Identification of Candidate Protein Biomarkers Associated with Domoic Acid Toxicosis in Cerebrospinal Fluid of California Sea Lions (Zalophus californianus).

Since 1998, California sea lion (Zalophus californianus) stranding events associated with domoic acid toxicosis (DAT) have consistently increased. Outside of direct measurement of domoic acid in bodily fluids at the time of stranding, there are no practical nonlethal clinical tests for the diagnosis of DAT that can be utilized in a rehabilitation facility. Proteomics analysis was conducted to discover candidate protein markers of DAT using cerebrospinal fluid from stranded California sea lions with acute DAT (n = 8), chronic DAT (n = 19), or without DAT (n = 13). A total of 2005 protein families were identified experiment-wide. A total of 83 proteins were significantly different in abundance across the three groups (adj. p < 0.05). MDH1, PLD3, ADAM22, YWHAG, VGF, and CLSTN1 could discriminate California sea lions with or without DAT (AuROC > 0.75). IGKV2D-28, PTRPF, KNG1, F2, and SNCB were able to discriminate acute DAT from chronic DAT (AuROC > 0.75). Proteins involved in alpha synuclein deposition were over-represented as classifiers of DAT, and many of these proteins have been implicated in a variety of neurodegenerative diseases. These proteins should be considered potential markers for DAT in California sea lions and should be prioritized for future validation studies as biomarkers.

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.

RETROSPECTIVE REVIEW OF PROPOFOL ANESTHESIA IN MULTIPLE ELASMOBRANCH SPECIES AT GEORGIA AQUARIUM, 2010-2022.

General anesthesia is often required in elasmobranch species for medical procedures. A variety of anesthetic drugs have been administered to elasmobranchs with wide variability in efficacy and safety. A retrospective review of 47 anesthetic procedures using IV propofol in eight different elasmobranch species at the Georgia Aquarium from 2010 to 2022 was performed. Cases involving seven sand tiger sharks (Carcharias taurus), four largetooth sawfish (Pristis perotteti), one longcomb sawfish (Pristis zijsron), four blacktip reef sharks (Carcharhinus melanopterus), three silvertip sharks (Carcharhinus albimarginatus), one sandbar shark (Carcharhinus plumbeus), five cownose rays (Rhinoptera bonasus), and one blotched fantail stingray (Taeniura meyeni) were evaluated. Induction dose of IV propofol (median: 2.5; 25-75%: 2.3-3.0; range: 1.7-4.0 mg/kg), time to desired effect (median: 4.0; 25-75%: 2.0-5.0; range: 0.5-15.0 min) and anesthetic duration (median: 76.0; 25-75%: 61.5-119.0; range: 27-216 min) were reported in all species. In six procedures (12.7%), maintenance of desired anesthetic plane required a supplemental dose of propofol IV (1 mg/kg) or addition of tricaine methanesulfonate (70 mg/L) as an immersion bath. The most common side effects were apnea and prolonged recovery. The IV propofol was efficacious and provided a procedural plane of anesthesia for a clinically relevant period of time in the majority of elasmobranch species, but observation for and management of complications is warranted.

Xenotransplantation of porcine progenitor cells in an epileptic California sea lion (Zalophus californianus): illustrative case.

BACKGROUND: Domoic acid (DA) is a naturally occurring neurotoxin harmful to marine animals and humans. California sea lions exposed to DA in prey during algal blooms along the Pacific coast exhibit significant neurological symptoms, including epilepsy with hippocampal atrophy. OBSERVATIONS: Here the authors describe a xenotransplantation procedure to deliver interneuron progenitor cells into the damaged hippocampus of an epileptic sea lion with suspected DA toxicosis. The sea lion has had no evidence of seizures after the procedure, and clinical measures of well-being, including weight and feeding habits, have stabilized. LESSONS: These preliminary results suggest xenotransplantation has improved the quality of life for this animal and holds tremendous therapeutic promise.

Cannabinoid receptor 1-labeled boutons in the sclerotic dentate gyrus of epileptic sea lions.

