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.

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.

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.

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.

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.

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.

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.

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.

Plasma concentrations of itraconazole following a single oral dose in juvenile California sea lions (Zalophus californianus).

The objective of this study was to establish a single-dose pharmacokinetic profile for orally administered itraconazole in California sea lions (Zalophus californianus). Twenty healthy rehabilitated juvenile California sea lions were included in this study. Itraconazole capsules were administered orally with food at a target dose of 5-10 mg/kg. Blood samples were collected from each animal at 0 hr and at two of the following timepoints: 0.5, 1, 2, 4, 6, 8, 12, 24, 48, and 72 hr. Quantitative analysis of itraconazole in plasma samples was performed by high-performance liquid chromatography. An average maximum concentration of 0.22 µg/ml ± 0.11 was detected 4 hr after administration. The average concentration fell to 0.12 µg/ml ± 0.11 by 6 hr and 0.02 µg/ml ± 0.02 at 12 hr. At no point did concentrations reach 0.5 µg/ml, the concentration commonly accepted for therapeutic efficacy. While this formulation was well tolerated by the sea lions, oral absorption was poor and highly variable among individuals. These data indicate that a single oral dose of itraconazole given as a capsule at 5-10 mg/kg, under the conditions used in this study, does not achieve therapeutic plasma concentrations in California sea lions.

EVALUATION OF SERIAL BLOOD LACTATE AND THE USE OF A POINT-OF-CARE LACTATE METER IN LIVE-STRANDED PINNIPEDS.

Live-stranded pinnipeds often present to rehabilitation centers systemically debilitated with dehydration and poor perfusion. In many terrestrial mammals, blood lactate elevation has been correlated with global tissue hypoxia and decreased circulating blood volume. Serial blood lactate measurements in companion animals and humans have been used to guide fluid resuscitation therapy and evaluate prognosis. The primary objective of this study was to evaluate the prognostic value of serial blood lactate levels in live-stranded pinnipeds in a rehabilitation setting. The secondary objectives were to evaluate the use of a point-of-care lactate meter, and potassium oxalate-sodium fluoride (gray-top) tubes for storing samples for lactate analysis in pinniped patients. Fifty-five live-stranded pinnipeds (30 northern elephant seals [Mirounga angustirostris], 21 Pacific harbor seals [Phoca vitulina richardsi], and four California sea lions [Zalophus californianus]) that presented to a rehabilitation center were manually restrained for blood collection and lactate measurement using a point-of-care analyzer (Lactate Plus™) prior to fluid or other medical therapy. Lactate measurements were repeated 72 hr post admission and prior to euthanasia or release. Plasma samples from gray-top tubes were frozen for 30-100 days at -80°C, then thawed and evaluated using both the LactatePlus and bench-top (ABL 815) analyzers. The point-of-care analyzer was successful in measuring lactate in pinnipeds. Gray-top tubes were effective at preserving lactate levels in frozen plasma samples for up to 100 days. Released animals had significantly greater lactate clearance at 72 hr (P = 0.039) than animals that died or were euthanized. Therefore, lactate clearance, determined by serial blood lactate measurements, may be useful for evaluating prognosis in live-stranded pinnipeds. Initial lactate and lactate values prior to euthanasia or release were not significantly associated with outcome. Given these findings, clinicians should interpret isolated lactate values in pinnipeds with caution.

Subconjunctival antimicrobial poloxamer gel for treatment of corneal ulceration in stranded California sea lions (Zalophus californianus).

OBJECTIVE: Corneal ulcers are commonly encountered in pinnipeds. Prolonged oral antibiotics and topical ophthalmic solutions may not be practical to administer, and novel treatment techniques are desired. Thermodynamic gels are a potential solution because they hold antimicrobials at the site of injection, slowly releasing drug. This study investigated the clinical efficacy of antibiotic-impregnated poloxamer gel in management of corneal ulceration. ANIMAL STUDIED: Twenty-six California sea lions undergoing rehabilitation at The Marine Mammal Center. PROCEDURES: A poloxamer gel mixed with 2% enrofloxacin was subconjunctivally injected in the treatment group. Control animals received oral doxycycline. Systemic anti-inflammatories and analgesics were administered as needed. Corneal examinations under general anesthesia were repeated weekly, and included sampling for bacterial culture and corneal cytology, collection of high-quality corneal images, and treatment administration until the ulcers were healed. RESULTS: There was no gross or histologic evidence of a localized tissue reaction to the gel administration in the conjunctiva, and no evidence of systemic reaction to therapy in animals that died due to unrelated causes during the study period (n = 17). In animals that experienced a superficial corneal ulcer involving only epithelium or superficial stroma (n = 12), all lesions resolved completely, in both treatment and control groups. Of those animals with deeper or more complex ulcers involving keratomalacia or descemetoceles (n = 15), four demonstrated complete lesion resolution (all four received gel treatment). CONCLUSIONS: This study demonstrates that subconjunctival antibiotic poloxamer gel administration is a safe and effective alternative therapeutic option to traditional treatments for superficial corneal ulceration in pinnipeds.

