Detection of Leptospira kirschneri in a short-beaked common dolphin (Delphinus delphis delphis) stranded off the coast of southern California, USA.

BACKGROUND: Pathogenic Leptospira species are globally important zoonotic pathogens capable of infecting a wide range of host species. In marine mammals, reports of Leptospira have predominantly been in pinnipeds, with isolated reports of infections in cetaceans. CASE PRESENTATION: On 28 June 2021, a 150.5 cm long female, short-beaked common dolphin (Delphinus delphis delphis) stranded alive on the coast of southern California and subsequently died. Gross necropsy revealed multifocal cortical pallor within the reniculi of the kidney, and lymphoplasmacytic tubulointerstitial nephritis was observed histologically. Immunohistochemistry confirmed Leptospira infection, and PCR followed by lfb1 gene amplicon sequencing suggested that the infecting organism was L.kirschneri. Leptospira DNA capture and enrichment allowed for whole-genome sequencing to be conducted. Phylogenetic analyses confirmed the causative agent was a previously undescribed, divergent lineage of L.kirschneri. CONCLUSIONS: We report the first detection of pathogenic Leptospira in a short-beaked common dolphin, and the first detection in any cetacean in the northeastern Pacific Ocean. Renal lesions were consistent with leptospirosis in other host species, including marine mammals, and were the most significant lesions detected overall, suggesting leptospirosis as the likely cause of death. We identified the cause of the infection as L.kirschneri, a species detected only once before in a marine mammal - a northern elephant seal (Mirounga angustirostris) of the northeastern Pacific. These findings raise questions about the mechanism of transmission, given the obligate marine lifestyle of cetaceans (in contrast to pinnipeds, which spend time on land) and the commonly accepted view that Leptospira are quickly killed by salt water. They also raise important questions regarding the source of infection, and whether it arose from transmission among marine mammals or from terrestrial-to-marine spillover. Moving forward, surveillance and sampling must be expanded to better understand the extent to which Leptospira infections occur in the marine ecosystem and possible epidemiological linkages between and among marine and terrestrial host species. Generating Leptospira genomes from different host species will yield crucial information about possible transmission links, and our study highlights the power of new techniques such as DNA enrichment to illuminate the complex ecology of this important zoonotic pathogen.

Systemic Erysipelas Outbreak among Free-Ranging Bottlenose Dolphins, San Diego, California, USA, 2022.

We diagnosed fatal Erysipelothrix rhusiopathiae sepsis in 3 stranded bottlenose dolphins (Tursiops truncatus) during summer 2022, in San Diego, California, USA. The previously undetected disease in this relatively small, regional population of dolphins most likely indicates an environmental or biological change in the coastal ocean or organisms.

Pathology of short-term dorsal fin tag-attachments in tagged and re-stranded short-beaked common dolphins Delphinus delphis on Cape Cod, MA.

Odontocetes are difficult to study in the wild, making tagging and remote tracking a valuable practice. However, evaluations of host responses at tagging sites have been primarily limited to visual observations in the field. Here we explore the macro- and microscopic pathology of dorsal fin tag attachments in 13 stranded and released short-beaked common dolphins Delphinus delphis from Cape Cod, MA that later re-stranded and died or were euthanized 1-28 d post-tagging. Tags were attached to stranded dolphins' dorsal fins using 2 methods: core biopsy or piercing. Grossly, the piercing method resulted in epidermal compression into the dermis. One tag site had a necrotic border 28 d after application. Grossly, the biopsy method resulted in minimal to no tissue reaction. Two tag sites had granulation tissue accumulation 4 and 12 d after tagging. Histopathologic findings for all tag types and animals consisted of focal epithelial loss, dermal edema, perivascular edema, inflammation and hyperplasia, and inter- and extracellular edema in the adjacent epidermis. Minor expected pathological changes given the procedure were also observed: superficial epidermal necrosis in 3 cases, and superficial bacterial colonization in 2 cases. There was no evidence of sepsis and tagging was not related to cause of re-stranding or death in any case. These gross and histopathologic findings support previous observational conclusions in small delphinids that with appropriate sterile technique, the impacts of single pin dorsal fin tagging on the animal can be minimal and localized. Of the 2 methods, core biopsy may be a better tagging method.