Pathology in the dentate gyrus, including sclerosis, is a hallmark of temporal lobe epilepsy, and reduced inhibition to dentate granule cells may contribute to epileptogenesis. The perisomatic-targeting axonal boutons of parvalbumin-expressing interneurons decrease in proportion with granule cells in temporal lobe epilepsy. In contrast, dendrite-targeting axonal boutons of somatostatin-expressing interneurons sprout exuberantly in temporal lobe epilepsy. A third major class of GABAergic interneurons expresses cannabinoid receptor type 1 (CB1) on their terminal boutons, but there is conflicting evidence as to whether these boutons are increased or decreased in temporal lobe epilepsy. Naturally occurring temporal lobe epilepsy in California sea lions, with unilateral or bilateral sclerosis, offers the benefit of neuroanatomy and neuropathology akin to humans, but with the advantage that the entirety of both hippocampi from control and epileptic brains can be studied. Stereological quantification in the dentate gyrus revealed that sclerotic hippocampi from epileptic sea lions had fewer CB1-labeled boutons than controls. However, the reduction in the number of granule cells was greater, resulting in increased CB1-labeled boutons per granule cell in sclerotic hippocampi at temporal levels. This suggests that although CB1-expressing boutons are decreased in sclerotic dentate gyri, surviving cells have enhanced innervation from these boutons in epileptic sea lions.

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.

Fetal mercury concentrations in central California Pacific harbor seals: Associated drivers and outcomes.

Mercury (Hg) is a well-known toxicant in wildlife and humans. High total Hg concentrations ([THg]) have been reported in central California harbor seals Phoca vitulina richardii. We evaluated the effects of presence/absence of early natal coat (lanugo), year (2012 to 2017), sex, stranding location, and trophic ecology (ẟ13C and ẟ15N values) on hair [THg] along coastal central California. Also examined were [THg] effects on growth rates of pups in rehabilitation and probability of release (e.g., successful rehabilitation). The [THg] ranged from 0.46-81.98 mg kg-1 dw, and ẟ15N and ẟ13C ranged from 13.6-21.5‰, and -17.2 to -13.0‰, respectively. Stranding location, year, and presence of lanugo coat were important factors explaining variation in [THg]. Seals from Sonoma and San Mateo County had higher [THg] than other locations. Seals with full or partial lanugo coat had lower [THg]. Seals from 2016 and 2017 had higher [THg] than those from 2015. Hair [THg] exceeded lower and upper toxicological thresholds (>20 mg kg-1 by year (5.88% to 23.53%); >30 mg kg-1 (0% to 12.31%)) with a pronounced increase from 2015 to 2016. Pups in 2017 had significantly higher odds ratio of [THg] above 20 mg kg-1 than pups of 2015, and pups in 2016 had significantly higher odds ratio than those from 2013 and 2015 (similar when using 30 mg kg-1). Pups in Sonoma County had the highest odds ratio for [THg] in lanugo above 20 mg kg-1. ẟ15N values were higher in 2015-2017, particularly relative to 2014, probably associated with the El Niño event. The [THg] was not a good predictor for probability of release and mass-specific growth rates in captivity. Further investigation of temporal trends of [THg] in harbor seals is warranted given the relatively high percentage of samples exceeding threshold values, particularly in the most recent sampling years.

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.

PHARMACOKINETICS OF SUBCUTANEOUS ALPHA LIPOIC ACID, A PROPOSED THERAPEUTIC AID FOR DOMOIC ACID INTOXICATION IN CALIFORNIA SEA LIONS (ZALOPHUS CALIFORNIANUS).

Domoic acid (DA) is a potent neurotoxin produced by certain marine algae that can cause neurologic and cardiac dysfunction by activating glutamate receptors. Glutamate receptor overexcitation results in continuous cellular activation, oxidative damage, and cell death. DA toxicosis causes disorientation and seizures, and antiseizure medications are the primary treatment. Alpha lipoic acid (ALA), a powerful antioxidant and glutathione precursor widely used in humans and dogs, can cross the blood-brain barrier to provide antioxidant availability to brain tissue. Hundreds of stranded California sea lions (CSL; Zalophus californianus) are diagnosed annually with DA toxicosis and thus are an appropriate animal in which to establish ALA dosing recommendations for treatment. The objective of this study was to determine the population pharmacokinetics of a single 10- or 20-mg/kg dose of ALA administered subcutaneously into the interscapular region to healthy rehabilitated CSL. Blood was collected at two time points between 15 min and 24 h after administration. Serum ALA concentrations were measured by liquid chromatography-mass spectrometry, and parameters were evaluated using a nonlinear mixed effects model. ALA was rapidly absorbed for each dose, peaking within 20 to 30 minutes, and t1/2 of 40 and 32 min (10 and 20 mg/kg, respectively), followed by an initial steep distribution phase and prolonged elimination phase. Peak concentration (CMAX) was 1,243 ng/ml (10-mg/ml dose) and 5,010 ng/ml (20-mg/ml dose). Serum from 13 CSLd with DA toxicosis treated with 10 mg/kg ALA for 1 to 9 d had measurable levels, and ALA was also measurable in cerebrospinal fluid from two treated CSLs. Therapeutic effects are noted with a CMAX of 4,000 to 5,000 ng/ml in humans; thus in CSLs, 20 mg/kg administered subcutaneously once daily may be sufficient to achieve a therapeutic level in this species. Determination of efficacy and optimal dosing interval and duration require additional investigation.

