DIAGNOSIS AND MANAGEMENT OF SUSPECTED CONGESTIVE HEART FAILURE SECONDARY TO DILATED CARDIOMYOPATHY IN A SAND TIGER SHARK (CARCHARIAS TAURUS) WITH ESTABLISHMENT OF PRELIMINARY NORMAL ECHOCARDIOGRAPHIC INDICES.

Elasmobranch cardiac anatomy and physiology has been well described; however, there is a dearth of information regarding cardiac disease. In support of a clinical case of suspected congestive heart failure in a 22-yr-old male sand tiger shark (Carcharias taurus), a study was undertaken to identify feasible echocardiographic imaging planes and preliminary indices for this species. Eleven echocardiograms were performed on six apparently healthy sand tiger sharks. Echocardiographic parameters are presented using descriptive statistics, including mean, median, standard deviation (SD), minimum and maximum values. These data were utilized for the diagnosis and clinical management of the affected shark. The shark initially presented with increased respiratory effort, dependent, peripheral edema, and anemia. Echocardiography revealed atrial, ventricular, and sinus venosus dilation. As congestive heart failure secondary to dilated cardiomyopathy was strongly suspected, therapy was initiated with oral benazepril and torsemide, and later pimobendan. After a year of therapy, clinical signs resolved. Cardiac size and function improved on echocardiography with a reduction in sinus venosus dilation, maximum and minimum atrial and ventricular inner diameters, and an increase in atrial and ventricular fractional shortening. Cardiac disease in elasmobranchs may be underdiagnosed, so it may be necessary to develop standardized ultrasound techniques and cardiac measurements for each species of elasmobranch managed within zoos and aquaria.

Development of a quantitative polymerase chain reaction assay for detection of the aetiological agents of piscine lactococcosis.

Piscine lactococcosis is an emergent bacterial disease that is associated with high economic losses in many farmed and wild aquatic species worldwide. Early and accurate detection of the causative agent of piscine lactococcosis is essential for management of the disease in fish farms. In this study, a TaqMan quantitative polymerase chain reaction (qPCR) targeting the 16S-23S rRNA internal transcribed spacer region was developed and validated. Validation of the qPCR was performed with DNA of previously typed L. petauri and L. garvieae recovered from different aquatic hosts from distinct geographical locations, closely related bacterial species and common pathogens in trout aquaculture. Further diagnostic sensitivity and specificity was investigated by screening of fish, water and faecal samples. The developed qPCR assay showed high specificity, sensitivity and accuracy in detection of L. petauri and L. garvieae with lack of signals from non-target pathogens, and in screening of rainbow trout (Oncorhynchus mykiss) posterior kidney and environmental samples. The detection limit of the qPCR was four amplicon copies. Moreover, the sensitivity of the qPCR assay was not affected by presence of non-target DNA from either fish or environmental samples. The robustness, specificity and sensitivity of the developed qPCR will facilitate fast and accurate diagnosis of piscine lactococcosis to establish appropriate control measures in fish farms and aquaria.

Artificial insemination and parthenogenesis in the whitespotted bamboo shark Chiloscyllium plagiosum.

Non-lethal methods for semen collection from elasmobranchs to better understand species reproduction has accompanied the development of artificial insemination. Ejaculates (n = 82) collected from whitespotted bamboo sharks Chiloscyllium plagiosum (n = 19) were assessed and cold-stored raw or extended at 4 °C. Females (n = 20) were inseminated with fresh or 24-48 h cold-stored raw or extended semen and paternity of offspring determined with microsatellite markers. Insemination of females with fresh semen (n = 10) resulted in 80 hatchlings and 27.6% fertility. Insemination of females with semen cold-stored 24 h (n = 4) and 48 h (n = 1) semen resulted in 17 hatchlings and fertilization rates of 28.1% and 7.1% respectively. Two females inseminated with fresh or cold-stored semen laid eggs that hatched from fertilization and parthenogenesis within the same clutch. Parthenogenesis rate for inseminated females was 0.71%. Results demonstrate artificial insemination with cold-stored semen can provide a strategy for transport of male genetics nationally and internationally, precluding the need to transport sharks. Production of parthenotes in the same clutch as sexually fertilized eggs highlights the prevalence of parthenogenesis in whitespotted bamboo sharks and poses important considerations for population management.

Testosterone and semen seasonality for the sand tiger shark Carcharias taurus†.

