Rookery through rehabilitation: Microbial community assembly in newborn harbour seals after maternal separation.

Microbial community assembly remains largely unexplored in marine mammals, despite its potential importance for conservation and management. Here, neonatal microbiota assembly was studied in harbour seals (Phoca vitulina richardii) at a rehabilitation facility soon after maternal separation, through weaning, to the time of release back to their native environment. We found that the gingival and rectal communities of rehabilitated harbour seals were distinct from the microbiotas of formula and pool water, and became increasingly diverse and dissimilar over time, ultimately resembling the gingival and rectal communities of local wild harbour seals. Harbour seal microbiota assembly was compared to that of human infants, revealing the rapid emergence of host specificity and evidence of phylosymbiosis even though these harbour seals had been raised by humans. Early life prophylactic antibiotics were associated with changes in the composition of the harbour seal gingival and rectal communities and surprisingly, with transient increases in alpha diversity, perhaps because of microbiota sharing during close cohabitation with other harbour seals. Antibiotic-associated effects dissipated over time. These results suggest that while early life maternal contact may provide seeding for microbial assembly, co-housing of conspecifics during rehabilitation may help neonatal mammals achieve a healthy host-specific microbiota with features of resilience.

Detecting signals of chronic shedding to explain pathogen persistence: Leptospira interrogans in California sea lions.

Identifying mechanisms driving pathogen persistence is a vital component of wildlife disease ecology and control. Asymptomatic, chronically infected individuals are an oft-cited potential reservoir of infection, but demonstrations of the importance of chronic shedding to pathogen persistence at the population-level remain scarce. Studying chronic shedding using commonly collected disease data is hampered by numerous challenges, including short-term surveillance that focuses on single epidemics and acutely ill individuals, the subtle dynamical influence of chronic shedding relative to more obvious epidemic drivers, and poor ability to differentiate between the effects of population prevalence of chronic shedding vs. intensity and duration of chronic shedding in individuals. We use chronic shedding of Leptospira interrogans serovar Pomona in California sea lions (Zalophus californianus) as a case study to illustrate how these challenges can be addressed. Using leptospirosis-induced strands as a measure of disease incidence, we fit models with and without chronic shedding, and with different seasonal drivers, to determine the time-scale over which chronic shedding is detectable and the interactions between chronic shedding and seasonal drivers needed to explain persistence and outbreak patterns. Chronic shedding can enable persistence of L. interrogans within the sea lion population. However, the importance of chronic shedding was only apparent when surveillance data included at least two outbreaks and the intervening inter-epidemic trough during which fadeout of transmission was most likely. Seasonal transmission, as opposed to seasonal recruitment of susceptibles, was the dominant driver of seasonality in this system, and both seasonal factors had limited impact on long-term pathogen persistence. We show that the temporal extent of surveillance data can have a dramatic impact on inferences about population processes, where the failure to identify both short- and long-term ecological drivers can have cascading impacts on understanding higher order ecological phenomena, such as pathogen persistence.

An unprecedented coastwide toxic algal bloom linked to anomalous ocean conditions.

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

Proteomic analysis of cerebrospinal fluid in California sea lions (Zalophus californianus) with domoic acid toxicosis identifies proteins associated with neurodegeneration.

Proteomic studies including marine mammals are rare, largely due to the lack of fully sequenced genomes. This has hampered the application of these techniques toward biomarker discovery efforts for monitoring of health and disease in these animals. We conducted a pilot label-free LC-MS/MS study to profile and compare the cerebrospinal fluid from California sea lions with domoic acid toxicosis (DAT) and without DAT. Across 11 samples, a total of 206 proteins were identified (FDR<0.1) using a composite mammalian database. Several peptide identifications were validated using stable isotope labeled peptides. Comparison of spectral counts revealed seven proteins that were elevated in the cerebrospinal fluid from sea lions with DAT: complement C3, complement factor B, dickkopf-3, malate dehydrogenase 1, neuron cell adhesion molecule 1, gelsolin, and neuronal cell adhesion molecule. Immunoblot analysis found reelin to be depressed in the cerebrospinal fluid from California sea lions with DAT. Mice administered domoic acid also had lower hippocampal reelin protein levels suggesting that domoic acid depresses reelin similar to kainic acid. In summary, proteomic analysis of cerebrospinal fluid in marine mammals is a useful tool to characterize the underlying molecular pathology of neurodegenerative disease. All MS data have been deposited in the ProteomeXchange with identifier PXD002105 (http://proteomecentral.proteomexchange.org/dataset/PXD002105).

