mammal Center. Necropsy revealed hemorrhagic fluid in the stomach and hepatomegaly. Histologically, there was evidence of multifocal necrotizing hepatitis and acute suppurative bronchopneumonia. Amphophilic intranuclear inclusions were found, and electron microscopy revealed virions morphologically classifiable as adenovirus.     

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.     

Urate nephrolithiasis in a northern elephant seal (Mirounga angustirostris) and a California sea lion (Zalophus californianus).

Nephrolithiasis has rarely been reported in marine mammals. During 2004 and 2005, two cases of nephrolithiasis were diagnosed during routine necropsy examination, one in a northern elephant seal (Mirounga angustirostris) and one in a California sea lion (Zalophus californianus). Nephroliths were found throughout both kidneys during necropsy examination, varying in size from 1-10 mm in diameter in the northern elephant seal and from 1-15 mm in diameter in the California sea lion. Necropsy and histopathology revealed nephroliths in association with renal pelvic dilation and pyelonephritis in both animals. In addition, hydronephrosis was noted in the sea lion. Nephroliths were composed of uric acid and ammonium urate in the northern elephant seal and of ammonium urate in the California sea lion. The underlying disease leading to nephrolith formation was not determined; however, it is hypothesized that unknown metabolic derangements due to morphologic or physiologic differences may have played a role. This is the first report of urate nephrolithiasis in the California sea lion and northern elephant seal.     

Surgical resection of an orbital fat prolapse in a California sea lion (Zalophus californianus)

A 23-year-old captive female California sea lion (Zalophus californianus) developed an inferiotemporal subconjunctival swelling of the right eye. Surgical excision of the mass was performed and a diagnosis of orbital fat prolapse was confirmed histopathologically. There was no recurrence of orbital fat prolapse at 15 months post operatively. This is the first known case of orbital fat prolapse in the California sea lion.     

Endocarditis associated with Escherichia coli in a sea lion (Zalophus californianus).

Endocarditis associated with Escherichia coli was diagnosed in a 2-year-old male California sea lion (Zalophus californianus). The diagnosis was based on light microscopic examination and bacterial isolation from the valvular lesion. This is the first case of bacterial endocarditis reported in a sea lion.     

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.     

Phylogenetic analysis of marine mammal herpesviruses

Five novel DNA-dependent DNA polymerase (Dpol) herpesviral sequences were generated using nested consensus polymerase chain reaction (PCR) in clinical samples from a harbor seal (Phoca vitulina), bottlenose dolphin (Tursiops truncatus), orca (Orcinus orca), California sea lion (Zalophus californianus), and a Phocid herpesvirus 2 (PhHV-2) isolate from a harbor seal (used as positive control). These novel sequences and other representative herpesvirus sequences were included in Bayesian and Maximum Likelihood analyses to illustrate the phylogeny of herpesviruses amongst the marine mammal host species and in comparison to those of other animals. All 19 novel and known marine mammal herpesviruses included in the analyses aligned with members of the Alphaherpesvirinae or Gammaherpesvirinae subfamilies. The novel harbor seal herpesvirus clustered with members of the Macavirus genus, subfamily Gammaherpesvirinae. The novel bottlenose dolphin herpesvirus clustered together in a monophyletic group with another delphinid alphaherpesvirus but could not be associated with an established genus. The orca herpesvirus also clustered with a delphinid alphaherpesvirus and formed a separate clade. The sea lion herpesvirus clustered with PhHV-2. PhHV-1 clustered with varicelloviruses and PhHV-2 clustered strongly in the Gammaherpesvirinae genus Percavirus. All cetacean gammaherpesviruses formed a monophyletic clade and could not be associated with an established gammaherpesviral genus.     

