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Beginning in December 2018, increased numbers of gray whale (Eschrichtius robustus) strandings were reported along the west coast of Mexico, the United States, and Canada, prompting declaration of a gray whale Unusual Mortality Event (UME) by the United States National Marine Fisheries Service. Although strandings declined in 2020 and 2021 from a peak in 2019, the UME is still ongoing as of fall 2023. Between 17 December 2018 and 31 December 2021, 503 animals stranded along the west coast of North America, with 226 strandings in Mexico, 71 in California, 12 in Oregon, 56 in Washington, 21 in British Columbia, and 117 in Alaska. These included 187 males, 167 females, and 149 whales of undetermined sex; and 193 adults, 194 subadults, 40 calves, 1 fetus, and 75 whales of undetermined age class. We report on 61 of the 503 carcasses (12%) that had external and internal gross necropsy and/or histopathology data: of these 61 whales, findings that contributed to death were identified in 33 (54%) whales. Sixteen of the 61 (26%) were severely emaciated. Gross lesions of blunt force trauma consistent with vessel strike were identified in 11 of the 61 animals (18%), only two of which were emaciated. Two whales (3%) were entangled at time of death, and one died from entrapment. Signs of killer whale (Orcinus orca) interaction were documented in 19 of the 61 animals; five were deemed from recent interactions and three (5%) likely contributed to mortality. A specific cause of death could not be identified in 28 of 61 whales (46%). Additionally, logistical challenges and the advanced state of decomposition of most examined carcasses precluded detection of potential infectious or toxic causes of morbidity or mortality. Up to 2016, the eastern North Pacific population of gray whale population had generally been increasing since the cessation of historic whaling and a prior UME in 1999-2000. However, recent abundance and calf production estimates have declined, a trend that overlaps the current UME. The relative contributions of carrying capacity, environmental change, prey shifts, and infectious, toxic, and other processes to the increased gray whale mortalities have not yet been resolved. Nevertheless, the marked temporal increase in strandings, including findings of malnutrition in some of the whales, along with low calf production, likely represent consequences of complex and dynamic ecological interactions in the ocean impacting the population., Competing Interests: The authors have no competing interests., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Introduction: Domoic acid (DA) is a glutaminergic excitatory neurotoxin that causes the morbidity and mortality of California sea lions (Zalophus californianus; CSL) and other marine mammals due to a suite of effects mostly on the nervous and cardiac systems. Between 1998 and 2019, 11,737 live-stranded CSL were admitted to The Marine Mammal Center (TMMC; Sausalito, CA, USA), over 2,000 of which were intoxicated by DA. A plethora of clinical research has been performed over the past 20 years to characterize the range of toxic effects of DA exposure on CSLs, generating the largest dataset on the effects of natural exposure to this toxin in wildlife., Materials and Methods: In this study, we review published methods for diagnosing DA intoxication, clinical presentation, and treatment of DA-intoxicated CSL and present a practical, reproducible scoring system called the neuroscore (NS) to help assess whether a DA-affected CSL is fit for release to the wild following rehabilitation. Logistic regression models were used to assess the relationships between outcome (released vs. euthanized or died) and multiple variables to predict the outcome for a subset of 92 stranded CSLs., Results: The largest proportion of DA-intoxicated CSLs was adult females (58.6%). The proportions of acute and chronic cases were 63.5 and 36.5% respectively, with 44% of affected CSL released and 56% either dying naturally or euthanized. The average time in rehabilitation was 15.9 days (range 0-169) for all outcomes. The best-performing model (85% accuracy; area under the curve = 0.90) assessing the relationship between outcome and predictor variables consisted of four variables: final NS, change in NS over time, whether the animal began eating in rehabilitation, and the state of nutrition on admission., Discussion: Our results provide longitudinal information on the symptomatology of CSL intoxicated by domoic acid and suggest that a behavioral scoring system is a useful tool to assess the fitness for the release of DA-intoxicated CSL., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 McClain, Field, Norris, Borremans, Duignan, Johnson, Whoriskey, Thompson-Barbosa and Gulland.)

Each year in the United States, thousands of sick, injured, or displaced wild animals are presented to individuals or organizations who have either a federal or state permit that allows them to care for these animals with the goal of releasing them back to the wild. The purpose of this review is to demonstrate the complexity of considerations rehabilitators and veterinarians face while trying to optimize the welfare of wild animals in need of care and rehabilitation. The process of rehabilitation is inherently stressful for wildlife. Maintaining an animal's welfare during the rehabilitation process-from initial contact and tria+ge to the animal's euthanasia, release, or captive placement-requires deliberate, timely and humane decision making. The welfare of wild animals can be improved by preventing human-related causes of admission, providing resources and support for wildlife rehabilitation (almost all rehabilitation in the United States is privately funded and access to veterinary care is often limited); further developing evidence-based wildlife rehabilitation methods and welfare measures, attracting more veterinary professionals to the field, harmonizing regulatory oversight with standards of care, training, and accountability, and increasing public education.

This study was designed to identify abnormalities in the electroencephalograms (EEGs) recorded from stranded California sea lions ( Zalophus californianus ) with suspected domoic acid (DA) toxicosis . Recordings from animals presenting for non-neurological issues were also obtained to better understand the normal EEG (background activity and transient events) in this species, as, to date, studies have focused on examining natural sleep in pinnipeds. Most animals were sedated for electrode placement and EEG acquisition with some receiving antiepileptic medications or isoflurane during the procedure. A total of 103 recordings were read and scored from 0 (normal) to 3 (severely abnormal). Epileptiform discharges, consisting of spikes, sharp waves, slow waves, and/or spike waves, were present in all EEGs with scores of 1, 2, or 3. The distribution of these events over the scalp varied. While often generalized, others were lateralized over one hemisphere, bifrontal, bioccipital, and/or bitemporal, while some discharges were multifocal. Findings were different between sea lions and occasionally changed within the EEG on a given sea lion. No clinical seizures were observed during the recording but a few sea lions had findings consistent with electroencephalographic seizures. When available, supporting diagnostic results obtained from magnetic resonance imaging (MRI) and/or necropsy/histopathology were described, as well as the status of those sea lions that recovered and were released with satellite tags., Competing Interests: SD is employed by Imaging Solutions, PLLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Williams, Haulena, Dennison, Waugh, Goldstein, Nutter, Bonn, Hoard, Laxer, Buckmaster, Gulland and Tharp.)

From 2011-2018, we conducted surveillance in marine mammals along the California coast for influenza A virus (IAV), frequently detecting anti-influenza antibodies and intermittently detecting IAV. In spring 2019, this pattern changed. Despite no change in surveillance intensity, we detected IAV RNA in 10 samples in March and April, mostly in nasal and rectal swabs from northern elephant seals (Mirounga angustirostris). Although virus isolation was unsuccessful, IAV sequenced from one northern elephant seal nasal swab showed close genetic identity with pandemic H1N1 IAV subclade 6B.1A.1 that was concurrently circulating in humans in the 2018/19 influenza season. This represents the first report of human A(H1N1)pdm09 IAV in northern elephant seals since 2010, suggesting IAV continues to spill over from humans to pinnipeds., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Plancarte et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Tattoo skin disease (TSD) is a poxviral dermatopathy diagnosed in cetaceans. We review the literature on TSD aetiology, clinical characteristics, pathology and epidemiology and evaluate immune responses against the virus. In addition, necropsy reports for fifty-five harbour porpoises ( Phocoena phocoena ), twenty-two Delphinidae and four Kogiidae stranded in northern California in 2018-2021 were checked for diagnostic tattoo lesions. TSD occurs in the Mediterranean, North and Barents Seas, as well as in the Atlantic, eastern Pacific and Indian Oceans in at least 21 cetacean species, with varying prevalence. Two cetacean poxvirus (CePV) clades are recognised: CePV-1 in odontocetes and CePV-2 in mysticetes. CePV-1 isolates were recovered from six Delphinidae and one Phocoenidae in the Americas, Europe and Hong Kong. Strains from Delphinidae are closely related. Among Phocoenidae, poxviruses were sampled only in harbour porpoises around the British Isles. CePV-2 isolates were obtained from southern right whales ( Eubalaena australis ) and a bowhead whale ( Balaena mysticetus ). In healthy animals, an immune response develops over time, with young calves protected by maternal immunity. Salinity and sea surface temperature do not seem to influence TSD prevalence in free-ranging cetaceans. High concentrations of immunotoxic halogenated organochlorines may cause a more severe clinical disease. Substitution and loss of genes involved in anti-viral immunity may favour CePV entry, replication and persistence in the epidermis. Off California, Delphinidae were less often (26.3%) affected by TSD than harbour porpoises (43.6%). Male porpoises were significantly more prone (58.1%) to show clinical disease than females (25%). Among males, TSD affected a high proportion of juveniles and subadults. TSD was not detected in the Kogiidae.