OPTIMIZATION OF FECAL FLOTATIONS IN MARINE PARASITOLOGY AND DETERMINATION OF THE SPECIFIC GRAVITY OF HELMINTH EGGS IN PINNIPEDS.

Recent studies have sought to optimize the fecal flotation procedure to improve the detection of helminth eggs in terrestrial domestic species. It is unclear, however, whether these efforts in optimization are applicable to parasite species of marine environments, and verification of veterinary diagnostic procedures is clinically important. It was hypothesized that the eggs belonging to the parasites of pinnipeds would have different specific gravities (SpG) than those belonging to terrestrial hosts. Fecal samples were collected from each of 25 stranded pinnipeds representing three species (Zalophus californianus, Phoca vitulina, Mirounga angustirostris), and modified double centrifugal flotations were performed on 1-g samples. Among the 22 California sea lions sampled, trematode, ascarid, and cestode eggs were detected in 17/22 (77%), 10/22 (45%), and 4/22 (18%) individuals, respectively. Sugar-gradient modified centrifugation flotations were then conducted on a subset of 10 samples from California sea lions to evaluate the distribution of eggs in fractions representing varying SpG. Higher numbers of ascarid eggs were found in fractions representing a lower SpG (1.00-1.15), whereas trematode eggs belonging to the genus Zalophotrema were found in significantly higher numbers in the fraction representing 1.25 (P < 0.05). In conclusion, the SpG of trematode, ascarid, and cestode eggs from pinnipeds appears to be similar to those from terrestrial hosts, but numerous factors may affect their ability to be detected using traditional diagnostic approaches. Further exploration into the nature of the variability noted may lead to improved diagnostics in marine parasitology.

THE USE OF EPHEDRINE TO TREAT ANESTHESIA-ASSOCIATED HYPOTENSION IN PINNIPEDS.

Hypotension is a common adverse effect of general anesthesia that has historically been difficult to measure in pinniped species due to technical challenges. A retrospective case review found seven pinniped cases that demonstrated anesthesia-associated hypotension diagnosed by direct blood pressure measurements during general anesthesia at The Marine Mammal Center (Sausalito, CA) between 2017 and 2019. Cases included five California sea lions (CSL: Zalophus californianus), one Hawaiian monk seal (HMS: Neomonachus schauinslandi), and one northern elephant seal (NES: Mirounga angustirostris). Patients were induced using injectable opioids, benzodiazepines, and anesthetics including propofol and alfaxalone. Excluding the HMS, all patients required supplemental isoflurane with a mask to achieve an anesthetic plane allowing for intubation. Each patient was maintained with inhalant isoflurane in oxygen for the duration of the anesthetic event. Each patient was concurrently administered continuous IV fluids and four patients received fluid boluses prior to administration of ephedrine. All hypotensive anesthetized patients were treated with IV ephedrine (0.05-0.2 mg/kg). The average initial systolic (SAP) and mean (MAP) arterial blood pressures for the CSL prior to ephedrine administration were 71 ± 14 mmHg and 48 ± 12 mmHg respectively. The average SAP and MAP for the CSL increased to 119 ± 32 mmHg and 90 ± 34 mmHg respectively within 5 m of ephedrine administration. The NES initial blood pressure measurement was 59/43 (50) (SAP/diastolic [MAP]) mmHg and increased to 80/51 (62) mmHg within 5 m. The initial HMS blood pressure was 79/68 (73) mmHg and increased to 99/78 (85) mmHg within 5 m following ephedrine administration. All patients recovered from anesthesia. These results support the efficacy of IV ephedrine for the treatment of anesthesia-associated hypotension in pinnipeds.

Use of potassium chloride for low-residue euthanasia of anesthetized California sea lions (Zalophus californianus) and northern elephant seals (Mirounga angustirostris) with life-threatening injury or disease.