Evaluation of viruses and their association with ocular lesions in pinnipeds in rehabilitation.

OBJECTIVE: To assess whether corneal lesions in stranded pinnipeds were associated with viral infections, and to identify the potential pathogen(s) associated with the lesions. ANIMALS STUDIED: Twenty-nine California sea lions (Zalophus californianus), 18 northern elephant seals (Mirounga angustirostris), and 34 Pacific harbor seals (Phoca vitulina richardsii). PROCEDURE: DNA and RNA were extracted from ocular swabs, corneal tissue, and aqueous humor and screened for herpesvirus, adenovirus, poxvirus, and calicivirus families by PCR. RESULTS: The results indicated a high overall prevalence of viruses, with adenoviruses and herpesviruses detected in all three host species. Three novel adenoviruses (PhAdV-1, PhAdV-2, OtAdV-2) and two novel herpesviruses (PhHV-6, OtHV-4) were detected. There were no statistical differences in the prevalence of viral infection or coinfection among groups of individuals with or without corneal lesions, nor were lesion type, onset, or presence of concurrent disease significantly associated with a viral infection. CONCLUSIONS: The results suggested that viral presence in ocular tissues was common, not significantly associated with ocular disease and thus should not preclude release of an otherwise healthy animal. We could not confirm a correlation of virus presence with lesion due to the high percentage of virus-positive, clinically normal animals. This implied that seals and sea lions can have ocular tissues infected with several viruses without having readily evident associated lesions. This difficulty in correlating viral presence, particularly herpesviruses, with ocular lesions was also a common finding in studies with terrestrial species and highlighted the difficulty of confirming a virus as a primary pathogen in ocular lesions.

LAPAROSCOPIC GASTROPEXY FOR CORRECTION OF A HIATAL HERNIA IN A NORTHERN ELEPHANT SEAL (MIROUNGA ANGUSTIROSTRIS).

A female northern elephant seal (Mirounga angustirostris) weaned pup presented with malnutrition. During rehabilitation, the seal developed regurgitation and reduced lung sounds on auscultation. Radiographs and endoscopy performed under sedation suggested a diaphragmatic hernia. A Type I (or sliding) hiatal hernia was confirmed with a positive contrast upper gastrointestinal study, revealing varying degrees of herniation of the gastric fundus through the diaphragm into the caudal thorax as well as esophageal reflux. The animal was treated preoperatively with an H2 antagonist and antinausea medication. A laparoscopic gastropexy was performed under general anesthesia. The animal recovered well postoperatively and resolution of clinical signs was achieved. The animal was released back into the wild 21 kg above admit weight. To our knowledge, we report here the first surgical correction of a hiatal hernia in a marine mammal.

PHARMACOKINETICS OF TRAMADOL HYDROCHLORIDE AND ITS METABOLITE O-DESMETHYLTRAMADOL FOLLOWING A SINGLE, ORALLY ADMINISTERED DOSE IN CALIFORNIA SEA LIONS (ZALOPHUS CALIFORNIANUS).

Tramadol is a synthetic, centrally acting, opiate-like analgesic that is structurally related to codeine and morphine. The objective of this study was to determine the pharmacokinetics of tramadol hydrochloride and its major active metabolite O-desmethyltramadol (M1) in the California sea lion (Zalophus californianus). A single dose of tramadol was administered orally in fish at 2 mg/kg to a total of 15 wild California sea lions admitted for rehabilitation. Twenty-four total blood samples were collected post drug administration at 10, 20, 30, and 45 min and at 1, 3, 5, 6, 8, 12, and 24 hr. Blood plasma was separated and stored at -80°C until analysis with high-performance liquid chromatography was performed to determine levels of tramadol and M1, the major active metabolite. The results indicate that the plasma levels of parent tramadol are low or negligible during the first 30-45 min and then reach the predicted mean maximum plasma concentration of 358 ng/ml at 1.52 hr. The M1 metabolite was not detectable in 21 of 24 plasma samples, below the level of quantification of 5 ng/ml in one sample, and detectable at 11 and 17 ng/ml in two of the samples. This study suggests that a 2 mg/kg dose would need to be administered every 6-8 hr to maintain concentrations of tramadol above the minimum human analgesic level for mild to moderate pain. Based on dosing simulations, a dose of 4 mg/kg q8 hr or q12 hr, on average, may represent an adequate compromise, but further studies are needed using a larger sample size. Pharmacodynamic studies are warranted to determine if tramadol provides analgesic effects in this species. The potential for tramadol toxicosis at any dose also has not been determined in this species.