Current prevalence of adult Uncinaria spp. in northern fur seal (Callorhinus ursinus) and California sea lion (Zalophus californianus) pups on San Miguel Island, California, with notes on the biology of these hookworms

A prevalence survey for hookworms (Uncinaria spp.) was done in northern fur seal (Callorhinus ursinus) and California sea lion (Zalophus californianus) pups on San Miguel Island, CA, in 2000. Intestines of dead pups were examined for adult hookworms in July. These parasites were found in 95% of 20 fur seal pups and 100% of 31 sea lion pups. The number of hookworms varied from 4 to 2142 (mean = 760) in fur seal pups and from 20 to 2634 (mean = 612) in sea lion pups. A direct relationship was evident between body condition and number of hookworms in the pups; that is, pups in poor condition had fewer hookworms than those in good condition. There was a decline in the number of hookworms in sea lion pups in 2000 compared to collections in 1996. Eggs of Uncinaria spp. were found in rectal feces (collected in late September and early October) of none of 35 (0%) live fur seal pups and 41 of 48 (85%) live sea lion pups. Packed cell volume values, determined for most of the same live pups, were essentially normal for C. ursinus but were much lower than normal for most Z. californianus. Hookworm larvae were not found in blubber of fur seal and sea lion pups or in rookery sand in July. Rookery sand, positive for live hookworm larvae when put in a refrigerator, was negative at removal 2.5 years later. The average number of eggs in utero of female hookworms was 285 for three specimens from a fur seal pup and 281 from three specimens from a sea lion pup. One hookworm larva was recovered from milk stripped from the teats of a stranded Z. californianus female at The Marine Mammal Center, Sausalito, CA.     

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.     

Isolation of San Miguel Sea Lion Virus from Samples of an animal food product produced from northern fur seal (Callorhinus ursinus) carcasses

A virus was isolated from California sea lions (Zalophus californianus californianus) and northern fur seals (Callorhinus ursinus) in 1972. It was later named San Miguel sea lion virus (SMSV). State and federal livestock disease control agencies became concerned, because SMSV was found to be indistinguishable from vesicular exanthema of swine virus and to cause (in laboratory trials) clinical signs in swine similar to those produced by vesicular exanthema of swine virus. Ground carcasses of northern fur seals, salvaged after harvesting pelts, are fed to mink on ranches in the United States. Domestic swine are kept on some of these same ranches. Samples withheld from lots of this seal carcass mink food were found to contain SMSV (serotype 5) in titers of 10(6.1) and 10(6.8) tissue culture infective doses.     

Neuronal migration defect: a case of subcortical heterotopia in a California sea lion

A 2 and a half-year-old male California sea lion (Zalophus californianus) presented with a history of intermittent generalized seizures. Magnetic resonance imaging revealed a large focal mass occupying the right cerebral hemisphere with moderate dilatation of the contralateral lateral ventricle. At necropsy, the right cerebral hemispheric white matter was expanded by numerous irregularly shaped, pale pink nodules up to 10 mm in diameter. The overlying cortex was characterized by increased numbers of small, poorly developed gyri with shallow, often indistinct, sulci (polymicrogyria). Microscopically, nodules were composed of neurons, oligodendroglia, microglia, and supporting neuropil and were well delineated from the surrounding white matter. The gross, histological, and immunohistochemical features of this lesion are consistent with a neuronal migration defect resulting in unilateral subcortical heterotopia.     

Use of laser rhinoscopy to treat a nasal obstruction in a captive California sea lion (Zalophus californianus).

Laser rhinoscopy was used to treat a nasal obstruction in a captive California sea lion (Zalophus californianus). The rehabilitated, adult, female sea lion developed mucopurulent, intermittent, bilateral nasal discharge and functional nasal obstruction 20 mo after acquisition by the Aquarium of the Pacific in Long Beach, California. A 3-mm-thick soft tissue structure spanning the region between the soft and hard palates, a deviated nasal septum, and several nasopharyngeal polyps were identified. Biopsies and cultures of the obstructive web showed ulcerative granulation tissue with suppurative inflammation, bacterial infection, and a partial section of an arthropod larva (not speciated). Laser rhinoscopy was performed to relieve the caudal nasopharyngeal obstruction and ablate the polyps. The sea lion appeared to breathe through the nares with lessened nasal discharge for a period of 6 wk after laser therapy, but within 8 wk the mucopurulent nasal discharge returned, the obstruction had reformed, and the sea lion was euthanized. Postmortem examination confirmed antemortem diagnoses of caudal nasopharyngeal obstruction secondary to inflammatory tissue; however, no additional sections of arthropod parasites were located microscopically.     