Nasopulmonary mites (NPMs) of the family Halarachnidae are obligate endoparasites that colonize the respiratory tracts of mammals. NPMs damage surface epithelium resulting in mucosal irritation, respiratory illness, and secondary infection, yet the role of NPMs in facilitating pathogen invasion or dissemination between hosts remains unclear. Using 16S rRNA massively parallel amplicon sequencing of six hypervariable regions (or "16S profiling"), we characterized the bacterial community of NPMs from 4 southern sea otters (Enhydra lutris nereis). This data was paired with detection of a priority pathogen, Streptococcus phocae, from NPMs infesting 16 southern sea otters and 9 California sea lions (Zalophus californianus) using nested conventional polymerase chain reaction (nPCR). The bacteriome of assessed NPMs was dominated by Mycoplasmataceae and Vibrionaceae, but at least 16 organisms with pathogenic potential were detected as well. Importantly, S. phocae was detected in 37% of NPM by nPCR and was also detected by 16S profiling. Detection of multiple organisms with pathogenic potential in or on NPMs suggests they may act as mechanical vectors of bacterial infection for marine mammals., Competing Interests: The authors have declared that no competing interests exist.

Background: Enteritis is a common cause of morbidity and mortality in lorikeets that can be challenging to diagnose and treat. In this study, we examine gut microbiota in two lorikeet flocks with enteritis (Columbus Zoo and Aquarium-CZA; Denver Zoo-DZ). Since 2012, the CZA flock has experienced repeated outbreaks of enteritis despite extensive diet, husbandry, and clinical modifications. In 2018, both CZA and DZ observed a spike in enteritis. Recent research has revealed that the gut microbiota can influence susceptibility to enteropathogens. We hypothesized that a dysbiosis, or alteration in the gut microbial community, was making some lorikeets more susceptible to enteritis, and our goal was to characterize this dysbiosis and determine the features that predicted susceptibility., Results: We employed 16S rRNA sequencing to characterize the cloacal microbiota in lorikeets (CZA n = 67, DZ n = 24) over time. We compared the microbiota of healthy lorikeets, to lorikeets with enteritis, and lorikeets susceptible to enteritis, with "susceptible" being defined as healthy birds that subsequently developed enteritis. Based on sequencing data, culture, and toxin gene detection in intestinal contents, we identified Clostridium perfringens type A (CZA and DZ) and C. colinum (CZA only) at increased relative abundances in birds with enteritis. Histopathology and immunohistochemistry further identified the presence of gram-positive bacilli and C. perfringens, respectively, in the necrotizing intestinal lesions. Finally, using Random Forests and LASSO models, we identified several features (young age and the presence of Rhodococcus fascians and Pseudomonas umsongensis) associated with susceptibility to clostridial enteritis., Conclusions: We identified C. perfringens type A and C. colinum associated with lorikeet necrohemorrhagic enteritis at CZA and DZ. Susceptibility testing of isolates lead to an updated clinical treatment plan which ultimately resolved the outbreaks at both institutions. This work provides a foundation for understanding gut microbiota features that are permissive to clostridial colonization and host factors (e.g. age, prior infection) that shape responses to infection., (© 2022. The Author(s).)

Mites from the family Halarachnidae Oudemans 1906 are obligate endoparasites that colonize the respiratory tracts of free-living and captive marine mammals. Infestations can range from mild to severe and result in respiratory tract irritation or impairment. Nasopulmonary acariasis was determined to be a contributing cause of death among several southern sea otters Enhydra lutris nereis Merriam 1904 in a longitudinal study of otter mortality, and proximity to Pacific harbor seals Phoca vitulina richardii Gray 1864 was a significant risk factor for sea otter infestation. Beyond scattered opportunistic reports, each halarachnid mite species' affinity for particular hosts and the extent of mite transmission between host species is poorly understood. We investigated the identity and prevalence of nasopulmonary mites from Pacific harbor seals, California sea lions Zalophus californianus Lesson 1828, northern elephant seals Mirounga angustirostris Gill 1866, northern fur seals Callorhinus ursinus Linnaeus 1758, and Guadalupe fur seals Arctocephalus philippii townsendi Merriam 1897 to complement published nasopulmonary mite findings from sympatric southern sea otters during a comparable timeframe. Halarachnid mite infestation was common among California sea lions (74.1%), northern fur seals (73.3%), and northern elephant seals (46.6%), but was less common among harbor seals (18.7%) and Guadalupe fur seals (8.8%). Observed host-mite relationships suggest a distinct host specificity, with genus Orthohalarachne infesting otariids, and genus Halarachne infesting phocids and lutrinids along the California coast. Harbor seals and southern sea otters were the primary hosts of H. halichoeri , but one nothern elephant seal was infested with both H. miroungae and a single H. halichoeri. We also present the first high-resolution SEM images for H. miroungae and O. attenuata and possible evidence for a new host record for H. halichoeri ., Competing Interests: None., (© 2021 The Authors.)

To understand susceptibility of wild California sea lions and Northern elephant seals to influenza A virus (IAV), we developed an ex vivo respiratory explant model and used it to compare infection kinetics for multiple IAV subtypes. We first established the approach using explants from colonized rhesus macaques, a model for human IAV. Trachea, bronchi, and lungs from 11 California sea lions, 2 Northern elephant seals, and 10 rhesus macaques were inoculated within 24 h postmortem with 6 strains representing 4 IAV subtypes. Explants from the 3 species showed similar IAV infection kinetics, with peak viral titers 48 to 72 h post-inoculation that increased by 2 to 4 log 10 PFU/explant relative to the inoculum. Immunohistochemistry localized IAV infection to apical epithelial cells. These results demonstrate that respiratory tissue explants from wild marine mammals support IAV infection. In the absence of the ability to perform experimental infections of marine mammals, this ex vivo culture of respiratory tissues mirrors the in vivo environment and serves as a tool to study IAV susceptibility, host range, and tissue tropism. IMPORTANCE Although influenza A virus can infect marine mammals, a dearth of marine mammal cell lines and ethical and logistical challenges prohibiting experimental infections of living marine mammals mean that little is known about IAV infection kinetics in these species. We circumvented these limitations by adapting a respiratory tract explant model first to establish the approach with rhesus macaques and then for use with explants from wild marine mammals euthanized for nonrespiratory medical conditions. We observed that multiple strains representing 4 IAV subtypes infected trachea, bronchi, and lungs of macaques and marine mammals with variable peak titers and kinetics. This ex vivo model can define infection dynamics for IAV in marine mammals. Further, use of explants from animals euthanized for other reasons reduces use of animals in research.

