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A juvenile, male tiger shark ( Galeocerdo cuvier) developed illness after capture in Florida waters and was euthanized. Gross lesions included mild skin abrasions, hepatic atrophy, and coelomic fluid. Histologically, gills contained multifocal lamellar epithelial cell necrosis and thromboses. Scattered gill and esophageal epithelial cells had large, basophilic, intracytoplasmic, and intranuclear inclusions. Ultrastructurally, lamellar epithelial cells contained arrays of intracytoplasmic viral particles and scattered intranuclear nucleocapsids. Capsulated virions were 148 ± 11 nm with an 84 ± 8 nm icosahedral nucleocapsid and an electron-dense core. Next-generation sequencing, quantitative polymerase chain reaction, and in situ hybridization performed on formalin-fixed tissue confirmed a herpes-like viral infection. The viral polymerase shared 24% to 31% protein homology with other alloherpesviruses of fish, indicating a divergent virus. This report documents the pathologic findings associated with a molecularly confirmed novel herpes-like virus in an elasmobranch.

Sand tiger sharks are an iconic large shark species held in aquaria worldwide. They rarely reproduce under managed care, with only seven aquaria reporting limited and sporadic success. For the first time in the Americas, a full-term young was born in an aquarium. The young was the result of breeding among a group of sharks purposefully brought together in 2016 for reproduction. Sharks were maintained in natural seawater and exposed to natural light and seasonal temperature fluctuations similar to their in situ counterparts. Decreased food consumption associated with breeding season and gestation was observed. Gestation time estimated from breeding observations and parturition was 321 days. Although the neonate was stillborn, this was a significant achievement. The husbandry details described within will be useful for other aquaria striving to support the reproduction of sand tiger sharks.

Southern sea otters Enhydra lutris nereis, a threatened marine mammal species, face numerous environmental and infectious disease challenges in their native habitat of coastal California, USA. However, there are few published cases describing neoplasia in sea otters despite their relatively long life span when cared for in aquarium settings. An 18 yr old neutered male southern sea otter, born and raised in human care, presented with an acute onset of seizures and dull mentation. Magnetic resonance imaging of the head revealed a large, central brain lesion. After no improvement with treatment, euthanasia was elected due to a poor prognosis. Grossly, a poorly demarcated, granular, tan mass expanded the cranial meninges in the longitudinal fissure at the level of the cruciate sulcus and extended into the underlying gray matter and superficial white matter. Histologically, the mass was composed of spindle cells, forming haphazardly arranged interlacing bundles and herringbone patterns, with a high mitotic count, moderate cellular pleomorphism, and prominent vascularization. Neoplastic cells demonstrated positive immunoreactivity for vimentin and negative immunoreactivity for smooth muscle actin, factor VIII-related antigen, S100, melan-A, E-cadherin, desmin, glial fibrillary acidic protein, and cytokeratin AE1/AE3. Based on gross, histologic, and immunohistochemical findings, the mass was most consistent with a primary intracranial fibrosarcoma (PIF). PIFs are a rare neoplasm in both humans and other animals with few reports in the veterinary literature. This is the first recorded case of a PIF in a sea otter.

Given the remote, rugged areas belugas typically inhabit and the low rehabilitation success rate with any cetacean, it is rare to have the opportunity to rescue a live-stranded beluga. The Alaska SeaLife Center cared for two stranded beluga calves with two different outcomes. In 2012, a neonatal male beluga calf (DL1202) stranded following intense storms in Bristol Bay. In 2017, a helicopter pilot discovered a stranded male beluga calf (DL1705) during a flight over Cook Inlet. The Alaska SeaLife Center transported both calves for rehabilitation and utilized supportive care plans based on those for other species of stranded cetaceans and care of neonatal belugas at zoological facilities. Diagnostics included complete blood counts, serum chemistries, microbial cultures, hearing tests, imaging and morphometric measurements to monitor systemic health. Treatments included in-pool flotation support; antimicrobials; gastrointestinal support; and close monitoring of respirations, urination, defecation and behaviour. After three weeks of supportive care, the Bristol Bay calf (DL1202) succumbed to sepsis secondary to a possible prematurity-related lack of passive transfer of antibodies. After seven weeks, the Cook Inlet calf (DL1705) recovered and all medications were discontinued. Unable to survive on his own, he was declared non-releasable and placed in long-term care at a zoological facility, to live with other belugas. Aspects and details from successful cases of cetacean critical care become important references especially vital for the survival of essential animals in small, endangered populations.

A juvenile, male tiger shark (

Display of whale sharks in public aquariums demands the largest aquarium habitats yet designed and presents a number of significant challenges. Nonetheless, these facilities offer extraordinary opportunities to advance our understanding of whale sharks in ways that are different from, and complementary to, field research. In this chapter, scientists, veterinarians and husbandry experts from two facilities that house whale sharks present data and lessons from their care in public aquariums, including acquisition, habitat design, feeding, behavior management, veterinary care, growth, and reproduction. Approaches to drawing blood are discussed and normal blood values for hematology, clinical chemistry, and fatty acid composition are presented. Future successful breeding efforts with whale sharks in public aquariums may prove the best and possibly even the only chance to answer some of the most pressing questions about reproduction in the world’s biggest fish. Beyond the scientific opportunities, public aquariums will continue to provide a way to raise the public profile of whale sharks because far more people visit these facilities than can (or should) participate in wildlife tourism with this species.

