Advances in research on the impacts of anti-submarine sonar on beaked whales.

Mass stranding events (MSEs) of beaked whales (BWs) were extremely rare prior to the 1960s but increased markedly after the development of naval mid-frequency active sonar (MFAS). The temporal and spatial associations between atypical BW MSEs and naval exercises were first observed in the Canary Islands, Spain, in the mid-1980s. Further research on BWs stranded in association with naval exercises demonstrated pathological findings consistent with decompression sickness (DCS). A 2004 ban on MFASs around the Canary Islands successfully prevented additional BW MSEs in the region, but atypical MSEs have continued in other places of the world, especially in the Mediterranean Sea, with examined individuals showing DCS. A workshop held in Fuerteventura, Canary Islands, in September 2017 reviewed current knowledge on BW atypical MSEs associated with MFAS. Our review suggests that the effects of MFAS on BWs vary among individuals or populations, and predisposing factors may contribute to individual outcomes. Spatial management specific to BW habitat, such as the MFAS ban in the Canary Islands, has proven to be an effective mitigation tool and mitigation measures should be established in other areas taking into consideration known population-level information.

Effects of polychlorinated biphenyls (PCB) on California sea lion (Zalophus californianus) lymphocyte functions upon in vitro exposure.

Polychorinated biphenyl (PCB) congeners are a cause for concern due to their persistence in the environment, their lipophilic properties that cause them to bio-accumulate in top predators, and their adverse effects on mammalian health. For example, the common urogenital carcinoma reported in California sea lions (Zalophus californianus) (CSL) is associated with high tissue levels of PCBs, but the mechanisms responsible for this association are unknown. This study investigated the effect of exposure to six PCB congeners and a congener mix at low and environmentally relevant concentrations on NK cell-like and T cell activity using in vitro assays on cryopreserved lymph node mononuclear cells isolated from dead CSL. Non dioxin-like congeners 153 and 180 increased lymphocyte proliferation at 5 and 10 ppm, while congener 138 decreased proliferation by up to 43% at 15 ppm. Dioxin-like PCBs 118 and 169 did not affect lymphocyte proliferation, while the effects of congener 105 depended on the mitogen concentration; these did not correlate with their predicted toxic equivalent factors. NK cell-like activity was affected only by the highest concentration of PCBs tested; it was increased by non-dioxin-like congeners 138 and 153, and decreased by dioxin-like congener 169. The PCB congener mix suggested that the effects of PCB congeners were not simply additive. Our results concur with effects of PCBs reported for other pinniped's lymphocytes and add further experimental support to the observation that dioxin-like PCBs are not the most toxic congeners for marine mammals, contrary to effects in other species. This is the first evidence of in vitro suppression of NK cell-like cytotoxicity by a dioxin-like congener in a pinniped. More importantly, the observed results suggest that PCBs can modulate the CSL immune system, increasing exposed individuals' susceptibility to viral and oncogenic challenges.

Oxidative stress and redistribution of glutamine synthetase in California sea lions (Zalophus californianus) with domoic acid toxicosis.

The aim of this study was to test the hypothesis that oxidative stress and glutamine synthetase (GS) redistribution occur in domoic acid (DA) toxicosis in California sea lions (CSLs, Zalophus californianus). Sections of archived hippocampi from seven control and 13 CSLs diagnosed with DA toxicosis were labelled immunohistochemically for GS and for two markers of oxidative stress, malondialdehyde (MDA) and 3-nitrotyrosine (NT). The distribution and intensity of labelling were compared with the pathological changes seen in haematoxylin and eosin-stained sections. Increased expression of MDA and NT occurred in neurons of the hippocampal formation of CSLs with lesions consistent with DA toxicosis. The degree of oxidative stress was not affected significantly by the chronicity or severity of hippocampal damage. In six out of seven CSLs with chronic effects of DA toxicosis, in addition to the normal glial distribution of GS, GS expression was very strong in some neurons of the subiculum. However, neuronal GS labelling was also seen in one control CSL, an effect that may have been due to previous exposure to DA. GS expression in neurons was associated with decreases in GS labelling in neighbouring glial cell processes. DA toxicosis therefore induces increased expression of markers of oxidative stress in neurons consistent with oxidative stress contributing to the initial DA insult and also the epilepsy that often develops in chronic DA toxicosis. GS redistribution occurred primarily in chronic DA toxicosis, perhaps leading to alterations of the glutamine-glutamate-GABA (gamma-aminobutyric acid) cycle and contributing to the excitotoxicity and seizures often seen in DA toxicosis.