During the 2018 breeding season, an outbreak of respiratory disease occurred among Galapagos sea lions (Zalophus wollebaeki) that inhabit rookeries near urban areas with introduced fauna such as dogs and cats. Several sea lions had nasal discharge and respiratory distress and were in poor body condition. Eighteen sea lions were captured for a general health assessment including collection of blood for serology and nasal discharge for culture and PCR. Samples were analyzed for 15 respiratory pathogens known to infect cats, dogs, and marine mammals. There was no evidence for interspecies pathogen transmission between Galapagos sea lions and domestic animals. Several bacterial pathogens associated with respiratory tract infection in the California sea lion (Zalophus californianus) were isolated. Mycoplasma spp. were identified by PCR in nasal discharge samples but were not the species commonly found in cats and dogs., (© Wildlife Disease Association 2021.)

Objective: To compare serum cardiac troponin I (cTnI) concentrations between sea otters with and without cardiomyopathy and describe 2 cases of cardiomyopathy with different etiologies., Animals: 25 free-ranging southern sea otters (Enhydra lutris nereis) with (n = 14; cases) and without (11; controls) cardiomyopathy and 17 healthy managed southern sea otters from aquariums or rehabilitation centers (controls)., Procedures: Serum cTnI concentration was measured in live sea otters. Histopathologic and gross necropsy findings were used to classify cardiomyopathy status in free-ranging otters; physical examination and echocardiography were used to assess health status of managed otters. Two otters received extensive medical evaluations under managed care, including diagnostic imaging, serial cTnI concentration measurement, and necropsy., Results: A significant difference in cTnI concentrations was observed between cases and both control groups, with median values of 0.279 ng/mL for cases and < 0.006 ng/mL for free-ranging and managed controls. A cutoff value of ≥ 0.037 ng/mL yielded respective sensitivity and specificity estimates for detection of cardiomyopathy of 64.3% and 90.9% for free-ranging cases versus free-ranging controls and 64.3% and 94.1% for free-ranging cases versus managed controls., Conclusions and Clinical Relevance: Cardiomyopathy is a common cause of sea otter death that has been associated with domoic acid exposure and protozoal infection. Antemortem diagnostic tests are needed to identify cardiac damage. Results suggested that serum cTnI concentration has promise as a biomarker for detection of cardiomyopathy in sea otters. Serial cTnI concentration measurements and diagnostic imaging are recommended to improve heart disease diagnosis in managed care settings.

Parapoxviruses cause nodular lesions on the skin and mucosal membranes of pinnipeds and infections by these viruses have been documented worldwide. Seal parapoxvirus is currently classified as a tentative species of the Parapoxvirus genus. Tissue or swab samples were analyzed from 11 pinnipeds of different host species undergoing rehabilitation on the east and west coasts of the United States of America (USA) that were positive for parapoxvirus. The aim of the study was to compare parapoxvirus sequences of fragments of the B2L, DNA polymerase, GIF and viral interleukin-10 ortholog ( vIL-10) genes and to examine the evolutionary relationship between viruses detected in different pinniped species and at different locations with other members of the Parapoxvirus genus, such as Orf virus (ORFV), Bovine papular stomatitis virus (BPSV) and Pseudocowpox virus (PCPV). The sequence analysis showed that the parapoxvirus sequences from the pinnipeds differed significantly from those found in terrestrial hosts and that they formed a separate cluster within the genus. Our results suggest that transmission of the same parapoxvirus strain is possible between different species, including between members of different families (phocids and otariids). Animals belonging to the same species but living in distant geographic locations presented genetically distant parapoxviruses. The findings of this study demonstrate that sealpox lesions in pinnipeds of different species are caused by viruses that belong to the Parapoxvirus genus but have significant genetic differences compared to the established virus species in terrestrial hosts, thus strongly supporting the classification of pinniped parapoxvirus as a new species of the genus., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Costa, Klein, Breines, Nollens, Matassa, Garron, Duignan, Schmitt, Goldstein and Tryland.)

Urogenital carcinoma in California sea lions ( Zalophus californianus ) is the most common cancer of marine mammals. Primary tumors occur in the cervix, vagina, penis, or prepuce and aggressively metastasize resulting in death. This cancer has been strongly associated with a sexually transmitted herpesvirus, otarine herpesvirus 1 (OtHV1), but the virus has been detected in genital tracts of sea lions without cancer and a causative link has not been established. To determine if OtHV1 has a role in causing urogenital carcinoma we sequenced the viral genome, quantified viral load from cervical tissue from sea lions with ( n = 95) and without ( n = 163) urogenital carcinoma, and measured viral mRNA expression using in situ mRNA hybridization (Basescope ® ) to quantify and identify the location of OtHV1 mRNA expression. Of the 95 sea lions diagnosed with urogenital carcinoma, 100% were qPCR positive for OtHV1, and 36% of the sea lions with a normal cervix were positive for the virus. The non-cancer OtHV1 positive cases had significantly lower viral loads in their cervix compared to the cervices from sea lions with urogenital carcinoma. The OtHV1 genome had several genes similar to the known oncogenes, and RNA in situ hybridization demonstrated high OtHV1 mRNA expression within the carcinoma lesions but not in normal cervical epithelium. The high viral loads, high mRNA expression of OtHV1 in the cervical tumors, and the presence of suspected OtHV1 oncogenes support the hypothesis that OtHV1 plays a significant role in the development of sea lion urogenital carcinoma.

A distinct ulcerative dermatitis known as "freshwater skin disease" is an emerging clinical and pathological presentation in coastal cetaceans worldwide. In Australia, two remarkably similar mortality events enabled the creation of a case definition based on pathology and environmental factors. The first affected a community of endemic Tursiops australis in the Gippsland Lakes, Victoria, while the second occurred among T. aduncus resident in the Swan-Canning River system, Western Australia. The common features of both events were (1) an abrupt and marked decrease in salinity (from > 30ppt to < 5ppt) due to rainfall in the catchments, with hypo-salinity persisting weeks to months, and (2) dermatitis characterized grossly by patchy skin pallor that progressed to variable circular or targetoid, often raised, and centrally ulcerated lesions covering up to 70% of the body surface. The affected skin was often colonized by a variety of fungal, bacterial and algal species that imparted variable yellow, green or orange discoloration. Histologic lesions consisted of epidermal hydropic change leading to vesiculation and erosion; alternately, or in addition, the formation of intra-epithelial pustules resulting in ulceration and hypodermal necrosis. Thus, the environmental factors and characteristic pathologic lesions, are necessary components of the case definition for freshwater skin disease.

Confronted with the challenge of understanding population-level processes, disease ecologists and epidemiologists often simplify quantitative data into distinct physiological states (e.g. susceptible, exposed, infected, recovered). However, data defining these states often fall along a spectrum rather than into clear categories. Hence, the host-pathogen relationship is more accurately defined using quantitative data, often integrating multiple diagnostic measures, just as clinicians do to assess their patients. We use quantitative data on a major neglected tropical disease (Leptospira interrogans) in California sea lions (Zalophus californianus) to improve individual-level and population-level understanding of this Leptospira reservoir system. We create a "host-pathogen space" by mapping multiple biomarkers of infection (e.g. serum antibodies, pathogen DNA) and disease state (e.g. serum chemistry values) from 13 longitudinally sampled, severely ill individuals to characterize changes in these values through time. Data from these individuals describe a clear, unidirectional trajectory of disease and recovery within this host-pathogen space. Remarkably, this trajectory also captures the broad patterns in larger cross-sectional datasets of 1456 wild sea lions in all states of health but sampled only once. Our framework enables us to determine an individual's location in their time-course since initial infection, and to visualize the full range of clinical states and antibody responses induced by pathogen exposure. We identify predictive relationships between biomarkers and outcomes such as survival and pathogen shedding, and use these to impute values for missing data, thus increasing the size of the useable dataset. Mapping the host-pathogen space using quantitative biomarker data enables more nuanced understanding of an individual's time course of infection, duration of immunity, and probability of being infectious. Such maps also make efficient use of limited data for rare or poorly understood diseases, by providing a means to rapidly assess the range and extent of potential clinical and immunological profiles. These approaches yield benefits for clinicians needing to triage patients, prevent transmission, and assess immunity, and for disease ecologists or epidemiologists working to develop appropriate risk management strategies to reduce transmission risk on a population scale (e.g. model parameterization using more accurate estimates of duration of immunity and infectiousness) and to assess health impacts on a population scale., Competing Interests: The authors have declared that no competing interests exist.