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 < 0.05) for in situ compared to aquarium sand tiger sharks. Testosterone was measured by an enzyme immunoassay validated for the species. Testosterone concentration was seasonal for both aquarium and in situ sharks with highest concentrations measured in spring and lowest in summer. In situ sharks had higher (P < 0.05) testosterone concentration in spring than aquarium sharks. This study demonstrated annual reproduction with spring seasonality for male sand tiger sharks through marked seasonal differences in testosterone and semen production. Lower testosterone and poorer semen quality was observed in aquarium sharks likely contributing to the species' limited reproductive success in aquariums.

Fatal Sarcocystis falcatula Infection in Three Penguins.

Sarcocystis falcatula is a well-known cause of fatal pneumonia in some birds, particularly Old World psittacines. Here we describe fatal sarcosystosis due to S. falcatula in 3 penguins (Family Spheniscidae) under managed care, including one African penguin (Spheniscus demersus), and two Southern rockhopper penguins (Eudyptes chrysocome). Randomly distributed foci of necrosis, inflammatory cell infiltrates, edema, and variable numbers of round to elongated protozoal schizonts were observed in sections of lung. Protozoal organisms exhibited strong immunoreactivity for Sarcocystis sp. antigen by immunohistochemistry. Apicomplexan and Sarcocystis genus-specific PCR assays and sequence analysis confirmed S. falcatula as the etiologic agent. These cases of fatal pneumonia attributed to S. falcatula expand the list of aberrant intermediate avian hosts, with particular implications for penguins.

Diagnosis and treatment of multi-species fish mortality attributed to Enteromyxum leei while in quarantine at a US aquarium.

Enteromyxum leei is an enteric myxozoan parasite of fish. This myxozoan has low host specificity and is the causative agent of myxozoan emaciation disease, known for heavy mortalities and significant financial losses within Mediterranean, Red Sea, and Asian aquaculture industries. The disease has rarely been documented within public aquaria and, to our knowledge, has never been confirmed within the USA. This case report describes an outbreak of E. leei in a population of mixed-species east African/Indo-Pacific marine fish undergoing quarantine at a public aquarium within the USA. Four of 16 different species of fish in the population, each of a different taxonomic family, were confirmed infected by the myxozoan through cloacal flush or intestinal wet mount cytology at necropsy. Clinical and histopathological findings in this case are similar to previous findings describing myxozoan emaciation disease, e.g. severe emaciation, cachexia, enteritis, and death. Sequence analysis of the 18S rDNA of intestinal samples from a powder blue tang Acanthurus leucosternon and an emperor angelfish Pomacanthus imperator confirmed the parasite to have 99-100% identity with other E. leei sequences. Spore morphology and ultrastructure were consistent with previous reports of E. leei. Treatment of clinically affected fish by oral administration of the coccidiostats amprolium and salinomycin led to reduction of mortalities and resolution of clinical signs. This case report highlights the importance of thorough examination and surveillance of fish during quarantine, particularly with respect to enteric myxozoans.

Multi-locus sequence typing provides epidemiological insights for diseased sharks infected with fungi belonging to the Fusarium solani species complex.

Fusarium spp. are saprobic moulds that are responsible for severe opportunistic infections in humans and animals. However, we need epidemiological tools to reliably trace the circulation of such fungal strains within medical or veterinary facilities, to recognize environmental contaminations that might lead to infection and to improve our understanding of factors responsible for the onset of outbreaks. In this study, we used molecular genotyping to investigate clustered cases of Fusarium solani species complex (FSSC) infection that occurred in eight Sphyrnidae sharks under managed care at a public aquarium. Genetic relationships between fungal strains were determined by multi-locus sequence typing (MLST) analysis based on DNA sequencing at five loci, followed by comparison with sequences of 50 epidemiologically unrelated FSSC strains. Our genotyping approach revealed that F. keratoplasticum and F. solani haplotype 9x were most commonly isolated. In one case, the infection proved to be with another Hypocrealian rare opportunistic pathogen Metarhizium robertsii. Twice, sharks proved to be infected with FSSC strains with the same MLST sequence type, supporting the hypothesis the hypothesis that common environmental populations of fungi existed for these sharks and would suggest the longtime persistence of the two clonal strains within the environment, perhaps in holding pools and life support systems of the aquarium. This study highlights how molecular tools like MLST can be used to investigate outbreaks of microbiological disease. This work reinforces the need for regular controls of water quality to reduce microbiological contamination due to waterborne microorganisms.

PHARMACOKINETICS OF ORALLY ADMINISTERED VORICONAZOLE IN AFRICAN PENGUINS (SPHENISCUS DEMERSUS) AFTER SINGLE AND MULTIPLE DOSES.