Brucella ceti sequence type 23, 26, and 27 infections in North American cetaceans.

Brucella ceti infection is associated with a variety of disease outcomes in cetaceans globally. Multiple genotypes of B. ceti have been identified. This retrospective aimed to determine if specific lesions were associated with different B. ceti DNA sequence types (STs). Characterization of ST was performed on 163 samples from 88 free-ranging cetaceans, including common bottlenose dolphin Tursiops truncatus (T.t.; n = 73), common short-beaked dolphin Delphinus delphis (D.d.; n = 7), striped dolphin Stenella coeruleoalba (n = 3), Pacific white-sided dolphin Lagenorhynchus obliquidens (n = 2), sperm whale Physeter macrocephalus (n = 2), and harbour porpoise Phocoena phocoena (n = 1), that stranded along the coast of the US mainland and Hawaii. ST was determined using a previously described insertion sequence 711 quantitative PCR. Concordance with 9-locus multi-locus sequence typing was assessed in a subset of samples (n = 18). ST 26 was most commonly identified in adult dolphins along the US east coast with non-suppurative meningoencephalitis (p = 0.009). Animals infected with ST 27 were predominately perinates that were aborted or died shortly after birth with evidence of in utero pneumonia (p = 0.035). Reproductive tract inflammation and meningoencephalitis were also observed in adult T.t. and D.d. with ST 27, though low sample size limited interpretation. ST 23 infections can cause disease in cetacean families other than porpoises (Phocoenidae), including neurobrucellosis in D.d. In total, 11 animals were potentially infected with multiple STs. These data indicate differences in pathogenesis among B. ceti STs in free-ranging cetaceans, and infection with multiple STs is possible.

A review of the toxicology of oil in vertebrates: what we have learned following the Deepwater Horizon oil spill.

In the wake of the Deepwater Horizon (DWH) oil spill, a number of government agencies, academic institutions, consultants, and nonprofit organizations conducted lab- and field-based research to understand the toxic effects of the oil. Lab testing was performed with a variety of fish, birds, turtles, and vertebrate cell lines (as well as invertebrates); field biologists conducted observations on fish, birds, turtles, and marine mammals; and epidemiologists carried out observational studies in humans. Eight years after the spill, scientists and resource managers held a workshop to summarize the similarities and differences in the effects of DWH oil on vertebrate taxa and to identify remaining gaps in our understanding of oil toxicity in wildlife and humans, building upon the cross-taxonomic synthesis initiated during the Natural Resource Damage Assessment. Across the studies, consistency was found in the types of toxic response observed in the different organisms. Impairment of stress responses and adrenal gland function, cardiotoxicity, immune system dysfunction, disruption of blood cells and their function, effects on locomotion, and oxidative damage were observed across taxa. This consistency suggests conservation in the mechanisms of action and disease pathogenesis. From a toxicological perspective, a logical progression of impacts was noted: from molecular and cellular effects that manifest as organ dysfunction, to systemic effects that compromise fitness, growth, reproductive potential, and survival. From a clinical perspective, adverse health effects from DWH oil spill exposure formed a suite of signs/symptomatic responses that at the highest doses/concentrations resulted in multi-organ system failure.

Polyphasic Rhabdomyositis in California Sea Lions (Zalophus Californianus): Pathology and Potential Causes.

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

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.

SARCOCYSTIS NEURONA-ASSOCIATED MENINGOENCEPHALITIS IN A PACIFIC WALRUS ( ODOBENDUS ROSMARUS DIVERGENS).

A 21-yr-old intact male walrus ( Odobendus rosmarus divergens) presented with acute onset of shifting lameness, initially associated with breeding behaviors. Further clinical signs manifested, including muscle tremors, anorexia, hematuria, and coughing. Diagnostics were limited, as the animal would not offer behaviors for voluntary sample collection. Signs were addressed with anti-inflammatories, anticonvulsants, and antibiotics. The walrus developed cluster seizures and ultimately, respiratory and cardiac arrest. Postmortem lesions included meningoencephalitis with intra- and extracellular protozoal zoites and schizonts, as well as interstitial pneumonia with intraendothelial protozoa. Immunolabeling of the protozoal organisms revealed Sarcocystis neurona. Previous S. neurona infections in an odobenid have not been reported. Protozoal infection should be considered in all species of captive marine mammals with nonspecific orthopedic, neurological, and respiratory clinical signs.