Florida manatees depend on freshwater environments as a source of drinking water and as warm-water refuges. These freshwater environments are in direct contact with human activities where glyphosate-based herbicides are being used. Glyphosate is the most used herbicide worldwide and it is intensively used in Florida as a sugarcane ripener and to control invasive aquatic plants. The objective of the present study was to determine the concentration of glyphosate and its breakdown product, aminomethylphosphonic acid (AMPA), in Florida manatee plasma and assess their exposure to manatees seeking a warm-water refuge in Crystal River (west central Florida), and in South Florida. We analyzed glyphosate’s and AMPA’s concentrations in Florida manatee plasma (n = 105) collected during 2009–2019 using HPLC-MS/MS. We sampled eight Florida water bodies between 2019 and 2020, three times a year: before, during and after the sugarcane harvest using grab samples and molecular imprinted passive Polar Organic Chemical Integrative Samplers (MIP-POCIS). Glyphosate was present in 55.8% of the sampled Florida manatees’ plasma. The concentration of glyphosate has significantly increased in Florida manatee samples from 2009 until 2019. Glyphosate and AMPA were ubiquitous in water bodies. The concentration of glyphosate and AMPA was higher in South Florida than in Crystal River, particularly before and during the sugarcane harvest when Florida manatees depend on warm water refuges. Based on our results, Florida manatees were chronically exposed to glyphosate and AMPA, during and beyond the glyphosate applications to sugarcane, possibly associated with multiple uses of glyphosate-based herbicides for other crops or to control aquatic weeds. This chronic exposure in Florida water bodies may have consequences for Florida manatees’ immune and renal systems which may further be compounded by other environmental exposures such as red tide or cold stress.

The objective of this study was to determine the pharmacokinetics of a single dose of meloxicam administered subcutaneously (SQ) to three species of sea turtles: loggerheads (Caretta caretta), Kemp's ridley (Lepidochelys kempii), and greens (Chelonia mydas). A dose of 1 mg/kg was given to the Kemp's ridleys and greens, whereas the loggerheads received 2 mg/kg. After SQ administration, the half-life (t1/2) of meloxicam administered at 1 mg/kg in the Kemp's ridleys was 5.51 hr but could not be determined in the greens. The half-life of meloxicam administered at 2 mg/kg in the loggerheads was 2.99 hr. The maximum concentration (Cmax) for meloxicam after SQ administration at 1 mg/kg in the Kemp's ridleys was 6.76 µg/ml and in the greens was 9.35 µg/ml. The Cmax in loggerheads for meloxicam after SQ administration at 2 mg/kg was 3.63 µg/mL. Meloxicam administered SQ at a dose of 1 mg/kg to the Kemp's ridley and greens provided measurable plasma concentrations of meloxicam for 48 and 120 hr, respectively, with no adverse side effects. In loggerheads, meloxicam administered SQ at a dose of 2 mg/kg provided measurable plasma levels of meloxicam for only 24 hr. Plasma levels of meloxicam of greater than 0.5 µg/ml are considered to be therapeutic in humans. Results suggested that administration of meloxicam SQ at 1 mg/kg in Kemp's ridleys and greens would result in plasma concentrations greater than 0.5 µg/ml for 12 and 120 hr, respectively. The administration of 2 mg/kg meloxicam to loggerhead turtles resulted in plasma concentrations greater than 0.5 µg/ml for only 4 hr.

Ecotourism opportunities in the marine environment often rely heavily on provisioning to ensure the viewing of cryptic species by the public. However, intentional feeding of wildlife can impact numerous aspects of an animals’ behavior and ecology. Southern stingrays (Hypanus americana) provisioned at Stingray City Sandbar (SCS) in Grand Cayman have altered diel activity patterns and decreased measures of health. This study looked at seasonal changes in stable isotope (SI) and fatty acid (FA) profiles of provisioned stingrays at SCS. Plasma δ15N was higher in male stingrays (11.86 ± 1.71‰) compared to females (10.70 ± 1.71‰). Lower values for δ15N in males and females were measured in October during low tourist season, suggesting stingrays may be forced to rely on native prey items to supplement the decreased amount of provisioned squid available during this time. Plasma FA profiles were significantly different between sexes and across sampling time points, with FAs 22:6n3, 16:0, 20:5n3, 18:1n3C, 18:0 and 18:1n9T contributing to dissimilarity scores between groups. Dietary FAs primarily contributed to differences between males and females lending further evidence to differences in foraging patterns at SCS, likely due to intraspecific competition. Further, canonical analysis of principal coordinates (CAP) analysis of FA profiles suggest similar diets during peak tourist season and differences in diet between males and females during the low season. This study demonstrates alterations in feeding ecology in stingrays at SCS which is of critical importance for effective management of the SCS aggregation.