Evidence for a genetic basis of urogenital carcinoma in the wild California sea lion.

Although neoplasia is a major cause of mortality in humans and domestic animals, it has rarely been described in wildlife species. One of the few examples is a highly prevalent urogenital carcinoma in California sea lions (CSLs). Although the aetiology of this carcinoma is clearly multifactorial, inbreeding depression, as estimated using levels of microsatellite multilocus heterozygosity, is identified as predictive for this neoplasia. On further analysis, this relationship appears to be largely driven by one marker, suggesting that a single locus might be associated with the occurrence of this disease in CSLs. In a case-control study, carcinoma was significantly associated with homozygosity at the Pv11 microsatellite locus. Pv11 was mapped to intron 9 of the heparanase 2 gene (HPSE2) locus, a very large gene encoding heparanase 2, which in humans is associated with multiple carcinomas. Correspondingly, immunohistochemical labelling in tissues was present in carcinoma cases within a single homozygous Pv11 genotype. To our knowledge, this is the first report of an individual locus being associated with cancer in any wildlife species. This adds emphasis to the study of HPSE2 in other species, including humans and will guide future studies on this sentinel species that shares much of its diet and environment with humans.

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.

Proteomic and Lipidomic Plasma Evaluations Reveal Biomarkers for Domoic Acid Toxicosis in California Sea Lions.

Domoic acid is a neurotoxin secreted by the marine diatom genus, Pseudo-nitzschia, during toxic algal bloom events. California sea lions ( Zalophus californianus ) are exposed to domoic acid through ingestion of fish that feed on toxic diatoms, resulting in a domoic acid toxicosis (DAT), which can vary from mild to fatal. Sea lions with mild disease can be treated if toxicosis is detected early after exposure, therefore, rapid diagnosis of DAT is essential but also challenging. In this work, we performed multi-omics analyses, specifically proteomic and lipidomic, on blood samples from 31 California sea lions. Fourteen sea lions were diagnosed with DAT based on clinical signs and postmortem histological examination of brain tissue, and 17 had no evidence of DAT. Proteomic analyses revealed three apolipoproteins with statistically significant lower abundance in the DAT individuals compared to the non-DAT individuals. These proteins are known to transport lipids in the blood. Lipidomic analyses highlighted 29 lipid levels that were statistically different in the DAT versus non-DAT comparison, 28 of which were downregulated while only one was upregulated. Furthermore, of the 28 downregulated lipids, 15 were triglycerides, illustrating their connection with the perturbed apolipoproteins and showing their potential for use in rapid DAT diagnoses. SYNOPSIS: Multi-omics evaluations reveal blood apolipoproteins and triglycerides are altered in domoic acid toxicosis in California sea lions.

Dynamics of Vibrio with virulence genes detected in Pacific harbor seals (Phoca vitulina richardii) off California: implications for marine mammal health.

Given their coastal site fidelity and opportunistic foraging behavior, harbor seals (Phoca vitulina) may serve as sentinels for coastal ecosystem health. Seals using urbanized coastal habitat can acquire enteric bacteria, including Vibrio that may affect their health. To understand Vibrio dynamics in seals, demographic and environmental factors were tested for predicting potentially virulent Vibrio in free-ranging and stranded Pacific harbor seals (Phoca vitulina richardii) off California. Vibrio prevalence did not vary with season and was greater in free-ranging seals (29 %, n = 319) compared with stranded seals (17 %, n = 189). Of the factors tested, location, turbidity, and/or salinity best predicted Vibrio prevalence in free-ranging seals. The relationship of environmental factors with Vibrio prevalence differed by location and may be related to oceanographic or terrestrial contributions to water quality. Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio cholerae were observed in seals, with V. cholerae found almost exclusively in stranded pups and yearlings. Additionally, virulence genes (trh and tdh) were detected in V. parahaemolyticus isolates. Vibrio cholerae isolates lacked targeted virulence genes, but were hemolytic. Three out of four stranded pups with V. parahaemolyticus (trh+ and/or tdh+) died in rehabilitation, but the role of Vibrio in causing mortality is unclear, and Vibrio expression of virulence genes should be investigated. Considering that humans share the environment and food resources with seals, potentially virulent Vibrio observed in seals also may be of concern to human health.