Protozoal-related mortalities in endangered Hawaiian monk seals Neomonachus schauinslandi.

Protozoal infections have been widely documented in marine mammals and may cause morbidity and mortality at levels that result in population level effects. The presence and potential impact on the recovery of endangered Hawaiian monk seals Neomonachus schauinslandi by protozoal pathogens was first identified in the carcass of a stranded adult male with disseminated toxoplasmosis and a captive monk seal with hepatitis. We report 7 additional cases and 2 suspect cases of protozoal-related mortality in Hawaiian monk seals between 2001 and 2015, including the first record of vertical transmission in this species. This study establishes case definitions for classification of protozoal infections in Hawaiian monk seals. Histopathology and immunohistochemistry were the primary diagnostic modalities used to define cases, given that these analyses establish a direct link between disease and pathogen presence. Findings were supported by serology and molecular data when available. Toxoplasma gondii was the predominant apicomplexan parasite identified and was associated with 100% of mortalities (n = 8) and 50% of suspect cases (n = 2). Incidental identification of sarcocysts in the skeletal muscle without tissue inflammation occurred in 4 seals, including one co-infected with T. gondii. In 2015, 2 cases of toxoplasmosis were identified ante-mortem and shared similar clinical findings, including hematological abnormalities and histopathology. Protozoal-related mortalities, specifically due to toxoplasmosis, are emerging as a threat to the recovery of this endangered pinniped and other native Hawaiian taxa. By establishing case definitions, this study provides a foundation for measuring the impact of these diseases on Hawaiian monk seals.

Fetal distress and in utero pneumonia in perinatal dolphins during the Northern Gulf of Mexico unusual mortality event.

An unusual mortality event (UME) involving primarily common bottlenose dolphins Tursiops truncatus of all size classes stranding along coastal Louisiana, Mississippi, and Alabama, USA, started in early 2010 and continued into 2014. During this northern Gulf of Mexico UME, a distinct cluster of perinatal dolphins (total body length <115 cm) stranded in Mississippi and Alabama during 2011. The proportion of annual dolphin strandings that were perinates between 2009 and 2013 were compared to baseline strandings (2000-2005). A case-reference study was conducted to compare demographics, histologic lesions, and Brucella sp. infection prevalence in 69 UME perinatal dolphins to findings from 26 reference perinates stranded in South Carolina and Florida outside of the UME area. Compared to reference perinates, UME perinates were more likely to have died in utero or very soon after birth (presence of atelectasis in 88 vs. 15%, p < 0.0001), have fetal distress (87 vs. 27%, p < 0.0001), and have pneumonia not associated with lungworm infection (65 vs. 19%, p = 0.0001). The percentage of perinates with Brucella sp. infections identified via lung PCR was higher among UME perinates stranding in Mississippi and Alabama compared to reference perinates (61 vs. 24%, p = 0.01), and multiple different Brucella omp genetic sequences were identified in UME perinates. These results support that from 2011 to 2013, during the northern Gulf of Mexico UME, bottlenose dolphins were particularly susceptible to late-term pregnancy failures and development of in utero infections including brucellosis.

Histomorphology of the bottlenose dolphin (Tursiops truncatus) pancreas and association of increasing islet ß-cell size with chronic hypercholesterolemia.