Parafilaroides decorus, also known as sea lion lungworm, is a metastrongyloid nematode that infects otariid hosts, such as the charismatic California sea lion, Zalophus californianus. P. decorus causes bronchointerstitial pneumonia, respiratory distress, reduced ability to swim, dive and hunt and as a result, increased mortality particularly in young animals. Respiratory disease is a leading cause of stranding and admission to rehabilitation centers on the Pacific coast. Low-coverage genomic sequencing of four P. decorus individuals analyzed through Galaxy's RepeatExplorer identified a novel repeat DNA family we employed to design a sensitive quantitative PCR (qPCR) assay for diagnosing infections from fecal or sputum samples. The assay detects as little as 10 fg of P. decorus DNA and a linear regression model developed using a standard curve can be used to estimate the concentration of P. decorus DNA in a sample, ± 0.015 ng. This knowledge can be leveraged to estimate the level of parasite burden, which can be used to design improved treatments for animals in rehabilitation. Improved treatment of infections will aid in more animals being successfully released back into the wild., Competing Interests: None., (© 2020 The Authors.)

Asymmetric genitalia and lateralized mating behaviors occur in several taxa, yet whether asymmetric morphology in one sex correlates or coevolves with lateralized mating behavior in the other sex remains largely unexplored. While lateralized mating behaviors are taxonomically widespread, among mammals they are only known in the harbor porpoise (Phocoena phocoena). Males attempt copulation by approaching a female exclusively on her left side. To understand if this unusual lateralized behavior may have coevolved with genital morphology, we quantified the shape of female and male harbor porpoise reproductive tracts using 2D geometric morphometrics and 3D models of the vaginal lumen and inflated distal penis. We found that the vaginas varied individually in shape and that the vaginas demonstrated both significant directional and fluctuating asymmetry. This asymmetry resulted from complex 3D spirals and vaginal folds with deep recesses, which may curtail the depth or direction of penile penetration and/or semen movement. The asymmetric shapes of the vaginal lumen and penis tip were both left-canted with similar angular bends that mirrored one another and correspond with the left lateral mating approach. We suggest that the reproductive anatomy of both sexes and their lateral mating behavior coevolved.

Mercury (Hg) poses a health risk to wildlife populations and has been documented at relatively high concentrations in many marine mammals, including wild-caught pinnipeds along the central California, US coast. We measured total Hg concentrations ([THg]) in hair and blood of live-stranded harbor seals (HS; Phoca vitulina ), California sea lions (CSL; Zalophus californianus ), and northern elephant seals (NES; Mirounga angustirostris ) in California to quantify species, temporal, and spatial variability in [THg] and assess the relationships between [THg] measured by different methods (blood vs. filter paper) and in different matrices (blood vs. hair). We compared [THg] with toxicologic thresholds of concern to aid in identification of at-risk individuals or groups and better understand how the use of different methods and matrices affects assumed toxicologic risk. There was a wide range of [THg] in blood (<0.01-1.13 μg/g) and hair (0.45-81.98 μg/g), and NES had higher [THg] compared with HS and CSL. All three species had individuals with [THg] that exceeded the lower threshold for one or both matrices, but only HS pups had [THg] exceeding upper thresholds. Spatial differences in [THg] were detected, with higher concentrations in HS pups from areas surrounding San Francisco Bay, but differences were dependent on sampling year and matrix. The relationship between [THg] in blood and filter paper ( r 2 =0.98) was strong, and differences had little influence on comparisons with toxicologic thresholds. Blood and hair [THg] were generally in agreement ( r 2 =0.72), but large mismatches for a few seals underscore the importance of combined sampling in adverse effects studies where accurate assessment of Hg exposure is crucial. The wide range of [THg] in stranded HS pups that exceeded published thresholds of concern makes them a promising candidate for adverse effects studies, particularly because different matrices represent Hg exposure across key developmental stages.

An adult female North American river otter ( Lontra canadensis ) presented with multiple intrathoracic masses identified histologically as squamous cell carcinoma. Immunohistochemical staining patterns for high- molecular-weight keratin, p40, p63, calretinin, and TTF-1, along with the gross and histologic findings, indicated a primary pleural squamous cell carcinoma as the most likely diagnosis.

A myositis syndrome has been recognized for more than a decade in California sea lions (CSLs; Zalophus californianus ) but a detailed description of the lesions and potential causes of this condition is lacking. The tissues of 136 stranded CSLs with rhabdomyositis were examined. Rhabdomyositis was considered incidental in 67% (91/136) of the CSLs, and a factor contributing to the animal stranding (significant rhabdomyositis) in 33% (45/136). Of the 91 cases with incidental rhabdomyositis, lesions consisted of a few small foci of lymphohistiocytic inflammation. Of the 45 cases with significant rhabdomyositis, 28 (62%) also presented with major comorbidities such as leptospirosis (2 animals) and domoic acid toxicosis (6 animals), whereas 17 (38%) had severe polyphasic rhabdomyositis as the only major disease process associated with mortality. In these animals, most striated muscles had multiple white streaks and diffuse atrophy. Microscopically, there was myofiber necrosis surrounded by lymphocytes and histiocytes admixed with areas of myofiber regeneration, and/or moderate to severe rhabdomyocyte atrophy usually adjacent to intact Sarcocystis neurona cysts. At the interface of affected and normal muscle, occasional T lymphocytes infiltrated the sarcoplasm of intact myocytes, and occasional myofibers expressed MHCII proteins in the sarcoplasm. S. neurona antibody titers and cyst burden were higher in animals with significant polymyositis antibody titers of (26125 ± 2164, 4.5 ± 1.2 cysts per section) and active myonecrosis than animals with incidental rhabdomyositis antibody titers of (7612 ± 1042, 1.7 ± 0.82 cysts per section). The presented findings suggest that S. neurona infection and immune-mediated mechanisms could be associated with significant polyphasic rhabdomyositis in CSLs.

The nematode lungworm, Otostrongylus circumlitis (OC), is a significant cause of northern elephant seal (NES; Mirounga angustirostris ) mortality at The Marine Mammal Center (TMMC, Sausalito, CA). The current lack of specific antemortem diagnostic tests for pre-patent OC infection in NES makes diagnosis, proper treatment, and assessment of efficacy of medications challenging. Severe inflammation and disseminated intravascular coagulation (DIC) develop rapidly and are difficult to treat once clinical signs develop. Certain blood inflammatory and hemostasis biomarkers for early diagnosis have recently been investigated. The objective of this study was to investigate the diagnostic performance of complete blood count, serum chemistry, acute phase proteins, protein electrophoresis, and coagulation parameters for diagnosis of OC clinical infection in NES. Samples from NES with OC infection confirmed by gross pathology with blood collected antemortem during clinical disease ( n = 9) and NES initially admitted for malnutrition and sampled shortly before release after successful rehabilitation ( n = 20) were included in the study. Using Receiver operator characteristic (ROC) curve analysis, the diagnostic performances (area under the curve [AUC]) of albumin (0.994), albumin:globulin ratio (0.983), serum amyloid A (0.972), activated partial thromboplastin time (0.936), total bilirubin (0.975), and gamma-glutamyl transferase (0.939) were high (AUC > 0.9). These results confirm systemic inflammation and DIC, and support previously reported clinical and gross pathological findings in NES infected with OC. In addition to AUC values, this study produced cut-off points, sensitivity, specificity, confidence intervals, and predictive values for analytes with high diagnostic performance. This data will be useful in the diagnosis and clinical management of OC-infected NES and will aid in assessment of treatment efficacy.