Background Host-specific microbiomes play an important role in individual health and ecology; in marine mammals, epidermal microbiomes may be a protective barrier between the host and its aqueous environment. Understanding these epidermal-associated microbial communities, and their ecological- or health-driven variability, is the first step toward developing health indices for rapid assessment of individual or population health. In Cook Inlet, Alaska, an endangered population of beluga whales (Delphinapterus leucas) numbers fewer than 300 animals and continues to decline, despite more than a decade of conservation effort. Characterizing the epidermal microbiome of this species could provide insight into the ecology and health of this endangered population and allow the development of minimally invasive health indicators based on tissue samples. Results We sequenced the hypervariable IV region of bacterial and archaeal SSU rRNA genes from epidermal tissue samples collected from endangered Cook Inlet beluga whales (n = 33) and the nearest neighboring population in Bristol Bay (n = 39) between 2012 and 2018. We examined the sequences using amplicon sequence variant (ASV)-based analyses, and no ASVs were associated with all individuals, indicating a greater degree of epidermal microbiome variability among beluga whales than in previously studied cetacean species and suggesting the absence of a species-specific core microbiome. Epidermal microbiome composition differed significantly between populations and across sampling years. Comparing the microbiomes of Bristol Bay individuals of known health status revealed 11 ASVs associated with potential pathogens that differed in abundance between healthy individuals and those with skin lesions or dermatitis. Molting and non-molting individuals also differed significantly in microbial diversity and the abundance of potential pathogen-associated ASVs, indicating the importance of molting in maintaining skin health. Conclusions We provide novel insights into the dynamics of Alaskan beluga whale epidermal microbial communities. A core epidermal microbiome was not identified across all animals. We characterize microbial dynamics related to population, sampling year and health state including level of skin molting. The results of this study provide a basis for future work to understand the role of the skin microbiome in beluga whale health and to develop health indices for management of the endangered Cook Inlet beluga whales, and cetaceans more broadly.

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.

Assessing the health of marine mammal sentinel species is crucial to understanding the impacts of environmental perturbations on marine ecosystems and human health. In Arctic regions, beluga whales, Delphinapterus leucas, are upper level predators that may serve as a sentinel species, potentially forecasting impacts on human health. While gene expression profiling from blood transcriptomes has widely been used to assess health status and environmental exposures in human and veterinary medicine, its use in wildlife has been limited due to the lack of available genomes and baseline data. To this end we constructed the first beluga whale blood transcriptome de novo from samples collected during annual health assessments of the healthy Bristol Bay, AK stock during 2012-2014 to establish baseline information on the content and variation of the beluga whale blood transcriptome. The Trinity transcriptome assembly from beluga was comprised of 91,325 transcripts that represented a wide array of cellular functions and processes and was extremely similar in content to the blood transcriptome of another cetacean, the bottlenose dolphin. Expression of hemoglobin transcripts was much lower in beluga (25.6% of TPM, transcripts per million) than has been observed in many other mammals. A T12A amino acid substitution in the HBB sequence of beluga whales, but not bottlenose dolphins, was identified and may play a role in low temperature adaptation. The beluga blood transcriptome was extremely stable between sex and year, with no apparent clustering of samples by principle components analysis and

Understanding the fundamental reproductive biology of a species is the first step toward identifying parameters that are critical for reproduction and for the development of assisted reproductive techniques. Ejaculates were collected from aquarium (n = 24) and in situ (n = 34) sand tiger sharks Carcharias taurus. Volume, pH, osmolarity, sperm concentration, motility, status, morphology, and plasma membrane integrity were assessed for each ejaculate. Semen with the highest proportion of motile sperm was collected between April and June for both in situ and aquarium sand tiger sharks indicating a seasonal reproductive cycle. Overall, 17 of 30 semen samples collected from aquarium sharks from April through June contained motile sperm compared to 29 of 29 of in situ sharks, demonstrating semen quality differences between aquarium and in situ sharks. Sperm motility, status, morphology, and plasma membrane integrity were significantly higher (P

International audience; Aspergillosis is a fungal infection caused by Aspergillus molds that can affect both humans and animals. Despite advances in diagnostics and therapy, medical management of this disease remains difficult. Expansion of the basic knowledge regarding its pathophysiology in animals is critical to aid in the identification of new bio-markers of infection for diagnosis and therapeutic targets. For such a purpose, proteomics can be used by addressing protein changes during various disease processes. In the present study, a mass spectrometry analysis based on isobaric tagging for relative and absolute quantitation (iTRAQ ®) was applied for direct identification and relative quantitation of proteins in blood collected from 32 Aspergillus-diseased common bottlenose dolphins (Tursiops truncatus, 32 samples) in comparison with blood from 55 other dolphins (55 samples from 41 clinically-normal controls and from 14 cetaceans with miscellaneous non-Aspergillus inflammation diseases) and ten convalescent dolphins (28 samples). Sixty-six and 40 proteins were found to be ≥2.0-fold over-and under-represented versus miscellaneous non-Aspergillus inflammatory dolphins, respectively, and most were confirmed vs. clinically-normal controls and convalescents. Many proteins which play a role in the adaptive immune response were identified, including MHC proteins and others involved in catalytic activity like the NADPH-ubi-quinone oxido-reductases. Overall, iTRAQ ® appears to be a convenient proteomic tool greatly suited for exploratory ex vivo studies focusing on pathophysiology. This technique should be considered as a preliminary step before validation of new diagnostic markers.