Bottlenose dolphins (Tursiops truncatus) can develop metabolic states mimicking prediabetes, including hyperinsulinemia, hyperlipidemia, elevated glucose, and fatty liver disease. Little is known, however, about dolphin pancreatic histomorphology. Distribution and area of islets, α, ß, and δ cells were evaluated in pancreatic tissue from 22 dolphins (mean age 25.7years, range 0-51). Associations of these measurements were evaluated by sex, age, percent high glucose and lipids during the last year of life, and presence or absence of fatty liver disease and islet cell vacuolation. The most common pancreatic lesions identified were exocrine pancreas fibrosis (63.6%) and mild islet cell vacuolation (47.4%); there was no evidence of insulitis or amyloid deposition, changes commonly associated with type 2 diabetes. Dolphin islet architecture appears to be most similar to the pig, where α and ß cells are localized to the central or periphery of the islet, respectively, or are well dispersed throughout the islet. Unlike pigs, large islets (greater than 10,000µm(2)) were common in dolphins, similar to that found in humans. A positive linear association was identified between dolphin age and islet area average, supporting a compensatory response similar to other species. The strongest finding in this study was a positive linear association between islet size, specifically ß-cells, and percent blood samples with high cholesterol (greater than 280mg/dl, R(2)=0.57). This study is the most comprehensive assessment of the dolphin pancreas to date and may help direct future studies, including associations between chronic hypercholesterolemia and ß-cell size.

Herpesvirus and calicivirus infection in a black-footed cat (Fels nigripes) family group following vaccination.

Preventive healthcare recommendations for zoo felids include vaccination against primary viral diseases that affect domestic felids. Although associated with a more-substantial immune response in their domestic counterparts, use of modified live virus vaccines (MLV) in nondomestic carnivores can result in vaccine-induced viral disease. This case report details a feline herpesvirus and calicivirus outbreak in two black-footed cat (Felis nigripes) kittens and their dam following use of an MLV in the kittens. Clinical signs included anorexia, nasal discharge, sneezing, and tachypnea. Patient history, clinical signs, real-time polymerase chain reaction results, and histopathologic evaluation of tissues confirmed the diagnosis. Although unable to definitively prove that the disease observed in these cases was caused by the MLV, there is strong temporal and circumstantial evidence that this was the case. This outbreak of feline upper respiratory infection following vaccination with a multivalent MLV resulted in significant morbidity and mortality, serving as an important reminder that recombinant or killed vaccines are the safest choice for use in nondomestic species, particularly in kittens.

Review of historical unusual mortality events (UMEs) in the Gulf of Mexico (1990-2009): providing context for the multi-year northern Gulf of Mexico cetacean UME declared in 2010.

An unusual mortality event (UME) was declared for cetaceans in the northern Gulf of Mexico (GoM) for Franklin County, Florida, west through Louisiana, USA, beginning in February 2010 and was ongoing as of September 2014. The 'Deepwater Horizon' (DWH) oil spill began on 20 April 2010 in the GoM, raising questions regarding the potential role of the oil spill in the UME. The present study reviews cetacean mortality events that occurred in the GoM prior to 2010 (n = 11), including causes, durations, and some specific test results, to provide a historical context for the current event. The average duration of GoM cetacean UMEs prior to 2010 was 6 mo, and the longest was 17 mo (2005-2006). The highest number of cetacean mortalities recorded during a previous GoM event was 344 (in 1990). In most previous events, dolphin morbillivirus or brevetoxicosis was confirmed or suspected as a causal factor. In contrast, the current northern GoM UME has lasted more than 48 mo and has had more than 1000 reported mortalities within the currently defined spatial and temporal boundaries of the event. Initial results from the current UME do not support either morbillivirus or brevetoxin as primary causes of this event. This review is the first summary of cetacean UMEs in the GoM and provides evidence that the most common causes of previous UMEs are unlikely to be associated with the current UME.

Multifocal Cryptococcus neoformans var. neoforms: infection, treatment, and monitoring by serial computed tomography in a Schmidt's red-tailed guenon (Cercopithecus ascanius schmidti).