Isolation of a novel adenovirus from California sea lions Zalophus californianus.

Viral hepatitis associated with adenoviral infection has been reported in California sea lions Zalophus californianus admitted to rehabilitation centers along the California coast since the 1970s. Canine adenovirus 1 (CAdV-1) causes viral hepatitis in dogs and infects a number of wildlife species. Attempts to isolate the virus from previous sea lion hepatitis cases were unsuccessful, but as the hepatitis had morphologic features resembling canine infectious hepatitis, and since the virus has a wide host range, it was thought that perhaps the etiologic agent was CAdV-1. Here, we identify a novel adenovirus in 2 stranded California sea lions and associate the infection with viral hepatitis and endothelial cell infection. Phylogenetic analysis confirmed the classification of the sea lion adenovirus in the Mastadenovirus genus with the most similarity to tree shrew adenovirus 1 (TSAdV-1, 77%). However, as the sea lion adenovirus appeared to be equally distant from the other Mastadenovirus species based on phylogenetic analysis, results indicate that it represents an independent lineage and species. Although sequences from this novel virus, otarine adenovirus 1 (OtAdV-1), show some similarity to CAdV-1 and 2, it is clearly distinct and likely the cause of the viral hepatitis in the stranded California sea lions.

Tumor morphology and immunohistochemical expression of estrogen receptor, progesterone receptor, p53, and Ki67 in urogenital carcinomas of California sea lions (Zalophus californianus).

Metastatic carcinoma of urogenital origin is a common cause of mortality in free-ranging California sea lions (Zalophus californianus). The etiology of this cancer is likely multifactorial, with viral infection, genetic factors, and exposure to environmental organochlorine contaminants possible contributing factors. In this study, expression of estrogen receptor alpha (ER alpha), progesterone receptor (PR), p53, and Ki67 were evaluated by immunohistochemistry in 12 sea lions with metastatic carcinoma, genital epithelial dysplasia, and intraepithelial neoplasia; 4 with genital epithelial dysplasia and intraepithelial neoplasia without metastases; and 6 control animals. Dysplastic and neoplastic lesions were identified in multiple areas of the cervix, vagina, penis, prepuce, and urethra in affected animals, suggesting multicentric development. Lesions were graded according to degree of epithelial dysplasia and infiltration and lesions of different grades were evaluated separately. Estrogen receptor expression was lower in intraepithelial lesions compared with normal genital epithelium, and expression in metastatic lesions was completely absent. There was progesterone receptor expression in neoplastic cells in intraepithelial lesions of all grades and in metastases, with no significant difference between lesion grades or between control and affected epithelium. Ki67 index and p53 expression increased with lesion grade and were higher in lesions than normal epithelium. Metastatic tumors exhibited highly variable morphology; however, proliferation index, ER alpha, PR, and p53 expression were similar in tumors with different patterns of growth. These results suggest that endogenous hormones, environmental contaminants that interact with steroid hormone receptors, and alterations in p53 may play a role in urogenital carcinogenesis in California sea lions.

Characterization of a degenerative cardiomyopathy associated with domoic acid toxicity in California sea lions (Zalophus californianus).

Domoic acid, produced by marine algae, can cause acute and chronic neurologic sequela in California sea lions (Zalophus californianus) from acute toxicity or sublethal exposure. Eight sea lions, representing acute and chronic cases, both sexes, and all age classes, were selected to demonstrate a concurrent degenerative cardiomyopathy. Critical aspects of characterizing the cardiomyopathy by lesion distribution and morphology were the development of a heart dissection and tissue-trimming protocol and the delineation of the cardiac conducting system by histomorphology and immunohistochemistry for neuron-specific protein gene product 9.5. Histopathologic features and progression of the cardiomyopathy are described, varying from acute to chronic active and mild to severe. The cardiomyopathy is distinguished from other heart lesions in pinnipeds. Based on histopathologic features, immunopositive staining for cleaved caspase-3, and comparison with known, similar-appearing cardiomyopathies, the proposed pathogenesis for the degenerative cardiomyopathy is the primary or at least initial direct interaction of domoic acid with receptors that are suspected to exist in the heart. l-Carnitine, measured in the heart and skeletal muscle, and troponin-I, measured in serum collected at the time of death from additional animals (n = 58), were not predictive of the domoic acid-associated cardiomyopathy. This degenerative cardiomyopathy in California sea lions represents another syndrome beyond central neurologic disease associated with exposure to domoic acid and may contribute to morbidity and mortality.