The California sea lion is one of the few wild mammals prone to develop cancer, particularly urogenital carcinoma (UGC), whose prevalence is currently estimated at 25% of dead adult sea lions stranded along the California coastline. Genetic factors, viruses and organochlorines have been identified as factors that increase the risk of occurrence of this pathology. Given that no cases of UGC have as yet been reported for the species along its distribution in Mexican waters, the potential relevance of contaminants for the development of urogenital carcinoma is highlighted even more as blubber levels of organochlorines are more than two orders of magnitude lower in the Gulf of California and Mexican Pacific than in California. In vitro studies have shown that organochlorines can modulate anti-viral and tumor-surveillance activities of NK and cytotoxic T-cells of marine mammals, but little is known about the activity of these effectors in live, free-living sea lions. Here, we examine leukocyte transcriptional profiles of free-ranging adult California sea lions for eight genes (Eomes, Granzyme B, Perforin, Ly49, STAT1, Tbx21, GATA3, and FoxP3) selected for their key role in anti-viral and tumor-surveillance, and investigate patterns of transcription that could be indicative of differences in ecological variables and exposure to two oncogenic viruses: sea lion type one gammaherpesvirus (OtHV-1) and sea lion papillomavirus type 1 (ZcPV-1) and systemic inflammation. We observed regional differences in the expression of genes related to Th1 responses and immune modulation, and detected clear patterns of differential regulation of gene expression in sea lions infected by genital papillomavirus compared to those infected by genital gammaherpesvirus or for simultaneous infections, similar to what is known about herpesvirus and papillomavirus infections in humans. Our study is a first approach to profile the transcriptional patterns of key immune effectors of free-ranging California sea lions and their association with ecological regions and oncogenic viruses. The observed results add insight to our understanding of immune competence of marine mammals, and may help elucidate the marked difference in the number of cases of urogenital carcinoma in sea lions from US waters and other areas of their distribution.

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.

A field study addressing causes of mortality in freshly dead northern elephant seals ( Mirounga angustirostris, Gill, 1866) was conducted on San Miguel Island, California, in February 2015. Necropsies were performed on 18 pups ranging in age from stillbirths to approximately 7 to 8 weeks. The primary gross diagnoses in these pups included trauma, myopathy, starvation/emaciation, infections, congenital anomalies, and perinatal mortality. However, 6 (33%) had a previously unrecognized myopathy characterized by multiple white streaks that were most obvious within the inner layer of the abdominal wall and the small innermost ventral intercostal muscles. Following histological examination, 2 more pups from San Miguel Island and 6 pups from The Marine Mammal Center (Sausalito, California) were found to have similar lesions. Histologically, the lesions within the skeletal muscles were characterized by a multifocal polyphasic, mild to severe, acute to subacute necrotizing myopathy with mineralization. Acute necrosis and degeneration characterized by pyknotic nuclei, eosinophilic cytoplasm and cytoplasmic vacuolization were found in smooth muscle myocytes within the urinary bladder and digestive system. Degeneration of myocytes was present in the tunica media of a few small- to medium-sized vessels and was characterized by a vacuolar degeneration and occasionally necrosis. This condition has been termed multifocal necrotizing myopathy. A cause of this myopathy was not identified.

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., (Copyright © 2018. Published by Elsevier Inc.)

Emergent hypermucoviscous (HMV) strains of Klebsiella pneumoniae have been reported in multiple marine mammal species; however, there is limited information regarding the epidemiology and pathogenesis of this infection in these species. We determined the prevalence of HMV K. pneumoniae in wild-caught and stranded marine mammal populations on the US Pacific Coast. Samples were collected from 270 free-ranging California sea lions (CSLs; Zalophus californianus) captured at three discrete sampling sites and from 336 stranded marine mammals of various species. We recovered HMV K. pneumoniae only from CSLs, with a prevalence of 1.5% (4 of 275) in stranded animals, compared with 1.1% (3 of 270) in wild-caught animals. We assessed the phenotypic and genotypic variability of recovered HMV K. pneumoniae isolates recovered from CSLs ( n=11) and of archival HMV and non-HMV isolates from stranded marine mammals ( n=19). All but two HMV isolates were of the K2 serotype, whereas none of the non-HMV isolates belonged to this serotype. Of the HMV isolates, 96% (24 of 25) were PCR positive for the HMV-associated gene p- rmpA, whereas 92% (23 of 25) were PCR positive for p- rmpA2. Genetic fingerprinting by repetitive extragenic palindromic PCR showed four discrete clusters, demonstrating genotypic variability that loosely correlated with phenotype. Antimicrobial susceptibility testing revealed all isolates from stranded CSLs were susceptible to ceftiofur, indicating this antimicrobial agent is an appropriate choice for treatment of HMV K. pneumoniae infections in stranded CSLs. Our culture assay could reliably detect HMV K. pneumoniae from concentrations as low as 10 2 colony-forming units per milligram of feces. We identified the presence of HMV K. pneumoniae in both wild-caught and stranded CSLs from the US Pacific Coast and highlight the need for further studies to evaluate the potential impact of this pathogen on marine mammal health.

The population of California sea lion Zalophus californianus (CSL) has steadily increased during the last several decades. Despite extensive research addressing CSL biology and ecology performed during the last decades, there has been a minimal number of published papers documenting their parasite fauna. Our objective was to analyze the actual list of the metazoan parasites reported from CSLs and add new data on the age-related differences in the prevalence and biodiversity of the parasite community. There have been 33 species recorded but this study considers only 24 of them valid. Among them, 11 species are specific parasites of CSLs and 13 species are not specific. Additional species represent accidental infections or misidentifications. In total, 6653 helminths and 847 mites were collected and identified from 34 CSLs for this study. Six species of nematodes, Anisakis simplex sensu lato s . l . (prevalence 41%; intensity 7.6), Contracaecum ogmorhini s . l . (38%; 269.6), Pseudoterranova decipiens s . l . (29%; 33), P. azarazi (9%; 2.7), Acanthocheilonema odendhali (15%; 3.5) and Parafilaroides decorus were found. Two species of cestodes, Diphyllobothrium sp. (38%; 8.5) and Anophryocephalus sp. (15%; 14.6) represent novel undescribed species. Two species of trematodes, Apophallus zalophi (18%; 19.7) and Zalophotrema hepaticum (12%; 39.2), and five species of acanthocephalans, Corynosoma obtuscens (68%; 100.8), C. strumosum (53%; 4.6), Andracantha phalacrocoracis (3%; 1), Andracantha sp. (9%; 4.3) and Profilicollis altmani (6%; 8.5) were found. Mites Orthohalarchne attenuata (prevalence 85%) were found in the nasal cavity, while O. diminuata (21%) parasitized in the trachea and bronchi. The highest levels of infection with nematodes and trematodes were found in adult CSLs (3-16 years old), whereas the highest level of infection with acanthocephalans was found in young CSLs (pups and yearlings).

A free-ranging, male, yearling Guadalupe fur seal ( Arctocephalus philippii townsendi) died due to multifocal verminous vasculitis with thrombosis and several embolic infarcts in liver, kidney, and brain. Nematodes extracted from lung blood vessels were identified as Parafilaroides decorus, a parasite normally found in alveoli of California sea lions ( Zalophus californianus).

