Air leak syndrome in animals: definition and pathogenesis.

Air leak syndrome (ALS) is described in human medicine as a constellation of clinical disorders including pneumomediastinum, pneumopericardium, pulmonary interstitial emphysema, pneumothorax, pneumoperitoneum, pneumoretroperitoneum and subcutaneous emphysema. The pathogenesis of ALS depends on the anatomy of the mediastinum and its associations with thoracic, abdominal and cervical connective tissues, as well as a physical phenomenon referred to as the Macklin effect. Various animal species develop diverse combinations of these lesions, although ALS has not been recognized in animals. However, this term aids pathologists in addressing this disease compilation. The aim of this retrospective study is to illustrate examples of ALS in animals by arbitrarily selecting 13 cases in dogs, cats, pinnipeds, sea otters and harbour porpoises. ALS can be classified into three groups based on aetiology: iatrogenic, secondary or spontaneous. Iatrogenic ALS was diagnosed in two cats with tracheal laceration following endotracheal intubation. Secondary ALS was identified in two dogs, one with acute respiratory distress syndrome and the other due to grass awn migration. Secondary ALS in pinnipeds was diagnosed following severe pulmonary parasitism, uraemic pneumonia and oesophageal perforation. The other marine mammals developed ALS following trauma. Spontaneous ALS was also diagnosed in one cat and one dog without any apparent predisposing causes.

Gray whale (Eschrichtius robustus) post-mortem findings from December 2018 through 2021 during the Unusual Mortality Event in the Eastern North Pacific.

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.

California sea lion (Zalophus californianus) lymph-node explant reveals involvement and possible transcriptional regulation of SLAM and nectin-4 during phocine distemper virus infection.

Phocine distemper virus (PDV) is a significant cause of mortality for phocid seals; however, the susceptibility of otariids to this virus is poorly understood. The authors used a lymph-node explant culture system from California sea lions (Zalophus californianus, CSL) to investigate: (1) the role of signaling lymphocyte activation molecule (SLAM) and nectin-4 in PDV infection and their cellular expression patterns, (2) if PDV induces transcriptional regulation of cell-entry receptors, and (3) the involvement of apoptosis in PDV infection. PDV replicated in the lymph-node explants with peak replication 3 days post-infection (dpi), but the replication was not sustained 4 to 5 dpi. The PDV+ cells co-localized SLAM and nectin-4. These cells expressed IBA1, indicating a histiocytic lineage. Comparison of receptor expression between infected and mock-infected lymph nodes suggested transcriptional downregulation of both receptors during the initial stage of infection and upregulation during the late stage of infection, but the values lack of statistical significance. Cleaved caspase-3+ cells were slightly increased in the infected lymph nodes compared with the mock-infected lymph node from 1 to 4 dpi, but without statistical significance, and a few apoptotic cells co-expressed PDV. The results suggest that lymph-node explants might be an important model to study PDV pathogenesis. CSLs have the potential to be infected with PDV, as they express both cell-entry receptors in histiocytes. The lack of statistical significance in the PDV replication, transcriptional regulation of viral receptors, and changes in apoptosis suggest that although CSL might be infected by PDV, they might be less susceptible than phocid species.

Population structure of pygmy (Kogia breviceps) and dwarf (Kogia sima) sperm whales in the Southern Hemisphere may reflect foraging ecology and dispersal patterns.

Little is known about the biology of pygmy (Kogia breviceps) and dwarf (K. sima) sperm whales as these animals are difficult to observe in the wild. However, both species strand frequently along the South African, Australian and New Zealand coastlines, providing samples for these otherwise inaccessible species. The use of DNA samples from tissue and DNA extracted from historical material, such as teeth and bone, allowed a first analysis of the population structure of both species in the Southern Hemisphere. A 279 base pair consensus region of the mitochondrial cytochrome b gene was sequenced for 96 K. breviceps (53 tissue and 43 teeth or bone samples) and 29 K. sima (3 tissue and 26 teeth or bone samples), and 26 and 12 unique haplotypes were identified, respectively. K. breviceps showed a higher nucleotide diversity of 0.82% compared to 0.40% in K. sima. Significant genetic differentiation was detected in the Southern Hemisphere between K. breviceps from South Africa and New Zealand (ФST = 0.042, p < 0.05). Mitochondrial control region sequences (505 bp) were available for 44 individuals (41 K. breviceps and 3 K. sima) for comparative purposes. A comprehensive global phylogenetic analysis (maternal lineage) of our sequences together with all available Kogia mtDNA sequences largely supported previously published phylogenetic findings, but highlighted some changed inferences about oceanic divergences within both species. The higher nucleotide diversity and low population differentiation observed in K. breviceps may result from its broad foraging ecology and wide distribution, which may indicate a more opportunistic feeding behaviour and tolerance towards a larger range of water temperatures than K. sima.

The symptomatology and diagnosis of domoic acid toxicosis in stranded California sea lions (Zalophus californianus): a review and evaluation of 20 years of cases to guide prognosis.

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.

Historical isolates of dolphin morbillivirus: origin and new data.