Pathological features of amyloidosis in stranded California sea lions (Zalophus californianus).

Amyloidosis was diagnosed in 26 stranded adult California sea lions between 1983 and 2006 by retrospective case analysis. The kidneys (92.3% of animals), blood vessels (80.7%) and thyroid glands (65.4%) were most commonly affected. Macroscopically, affected kidneys were swollen, with pale tan cortices and loss of corticomedullary differentiation. Amyloid deposits in the kidney were located in the glomeruli, blood vessels, and peritubular interstitium, most prominently in the outer stripe of the medulla. The amyloid deposits were identified as type amyloid A (AA) by potassium permanganate staining and immunolabelling with antibodies against AA protein. Concurrent diseases, including inflammatory processes and genital carcinoma, were common in affected animals. Serum amyloid A concentrations were high (>1200 microg/ml) in six of seven affected sea lions, while the median value in clinically healthy animals was <10 microg/ml. These findings suggest that renal amyloidosis contributes to morbidity and mortality in stranded adult California sea lions.

Prevalence and pathology of lungworm infection in bottlenose dolphins Tursiops truncatus from southwest Florida.

Parasitism of the respiratory system is a relatively common finding in stranded cetaceans; however, no systematic investigations regarding the severity, distribution, and clinical consequences of these infections in bottlenose dolphins Tursiops truncatus have been conducted previously. The present study determined the prevalence of lungworm infections in dead stranded (n=22) and live bottlenose dolphins (n=44) from southwestern Florida, USA, during the period from 2003 to 2005. Dead stranded bottlenose dolphins were necropsied and lungs were examined visually, by palpation, and histologically for lesions consistent with verminous pneumonia. When present, nematodes were counted, measured, and identified to species based upon their morphology. Dolphin feces and blowhole swabs were collected and examined for nematode larvae. Lungworm prevalence was 77% in dead animals (n=22). The lesions in most cases were mild, chronic, and not the primary cause of death. Only 13% of dead animals examined had patent infections, with larvae present in blowhole and fecal cytology, and only 18% of animals had intact worms present at necropsy, with a geometric mean intensity of infection of 22.6 worms animal(-1). Intact worms were identified as either Halocercus lagenorhynchi or Skrjabinalius cryptocephalus. The highest prevalence of active infections was found in neonates and calves, including 1 stillborn calf. For free-ranging animals, all blowhole swabs (n=44) were negative, and fecal cytology (n=22) showed a 3% prevalence of patent infection. Findings from the present study support the theory that bottlenose dolphins can be infected transplacentally by lungworms. The impact that such infections may have on neonatal survival is unknown; however, these infections could increase neonatal mortality.

Changes in blubber contaminant concentrations in California sea lions (Zalophus californianus) associated with weight loss and gain during rehabilitation.

California sea lions have high levels of persistent organic pollutants (POPs) in their blubber. Animals affected by domoic acid fast and refeed during their rehabilitation. We studied the effect of decreases in total body mass (16 +/- 7% of initial body mass) on blubber POP contaminant concentrations and estimated POP burdens during fasting (12 +/- 5 days) in 19 California sea lions. The effect of refeeding (92 +/- 8% of initial body mass) was also investigated. Significant increases in the concentration of all POPs were found over the mass loss period and decreases during mass gain. A basic mass balance model indicated that the changes did not conform to a simple concentrating and diluting pattern and a proportion of the contaminants were lost from the lipid pool. During mass loss, the lower chlorinated polychlorinated biphenyl congeners, chlordanes, and hexachlorocyclohexanes were lost at a higher rate than the other contaminant classes (particularly polybrominated diphenyl ethers). During mass gain the behavior of all contaminant classes was more consistent with the dilution model. These results indicate the importance of considering the energetic contextwhen sampling blubberfor long-term contaminant monitoring and suggest an initial approach to adjust for such differences.

Novel symptomatology and changing epidemiology of domoic acid toxicosis in California sea lions (Zalophus californianus): an increasing risk to marine mammal health.