Urogenital carcinoma is common in wild California sea lions ( Zalophus californianus) along the west coast of the US. From 1979 to 1994, this cancer was observed in 18% (66/370) of necropsied subadult and adult sea lions at The Marine Mammal Center in Sausalito, California. A retrospective review of records from 1 January 2005 to 31 December 2015 was performed to characterize prevalence and characteristics of cancer over this decade. Fourteen percent (263/1917) of necropsied sea lions had cancer, of which 90% (237/263) were urogenital carcinoma. The prevalence of urogenital carcinoma was significantly higher in adults compared to juveniles and subadults. Advanced-stage disease with metastases was identified histologically in 78% (182/232) of cases and was the cause of death in 95% (172/182) of these cases. Metastases were most common in lung and lymph nodes, and hydronephrosis, secondary to ureter obstruction by metastases, was identified in 62% (114/185) of animals with advanced disease. No significant temporal change in prevalence was detected over the decade, and this highly aggressive, fatal cancer remains common in stranded California sea lions.

To date, most studies on pinniped immunoglobulins have focused on circulating antibodies. However, systemic and local immune activities differ in terms of maturation, intensity, and types of effectors that participate. Here, we examined levels of three immunoglobulin isotypes, IgG, IgM and IgA, in the blood and mucosal membranes of free-living California sea lion pups. We investigated whether age, body condition and sex influenced their concentration. Isotype levels varied among tissues, with age-related patterns that could be indicative of differential regulation along development. Serum IgG and IgA increased linearly with age, reaching adult levels at five months of age, while IgM remained unchanged. Mucosal isotypes tended to be low in newborns and remained so until five months of age. Regardless of age, pups with better condition tended to have higher anal IgG levels and higher genital IgA levels, suggesting that their synthesis and transport to the mucosal membranes is costly. Intersex differences were only observed in the genital mucosa, where all isotypes differed between male and female pups, regardless of age, presumably due to histological and anatomical differences., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

We developed a stochastic susceptible-exposed-infectious-removed (SEIR) model to simulate a range of plausible morbillivirus outbreak scenarios in a randomly mixing population of 170 endangered Hawaiian monk seals (Neomonachus schauinslandi). We then modeled realistic vaccination and quarantine measures to determine the potential efficacy of such mitigation efforts. Morbillivirus outbreaks represent substantial risk to monk seals-91% of simulated baseline outbreaks grew (R 0 >1), and in one-third of the scenarios all, or nearly all, individuals were infected. Simulated vaccination efforts in response to an outbreak were not effective in substantially reducing infections, largely because of the prolonged interval between vaccination and immunity. Prophylactic vaccination, in contrast, could be an effective tool for preventing outbreaks. Herd immunity is practically achievable because of the small sizes of monk seal populations and the animals' accessibility on shore. Adding realistic spatial structure to the model, as informed by movement of seals tracked in the main Hawaiian Islands with the use of telemetry, greatly reduced the simulated impact of outbreaks (≤10 seals were infected in 62% of spatially structured simulations). Although response vaccination remained relatively ineffective, spatial segregation allowed herd immunity to be achieved through prophylactic vaccination with less effort. In a randomly mixing population of 170 seals, 86% would need to be vaccinated to achieve herd immunity in 95% of simulated outbreaks, compared to only approximately 60% in three spatially segregated subgroups with the same combined abundance. Simulations indicate that quarantining a modest number (up to 20) of ill seals has the potential to extinguish even fast-growing outbreaks rapidly. The efficacy of quarantine, however, is highly dependent upon rapid detection and response. We conclude that prophylactic vaccination combined with a quarantine program supported by vigilant surveillance and rapid, reliable diagnosis could greatly mitigate the threat of a morbillivirus outbreak in Hawaiian monk seals.

We used surveillance data collected in California before, concurrent with, and subsequent to an outbreak of highly pathogenic (HP) clade 2.3.4.4 influenza A viruses (IAVs) in 2014-2015 to (i) evaluate IAV prevalence in waterfowl, (ii) assess the evidence for spill-over infections in marine mammals and (iii) genetically characterize low-pathogenic (LP) and HP IAVs to refine inference on the spatiotemporal extent of HP genome constellations and to evaluate possible evolutionary pathways. We screened samples from 1496 waterfowl and 1142 marine mammals collected from April 2014 to August 2015 and detected IAV RNA in 159 samples collected from birds (n=157) and pinnipeds (n=2). HP IAV RNA was identified in three samples originating from American wigeon (Anas americana). Genetic sequence data were generated for a clade 2.3.4.4 HP IAV-positive diagnostic sample and 57 LP IAV isolates. Phylogenetic analyses revealed that the HP IAV was a reassortant H5N8 virus with gene segments closely related to LP IAVs detected in mallards (Anas platyrhynchos) sampled in California and other IAVs detected in wild birds sampled within the Pacific Americas Flyway. In addition, our analysis provided support for common ancestry between LP IAVs recovered from waterfowl sampled in California and gene segments of reassortant HP H5N1 IAVs detected in British Columbia, Canada and Washington, USA. Our investigation provides evidence that waterfowl are likely to have played a role in the evolution of reassortant HP IAVs in the Pacific Americas Flyway during 2014-2015, whereas we did not find support for spill-over infections in potential pinniped hosts.

Tissues of stranded California sea lions ( Zalophus californianus) naturally infected with a hyperviruluent strain of Klebsiella pneumoniae were examined by histopathology and immunohistochemistry against the K. pneumoniae K2 capsular antigen. In 7 of 8 animals, there was severe purulent bronchopneumonia, sometimes complicated by fibrinonecrotizing pleuritis with pyothorax. In affected areas of lung, large numbers of degenerate neutrophils and macrophages were admixed with rare large extracellular and intracellular gram-negative bacilli surrounded by a prominent capsule. Through serotyping, polymerase chain reaction, sequencing, and immunohistochemistry, these bacteria were confirmed to be a K2 serotype of K. pneumoniae. The same bacteria were identified through double immunolabeling within macrophages in blood vessels, lymph nodes, spleen, and liver. Intact K. pneumoniae were identified on epithelial surfaces of the nasopharyngeal, tracheal, and small intestine mucosae and within distal renal tubules. Our findings indicate that hypervirulent K. pneumoniae causes severe respiratory disease and intrahistiocytic bacteremia in California sea lions.

The past decades have been characterized by a growing number of climatic anomalies. As these anomalies tend to occur suddenly and unexpectedly, it is often difficult to procure empirical evidence of their effects on natural populations. We analysed how the recent sea surface temperature (SST) anomaly in the northeastern Pacific Ocean affects body condition, nutritional status, and immune competence of California sea lion pups. We found that pup body condition and blood glucose levels of the pups were lower during high SST events, although other biomarkers of malnutrition remained unchanged, suggesting that pups were experiencing early stages of starvation. Glucose-dependent immune responses were affected by the SST anomaly; specifically, pups born during high SST events had lower serum concentrations of IgG and IgA, and were unable to respond to an immune challenge. This means that not only were pups that were born during the SST anomaly less able to synthesize protective antibodies; they were also limited in their ability to respond rapidly to nonspecific immune challenges. Our study provides empirical evidence that atypical climatic conditions can limit energetic reserves and compromise physiological responses that are essential for the survival of a marine top predator.

Urogenital carcinoma is a highly metastatic cancer affecting California sea lions ( Zalophus californianus ). The disease has high prevalence amongst stranded animals, and is one of the most commonly observed cancers in wildlife. The genital localisation of primary tumours suggests the possibility that coital transmission of an infectious agent could underlie this disease. Otarine herpesvirus type 1 has been associated with lesions, however a causative role for this virus has not been confirmed. We investigated the possibility that urogenital carcinoma might be clonally transmissible, spread by the direct transfer of cancer cells. Analysis of sequences at the mitochondrial DNA control region in seven matched tumour and host pairs confirmed that tumour genotypes were identical to those of their matched hosts and did not show similarity with tumours from other individuals. Thus our findings suggest that urogenital carcinoma in California sea lions is not clonally transmitted, but rather arises from transformed host cells., Competing Interests: Competing interests: No competing interests were disclosed.