Dolphin morbillivirus (DMV) was isolated in striped dolphins Stenella coeruleoalba from the Mediterranean Sea stranded along the coast of Spain during a lethal epidemic that killed thousands of individuals in 1990-1992. Though some of these isolates (MUC, 16A and the reference strain) have been extensively characterised, details on their origin were not reported in the literature, and records for these isolates are often difficult to trace and are, sometimes, erroneous. Here, we provide unpublished biological and histopathological data for these isolates, summarize the literature on their characterization and make suggestions for future studies.

Human influenza A virus H1N1 in marine mammals in California, 2019.

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.

DEVELOPMENT AND VALIDATION OF A NOVEL DUPLEX PROBE-HYBRIDIZATION QUANTITATIVE PCR FOR LYMPHOMA-ASSOCIATED MIROUNGINE GAMMAHERPESVIRUS 3 IN NORTHERN ELEPHANT SEALS (MIROUNGA ANGUSTIROSTRIS).

Recently, a novel gammaherpesvirus, miroungine gammaherpesvirus 3 (MirGHV3), was described in two juvenile elephant seals (Mirounga angustirostris) with diffuse large B-cell lymphoma. We developed and validated a quantitative (q)PCR for rapid detection of MirGHV3 and investigated its potential association with lymphoma. We developed a duplex probe-hybridization qPCR with MirGHV3 DNA polymerase (pol) as the target gene. Each primer-probe combination was cross-validated against the others. Interference was not seen when they were run in the same well as a duplex assay. Twenty-three samples from seven northern elephant seals were tested using the duplex assay. Viral DNA was detected by the assay in 9 of 9 (100%) tissues affected by lymphoma and in 6 of 14 (43%) samples from tissues unaffected by lymphoma. There was a strong correlation between viral copies detected with each of the assays (P=0.0002). Viral load was significantly higher in tissues affected by lymphoma than in those unaffected (P<0.0001). Excluding the virus-negative samples, viral load was still significantly higher in tissues affected by lymphoma than in those unaffected (P=0.0004). This is consistent with a potential role of MirGHV3 in oncogenesis in northern elephant seals, although more studies are needed to determine this definitively. The qPCR developed has utility for further investigations of MirGHV3.

Tattoo Skin Disease in Cetacea: A Review, with New Cases for the Northeast Pacific.

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 (Acari: Halarachnidae) as potential vectors of bacterial pathogens, including Streptococcus phocae, in marine mammals.

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.

Diffuse large B cell lymphoma and a novel gammaherpesvirus in northern elephant seals Mirounga angustirostris.

Two emaciated male northern elephant seal (NES) Mirounga angustirostris pups were admitted to The Marine Mammal Center (Sausalito, California, USA) and treated for malnutrition. Complete blood counts showed a progressive moderate to marked leukocytosis characterized by a predominance of large monomorphic mononuclear cells of probable lymphoid origin, frequently with flower-shaped nuclei. Both seals were euthanized due to suspected lymphoid neoplasia. At necropsy, most lymph nodes in both pups were markedly enlarged, some with distinct white nodules, the spleens were diffusely enlarged, and the intestinal mucosae were thickened. Histopathologic features consistent with disseminated large cell lymphoma were identified to varying degrees of severity in lymph nodes, bone marrow, liver, tonsils, spleen, liver, intestines, kidneys, lower urinary tract, and several other organs. Immunohistochemical staining of neoplastic cells was most consistent with B lymphocyte origin, with most cells staining positively for Pax 5 and CD20 with admixed small CD3-positive T lymphocytes and CD204-positive macrophages. PCR and sequencing identified a novel gammaherpesvirus, herein called miroungine gammaherpesvirus 3, from affected tissues. This virus is in a clade outside of named genera that utilize hosts in the suborder Caniformia. The present study is the first description of diffuse large B cell lymphoma with leukemic manifestation and concomitant detection of a novel gammaherpesvirus in free-living NESs. Further research regarding the prevalence of this new gammaherpesvirus and its associated pathogenesis in this species is indicated.

Contagious Ecthyma Dermatitis as a Portal of Entry for Erysipelothrix rhusiopathiae in Muskoxen (Ovibos moschatus) of the Canadian Arctic.

Erysipelothrix rhusiopathiae was detected immunohistochemically in contagious ecthyma (orf virus) dermatitis in two muskoxen (Ovibos moschatus), harvested and found dead in 2014 and 2015, respectively, on Victoria Island, Canada. This may help target further research on E. rhusiopathiae epidemiology and mechanisms of infection in muskoxen, recently associated with widespread mortalities in Canada's Arctic.

Genetics and pathology associated with Klebsiella pneumoniae and Klebsiella spp. isolates from North American Pacific coastal marine mammals.