Harmful algal blooms are increasing worldwide, including those of Pseudo-nitzschia spp. producing domoic acid off the California coast. This neurotoxin was first shown to cause mortality of marine mammals in 1998. A decade of monitoring California sea lion (Zalophus californianus) health since then has indicated that changes in the symptomatology and epidemiology of domoic acid toxicosis in this species are associated with the increase in toxigenic blooms. Two separate clinical syndromes now exist: acute domoic acid toxicosis as has been previously documented, and a second novel neurological syndrome characterized by epilepsy described here associated with chronic consequences of previous sub-lethal exposure to the toxin. This study indicates that domoic acid causes chronic damage to California sea lions and that these health effects are increasing.

Otarine Herpesvirus-1, not papillomavirus, is associated with endemic tumours in California sea lions (Zalophus californianus).

The purpose of this study was to determine if Otarine Herpesvirus-1 (OtHV-1) is associated with the presence of urogenital carcinomas in California sea lions. Polymerase chain reaction (PCR) analysis with primers specific for OtHV-1 was used to compare the prevalence of OtHV-1 infection in 15 sea lions affected by urogenital carcinoma with that of age-matched and juvenile tumour-free animals, and animals with tumours of non-urogenital origin. The herpesvirus was more prevalent (100%) and more widespread in the 15 animals with urogenital carcinoma than in 25 control animals, and was most often found in the urogenital tissue (vagina and prostate) and in the draining lymph nodes. Moreover, OtHV-1 DNA was not found in any juvenile animal, or in the neoplastic tissues of animals with non-urogenital tumours. Papillomavirus-specific PCR analysis of urogenital carcinoma tissues detected papillomavirus sequences in only one carcinomatous tissue. Further studies are needed to determine if OtHV-1 contributes to oncogenesis in the California sea lion; these data show, however, that OtHV-1 is associated with urogenital carcinomas, is preferentially present in urogenital tissues, and may be sexually transmitted. Papillomaviruses, which are known to contribute to urogenital tumours in other species, did not appear to be associated with the sea lion carcinomas.

Fetus in fetu in a harbor seal (Phoca vitulina richardi): histopathologic, genetic, and toxicologic analysis.

A young harbor seal (Phoca vitulina richardi), stranded on the coast of California, was found to have a 20-cm-diameter cranial cervical mass. Surgical excision revealed the subcutaneous mass to be covered in haired skin with multiple glabrous areas and structures resembling a jaw with tooth buds, eyelids, and a tail. The mass deformed the host pup's skull. Histologic examination revealed a complete vertebra in the tail, teeth in the jaw, and areas resembling tongue and larynx. Class 1 MHC sequences amplified by polymerase chain reaction from the mass and the host twin were identical. The mass was diagnosed as a fetus in fetu, a rare congenital anomaly in which 1 conjoined twin is completely enclosed in the body of the other twin. The host pup died, and no additional defects were found; however, blubber levels of persistent organic pollutants were high. The cause of the congenital anomaly in this pup is uncertain.

Tissue distribution of phocine herpesvirus-1 (PhHV-1) in infected harbour seals (Phoca vitulina) from the central Californian coast and a comparison of diagnostic methods.

The polymerase chain reaction (PCR) was used to determine the tissue distribution of phocine herpesvirus-1 (PhHV-1) DNA in 20 stranded Pacific harbour seals (17 pups and three seals older than one year) that died during rehabilitation. The aim was to begin to define stages of infection and to investigate the relation between the presence of PhHV-1 in tissues, histological lesions and serology. PhHV-1 DNA was detected in a wide range of tissues from 10/17 pups and 3/3 subadults or adults. Different clinical patterns emerged from the examination of ante- and post-mortem samples. These patterns probably represented pups with active PhHV-1 infection, pups recovering from infection, and older harbour seals with chronic, reactivated infection. As PhHV-1 DNA was detected in tissues in the absence of typical histological lesions in seven seals and in the absence of PhHV-1 specific antibodies in four seals, it is clear that both histological examination and serology underestimate the presence of infection. These results showed that infection can occur in the absence of obvious disease and that seroconversion may be associated with clinical recovery.

Contaminant loads and hematological correlates in the harbor seal (Phoca vitulina) of San Francisco Bay, California.

