PRIMARY HEPATIC NEOPLASIA IN THREE ADULT BAMBOO SHARKS (CHILOSCYLLIUM PLAGIOSUM AND CHILOSCYLLIUM PUNCTATUM) IN AN AQUARIUM POPULATION.

Neoplasia in elasmobranchs is uncommonly documented. This report describes primary hepatic neoplasia in three adult female bamboo sharks under managed care: biliary adenocarcinoma in a whitespotted bamboo shark (Case 1; Chiloscyllium plagiosum), biliary adenocarcinoma in a brownbanded bamboo shark (Case 2; Chiloscyllium punctatum), and hepatocellular carcinoma in a whitespotted bamboo shark (Case 3). Case 1 presented with extensive cutaneous papillomas and was electively euthanized. At necropsy, a 4-cm-diameter, pale-tan, firm hepatic mass was identified and diagnosed histologically as a biliary adenocarcinoma. Case 2 presented with decreasing body condition despite normal food intake. Coelomic ultrasound and exploratory surgery revealed several large masses in both hepatic lobes, and the patient was euthanized. At necropsy, nine, 1-6-cm-diameter, black to tan, firm hepatic masses were identified and diagnosed histologically as biliary adenocarcinoma with branchial intravascular neoplastic emboli. Case 3 presented for routine health examination and was euthanized for diagnostic purposes after coelomic ultrasound revealed multiple hepatic masses. At necropsy, two 1-3-cm-diameter, brown- and-black mottled, firm hepatic masses were identified and diagnosed histologically as hepatocellular carcinoma. Immunohistochemistry was performed in two of these cases and was noncontributory, likely because of a lack of cross reactivity between antibodies (antipancytokeratin) and elasmobranch tissues.

DEXMEDETOMIDINE AND MIDAZOLAM INTRAMUSCULAR SEDATION IN BROWNBANDED BAMBOO SHARKS (CHILOSCYLLIUM PUNCTATUM).

Bamboo sharks are some of the most common elasmobranch species in zoos and aquaria and are frequently sedated for medical exams, treatments, and research. This study investigated the use of an IM sedation protocol of a single dose of dexmedetomidine (0.05 mg/kg) and midazolam (2.0 mg/kg) in brownbanded bamboo sharks (Chiloscyllium punctatum). Sharks were serially monitored every 5 min for heart rate, branchial beats, righting reflex, coelomic response, cloacal response, pelvic fin reflex, response to noxious stimulus, voluntary movement, and ability to swim. This sedation dose was effective at rapidly and significantly decreasing responses to tactile and noxious stimuli with minimal respiratory depression and was quickly reversible with atipamezole (0.5 mg/kg) and flumazenil (0.05 mg/kg). Sedated sharks developed a mild metabolic acidosis evidenced by a significant increase in lactic acid (mean < 0.37 mmol/L presedation, 4.2 mmol/L after reversal) and decrease in blood pH (mean 7.464 presedation, 7.277 after reversal); however, clinical intervention was not required. This protocol should be further investigated in different elasmobranch species but is promising for providing sedation for noninvasive procedures in brownbanded bamboo sharks.

INTRAVENOUS CONTRAST-ENHANCED COMPUTED TOMOGRAPHY IN ADULT KOI (CYPRINUS CARPIO).

IV iodinated contrast is undocumented in koi (Cyprinus carpio). IV contrast-enhanced computed tomography (CT) provides detailed imaging of the vascular structures and parenchymal organs. The goals of this study were to (1) document feasibility of IV contrast into the caudal vein, (2) determine distribution of contrast media and conspicuity of organs, (3) evaluate for differences in contrast uptake with two different doses of IV contrast, and (4) evaluate for contraindications of IV iodinated contrast in a small cohort of fish. Thirty adult koi were split into three groups (n = 10) in a prospective interrupted time series design with nonequivalent groups, receiving 480 mg iodine per kg (mg I/kg), 800 mg I/kg, and saline. Contrast doses were selected based on the range of doses used in other species. Half of the fish given IV contrast (n = 10) were imaged with CT. Physical exams, blood smears, and biochemistries were performed on all fish. Half of the fish in each group were euthanized 24 h after injection and the other half two weeks later. Necropsies and histopathology were performed to evaluate pathology associated with contrast. For the CT examinations, precontrast, immediate postcontrast, and 5-m delayed postcontrast CT scans were obtained. Contrast in coelomic organs and circulatory system was measured using Hounsfield units. The caudal kidney was the most contrast-enhancing organ. Both doses produced good vascular enhancement and similar distribution. The delayed postcontrast scans showed repeatable parenchymal enhancement of organs. No mortality was encountered. No abnormalities were detected on blood smears, necropsies, or histopathology. Results suggest IV contrast administration is feasible, both contrast doses provided valuable anatomical information in the study with CT of coelomic organs, and no contraindications of contrast administration were detected.