Endocrine changes in harbor seal (Phoca vitulina) pups undergoing rehabilitation.

Rehabilitating pinniped pups are often admitted to care centers as neonates and generally lack maternal investment and are in poor body condition. Upon admittance to a rehabilitation facility, pups are typically fed a milk replacement formula via gavage, which is switched to frozen fish upon weaning. While rehabilitation has been successful in terms of recovery and release, preweaning growth rates in captivity are consistently lower than in the wild. Indicators of stress (cortisol and total thyroxine; TT4), and standard morphometrics, of harbor seal pups in rehabilitation (n = 20) were determined for both preweaned and weaned pups. Hormone concentrations and standard morphometrics from pups in care were compared with free-ranging harbor seal pups (n = 59). Pups in rehabilitation gained mass on both milk and fish diets. Preweaned pups had greater mean serum cortisol and similar TT4 concentrations than weaned pups. Free-ranging harbor seal pups were heavier and longer than preweaned and weaned pups in rehabilitation. The free-ranging pups had the lowest cortisol and highest TT4 concentrations of any of the pups. These results suggest that weaned pups that have undergone rehabilitation are not physiologically equivalent to free-ranging weaned pups. Additional research is needed regarding physiological changes in endocrine values during early development under captive care conditions. This information should be useful to marine mammal rehabilitation centers in their development of care protocols and release criteria for rehabilitating harbor seal pups.

Pharmacokinetics of ceftiofur crystalline-free acid (EXCEDE sterile suspension) administered via intramuscular injection in wild California sea lions (Zalophus californianus).

The pharmacokinetics of ceftiofur crystalline-free acid (EXCEDE Sterile Suspension, 200 mg ceftiofur equivalents/ml) were determined for the California sea lion (Zalophus californianus). A single dose of EXCEDE was administered intramuscularly at 6.6 mg/kg to 12 wild California sea lions during rehabilitation. The first 10 animals were each assigned to two blood collection time points, with a total of 10 time points at: 6, 12, 24, 48, 72, 96, 120, 144, 168, and 192 hr after administration of the drug. An additional two animals were sampled 1, 2, 3, 4, 5, and 6 hr postinjection. Plasma was separated within 10 min of blood collection and stored at -20 degrees C until analysis. Plasma concentrations of ceftiofur, desfuroylceftiofur, and related metabolites, were determined using liquid chromatography with tandem mass spectrometry (MS). Maximum plasma concentrations of ceftiofur and related metabolites were observed 24 hr postdosing with a mean concentration of 3.6 microg/ml. The half life (60 hr) and area under the curve (270 microg x hr/ml) were also determined. These data indicate that a single dose of EXCEDE at 6.6 mg/kg i.m. would likely maintain a mean plasma drug level >0.6 microg/ml for 5 days and >0.5 microg/ml for 8 days.

Pinniped electroencephalography: Methodology and findings in California sea lions (Zalophus californianus).

This study was designed to identify abnormalities in the electroencephalograms (EEGs) recorded from stranded California sea lions (Zalophus californianus) with suspected domoic acid (DA) toxicosis. Recordings from animals presenting for non-neurological issues were also obtained to better understand the normal EEG (background activity and transient events) in this species, as, to date, studies have focused on examining natural sleep in pinnipeds. Most animals were sedated for electrode placement and EEG acquisition with some receiving antiepileptic medications or isoflurane during the procedure. A total of 103 recordings were read and scored from 0 (normal) to 3 (severely abnormal). Epileptiform discharges, consisting of spikes, sharp waves, slow waves, and/or spike waves, were present in all EEGs with scores of 1, 2, or 3. The distribution of these events over the scalp varied. While often generalized, others were lateralized over one hemisphere, bifrontal, bioccipital, and/or bitemporal, while some discharges were multifocal. Findings were different between sea lions and occasionally changed within the EEG on a given sea lion. No clinical seizures were observed during the recording but a few sea lions had findings consistent with electroencephalographic seizures. When available, supporting diagnostic results obtained from magnetic resonance imaging (MRI) and/or necropsy/histopathology were described, as well as the status of those sea lions that recovered and were released with satellite tags.

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.

The fecal viral flora of California sea lions.