Blood and blubber were sampled from live-captured beluga whales in Bristol Bay, Alaska. Progesterone levels were used to assess pregnancy status. For most cases, blubber progesterone levels correlated with serum, indicating that pregnancy can be assessed using a remote biopsy and thereby providing a valuable tool to better understand reproduction dynamics from populations that cannot be readily captured for examination., The Cook Inlet population of beluga whales (Delphinapterus leucas) is listed as endangered and continues to decline for largely unknown reasons; however, there is some evidence that poor reproductive success is a contributing factor. Pregnancy is difficult to detect through observation, and, there is reluctance to capture endangered beluga whales for reproductive tract imaging via ultrasound or to obtain suitable samples for pregnancy assessments. An endocrine analysis of blubber biopsies collected by remote darting could represent a minimally invasive way to identify pregnant females and compare pregnancy rates among years or populations. Studies have validated the use of blubber biopsies to identify pregnant females in other cetacean species, but not beluga whales; therefore, validation of blubber progesterone levels to proven tests that reliably detect pregnancy was needed for this species. As part of a larger study, we sampled blood and blubber from live-captured beluga whales (21 females, 26 males) in Bristol Bay, Alaska. Progesterone levels were determined in serum samples obtained from all animals and in blubber samples from a subset (14 females, 13 males) to determine pregnancy status, estimate the stage of pregnancy, and evaluate the suitability of using blubber alone for these assessments. In general, there was distinct separation of high levels of progesterone in serum and blubber for presumed pregnant females and low levels for males and presumed non-pregnant females. Blubber progesterone levels in two females (14% of females tested) were intermediate (i.e. ambiguous); their corresponding serum levels were consistent with being pregnant in one case and not being pregnant in the other. Except for these two intermediate values, pregnancy status of beluga whales could be determined from blubber alone, thereby providing a valuable tool to better understand reproduction dynamics from populations that cannot be captured for examination.

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.

Aspergillosis is a common respiratory fungal disease in penguins managed under human care. Triazole antifungal drugs, including itraconazole, are most commonly used for treatment; however, itraconazole treatment failures from drug resistance are becoming more common, requiring newer treatment options. Voriconazole, a newer triazole, is being used more often. Until recently, no voriconazole pharmacokinetic studies had been performed in penguins, leading to empiric dosing based on other avian studies. This has led to increased anecdotal reporting of apparent voriconazole toxicity in penguins. This report describes 18 probable and 6 suspected cases of voriconazole toxicity in six penguin species from nine institutions: 12 African penguins (Spheniscus demersus), 5 Humboldt penguins (Spheniscus humboldti), 3 Magellanic penguins (Spheniscus magellanicus), 2 gentoo penguins (Pygoscelis papua papua), 1 macaroni penguin (Eudyptes chrysolophus), and 1 emperor penguin (Aptenodytes forsteri). Observed clinical signs of toxicity included anorexia, lethargy, weakness, ataxia, paresis, apparent vision changes, seizure-like activity, and generalized seizures. Similar signs of toxicity have also been reported in humans, in whom voriconazole therapeutic plasma concentration for Aspergillus spp. infections is 2-6 μg/ml. Plasma voriconazole concentrations were measured in 18 samples from penguins showing clinical signs suggestive of voriconazole toxicity. The concentrations ranged from 8.12 to 64.17 μg/ml, with penguins having plasma concentrations above 30 μg/ml exhibiting moderate to severe neurologic signs, including ataxia, paresis, and seizures. These concentrations were well above those known to result in central nervous system toxicity, including encephalopathy, in humans. This case series highlights the importance of species-specific dosing of voriconazole in penguins and plasma therapeutic drug monitoring. Further investigation, including pharmacokinetic studies, is warranted. The authors recommend caution in determining voriconazole dosages for use in penguin species.

Aspergillosis is a fungal infection with high mortality and morbidity rates. As in humans, its definitive diagnosis is difficult in animals, and thus new laboratory tools are required to overcome the diagnostic limitations due to low specificity and lack of standardization. In this study of common bottlenose dolphins (Tursiops truncatus), we evaluated the diagnostic performance of a new commercial immunoblot kit that had been initially developed for the serologic diagnosis of chronic aspergillosis in humans. Using this in a quantitative approach, we first established its positive cutoff within an observation cohort of 32 serum samples from dolphins with "proven" or "probable" diagnosis of aspergillosis and 55 negative controls. A novel enzyme-linked immunosorbent assay (ELISA) test was also developed for detecting anti-Aspergillus antibodies, and results were compared between the two assays. Overall, the diagnostic performance of immunoblot and ELISA were strongly correlated (P < .0001). The former showed lower sensitivity (65.6% versus 90.6%), but higher specificity (92.7% vs. 69.1%), with no cross-reaction with other fungal infections caused by miscellaneous non-Aspergillus genera. When assessing their use in a validation cohort, the immunoblot kit and the ELISA enabled positive diagnosis before mycological cultures in 42.9% and 33.3% subjects addressed for suspicion of aspergillosis, respectively. There was also significant impact of antifungal treatment on the results of the two tests (P < .05). In all, these new serological methods show promise in aiding in the diagnosis of aspergillosis in dolphins, and illustrate the opportunity to adapt commercial reagents directed for human diagnostics to detect similar changes in other animals.