OBJECTIVE: To demonstrate the efficacy of potassium chloride (KCl) for low-residue euthanasia of anesthetized pinnipeds in field settings for which carcass retrieval for disposal is not feasible. ANIMALS: Stranded, free-ranging California sea lions (CSLs; Zalophus californianus; n = 17) and northern elephant seals (NESs; Mirounga angustirostris; 6) with life-threatening injury or disease between May and August 2020. PROCEDURES: Each animal was anesthetized and then received a lethal dose of KCl solution administered by IV or intracardiac injection. The effective KCl dose; durations to cardiac arrest, respiratory arrest, and pupil dilation; and presence or absence of agonal breaths, muscle fasciculations, or skeletal movements were recorded. RESULTS: Mean effective dose of KCl was 207.4 mg/kg (94.3 mg/lb) for the 17 CSLs and 209.1 mg/kg (95.0 mg/lb) for 5 of 6 NESs (1 outlier NES was excluded). The range in duration from the beginning of KCl injection to cardiac arrest was 0 to 6 minutes, to pupil dilation was 0 to 5 minutes, and to respiratory arrest was 0 to 5 minutes. Muscle fasciculations, skeletal movements, and agonal breaths were observed in both species during and after KCl administration. CONCLUSIONS AND CLINICAL RELEVANCE: The use of KCl provides an effective, low-residue method of euthanasia in anesthetized CSLs and NESs. Our recommended dose for these species is 250 mg KCl/kg (113.6 mg KCl/lb) delivered by intracardiac injection. Compared with euthanasia by barbiturate overdose, the use of KCl reduces the potential for secondary intoxication of scavengers and is appropriate in field scenarios in which the carcass cannot be retrieved for disposal.

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.

TRANSTHORACIC ECHOCARDIOGRAPHIC EVALUATION AND SERUM CARDIAC TROPONIN VALUES IN ANESTHETIZED HEALTHY FEMALE SOUTHERN SEA OTTERS (ENHYDRA LUTRIS NEREIS).

Information about antemortem cardiac evaluation in sea otters (Enhydra lutris) is limited, despite well-established clinical care and rehabilitation procedures and a reported elevated risk of cardiac disease for this species. Serum cardiac troponin I (cTnI) concentration and echocardiographic assessment are two ways of screening for and diagnosing cardiac disease. However, no baseline data or reference intervals for either evaluation are published for sea otters. The objectives of this prospective study were to establish serum cTnI concentrations and echocardiographic technique and quantitative measurements in anesthetized healthy female southern sea otters (Enhydra lutris nereis) (n=15). Serum cTnI values were assessed by a high-sensitivity assay. Serum cTnI concentration ranged from <0.006 to 0.038 ng/ml. A complete echocardiogram, including two-dimensional and M-mode modalities, was performed. Echocardiographic measurements for left atrial size, aorta size, left ventricular structure, and left ventricular function were reported. The median left atrial size to aorta ratio was 1.22 (range 0.80-1.59) in short-axis and 1.70 (range 1.39-2.15) in long-axis. The median left ventricular internal dimension was 3.53 cm (range 2.87-4.92 cm) when assessed in two dimensions and 3.58 cm (range 2.80-4.48 cm) by M-mode. Serum concentrations of cTnI and transthoracic echocardiography may represent valuable tools for the antemortem diagnosis of cardiac disease in sea otters.

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.

Occurrence of Mycoplasmas in Galapagos Sea Lions (Zalophus wollebaeki) and their Association with Other Respiratory Pathogens.

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.

An MRI protocol for anatomical and functional evaluation of the California sea lion brain.

BACKGROUND: Domoic acid (DOM) is a neurotoxin produced by some harmful algae blooms in coastal waters. California sea lions (Zalophus californianus) exposed to DOM often strand on beaches where they exhibit a variety of symptoms, including seizures. These animals typically show hippocampal atrophy on MRI scans. NEW METHOD: We describe an MRI protocol for comprehensive evaluation of DOM toxicosis in the sea lion brain. We intend to study brain development in pups exposed in utero. The protocol depicts the hippocampal formation as the primary region of interest. We include scans for quantitative morphometry, functional and structural connectivity, and a cerebral blood flow map. RESULTS: High-resolution 3D anatomical scans facilitate post hoc slicing in arbitrary planes and accurate morphometry. We demonstrate the first cerebral blood flow map using MRI, and the first structural tractography from a live sea lion brain. COMPARISON WITH EXISTING METHODS: Scans were compared to prior anatomical and functional studies in live sea lions, and structural connectivity in post mortem specimens. Hippocampal volumes were broadly in line with prior studies, with differences likely attributable to the 3D approach used here. Functional connectivity of the dorsal left hippocampus matched that found in a prior study conducted at a lower magnetic field, while structural connectivity in the live brain agreed with findings observed in post mortem studies. CONCLUSIONS: Our protocol provides a comprehensive, longitudinal view of the functional and anatomical changes expected to result from DOM toxicosis. It can also screen for other common neurological pathologies and is suitable for any pinniped that can fit inside an MRI scanner.