An expanding body of research indicates that exposure to contaminants may impact marine mammal health, thus possibly contributing to population declines. The harbor seal population of the San Francisco Bay (SFB), California, has suffered habitat loss and degradation, including decades of environmental contamination. To explore the possibility of contaminant-induced health alterations in this population, blood levels of polychlorinated biphenyls (PCBs), dichlorodiphenyldichloroethylene (DDE), and polybrominated diphenyl ethers (PBDEs) were quantified in free-ranging seals; relationships between contaminant exposure and several key hematological parameters were examined; and PCB levels in the present study were compared with levels determined in SFB seals a decade earlier. PCB residues in harbor seal blood decreased during the past decade, but remained at levels great enough that adverse reproductive and immunological effects might be expected. Main results included a positive association between leukocyte counts and PBDEs, PCBs, and DDE in seals, and an inverse relationship between red blood cell count and PBDEs. Although not necessarily pathologic, these responses may serve as sentinel indications of contaminant-induced alterations in harbor seals of SFB, which, in individuals with relatively high contaminant burdens, might include increased rates of infection and anemia.

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.

Pleomorphic rhabdomyosarcoma with pulmonary metastasis in a stranded Steller (northern) sea lion (Eumetopias jubatus).

A Steller (northern) sea lion (SSL), stranded in northern California in July 2000 had an anaplastic pleomorphic rhabdomyosarcoma in the latissimus dorsi muscle, with pulmonary metastasis. Diagnosis was based on light and electron microscopy and immunohistochemistry. Death was attributed to multiple parasitic and bacterial lesions. The SSL is of special concern because, for unknown reasons, the global population has declined by 50% over the last decade. Published post-mortem data, however, are scarce. This case report highlights several disease conditions that affect this species and is the first report of a malignant neoplasm in a free-ranging SSL.

Meningoencephalitis associated with an unidentified apicomplexan protozoan in a Pacific harbor seal.

A Pacific harbor seal (Phoca vitulina richardsii) was found on the central California coast with neurologic signs and labored breathing, which were unresponsive to treatment. Necropsy revealed a nonsuppurative necrotizing meningoencephalitis, a multilocular thymic cyst, and nonsuppurative cystitis and renal pyelitis. Microscopic examination revealed protozoans in the brain, thymic cyst, and bladder mucosa. Ultrastructurally, the protozoal tachyzoites were different from those of Neospora caninum, Toxoplasma gondii, and Sarcocystis neurona; the rhoptries were small and had electron-dense contents, and the organism divided by endodyogeny. Specific antibodies were not detected in serum using agglutination (N. caninum, T. gondii) and immunoblot assays (S. neurona). Immunohistochemistry for these organisms was negative. Polymerase chain reaction on brain tissue using specific primers did not amplify T. gondii deoxyribonucleic acid. The meningoencephalitis in this seal thus appears to have been caused by a novel protozoan.

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).

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.

Isolation and characterization of two parasitic protozoa from a Pacific harbor seal (Phoca vitulina richardsi) with meningoencephalomyelitis.

Two species of protozoans were isolated from a harbor seal with fatal meninogoencephalitis. Serologic reactivity was detected to both Sarcocystis neurona and Toxoplasma gondii. Parasites associated with brain inflammation and necrosis reacted only with immunohistochemical stains utilizing polyclonal antisera raised against Sarcocystis neurona. However, 2 distinct parasites were observed in cell cultures derived from the seal's brain tissue. These parasites were separated by mouse passage and limiting dilution. Purified zoites from 1 isolate (HS1) reacted strongly with polyclonal antiserum to S. neurona and with the harbor seal's own serum (1:2,560 for each) on indirect immunofluorescent antibody tests (IFAT), but weakly to antisera to T. gondii and Neospora caninum (1:40). Zoites from the second isolate (HS2) reacted positively with T. gondii polyclonal antiserum (1:81,920) and with the harbor seal's own serum (1:640), but weakly to S. neurona and N. caninum antisera (1:80 or less). Amplification and sequence analysis of protozoal DNA encoding portions of the 18s ribosomal RNA (18s rDNA) and the adjacent first internal transcribed spacer (ITSI) were performed for both isolates, and resulting sequences were compared to those from similar protozoans. Based on molecular characterization, parasite morphology, serologic reactivity, histology, and immunohistochemistry, HS1 was indistinguishable from S. neurona, and HS2 was indistinguishable from T. gondii.