WHOLE BLOOD TRANSFUSIONS TO TREAT SEVERE ANEMIA IN SEVEN COWNOSE RAYS ( RHINOPTERA BONASUS) AND ONE SHORT-TAIL STINGRAY ( DASYATIS BREVICAUDATA).

Blood transfusions can provide life-saving treatment to severely anemic animals. Due to limited availability and the difficulty of storing whole blood and blood products, such as fresh frozen plasma and packed red blood cells, exotic animals often receive fresh whole blood transfusions. Little is known about elasmobranch blood types and transfusions. Conspecific cross-matches within several different elasmobranch species were negative, indicating that in an emergency situation a single whole blood transfusion may be possible without causing a transfusion reaction. Experimental transfusions between healthy conspecific Atlantic rays ( Dasyatis sabina) showed no adverse reactions and autotransfusions in marbled electric rays ( Torpedo marmorata) were successful. There are no published reports of blood transfusions performed on clinically abnormal elasmobranchs. The following case series documents blood transfusions performed on seven cownose rays ( Rhinoptera bonasus) and one short-tail stingray ( Dasyatis brevicaudata). All rays were treated with the same protocol, which included pretreatment with steroids and antibiotics followed by an intravenous transfusion of freshly collected, heparinized, whole blood. Three animals survived and currently exhibit no abnormal clinical signs. Two animals died 55 days and 100 days post transfusion. Three animals died 2-22 days post transfusion. Although complications from blood transfusions could not be ruled out, all five animals that died had other health problems that likely contributed to their demise. All eight animals would almost certainly have died without a blood transfusion as they were severely anemic and moribund at the time of presentation. The methods described in this paper may be useful in the treatment of severely anemic elasmobranchs and this is the first report of blood transfusions in clinically abnormal elasmobranchs.

MEDICAL MANAGEMENT OF COELOMIC DISTENTION, ABNORMAL SWIMMING, SUBSTRATE RETENTION, AND HEMATOLOGIC CHANGES IN A REEF MANTA RAY (MANTA ALFREDI).

An adult, female, reef manta ray (Manta alfredi) was evaluated for abnormal swimming and coelomic distention after colliding with a floating dock in an exhibit. Initial clinical signs included abnormal posture and swimming. Clinical signs progressed to include muscle wasting, poor body condition, and coelomic distention. Further diagnostics revealed excessive exhibit substrate within the gastrointestinal tract, gastric ulcers, free coelomic fluid, possible spinal lesion, possible uterine abnormality, and hematologic changes. The ray was treated with repeated gastroscopic examinations to remove substrate from the stomach. Psyllium and mineral oil were administered in gel food to assist with passage of substrate through the gastrointestinal tract. Gastric ulcers were treated with sucralfate. Vitamin B complex, iron dextran, and Yunnan Paiyao were used to treat the anemia. Amikacin and sulfadimethoxine-ormetoprim were administered for suspected bacterial and coccidial infections, respectively. Over the course of 11 mo the ray returned to normal health.

COMPARISON OF PRE- AND POSTQUARANTINE BLOOD CHEMISTRY AND HEMATOLOGY VALUES FROM WILD-CAUGHT COWNOSE RAYS (RHINOPTERA BONASUS).

Though one of the most widely kept elasmobranchs in human care, the cownose ray (CNR; Rhinoptera bonasus ), remains a species with minimal published information on hematologic reference intervals. As part of a larger study investigating the health and nutrition of the CNR, this study established a preliminary data set of plasma chemistry and hematology values specific to animals recently caught from the wild and compared this data set (intake sample) to values obtained following a period of quarantine (27-40 days) in an aquarium (exit sample). Blood samples were collected from 47 wild female (n = 46) and male (n = 1) CNR caught in pound nets off the coast of North Carolina and South Carolina. Differences between intake and exit values were analyzed. Due to the preponderance of female animals, data were not analyzed for sex differences. Plasma biochemical profiles were performed and analyzed. A select number of complete blood cell counts were performed (n = 24 from 12 animals). Statistically significant differences (P < 0.05) specific to time of sampling were determined for packed cell volume, total solids, blood urea nitrogen, sodium, chloride, potassium, phosphorus, cholesterol, glucose, and aspartate aminotransferase. Values reported are a significant expansion on the existing limited data for CNRs and will serve as a reference for health assessment of individuals both in the wild and in exhibit populations.

PLASMA PROTEIN ELECTROPHORESIS AND SELECT ACUTE PHASE PROTEINS IN HEALTHY BONNETHEAD SHARKS (SPHYRNA TIBURO) UNDER MANAGED CARE.