We characterize Brucella infection in a wild southern sea otter ( Enhydra lutris nereis) with osteolytic lesions similar to those reported in other marine mammals and humans. This otter stranded twice along the central California coast, US over a 1-yr period and was handled extensively at two wildlife rehabilitation facilities, undergoing multiple surgeries and months of postsurgical care. Ultimately the otter was euthanized due to severe, progressive neurologic disease. Necropsy and postmortem radiographs revealed chronic, severe osteoarthritis spanning the proximal interphalangeal joint of the left hind fifth digit. Numerous coccobacilli within the joint were strongly positive on Brucella immunohistochemical labelling, and Brucella sp. was isolated in pure culture from this lesion. Sparse Brucella-immunopositive bacteria were also observed in the cytoplasm of a pulmonary vascular monocyte, and multifocal granulomas were observed in the spinal cord and liver on histopathology. Findings from biochemical characterization, 16S ribosomal DNA, and bp26 gene sequencing of the bacterial isolate were identical to those from marine-origin brucellae isolated from cetaceans and phocids. Although omp2a gene sequencing revealed 100% homology with marine Brucella spp. infecting pinnipeds, whales, and humans, omp2b gene sequences were identical only to pinniped-origin isolates. Multilocus sequence typing classified the sea otter isolate as ST26, a sequence type previously associated only with cetaceans. Our data suggest that the sea otter Brucella strain represents a novel marine lineage that is distinct from both Brucella pinnipedialis and Brucella ceti. Prior reports document the zoonotic potential of the marine brucellae. Isolation of Brucella sp. from a stranded sea otter highlights the importance of wearing personal protective equipment when handling sea otters and other marine mammals as part of wildlife conservation and rehabilitation efforts.

California sea lion adenovirus 1 (CSLAdV-1) has been associated with hepatitis and enteritis in several wild and captive populations of diverse pinniped species. Currently available tests have been limited to pan-adenoviral polymerase chain reaction (PCR) followed by sequencing. We present the development of a quantitative probe-hybridization PCR (qPCR) assay for rapid, sensitive, and specific detection of this virus in California sea lions ( Zalophus californianus) and other pinnipeds. This assay did not amplify other mammalian adenoviruses and is able to detect consistently down to 10 viral copies per well. Compared with the gold standard conventional pan-adenovirus PCR/sequencing assay, diagnostic sensitivity and specificity of 100% and 88.2% were found, respectively. The lower diagnostic specificity of this qPCR assay may be the result of the lower limit of detection of this assay compared with the gold standard rather than the result of detection of true false-positives.

A coastwide bloom of the toxigenic diatom Pseudo-nitzschia in spring 2015 resulted in the largest recorded outbreak of the neurotoxin, domoic acid, along the North American west coast. Elevated toxins were measured in numerous stranded marine mammals and resulted in geographically extensive and prolonged closures of razor clam, rock crab, and Dungeness crab fisheries. We demonstrate that this outbreak was initiated by anomalously warm ocean conditions. Pseudo-nitzschia australis thrived north of its typical range in the warm, nutrient-poor water that spanned the northeast Pacific in early 2015. The seasonal transition to upwelling provided the nutrients necessary for a large-scale bloom; a series of spring storms delivered the bloom to the coast. Laboratory and field experiments confirming maximum growth rates with elevated temperatures and enhanced toxin production with nutrient enrichment, together with a retrospective analysis of toxic events, demonstrate the potential for similarly devastating ecological and economic disruptions in the future.

The establishment of clinical pathology baseline data is critical to evaluate temporal and spatial changes in marine mammal groups. Despite increased availability of studies on hematology and biochemistry of marine mammals, reference ranges are lacking for many populations, especially among fur seal species. During the austral summers of 2014 and 2015, we evaluated basic hematologic and biochemical parameters in clinically healthy, physically restrained South American fur seal ( Arctocephalus australis ) lactating females and 2-mo-old pups. We also assessed the temporal variation of hematology parameters on the pups during their first 2 mo of life. Reference ranges of lactating females were similar to those previously reported in other fur seal species. In the case of pups, reference ranges are similar to values previously reported in sea lion species. As expected, most biochemical and hematologic values differ significantly between adult females and pups. As in other otariids, South American fur seals pups are born with higher values of total red blood cells, hemoglobin, and packed cell volume, and lower numbers of total leukocytes, neutrophils, lymphocytes, and eosinophils. To the best of our knowledge, data on hematology reference values for South American fur seals has not been previously reported and is useful for continued health monitoring of this species, as well as for comparisons with other otariid groups.

Free-ranging California sea lions ( Zalophus californianus ) with marine debris entanglements were darted with a combination of medetomidine, butorphanol, and midazolam by using acoustic transmitter-equipped darts. Of the 15 animals sedated, 13 (87%) reentered the water and were tracked by using a unidirectional hydrophone. Sea lions that entered the water continued to surface and breathe postsedation. There were three mortalities (20%) during the course of this study due to the following: suspected drowning caused by entrapment under a dock, overdose due to inaccurate weight estimation, and trauma caused by a dart puncturing the animal's abdomen. The drug combination, new dart design, and tracking techniques allowed for successful remote sedation and capture of California sea lions in high-risk situations and improved our ability to determine the final outcome for all cases. These methods allow targeting and capture of individual animals, while minimizing disturbance to other animals.

The abundance of California sea lions (Zalophus californianus) (CSLs) and Guadalupe fur seals (Arctocephalus philippii townsendi) (GFSs) from the San Benito Archipelago (SBA) was determined through nine monthly surveys in 2014-2015. Assessment of their foraging habits was examined based on the isotopic analysis of pups (maternal indicators) (SIAR/SIBER-R). Environmental variability between 2014 and 2015 was also analyzed, in terms of sea surface temperature (SST) and chlorophyll (Chl-a) concentration. Both otariids reached their highest abundance in July of both years; however, relative to 2014, the 2015 survey showed a 59.7% decline in the total GFS abundance and a 42.9% decrease of GFS pups, while total CSL abundance decreased 52.0% and CSL pup presence decreased in 61.7%. All monthly surveys for both otariids showed a similar trend (>50% decrease in 2015). Compared to 2014, the 2015 GFSs isotopic niche was three times larger (2.0 in 2015, 0.6 in 2014) and the δ13C was significantly lower. CSLs also showed significantly lower δ13C and higher δ15N in 2015. Interannual segregation was greater for CSLs, and their pup body mass was also significantly lower during the 2015 breeding season (mean = 8.7 kg) than in the same season of 2014 (mean = 9.9 kg). The decrease in δ13C for both otariids reflected a more oceanic foraging; most likely associated with the decline in primary productivity in surrounding areas to the SBA, related to a higher SST caused by the 2015 ENSO, with a subsequent increase in foraging effort. These would explain the fewer observed individuals on land, especially pups, which showed diminished body condition (CSLs). This study highlights the importance of marine mammals as sentinel species that respond dynamically to changes in environment, providing valuable information on the effect of ENSO on pinnipeds in Mexican waters.

An unusually high prevalence of metastatic urogenital carcinoma has been observed in free-ranging California sea lions stranded off the coast of California in the past two decades. No cases have been reported for sea lions in the relatively unpolluted Gulf of California. We investigated occurrence of genital epithelial transformation in 60 sea lions (n=57 pups and 3 adult females) from the Gulf of California and examined whether infection by a viral pathogen previously found to be associated with urogenital carcinoma accounted for such alterations. We also explored the contribution of MHC class II gene expression on transformation. Cellular alterations, such as squamous cell atypia (ASC), atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesions were observed in 42% of the pups and in 67% of the adult females. Normal genital epithelium was more common in male than female pups. ASC was five times more likely to occur in older pups. Epithelial alterations were unrelated to infection by the potentially oncogenic otarine type I gammaherpesvirus (OtHV-1), but ASCUS was more common in pups with marked and severe inflammation. Expression of MHC class II DRB loci (Zaca DRB-D) by peripheral antigen-presenting leucocytes showed a slightly 'protective' effect for ASC. We propose that transformation of the California sea lion genital epithelium is relatively common in young animals, increases with age and is probably the result of infection by an unidentified pathogen. Expression of a specific MHC class II gene, suggestive of presentation of specific antigenic peptides to immune effectors, appears to lower the risk of transformation. Our study provides the first evidence that epithelial transformation of the California sea lion genital tract is relatively common, even from an early age, and raises questions regarding differences in sea lion cancer-detection and -repair success between geographical regions.