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, β 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.

Objective—To evaluate the efficacy and safety of a combination of dexmedetomidine, butorphanol, and midazolam administered IM for anesthesia in captive Asian small-clawed otters (Aonyx cinereus) and to compare this combination with a combination of ketamine and midazolam. Design—Prospective crossover study. Animals—10 captive Asian small-clawed otters. Procedures—A combination of either dexmedetomidine (0.03 mg/kg [0.014 mg/lb]), butorphanol (0.2 mg/kg [0.091 mg/lb]), and midazolam (0.15 mg/kg [0.068 mg/lb]) or ketamine (10.1 mg/kg [4.59 mg/lb]) and midazolam (0.3 mg/kg [0.14 mg/lb]) was administered IM to otters for immobilization to allow scheduled wellness examinations. Otters were intubated and administered 100% oxygen during the examination. Anesthesia was supplemented with isoflurane in oxygen if necessary. Routine medical procedures, including blood collection, radiography, echocardiography, dental scaling, vaccinations, and contraception administration, were performed as indicated during the immobilization. Physiologic, clinicopathologic, and anesthetic variables were recorded and compared. Otters given dexmedetomidine-butorphanol-midazolam were administered atipamezole (0.2 mg/kg [0.091 mg/lb]), naltrexone (0.6 mg/kg [0.27 mg/lb]), and flumazenil (0.05 mg/kg [0.023 mg/lb]) IM at the completion of the examination. Results—The need for and duration of isoflurane administration were greater for ketamine-midazolam anesthesia, compared with dexmedetomidine-butorphanol-midazolam anesthesia. Recoveries were shorter and subjectively smoother with dexmedetomidine-butorphanol-midazolam. Heart rates were significantly higher during ketamine-midazolam anesthesia. Regardless of protocol, all otters developed hypothermia and hypercapnia during anesthesia. Conclusions and Clinical Relevance—Both protocols were safe and effective for this species, but the reversible nature of dexmedetomidine-butorphanol-midazolam resulted in more rapid recoveries than did ketamine-midazolam. Otters anesthetized with ketamine-midazolam may require additional anesthetic medications for routine examinations, and assisted ventilation and thermal support may be of benefit with either protocol.

Acute phase proteins (APPs) have been studied in many companion and large animals and have been reported to have a differential sensitivity to traditional markers of inflammation. Studies have been performed indicating the conservation of these proteins as well as the application and cross-reactivity of automated assays among different species, but few reports have detailed APPs in marine mammal species. In the present study, automated assays were utilized to generate reference intervals for C-reactive protein, haptoglobin, and serum amyloid A using 44 serum samples from healthy Atlantic bottlenose dolphins ( Tursiops truncatus). A total of 25 samples were obtained from dolphins under human care and 19 samples were obtained from free-ranging dolphins. Mild yet statistically significant differences were observed in levels of haptoglobin and serum amyloid A between these groups. The reference intervals from the combined groups were as follows: C-reactive protein 3.1–19.7 mg/l, haptoglobin 0–0.37mg/ml, and serum amyloid A 17.5–42.9 mg/l. These baseline data should provide an important foundation for future studies of the application of APP quantitation in monitoring the health and stressors of dolphins under human care and with live capture of free-ranging dolphins.

Serum protein electrophoresis (SPEP) has been recognized as an important tool in human and veterinary medicine. The present study investigated the use of SPEP in serum samples from healthy Atlantic bottlenose dolphins (Tursiops truncatus). Fraction delimitation was defined for the standardization of use by other laboratories. The imprecision of this method was comparable to reports in other species. A significant difference between albumin levels determined by SPEP and the traditional chemistry analyzer method (bromcresol green [BCG]) was observed (BCG = 3.38 ± 0.46 g/dL, and SPEP = 3.74 ± 0.43 g/dL, p < 0.0001). Bland-Altman analysis also showed that these two methods were not identical. Notably, several differences were observed between SPEP-derived values using samples from dolphins under human care vs free-ranging dolphins. The total protein was significantly increased in serum from free-ranging dolphins, and the A/G ratio was found to be significantly decreased (under human care: 1.91 ± 0.39 g/dL, free ranging: 1.07 ± 0.39 g/dL, p ≤ 0.05). The latter change was related to a significantly lower albumin fraction and 2.3fold increase in gamma globulins. In total, this study provides method standardization and preliminary data toward the generation of reference intervals for this species.