Systemic phaeohyphomycosis in splitnose rockfish (Sebastes diploproa) caused by Devriesia sp.

A novel pathogen was documented after two wild-caught, juvenile, splitnose rockfish presented with buphthalmia, grey corneal endothelial plaques and evidence of uveitis. Cytologic evaluation of ocular contents revealed fungal hyphae. Histologic evaluation identified multiple fungal granulomas and granulomatous inflammation in the globes, periocular tissue and heart. Fungi were slender, hyphenated and branched at angles, had parallel cell walls and had brown pigmentation in haematoxylin- and eosin-stained sections. Both fish were diagnosed with phaeohyphomycosis. Culture with nuclear ribosomal RNA internal transcribed spacer (ITS) segment identification further classified the fungus as Devriesia sp., which has not been previously documented as a cause of disease in animals.

Non-lethal diagnostic methods for koi herpesvirus in koi Cyprinus carpio.

Cyprinid herpesvirus 3, also known as koi herpesvirus (KHV), is a viral pathogen responsible for mass mortalities of carp worldwide. In this study, we compared the sensitivity and specificity of ELISA and quantitative PCR (qPCR) methods for the diagnosis of KHV in experimentally infected koi Cyprinus carpio over an 11 mo period. Koi were exposed to KHV at 18 ± 1°C (permissive temperatures for KHV disease) in laboratory-controlled conditions. At 21 d post challenge, the temperature in the system was decreased to <15°C (non-permissive temperature for KHV disease), and fish were monitored for the following 11 mo. At different time points throughout the study, samples of blood and gills were collected from exposed and control koi and subjected to qPCR and ELISA. Survival proportions of 53.3 and 98.8% in exposed and control treatments, respectively, were recorded at the end of the challenge. Traditional receiver-operating characteristic analysis was used to compare the sensitivity of the ELISA and blood and gill qPCR during permissive and non-permissive temperatures. ELISA was superior to qPCR of gills and whole-blood samples in detecting previous exposure to KHV. Similar results were obtained in a second experiment exposing koi to KHV and inducing persistent infection at >30°C (non-permissive temperature for KHV disease). Finally, KHV ELISA specificity was confirmed using cyprinid herpesvirus 1-exposed koi through a period of 3 mo. This study demonstrates that the combination of ELISA and gill qPCR should be recommended in the diagnosis of KHV exposure of suspected carrier-state fish.

Validation of environmental DNA sampling for determination of Ceratonova shasta (Cnidaria: Myxozoa) distribution in Plumas National Forest, CA.

Ceratonova shasta is the etiological agent of myxozoan-associated enteronecrosis in North American salmonids. The parasite's life cycle involves waterborne spores and requires both a salmonid fish and a freshwater fabriciid annelid. The success and survival of annelids can be enhanced by flow moderation by dams, and through the erosion of fine sediments into stream channels following wildfires. In this study, the presence of C. shasta environmental/ex-host DNA (eDNA) in river water and substrate samples collected from areas affected by recent fire activity in California, USA, was investigated. Additionally, DNA loads in the environment were compared to C. shasta infection in sentinel-exposed rainbow trout (Oncorhynchus mykiss). Significant associations between C. shasta detection in environmental samples and location within a wildfire perimeter (p = 0.002), between C. shasta detection in sentinel fish and exposure location within a wildfire perimeter (p = 0.015), and between C. shasta detection in fish and locations where water temperature was above the median (p < 0.001) were observed. Additionally, a higher prevalence of C. shasta infection in fish was detected where C. shasta was also detected in environmental samples (p < 0.001). Results suggest that pathogen eDNA sampling can be used as a non-invasive, rapid, specific, and sensitive method for establishing risk of C. shasta infection in wild populations. Knowledge of the complete life cycle of the target parasite, including ecology of each host, can inform the choice of eDNA sampling strategy. Environmental DNA sampling also revealed a novel species of Ceratonova, not yet observed in a host.

Immunohistochemical analysis of pigment cell tumors in two cyprinid species.