Southern sea otters (SSO: Enhydra lutris nereis) are a federally-listed threatened subspecies found almost exclusively in California, USA. Despite their zoonotic potential and lack of host specificity, K. pneumoniae and Klebsiella spp. have largely unknown epizootiology in SSOs. Klebsiella pneumoniae is occasionally isolated at necropsy, but not from live SSOs. Hypermucoviscous (HMV) K. pneumoniae strains are confirmed pathogens of Pacific Basin pinnipeds, but have not been previously isolated from SSOs. We characterized the virulence profiles of K. pneumoniae isolates from necropsied SSOs, evaluated killing of marine mammal K. pneumoniae following in vitro exposure to California sea lion (CSL: Zalophanus californianus) whole blood and serum, and characterized lesion patterns associated with Klebsiella spp. infection in SSOs. Four of 15 SSO K. pneumoniae isolates were HMV and all were recovered from SSOs that stranded during 2005. Many K. pneumoniae infections were associated with moderate to severe pathology as a cause of death or sequela. All HMV infections were assessed as a primary cause of death or as a direct result of the primary cause of death. Klebsiella-infected SSOs exhibited bronchopneumonia, tracheobronchitis and/or pleuritis, enteritis, Profilicollis sp. acanthocephalan peritonitis, septic peritonitis, and septicemia. All SSO HMV isolates were capsular type K2, the serotype most associated with HMV infections in CSLs. Multiplex PCR revealed two distinct virulence gene profiles within HMV isolates and two within non-HMV isolates. In vitro experiments investigating CSL whole blood and serum killing of K. pneumoniae suggest that HMV isolates are more resistant to serum killing than non-HMV isolates.

Clinical signs, treatment, and outcome for California sea lions (Zalophus californianus) with Sarcocystis-associated polyphasic rhabdomyositis.

OBJECTIVE: To describe clinical signs, treatment, and outcome for California sea lions (Zalophus californianus) with Sarcocystis-associated polyphasic rhabdomyositis. ANIMALS: 38 free-ranging juvenile to adult California sea lions examined at a rehabilitation center in California between September 2015 and December 2017. PROCEDURES: Medical records at The Marine Mammal Center were reviewed to identify sea lions in which sarcocystosis had been diagnosed. RESULTS: Clinical signs were highly variable and associated with polyphasic rhabdomyositis attributed to Sarcocystis neurona infection. Generalized severe muscle wasting, respiratory compromise, and regurgitation secondary to megaesophagus were the most profound clinical findings. Respiratory compromise and megaesophagus were associated with a poor prognosis. Eight of the 38 sea lions were treated and released to the wild, and 2 subsequently restranded and were euthanized. Two additional animals received no targeted treatment and were released. The remaining 28 animals were either euthanized or died during treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that unlike other marine mammals, which typically develop encephalitis, California sea lions with sarcocystosis often have polyphasic rhabdomyositis with highly variable clinical signs and that extensive diagnostic testing may be required to confirm the diagnosis. Treatment with an antiprotozoal drug in combination with corticosteroids may resolve clinical disease, but the prognosis is guarded.

Nasopulmonary mites (Halarachnidae) of coastal Californian pinnipeds: Identity, prevalence, and molecular characterization.

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.

Exploration of serum cardiac troponin I as a biomarker of cardiomyopathy in southern sea otters (Enhydra lutris nereis).

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.

A Comparison of Parapoxviruses in North American Pinnipeds.

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.

Respiratory Tract Explant Infection Dynamics of Influenza A Virus in California Sea Lions, Northern Elephant Seals, and Rhesus Macaques.

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 log10 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.

CLASSIFICATION AND REGRESSION TREE ANALYSIS FOR PREDICTING PROGNOSIS IN WILDLIFE REHABILITATION: A CASE STUDY OF LEPTOSPIROSIS IN CALIFORNIA SEA LIONS (ZALOPHUS CALIFORNIANUS).

The spirochete bacterium Leptospira interrogans serovar Pomona is enzootic to California sea lions (CSL; Zalophus californianus) and causes periodic epizootics. Leptospirosis in CSL is associated with a high fatality rate in rehabilitation. Evidence-based tools for estimating prognosis and guiding early euthanasia of animals with a low probability of survival are critical to reducing the severity and duration of animal suffering. Classification and regression tree (CART) analysis of clinical data was used to predict survival outcomes of CSL with leptospirosis in rehabilitation. Classification tree outputs are binary decision trees that can be readily interpreted and applied by a clinician. Models were trained using data from cases treated from 2017 to 2018 at The Marine Mammal Center in Sausalito, CA, and tested against data from cases treated from 2010 to 2012. Two separate classification tree analyses were performed, one including and one excluding data from euthanized animals. When data from natural deaths and euthanasias were included in model-building, the best classification tree predicted outcomes correctly for 84.7% of cases based on four variables: appetite over the first 3 days in care, and blood urea nitrogen (BUN), creatinine, and sodium at admission. When only natural deaths were included, the best model predicted outcomes correctly for 87.6% of cases based on BUN and creatinine at admission. This study illustrates that CART analysis can be successfully applied to wildlife in rehabilitation to establish evidence-based euthanasia criteria with the goal of minimizing animal suffering. In the context of a large epizootic that challenges the limits of a facility's capacity for care, the models can assist in maximizing allocation of resources to those animals with the highest predicted probability of survival. This technique may be a useful tool for other diseases seen in wildlife rehabilitation.

Unlocking the Role of a Genital Herpesvirus, Otarine Herpesvirus 1, in California Sea Lion Cervical Cancer.

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.