A 9-yr-old, female, intact Schmidt's red-tailed guenon (Cercopithecus ascanius schmidti) presented with an acute swelling, consistent with an abscess over the right hip. Cytology, culture, and polymerase chain reaction (PCR) results from material within the abscess and serum titers were all indicative of a Cryptococcus neoformans infection. Thoracic radiographs and computed tomography (CT) revealed a consolidated right caudal lung lobe associated with an endobronchial mass. Pulmonary Cryptococcus infection and systemic disease was confirmed with positive brush cytology and bronchoalveolar lavage (BAL). Culture and sensitivity, minimum inhibitory concentration values, and serum fluconazole level results were used to develop and monitor treatment efficacy for this animal. Sequential thoracic radiographs, CT images, bronchoscopy, BAL, and serology were used to monitor response to therapy. Five months after initial diagnosis, pregnancy was confirmed and the frequency of immobilizations for recheck serology and diagnostic imaging was decreased to lessen radiation exposure and physiologic stress to the dam and fetus. Long-term administration of oral fluconazole was well accepted and effective at treating the multifocal C. neoformans infection. No long-term adverse effects of therapy were observed, and repeated diagnostics were well tolerated. Advanced imaging modalities and serum drug levels were critical to the assessment, monitoring, and treatment of infection in this animal and should be considered for other cases.

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

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

Cardiac assessments of bottlenose dolphins (Tursiops truncatus) in the Northern Gulf of Mexico following exposure to Deepwater Horizon oil.

The Deepwater Horizon (DWH) oil spill profoundly impacted the health of bottlenose dolphins (Tursiops truncatus) in Barataria Bay, LA (BB). To comprehensively assess the cardiac health of dolphins living within the DWH oil spill footprint, techniques for in-water cardiac evaluation were refined with dolphins cared for by the U.S. Navy Marine Mammal Program in 2018 and applied to free-ranging bottlenose dolphins in BB (n = 34) and Sarasota Bay, Florida (SB) (n = 19), a non-oiled reference population. Cardiac auscultation detected systolic murmurs in the majority of dolphins from both sites (88% BB, 89% SB) and echocardiography showed most of the murmurs were innocent flow murmurs attributed to elevated blood flow velocity [1]. Telemetric six-lead electrocardiography detected arrhythmias in BB dolphins (43%) and SB dolphins (31%), all of which were considered low to moderate risk for adverse cardiac events. Echocardiography showed BB dolphins had thinner left ventricular walls, with significant differences in intraventricular septum thickness at the end of diastole (p = 0.002), and left ventricular posterior wall thickness at the end of diastole (p = 0.033). BB dolphins also had smaller left atrial size (p = 0.004), higher prevalence of tricuspid valve prolapse (p = 0.003), higher prevalence of tricuspid valve thickening (p = 0.033), and higher prevalence of aortic valve thickening (p = 0.008). Two dolphins in BB were diagnosed with pulmonary arterial hypertension based on Doppler echocardiography-derived estimates and supporting echocardiographic findings. Histopathology of dolphins who stranded within the DWH oil spill footprint showed a significantly higher prevalence of myocardial fibrosis (p = 0.003), regardless of age, compared to dolphins outside the oil spill footprint. In conclusion, there were substantial cardiac abnormalities identified in BB dolphins which may be related to DWH oil exposure, however, future work is needed to rule out other hypotheses and further elucidate the connection between oil exposure, pulmonary disease, and the observed cardiac abnormalities.

Polyomavirus infection in a free-ranging California sea lion (Zalophus californianus) with intestinal T-cell lymphoma.

An adult female California sea lion (Zalophus californianus) that stranded in central California was found to have a small glossal polypoid mass on gross necropsy. Histologically, the mass was consistent with a fibropapilloma, and intranuclear inclusions were found within endothelial cells lining small arterioles within the mass. Electron microscopy revealed 40-nm virions within endothelial intranuclear inclusions. Rolling circle amplification was used to obtain a partial viral genomic sequence. Sequence analysis identified the virus as a novel polyomavirus, tentatively named California sea lion polyomavirus 1. In addition, the sea lion had a severely thickened small intestine and swollen pale kidneys on gross examination. Severe renal amyloidosis with chronic interstitial nephritis was diagnosed histologically as well as T-cell intestinal lymphoma, which was confirmed via immunophenotyping and molecular clonality. The relationship, if any, between polyomavirus infection and the other disease processes in this sea lion is not known, but it is considered unlikely that the polyomavirus induced the lymphoma.

Complex Virome in a Mesenteric Lymph Node from a Californian Sea Lion (Zalophus Californianus) with Polyserositis and Steatitis.