New marine calicivirus serotype infective for swine

A new serotype of calicivirus was isolated from California sea lions (Zalophus californianus) with severe vesicular disease. Neutralizing antibodies were found in 27 of 82 (32.9%) serum samples from California sea lions and in 15 of 146 (10.3%) serum samples from Steller sea lions (Eumetopias jubatus) tested. The seropositive animals were widely dispersed along the margins of the eastern Pacific basin, from the Bering Sea to the Santa Barbara Channel. Seropositive samples were found from as early as 1976 through the present time. This new calicivirus serotype, San Miguel sea lion virus type 13, was inoculated into weaned pigs, resulting in induction of severe vesicular disease, which spread to all pigs, including uninoculated pen contacts. Virus was continually shed by most of the pigs throughout the 2-week duration of the experiment.     

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.     

Acute viral hepatitis in California sea lions

Acute viral hepatitis was diagnosed in five California sea lions (Zalophus californianus) stranded along the Los Angeles coast. Light microscopy revealed large nuclear inclusion bodies in hepatocytes. Electron microscopy provided evidence that these inclusion bodies were composed of adenovirus-like virions. Attempts to grow the virus in cell culture systems were unsuccessful.     

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.     

Risk factors associated with development of poxvirus lesions in hospitalized California sea lions

OBJECTIVE: To identify risk factors that may predispose California sea lions (Zalophus californianus) to development of cutaneous poxvirus nodules during hospitalization in a rehabilitation center. DESIGN: Retrospective case-control study. ANIMALS: 90 California sea lions admitted to a rehabilitation center. PROCEDURE: Hospital records of 275 stranded California sea lions admitted to the rehabilitation center between January 1 and December 31, 2002, were reviewed. All California sea lions (n = 18) that developed > or = 1 cutaneous poxvirus nodule during hospitalization were classified as cases. Seventy-two California sea lions that did not develop poxvirus lesions during hospitalization were randomly selected (control group). The frequencies of various exposure factors prior to admission, at admission, and during hospitalization for cases and control sea lions were compared by use of logistic regression. RESULTS: California sea lions that had previously been admitted to the rehabilitation center were 43 times as likely to develop poxvirus lesions as sea lions admitted for the first time; those with high band neutrophil counts (> 0.69 X 10(3) bands/microL) at admission were 20 times less likely to develop poxvirus lesions than sea lions with counts within reference limits. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that sea lions with a history of prior hospitalization or band neutrophil counts within reference limits at admission were more likely to develop poxvirus lesions during hospitalization. Sea lions with histories of hospitalization should be kept in quarantine and infection control measures implemented to help prevent disease transmission to attending personnel and other hospitalized animals.     

Clinical relevance of novel Otarine herpesvirus-3 in California sea lions (Zalophus californianus): lymphoma,esophageal ulcers,and strandings

Herpesviruses have been recognized in marine mammals, but their clinical relevance is not always easy to assess. A novel otarine herpesvirus-3 (OtHV3) was detected in a geriatric California sea lion (Zalophus californianus), and using a newly developed quantitative PCR assay paired with histology, OtHV3 was associated with esophageal ulcers and B cell lymphoblastic lymphoma in this animal. The prevalence and quantities of OtHV3 were then determined among buffy coats from 87 stranded and managed collection sea lions. Stranded sea lions had a higher prevalence of OtHV3 compared to managed collection sea lions (34.9% versus 12.5%; p = 0.04), and among the stranded sea lions, yearlings were most likely to be positive. Future epidemiological studies comparing the presence and viral loads of OtHV3 among a larger population of California sea lions with and without lymphoid neoplasia or esophageal ulcers would help elucidate the relevance of OtHV3-associated pathologies to these groups.