Infectious marine diseases can decimate populations and are increasing among some taxa due to global change and our increasing reliance on marine environments. Marine diseases become emergencies when significant ecological, economic or social impacts occur. We can prepare for and manage these emergencies through improved surveillance, and the development and iterative refinement of approaches to mitigate disease and its impacts. Improving surveillance requires fast, accurate diagnoses, forecasting disease risk and real-time monitoring of disease-promoting environmental conditions. Diversifying impact mitigation involves increasing host resilience to disease, reducing pathogen abundance and managing environmental factors that facilitate disease. Disease surveillance and mitigation can be adaptive if informed by research advances and catalysed by communication among observers, researchers and decision-makers using information-sharing platforms. Recent increases in the awareness of the threats posed by marine diseases may lead to policy frameworks that facilitate the responses and management that marine disease emergencies require., (© 2016 The Author(s).)

Otarine herpesvirus 1 (OtHV1) is strongly associated with California sea lion (CSL, Zalophus californianus) urogenital carcinoma, the most common cancer documented in marine mammals. In addition to CSL, OtHV1 has also been found in association with carcinoma in South American fur seals (Arctocephalus australis), demonstrating it can infect related species. Northern fur seals (NFS, Callorhinus ursinus) are sympatric with CSL, and copulation between these species has been observed; yet, there are no reports of urogenital carcinoma in NFS. We describe a new Otarine herpesvirus found in vaginal swabs from NFS, herein called OtHV4. Partial sequencing of the polymerase gene and the glycoprotein B gene revealed OtHV4 is closely related to OtHV1, with 95% homology in the region of polymerase sequenced, and phylogenetic analyses demonstrate that they are sister taxa. An OtHV4-specific hydrolysis probe quantitative PCR was developed and validated, and its use on vaginal swabs revealed 16 of 50 (32%) wild adult female NFS were positive for OtHV4. The identification of a virus highly similar to the carcinoma-associated OtHV1 in a sympatric species without carcinoma suggests that comparative genomics of OtHV1 and OtHV4 may identify candidate viral oncogenes.

Objective: To review cases of bites and contact abrasions in open-water swimmers from California sea lions (Zalophus californianus) and harbor seals (Phoca vitulina richardii)., Methods: Open-water swimmers from a San Francisco swimming club were questioned about encounters with pinnipeds (seals and sea lions) that resulted in bites or contact abrasions. When possible, wounds were documented with photographs. Medical follow-up and treatment complications were also reviewed., Results: From October 2011 to December 2014, 11 swimmers reported bites by a sea lion (n = 1), harbor seal (n = 7), or unidentified pinniped (n = 3). Ten of the encounters occurred in San Francisco Bay; 1 occurred in the Eld Inlet, in Puget Sound, near Olympia, WA. None of the swimmers were wearing wetsuits. All bites involved the lower extremities; skin was broken in 4 of 11 bites and antibiotics were prescribed in 3 cases. One swimmer, who was bitten by a harbor seal, also had claw scratches. A treatment failure occurred with amoxicillin/clavulanate in another swimmer who was bitten by an unidentified pinniped; the wound healed subsequently with doxycycline, suggesting an infection with Mycoplasma spp. There were no long-lasting consequences from any of the bites. The majority of cases occurred at low tide, and bumping of the swimmer by the animal before or after a bite was common, but no clear tide or attack pattern was identified., Conclusions: Bites and contact abrasions from sea lions and harbor seals are reported infrequently in open-water swimmers and typically involve the lower extremities. Because of the risk of Mycoplasma infection, treatment with a tetracycline is recommended in pinniped bites with signs of infection or serious trauma. Attempting to touch or pet sea lions or seals is inadvisable and prohibited by the Marine Mammal Protection Act. Swimmers should leave the water as soon as possible after a bite or encounter., (Copyright © 2015 Wilderness Medical Society. Published by Elsevier Inc. All rights reserved.)

Tissue-cyst forming coccidia in the family Sarcocystidae are etiologic agents of protozoal encephalitis in marine mammals including the federally listed Southern sea otter (Enhydra lutris). California sea lions (Zalophus californianus), whose coastal habitat overlaps with sea otters, are definitive hosts for coccidian protozoa provisionally named Coccidia A, B and C. While Coccidia A and B have unknown clinical effects on aquatic wildlife hosts, Coccidia C is associated with severe protozoal disease in harbor seals (Phoca vitulina). In this study, we conducted surveillance for protozoal infection and fecal shedding in hospitalized and free-ranging California sea lions on the Pacific Coast and examined oocyst morphology and phenotypic characteristics of isolates via mouse bioassay and cell culture. Coccidia A and B were shed in similar frequency, particularly by yearlings. Oocysts shed by one free-ranging sea lion sampled at Año Nuevo State Park in California were previously unidentified in sea lions and were most similar to coccidia infecting Guadalupe fur seals (Arctocephalus townsendi) diagnosed with protozoal disease in Oregon (USA). Sporulated Coccidia A and B oocysts did not replicate in three strains of mice or in African green monkey kidney cells. However, cultivation experiments revealed that the inoculum of fecally-derived Coccidia A and B oocysts additionally contained organisms with genetic and antigenic similarity to Sarcocystis neurona; despite the absence of detectable free sporocysts in fecal samples by microscopic examination. In addition to the further characterization of Coccidia A and B in free-ranging and hospitalized sea lions, these results provide evidence of a new role for sea lions as putative mechanical vectors of S. neurona, or S. neurona-like species. Future work is needed to clarify the distribution, taxonomical status, and pathogenesis of these parasites in sea lions and other marine mammals that share their the near-shore marine environment.

Marine mammals are often cited as "sentinels of ocean health" yet accessible, synthesized data on their health changes that could effectively warn of ocean health changes are rare. The objectives of this study were to 1) perform a systematic review of published cases of marine mammal disease to determine spatial and temporal trends in disease from 1972-2012, including changes in regions and taxa affected and specific causes; and 2) compare numbers of published cases of neoplasia with known, hospital-based neoplasia records to explore the causes of discrepancy between numbers of published cases and true disease trends. Peer-reviewed literature was compiled, and data were collected from The Marine Mammal Center database in Sausalito, California for comparison of numbers of neoplasia cases. Toxicoses from harmful algal blooms appear to be increasing. Viral epidemics are most common along the Atlantic U.S. coastline, while bacterial epidemics, especially leptospirosis, are most common along the Pacific coast. Certain protozoal and fungal zoonoses appear to be emerging, such as Toxoplasma gondii in southern sea otters in California, and Cryptococcus gattii in cetaceans in the Pacific Northwest. Disease reports were most common from California where pinniped populations are large, but increased effort also occurs. Anthropogenic trauma remains a large threat to marine mammal health, through direct mortality and indirect chronic disease. Neoplasia cases were under-reported from 2003-2012 when compared to true number of cases, and over-reported in several years due to case duplication. Peer-reviewed literature greatly underestimates the true magnitude of disease in marine mammals as it focuses on novel findings, fails to reflect etiology of multifactorial diseases, rarely reports prevalence rather than simple numbers of cases, and is typically presented years after a disease first occurs. Thus literature cannot guide management actions adequately, nor inform indices of ocean health. A real-time, nationally centralized system for reporting marine mammal disease data is needed to be able to understand how marine mammal diseases are changing with ecosystem changes, and before these animals can truly be considered 'sentinels of ocean health'.