An emaciated subadult free-ranging California sea lion (Csl or Zalophus californianus) died following stranding with lesions similar to 11 other stranded animals characterized by chronic disseminated granulomatous inflammation with necrotizing steatitis and vasculitis, involving visceral adipose tissues in the thoracic and peritoneal cavities. Histologically, affected tissues had extensive accumulations of macrophages with perivascular lymphocytes, plasma cells, and fewer neutrophils. Using viral metagenomics on a mesenteric lymph node six mammalian viruses were identified consisting of novel parvovirus, polyomavirus, rotavirus, anellovirus, and previously described Csl adenovirus 1 and Csl bocavirus 4. The causal or contributory role of these viruses to the gross and histologic lesions of this sea lion remains to be determined.

Echolocating Whales and Bats Express the Motor Protein Prestin in the Inner Ear: A Potential Marker for Hearing Loss.

Prestin is an integral membrane motor protein located in outer hair cells of the mammalian cochlea. It is responsible for electromotility and required for cochlear amplification. Although prestin works in a cycle-by-cycle mode up to frequencies of at least 79 kHz, it is not known whether or not prestin is required for the extreme high frequencies used by echolocating species. Cetaceans are known to possess a prestin coding gene. However, the expression and distribution pattern of the protein in the cetacean cochlea has not been determined, and the contribution of prestin to echolocation has not yet been resolved. Here we report the expression of the protein prestin in five species of echolocating whales and two species of echolocating bats. Positive labeling in the basolateral membrane of outer hair cells, using three anti-prestin antibodies, was found all along the cochlear spiral in echolocating species. These findings provide morphological evidence that prestin can have a role in cochlear amplification in the basolateral membrane up to 120-180 kHz. In addition, labeling of the cochlea with a combination of anti-prestin, anti-neurofilament, anti-myosin VI and/or phalloidin and DAPI will be useful for detecting potential recent cases of noise-induced hearing loss in stranded cetaceans. This study improves our understanding of the mechanisms involved in sound transduction in echolocating mammals, as well as describing an optimized methodology for detecting cases of hearing loss in stranded marine mammals.

Standardization of Dolphin Cardiac Auscultation and Characterization of Heart Murmurs in Managed and Free-Ranging Bottlenose Dolphins (Tursiops truncatus).

Cardiac auscultation is an important, albeit underutilized tool in aquatic animal medicine due to the many challenges associated with in-water examinations. The aims of this prospective study were to (1) establish an efficient and repeatable in-water cardiac auscultation technique in bottlenose dolphins (Tursiops truncatus), (2) describe the presence and characterization of heart murmurs detected in free-ranging and managed dolphins, and (3) characterize heart murmur etiology through echocardiography in free-ranging dolphins. For technique development, 65 dolphins cared for by the Navy Marine Mammal Program (Navy) were auscultated. The techniques were then applied to two free-ranging dolphin populations during capture-release health assessments: Sarasota Bay, Florida (SB), a reference population, and Barataria Bay, LA (BB), a well-studied population of dolphins impacted by the Deepwater Horizon oil spill. Systolic heart murmurs were detected at a frequent and similar prevalence in all dolphin populations examined (Navy 92%, SB 89%, and BB 88%), and characterized as fixed or dynamic. In all three populations, sternal cranial and left cranial were the most common locations for murmur point of maximal intensity (PMI). An in-water transthoracic echocardiogram technique was refined on a subset of Navy dolphins, and full echocardiographic exams were performed on 17 SB dolphins and 29 BB dolphins, of which, 40 had murmurs. Spectral Doppler was used to measure flow velocities across the outflow tracts, and almost all dolphins with audible murmurs had peak outflow velocities ≥1.6 m/s (95%, 38/40); three dolphins also had medium mitral regurgitation which could be the source of their murmurs. The presence of audible murmurs in most of the free-ranging dolphins (88%) was attributed to high velocity blood flow as seen on echocardiography, similar to a phenomenon described in other athletic species. These innocent murmurs were generally characterized as Grade I-III systolic murmurs with PMI in the left or sternal cranial region. This study is the first to describe an efficient technique for in-water dolphin cardiac auscultation, and to present evidence that heart murmurs are common in bottlenose dolphins.