A 19-year-old male African penguin ( Spheniscus demersus ) was presented with coelomic distention after a 6-week history of lethargy and decreased appetite. Results of radiographs showed loss of coelomic detail, and ultrasound and computed tomography results revealed coelomic fluid and dilated hepatic veins. Echocardiography revealed moderate right atrial enlargement. Findings were consistent with right-sided cardiac disease. Treatment with furosemide initially reduced ascites, but the clinical condition worsened weeks later and enalapril, pimobendan, and sildenafil were added to the medical therapy. At 12 weeks after presentation, results of an echocardiogram revealed persistent right atrioventricular valve regurgitation, moderate ascites, and dilation of hepatic veins. Clinical signs of right heart failure were managed through adjustments in medical therapy and coelomic fluid aspiration, but the bird died 18 weeks after initial presentation. Gross and microscopic findings were consistent with valvular insufficiency and right-sided heart failure. To our knowledge, this case is the first documented report of cardiac disease in an African penguin.

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.

We developed a long-term tagging method that can be used to understand species assemblages and social groupings associated with large marine fishes such as the Sand Tiger shark Carcharias taurus. We deployed internally implanted archival VEMCO Mobile Transceivers (VMTs; VEMCO Ltd. Nova Scotia, Canada) in 20 adult Sand Tigers, of which two tags were successfully recovered (10%). The recovered VMTs recorded 29,646 and 44,210 detections of telemetered animals respectively. To our knowledge, this is the first study to demonstrate a method for long-term (~ 1 year) archival acoustic transceiver tag implantation, retention, and recovery in a highly migratory marine fish. Results show low presumed mortality (n = 1, 5%), high VMT retention, and that non-lethal recovery after almost a year at liberty can be achieved for archival acoustic transceivers. This method can be applied to study the social interactions and behavioral ecology of large marine fishes.

During a 4 mo epizootic, 100% of 152 lined seahorses Hippocampus erectus in 3 separate groups died while in quarantine following shipment to a public aquarium. Twelve animals with skin depigmentation and ulceration were received by the Aquatic Pathology Service, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA, for diagnostic evaluation. Microscopically, lesions in 11 seahorses included multifocal epithelial necrosis and ulceration associated with 2 to 7 µm diameter, branching, septate fungal hyphae, typically accompanied by deeper infiltration into underlying skeletal muscle. Angioinvasion, with vascular thrombosis and tissue infarction, was a prominent feature in multiple animals. Fungal invasion of one or more internal organs was observed in 4 animals. Hyphae appeared to course freely through tissues and elicited little or no inflammatory response. Fusariosis has been reported sporadically in fish and other aquatic organisms, but identification has often been limited to the genus level based solely on morphologic features. Morphologic characteristics of the fungus isolated from this case were consistent with the Fusarium solani species complex (FSSC), which includes over 50 members that can only be identified definitively using DNA sequence data. A 3-locus typing scheme identified the isolate as a distinct species/haplotype, designated FSSC 12-a, belonging to a specific lineage that appears adapted to aquatic environments and disease in marine animals. Empirical treatment with itraconazole failed to stop mortalities, and subsequent in vitro antifungal susceptibility data explained a lack of clinical efficacy for this agent. Effective treatment in human medicine has similarly been limited by poor susceptibility to several classes of antifungal compounds.

In this report, two cases of systemic mycosis in captive sharks are characterized. These cases were progressive and ultimately culminated in terminal disease. Paecilomyces lilacinus, an uncommon pathogen in human and veterinary medicine, was associated with areas of necrosis in the liver, heart, and gill in a great hammerhead shark (Sphyrna mokarran). Fungal growth was observed from samples of kidney, spleen, spinal fluid, and coelomic cavity swabs. Dual fungal infection by Exophiala pisciphila and Mucor circinelloides was diagnosed in a juvenile zebra shark (Stegostoma fasciatum). Both fungi were present in the liver, with more severe tissue destruction associated with E. pisciphila. E. pisciphila also produced significant necrosis in the spleen and gill, while M. circinelloides was associated with only minimal tissue changes in the heart. Fungal cultures from liver, kidney, and spleen were positive for both E. pisciphila and M. circinelloides. Identification of P. lilacinus and M. circinelloides was based on colonial and hyphal morphology. E. pisciphila was identified by sequence analysis of the 28S rRNA D1/D2 region and the internal transcribed spacer (ITS) region between the 18S and 28S rRNA subunit. These cases, and a lack of information in the literature, highlight the need for further research and diagnostic sampling to further characterize the host-pathogen interaction between elasmobranchs and fungi.

A captive adult crevice kelpfish, Gibbonsia montereyensis, developed a cutaneous mass, approximately 9 × 7 mm on the right side of the head in an area of nonscaled skin. Following surgical debulking, examination of both impression smears and histologic sections of the tumor revealed a predominant population of round to spindloid to polygonal cells with a moderate amount of lightly basophilic cytoplasm. The cytoplasm was filled with round, variably-sized reddish-brown granules that often obscured the nucleus. Nuclei were round to ovoid with coarsely granular chromatin. There was minimal anisocytosis and anisokaryosis. The cytoplasmic granules in histologic sections were weakly positive by the Fontana-Masson method, and staining was eliminated with melanin bleach. Immunohistochemical staining was strongly positive with a murine monoclonal antibody for melan A. As the specificity of melan A for melanophores is not clearly defined in nonmammalian species, the tumor was examined by transmission electron microscopy. Melanophores were not detected. Instead, neoplastic cells were filled with numerous intracytoplasmic organelles with triple-limiting membranes composed of concentric lamellae; these structures were most compatible with pterinosomes, which are the pigment-containing organelles of cells called xanthophores and erythrophores. As both of these organelles are ultrastructurally indistinguishable and as kelpfish skin is known to contain both xanthophores and erythrophores, a diagnosis of a mixed pigment cell tumor or chromatophoroma was made. As the tumor was grossly reddish-brown, the possibility of a neoplastic population of only erythrophores could not be excluded. Pigment cell tumors, arising from cells of the embryonic neural crest, are common in reptiles and bony fish.