California sea lions are one of the major marine mammal species along the Pacific coast of North America. Sea lions are susceptible to a wide variety of viruses, some of which can be transmitted to or from terrestrial mammals. Using an unbiased viral metagenomic approach, we surveyed the fecal virome in California sea lions of different ages and health statuses. Averages of 1.6 and 2.5 distinct mammalian viral species were shed by pups and juvenile sea lions, respectively. Previously undescribed mammalian viruses from four RNA virus families (Astroviridae, Picornaviridae, Caliciviridae, and Reoviridae) and one DNA virus family (Parvoviridae) were characterized. The first complete or partial genomes of sapeloviruses, sapoviruses, noroviruses, and bocavirus in marine mammals are reported. Astroviruses and bocaviruses showed the highest prevalence and abundance in California sea lion feces. The diversity of bacteriophages was higher in unweaned sea lion pups than in juveniles and animals in rehabilitation, where the phage community consisted largely of phages related to the family Microviridae. This study increases our understanding of the viral diversity in marine mammals, highlights the high rate of enteric viral infections in these highly social carnivores, and may be used as a baseline viral survey for comparison with samples from California sea lions during unexplained disease outbreaks.

Risk factors associated with perianesthetic mortality of stranded free-ranging California sea lions (Zalophus californianus) undergoing rehabilitation.

The objective of this study was to identify risk factors associated with perianesthetic mortality of stranded free-ranging California sea lions (Zalophus californianus) undergoing rehabilitation. Hospital records of California sea lions that underwent heavy sedation or general anesthesia from 2004 through 2008 were reviewed, including records from 419 anesthetic events. Procedures that resulted in death during or in the subsequent 72 hr of anesthesia were classified as cases (n = 15). Procedures in which the animal survived were classified as controls (n = 334). Procedures that resulted in euthanasia (n = 70) were removed from subsequent analysis. The following risk factors were reviewed: gender, age class, health status, duration of anesthetic period, atropine premedication, induction protocols, maintenance protocols, and history of prior anesthesia. The prevalence of fatalities during anesthesia was 3.4% (n = 12) over the 5-yr period. With the inclusion of animals that died within 72 hr after anesthesia, the total mortality prevalence rose to 4.3% (n = 15). The most common time of death was during anesthetic maintenance. Health status was the single best predictor of anesthetic outcome, and sea lions premedicated with atropine had increased odds of anesthetic-related death.

Antemortem diagnosis of a ventricular septal defect in a California sea lion Zalophus californianus.

A yearling California sea lion Zalophus californianus stranded in poor body condition, and on physical examination a heart murmur was audible bilaterally. The sea lion was diagnosed with a left-to-right shunting membranous ventricular septal defect (VSD) using B-mode, color-flow Doppler and continuous-wave Doppler echocardiography. A left-to-right intracardiac shunting lesion was confirmed during cardiac angiographic computed tomography. The VSD defect was verified during the necropsy examination. On histologic examination concurrent mild multifocal myocarditis with focal mild ventricular free-wall myocardial necrosis were identified. A specific cause for the myocarditis and myocardial necrosis was not found, and association with the VSD and resultant myocardial dysfunction was presumed. This is the first report of the antemortem diagnosis of a VSD in a marine mammal and the first report of a VSD in a California sea lion.

Using PacBio SMRT data for identification of class I MHC alleles in a wildlife species, Zalophus californianus (California sea lion).

High allelic polymorphism and association with disease susceptibility has made the genes encoding major histocompatibility complex (MHC) antigen presentation molecules in humans, domesticated animals, and wildlife species of wide interest to ecologists, evolutionary biologists, and health specialists. The often multifaceted polygenism and extreme polymorphism of this immunogenetic system have made it especially difficult to characterize in non-model species. Here we compare and contrast the workflows of traditional Sanger sequencing of plasmid-cloned amplicons to Pacific Biosciences SMRT circular consensus sequencing (CCS) in their ability to capture alleles of MHC class I in a wildlife species where characterization of these genes was absent. We assessed two California sea lions (Zalophus californianus), a species suffering from a high prevalence of an aggressive cancer associated with a sexually transmitted gamma herpesvirus. In this pilot study, SMRT CCS proved superior in identifying more alleles from each animal than the more laborious plasmid cloning/Sanger workflow (12:7, 10:7), and no alleles were identified with the cloning/Sanger approach that were not identified by SMRT CCS. We discuss the advantages and disadvantages of each approach including cost, allele rarefaction, and sequence fidelity.

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.