Aspergillosis is a common respiratory fungal disease in African penguins ( Spheniscus demersus ) under managed care, and treatment failures with itraconazole due to drug resistance are increasingly common, leading to recent use of voriconazole. Empirical dosing with voriconazole based on other avian studies has resulted in adverse clinical drug effects in penguins. The objective of this study was to determine oral voriconazole pharmacokinetics (PK) in African penguins (n = 18). Single and once daily multiple oral doses of 5 mg/kg voriconazole were evaluated with a 4-mo washout period between trials. Plasma voriconazole concentrations were determined via high-performance liquid chromatography. Data was modeled using 3-compartamental population methodologies that supported first-order elimination. Observed mean peak concentration (1.89 µg/ml) after single dosing PK analysis was determined within the first hour following voriconazole administration. In the multiple-dose trial average plasma voriconazole concentrations were significantly higher on days 4 and 7 as compared with day 2. The mean estimates for volume of distribution (V/F) and clearance (Cl/F) for the multiple-dose study were 3.34 L and 0.18 L/hr, respectively. Monte Carlo simulations determined the median area under the curve (AUC0-24) at 84 hr was 37.7 µg·h/ml. As this assessment was comparable with the average AUC in humans receiving the recommended human oral dosage 200 mg b.i.d., it suggests that 5 mg/kg p.o. s.i.d. could be a safe and effective regimen in African penguins for treatment of aspergillosis. However, due to potential drug accumulation and subsequent toxicity, therapeutic drug monitoring with dosage adjustments is recommended to individualize dosing.

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

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

VORICONAZOLE TOXICITY IN MULTIPLE PENGUIN SPECIES.

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.

Spongiosis Hepatis in a Wild Aquarium-Maintained Red Irish Lord.

An aquarium-maintained female Red Irish Lord Hemilepidotus hemilepidotus presented with severe coelomic distension. The fish was anesthetized for ultrasonographic examination, which highlighted multiple cyst-like lesions in the liver and a distended ovary that was filled with follicles and an inspissated egg mass. Multiple exploratory celiotomies were performed for egg mass removal, liver biopsy, ovariosalpingectomy, and body wall rupture repair. Fourteen weeks after original presentation, and subsequent to 2 weeks of anorexia, the fish died. At necropsy, the liver was severely enlarged and distorted by multiple, coalescing, cyst-like spaces with no grossly normal liver parenchyma. The spleen also contained a raised cyst-like structure. Microscopically, the liver had well-demarcated foci of hepatocyte loss with retained meshworks of interconnected, perisinusoidal stellate cells. The fluid-filled spaces surrounded by stellate cells were not lined by epithelium or endothelium. The spleen had similar fluid-filled spaces formed of stellate cells. The cyst-like lesions in the liver were consistent with spongiosis hepatis; however, the concurrent development of a morphologically comparable lesion in the spleen is not typical of spongiosis hepatis cases. This case may represent the first report of spontaneously occurring spongiosis hepatis in a fish maintained in a public aquarium, as well as the first report in a fish of spongiosis hepatis-like lesions in an organ other than the liver.

Retroperitoneal hemangiosarcoma in a common carp Cyprinus carpio: a case report.

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.

Assessment of acid-base derangements among bonnethead (Sphyrna tiburo), bull (Carcharhinus leucas), and lemon (Negaprion brevirostris) sharks from gillnet and longline capture and handling methods.

Blood gasses of wild bonnethead, bull, and lemon sharks were measured with the i-STAT clinical analyzer with the CG4+ cartridge immediately after capture; and again immediately prior to release after tagging, handling and morphometric measurements were taken. Relative reference ranges of post-capture status were established. Among species, stress response to capture was similar for all parameters; however, pH declined and lactate concentrations rose over time, indicating continued insult from capture and/or response to additional handling stress. pCO(2) rose faster for S. tiburo than for C. leucas, and lactate concentrations rose faster for S. tiburo than for N. brevirostris. All species caught in gillnets experienced lower pH and higher lactate concentrations than on longlines. Discriminant analysis justified the use of blood gas analysis to assess physiological stress induced by different capture methods. From these results, we recommend 1) that gear be monitored closely and sharks be removed immediately, or suboptimally, that gear is deployed for the shortest soak time possible; 2) longline over gillnet gear; and 3) extra caution with sensitive species (e.g., S. tiburo), which may include the administration of blood buffers and other therapeutics if a shark is beyond the limits of relative reference ranges reported here.

Chromatophoroma in a crevice kelpfish (Gibbonsia montereyensis).

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.