Hematology can be a useful tool for monitoring health status, detecting illness, and following the progress of disease and response to therapy. Despite advances in fish medicine in recent years, interpretation of fish hematology often is hampered by a lack of meaningful reference values and the bewildering diversity of fish species. A multitude of intrinsic and extrinsic factors cause normal and abnormal variation in hematologic data. This article provides an overview of some of the hematologic abnormalities in fish induced by infectious agents and environmental, husbandry, and nutritional issues.

Although lungworms are known to infect many mammalian species and a few are known to infect snakes, lizards, and birds, previously none were known to infect chelonians. This study documents the first three known cases of lungworms in loggerhead sea turtles, Caretta caretta. It is unlikely that the lungworms were the primary cause of illness in any of the cases, and they may be only contributory or even incidental. Changes observed in the two cases that died included tracheal and bronchial epithelial hyperplasia and goblet cell hyperplasia. Lesions caused directly by the parasites seem to be restricted to the upper respiratory tree (trachea and main bronchi), but changes in the lungs themselves may be caused by the debris produced by the worms. Although neither case was successfully treated for the lungworms prior to death, it would appear that oxfendazole may be an effective treatment and has been used in the third case that is still undergoing rehabilitation.

Our study was undertaken to assess the application of semiautomated methods available at the reference laboratory level for the evaluation of plasma protein and cholesterol via electrophoresis in samples from cownose rays ( Rhinoptera bonasus). Three groups of animals were assessed: clinically normal, clinically abnormal, and parasitized with leeches. As reported previously, the albumin band was negligible; the protein electrophoretograms were dominated by a large beta-globulin fraction. While the group of samples from the leech-parasitized rays did not show any large differences, the abnormal group exhibited significantly elevated total solids and cholesterol levels. The latter was related to a significant increase in very low density lipoprotein levels. The results demonstrate the potential application of these laboratory methods in quantitation of plasma proteins and cholesterol fractions in subclass Elasmobranchii.

A 7.5 kg common carp Cyprinus carpio presented with prominent localized swelling in the caudal right coelomic area, identified by ultrasound as a fluid filled mass. Fine needle aspirate (FNA) and culture results suggested a sterile seroma. Centesis removed 290 ml of serosanguinous fluid that returned within days. Recheck ultrasonography revealed a solid component within the cavity. Radiography demonstrated irregular lysis and misalignment of vertebrae adjacent to the mass, most suggestive of bacterial osteitis or neoplasia. Treatment with antibiotics followed for 2 mo but failed to resolve the lesion. Repeated radiography and ultrasonography showed progressive enlargement of the mass, with vertebral lysis and invasion characteristic of neoplasia. Ultrasound-guided FNA of the solid component of the mass was non-diagnostic. Euthanasia was elected due to the poor clinical response and primary differential of neoplasia. Post-mortem MRI and CT confirmed a retroperitoneal soft tissue mass, partially surrounded by a fluid-filled cavity, causing vertebral lysis and infiltration of the spinal canal. Expansion of the mass caused severe muscle loss and an associated elevation in creatine kinase (>120000 U l-1). Necropsy results corroborated the MRI and CT findings, revealing a retroperitoneal, multilobular, red and tan mass causing dorsal displacement of the vertebral column, with vertebral lysis, pathologic fracture and invasion of the spinal canal. Histopathologic examination revealed a locally aggressive neoplasm exhibiting multiple patterns of growth, including endothelial lined vascular channels and solid areas formed by more pleomorphic polygonal and spindle cells, consistent with hemangiosarcoma.

A novel surgical technique was used in the routine castrations of two intact male southern sea otters, Enhydra lutris nereis, housed at the Georgia Aquarium (Atlanta, Georgia, USA). This technique involved incising the parietal vaginal tunic to allow placement of double ligation of the ductus deferens, testicular artery, and pampiniform plexus en masse. After ligating and transecting these structures, they were introduced back into the tunic, which was closed with a circumferential ligature. The incision site was closed in a routine manner. Both otters recovered well from the procedure. One otter had mild cutaneous dehiscence postoperatively, and the other had no obvious complications. Benefits of this procedure include reduced risk of ligature slippage or loosening and resultant hemorrhage, as provided by the traditional open portion of the castration, and decreased postoperative swelling, as provided by the closed part of the castration. To the authors' knowledge, this is the first time this technique has been described for use in sea otters.