Assessment of clinical outcomes associated with mercury concentrations in harbor seal pups (Phoca vitulina richardii) in central California.

Monomethyl mercury (MeHg+) from the diet can cause mild to severe neurotoxicosis in fish-eating mammals. Chronic and low-level in utero exposure also can be neurotoxic, as documented in laboratory animal studies and epidemiologic investigations. In free-ranging animals, it is challenging to study low-level exposure related neurotoxicosis, and few studies have investigated the relationship between mercury (Hg) and adverse outcomes in wild populations. Relative to Hg concentrations on admission we evaluated different types of behaviors for 267 Pacific harbor seal (HS; Phoca vitulina richardii) pups at The Marine Mammal Center from 2015 to 2019 during rehabilitation after stranding and maternal separation. Admitted HS pups underwent a clinical exam; including sex and weight determination, and hair (partly lanugo grown in utero) and blood samples were collected for total Hg concentration ([THg]) determination. All pups were monitored weekly (behavior assessments included response to tactile stimulation, movement, swimming, interactions with other seals, hand feeding, and feeding independently), and days in rehabilitation and survival were recorded. There was a significant negative correlation between [THg] and responses to tactile stimulation and movements, measured in both hair and whole blood (p < 0.05). This relationship was found both during the intensive care unit (ICU) stage, and during the pool stage of rehabilitation. Additionally, there was a significant association between greater [THg] and number of days spent in rehabilitation, although there was no relationship between [THg] and survival. There was a significant sex difference, with greater [THg] in female pups, which contrasts with previously published findings in juvenile and adult harbor seals. Our findings support small, but significant associations between gestational THg exposure and clinical effects for tactile sensory response and movement, and longer rehabilitation durations for HS pups, although there was considerable variability among animals.

CLINICAL AND MICROBIOLOGICAL CHARACTERIZATION OF LYMPH NODE ABSCESSATION IN PUP AND YEARLING CALIFORNIA SEA LIONS (ZALOPHUS CALIFORNIANUS) UNDERGOING REHABILITATION IN A MARINE MAMMAL CENTER.

Severe focal to multifocal abscessation of the axillary and inguinal lymph nodes is an important cause of morbidity and mortality in stranded pup and yearling California sea lions (Zalophus californianus; CSLs) at The Marine Mammal Center (TMMC). A retrospective case review was conducted of all pup and yearling CSLs with axillary and/or inguinal lymph node abscesses admitted to this California rehabilitation center between January 2015 and December 2019 (n = 162). Clinical data and cultured isolates were evaluated to (1) characterize the clinical syndrome, (2) report isolated pathogens, and (3) investigate factors associated with clinical outcome (survival to release versus death). Of the 162 CSLs admitted with at least one axillary or inguinal lymph node abscess, almost all were in poor body condition, and overall mortality rate was 63% (102 of 165). Mortality rate was lower for animals presenting with a single abscess and abscess(es) in the inguinal location only; odds of death were significantly lower for animals that had their abscesses surgically drained (P = 0.029) and those that received antimicrobials (P = 0.037). Hematology and serum chemistry parameters at the time of abscess diagnosis reflected inflammation and malnutrition. Radiographic findings associated with abscesses from 45 cases included soft tissue swelling (n = 40), intralesional gas (n = 19), and osteomyelitis (n = 3). Ninety bacterial isolates were recovered from aerobic (n = 48) and anaerobic cultures (n = 17), 48.9% of which were gram negative. The most common gram-negative organisms were Escherichia coli (n = 15), Proteus spp. (n = 8), and Bacteroides ureolyticus (n = 7), and the most common gram-positive organisms were Streptococcus phocae (n = 10) and Staphylococcus spp. (n = 9). Management of lymph node abscesses via surgical drainage and multimodal systemic antimicrobial therapy with a broad-spectrum of activity may be considered to improve survival of these cases.