Preventative health care of elasmobranchs is an important but understudied field of aquatic veterinary medicine. Evaluation of inflammation through the acute phase response is a valuable tool in health assessments. To better assess the health of bonnethead sharks ( Sphyrna tiburo ) under managed care, normal reference intervals of protein electrophoresis (EPH) and the acute phase proteins, C-reactive protein (CRP) and haptoglobin (HP), were established. Blood was collected from wild caught, captive raised bonnethead sharks housed at public aquaria. Lithium heparinized plasma was either submitted fresh or stored at -80°C prior to submission. Electrophoresis identified protein fractions with migration characteristics similar to other animals with albumin, α-1 globulin, α-2 globulin, ß globulin, and γ globulin. These fractions were classified as fractions 1-5 as fractional contents are unknown in this species. Commercial reagents for CRP and HP were validated for use in bonnethead sharks. Reference intervals were established using the robust method recommended by the American Society for Veterinary Clinical Pathology for the calculation of 90% reference intervals. Once established, the diagnostic and clinical applicability of these reference intervals was used to assess blood from individuals with known infectious diseases that resulted in systemic inflammation and eventual death. Unhealthy bonnethead sharks had significantly decreased fraction 2, fraction 3, and fraction 3:4 ratio and significantly increased fraction 5, CRP, and HP. These findings advance our understanding of elasmobranch acute phase inflammatory response and health and aid clinicians in the diagnosis of inflammatory disease in bonnethead sharks.

Surveillance for zoonotic and selected pathogens in harbor seals Phoca vitulina from central California.

The infection status of harbor seals Phoca vitulina in central California, USA, was evaluated through broad surveillance for pathogens in stranded and wild-caught animals from 2001 to 2008, with most samples collected in 2007 and 2008. Stranded animals from Mendocino County to San Luis Obispo County were sampled at a rehabilitation facility: The Marine Mammal Center (TMMC, n = 175); wild-caught animals were sampled at 2 locations: San Francisco Bay (SF, n = 78) and Tomales Bay (TB, n = 97), that differed in degree of urbanization. Low prevalences of Salmonella, Campylobacter, Giardia, and Cryptosporidium were detected in the feces of stranded and wild-caught seals. Clostridium perfringens and Escherichia coli were more prevalent in the feces of stranded (58% [78 out of 135] and 76% [102 out of 135]) than wild-caught (42% [45 out of 106] and 66% [68 out of 106]) seals, whereas Vibrio spp. were 16 times more likely to be cultured from the feces of seals from SF than TB or TMMC (p < 0.005). Brucella DNA was detected in 3.4% of dead stranded harbor seals (2 out of 58). Type A influenza was isolated from feces of 1 out of 96 wild-caught seals. Exposure to Toxoplasma gondii, Sarcocystis neurona, and type A influenza was only detected in the wild-caught harbor seals (post-weaning age classes), whereas antibody titers to Leptospira spp. were detected in stranded and wild-caught seals. No stranded (n = 109) or wild-caught (n = 217) harbor seals had antibodies to phocine distemper virus, although a single low titer to canine distemper virus was detected. These results highlight the role of harbor seals as sentinel species for zoonotic and terrestrial pathogens in the marine environment.

Cloacolithiasis and intestinal lymphosarcoma in an African black-footed penguin (Spheniscus demersus).

A 13-yr-old male African black-footed penguin (Spheniscus demersus) presented thrice over 7 mo with gastrointestinal obstruction secondary to cloacolithiasis. Clinical signs consistently resolved with cloacolith removal and supportive care. However, 10 mo after initial presentation, it presented with similar signs, plus significant weight loss. No cloacolith was found, and it subsequently died. Significant gross findings included bilateral cecal masses, colonic perforation, and marked secondary coelomitis, multifocal tan to pale hepatic nodules, and pale kidneys with miliary white foci. Histopathologic diagnoses were intestinal lymphosarcoma with hepatic and renal metastases, secondary intestinal rupture, and subacute severe bacterial coelomitis. To the authors' knowledge, this is the first full report of either cloacolithiasis or lymphosarcoma in a penguin.

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.

Response of Northern Elephant Seal platelets to pressure and temperature changes: a comparison with human platelets.

Mammalian blood platelets are activated by physiological agonists such as collagen or thrombin, or by physical stimuli such as cold temperatures and rapid decompression. Marine mammals regularly experience cold temperatures, high pressures and rapid decompression while diving, yet do not appear to suffer from thrombotic events during routine dive activity. We evaluated the effects of cold temperature and high pressure excursions on Northern Elephant Seal (NES) platelets and compared NES platelet response to that of human platelets subjected to identical stimuli. NES platelets undergo cold-induced activation when chilled to 4 °C, and 3 distinct phase transitions can be measured using Fourier Transform Infrared Spectroscopy. NES platelet membrane lipid composition was determined using thin layer chromatography and NES platelets were found to have three times the amount of cholesterol (21% by weight) as human platelets. When exposed to high pressure-rapid decompression excursion, NES platelets did not undergo morphological shape change nor bind increased amounts of fibrinogen, while human platelets were significantly activated by the same excursion. These results demonstrate that while NES platelets are activated by the physical stimulus of cold temperatures, they are resistant to decompression-induced activation. We suggest that the composition of NES platelet membranes may play an important role in preventing pressure-related activation.