This report details 2 cases of epitheliocystis in spotted eagle rays Aetobatus narinari associated with a novel Chlamydiales 16S rDNA signature sequence. Epitheliocystis is a common disease of variable severity affecting >50 species of wild and cultured freshwater and marine teleosts. Disease in elasmobranchs is rarely reported and descriptions are limited. Occurring in gill and skin epithelium, lesions are characterized by large hypertrophied cells with basophilic inclu- sions containing Gram-negative, chlamydia-like bacteria. Acute lethargy, labored respiration, and abnormal swimming developed in a captive spotted eagle ray following an uneventful quarantine period, and mild epitheliocystis lesions were found microscopically. Three months later, a second animal exhibited similar signs. A gill clip revealed myriad spherical bodies identical to the previous case, and treatment with chloramphenicol and oxytetracycline was initiated. Despite therapy, res- piration became irregular and euthanasia was elected. Histologically, epitheliocystis inclusions up to 200 µm filled approximately 80% of lamellar troughs. Multifocal mild hypertrophy and hyper - plasia of lamellar tips was accompanied by mild to moderate infiltrates of granulocytes and lym - phocytes. Electron microscopy revealed a homogeneous population of elongate chlamydia-like bacterial forms similar in size and morphology to the primary long cells described in teleosts. Immunohistochemical staining with a polyclonal anti-chlamydial lipopolysaccharide antibody was positive. Sequence analysis of a unique 296 bp Chlamydiales signature sequence amplicon isolated from the rays showed greatest homology (85 to 87%) to 'Candidatus Piscichlamydia salmonis'.

Infections caused by Piscirickettsia-like organisms (PLO) appeared in Taiwanese tilapia, Oreochromis spp., culture in the early 1990s (Chen et al. 1994; Chern & Chao 1994). Additional reports quickly appeared making it evident that PLO posed an emerging threat to a range of fish species in both fresh and salt water (Mauel & Miller 2002). Early reports were based largely on gross and histologic features of the disease and failure of the organisms to react serologically or in PCR assays for Piscirickettsia salmonis. Improved culture and molecular detection methods determined later PLO cases to be caused by Francisella spp. bacteria capable of survival and growth within macrophages (Ottem et al. 2009; Birkbeck, Feist & Verner-Jeffreys 2011; Colquhoun & Duodu 2011). Taxonomic clarification of the pleomorphic 0.5to 1.7-lm bacteria indicates a single species, Francisella noatunensis, for all fish isolates. Two subspecies are recognized, F. noatunensis subsp. noatunensis (Fnn) and F. noatunensis subsp. orientalis (Fno), with apparent predilections for cold and warmwater fish, respectively (Ottem et al. 2009). Regardless of the bacterial subspecies involved, infections share clinical and pathologic features, including acute to chronic mortalities accompanied by non-specific signs. Most notable grossly is splenomegaly and renomegaly, with whitish-tan nodules corresponding histologically to granulomas typified by vacuolated macrophages containing the bacteria (Birkbeck et al. 2011; Colquhoun & Duodu 2011). Some granulomas develop cuffs of lymphocytes (Mauel et al. 2007) and with time may undergo central necrosis and fibrous encapsulation (Hsieh et al. 2006; Birkbeck et al. 2011). The spleen and kidney are most severely affected, although lesions have been described in all organ systems (Birkbeck et al. 2011; Colquhoun & Duodu 2011). Evidence for a PLO induced disease, suggestive of francisellosis, in the continental United States was reported from California in 1998 involving white seabass, Atractoscion nobilis (Ayres) (Chen et al. 2000). To the best of our knowledge, however, francisellosis has not emerged in the marine tropical fish trade in this country, although movements of live fish are likely involved in dissemination of the agent, and it has been predicted that the number of affected species will continue to rise (Birkbeck et al. 2011). This report describes Fno infections diagnosed in Indo-Pacific reef fish following international shipment to and distribution in the United States from Asian waters. Correspondence E Soto, Ross University, School of Veterinary Medicine, Department of Pathobiology, P.O. Box 334, Basseterre, St. Kitts, West Indies (e-mail: esoto@rossvet.edu.kn)

Sex-steroid hormones are essential for normal reproductive activity in both sexes in all vertebrates. Estrogens are required for ovarian differentiation during a critical developmental stage and promote the growth and differentiation of the female reproductive system following puberty. Recent studies have shown that environmental estrogens influence the developing reproductive system as well as gametogenesis, especially in males. To understand the molecular mechanisms of estrogen actions and to evaluate estrogen receptor-ligand interactions in Elasmobranchii, we cloned a single estrogen receptor (ESR) from two shark species, the cloudy catshark (Scyliorhinus torazame) and whale shark (Rhincodon typus) and used an ERE-luciferase reporter assay system to characterize the interaction of these receptors with steroidal and other environmental estrogens. In the transient transfection ERE-luciferase reporter assay system, both shark ESR proteins displayed estrogen-dependent activation of transcription, and shark ESRs were more sensitive to 17beta-estradiol compared with other natural and synthetic estrogens. Further, the environmental chemicals, bisphenol A, nonylphenol, octylphenol and DDT could activate both shark ESRs. The assay system provides a tool for future studies examining the receptor-ligand interactions and estrogen disrupting mechanisms in Elasmobranchii.