Pigment cell tumors, also known as chromatophoromas, are cutaneous spindle cell neoplasms originating from pigment cells (chromatophores) in the dermis of teleosts, amphibians, and reptiles. Chromatophoromas share similar histologic morphology to other spindle cell tumors and are not always pigmented. Therefore, immunohistochemical analysis may be useful in distinguishing these neoplasms from tumors of other cellular origin when poorly pigmented. We performed 3 immunohistochemistry assays (PNL-2, melan A, and SOX10) on 8 cutaneous neoplasms from 8 teleosts diagnosed as chromatophoromas based on histologic morphology. Semiquantitative analysis of immunoreactivity was evaluated on each immunohistochemical assay using a 0-3 scale. PNL-2 exhibited mild-to-moderate (1 or 2) immunoreactivity in 7 of the cases, and resident chromatophores (internal control) were also immunoreactive in these cases. Melan A exhibited mild-to-moderate (1 or 2) immunoreactivity in 4 cases (and with resident chromatophores in these cases); SOX10 was not immunoreactive in any cases. Our results indicate that PNL-2 may be a useful marker in teleosts to distinguish tumors of chromatophore origin. Melan A could also be useful, but appears to be less sensitive, and SOX10 is likely not a useful marker for these neoplasms in teleosts.

Evaluation of diagnostic coelioscopy in koi (Cyprinus carpio).

OBJECTIVE To describe a technique for endoscopic evaluation of the coelomic viscera of koi (Cyprinus carpio) and to evaluate the ability to visually examine coelomic structures by use of an approach cranial or caudal to the pelvic girdle. ANIMALS 16 subadult koi. PROCEDURES Koi were anesthetized with buffered tricaine methanesulfonate. Coelioscopic examination was performed via a ventral midline incisional approach cranial or caudal to the pelvic girdle. A 2.7-mm × 18-cm 30° oblique endoscope within a 4.8-mm operating sheath and infusion of saline (0.9% NaCl) solution was used. Ease of entry into the coelomic cavity and visual examination of structures were scored for each fish. Fish were euthanized 2 or 8 weeks after the procedure, and necropsy was performed. RESULTS The coelioscopic procedure was tolerated well, and all koi recovered uneventfully. For all fish, ease of entry and visual examination scores of the liver, intestines, gonads, heart, and anterior kidney were satisfactory to excellent. Visual examination of the posterior kidney and swim bladder was satisfactory to difficult, whereas the spleen and gallbladder were not visually identified. No significant differences were noted in entry or visual examination scores between the cranial and caudal approaches or between sexes. Minor complications included mild hemorrhage, rupture of the gonadal capsule, formation of adhesions between the viscera and incision site, and delayed healing of the incision. CONCLUSIONS AND CLINICAL RELEVANCE Diagnostic coelioscopy of koi appeared to be safe and effective. This procedure could have potential for use in examination of coelomic structures and disease diagnosis.

OUTBREAK AND TREATMENT OF CARP EDEMA VIRUS IN KOI ( CYPRINUS CARPIO) FROM NORTHERN CALIFORNIA.

Carp edema virus (CEV) is the causative agent of carp edema virus disease (CEVD), also referred to as koi sleepy disease, which is an emerging disease of global concern that may cause high rates of morbidity and mortality in common carp and ornamental koi ( Cyprinus carpio). This article reports the third confirmed outbreak of CEVD in California. In June 2015, three koi presented with clinical signs of cutaneous lesions, severe lethargy, and signs of hypoxia. All fish tested positive for CEV by polymerase chain reaction (PCR). Euthanasia and complete necropsy were performed on two fish. The most significant necropsy findings included necrotizing branchitis with marked interstitial edema, multifocal cutaneous ulcerations, and severe cutaneous edema. Treatment of the pond with 0.3-0.5% salt was recommended to the owner. Approximately 7 wk later, a recheck visit was made to the pond. No mortalities had been noted since the initiation of the salt treatment. Physical examination revealed a vast improvement but not complete elimination of the clinical signs of hypoxia and intermittent lethargy in the affected fish. Gill biopsy samples from the two most affected fish were tested and remained PCR positive for CEV. Subsequent recheck visits over 11 mo postdiagnosis and initiation of treatment showed continued improvement in most fish. Gill samples from all fish in the pond ( n = 9) were repeatedly tested by quantitative PCR for CEV, and all samples were negative. This case series further confirms the global spread of CEV and the need for practitioners to be vigilant for outbreaks of this disease. If CEVD is suspected, treatment with 0.3-0.5% salt can be recommended to potentially mitigate the effects of this disease. However, fish may remain potential carriers of this pathogen, and strict biosecurity measures should continue to be enforced for any pond that has had a confirmed CEV outbreak.

HYPERMUCOVISCOUS KLEBSIELLA PNEUMONIAE ISOLATES FROM STRANDED AND WILD-CAUGHT MARINE MAMMALS OF THE US PACIFIC COAST: PREVALENCE, PHENOTYPE, AND GENOTYPE.

Emergent hypermucoviscous (HMV) strains of Klebsiella pneumoniae have been reported in multiple marine mammal species; however, there is limited information regarding the epidemiology and pathogenesis of this infection in these species. We determined the prevalence of HMV K. pneumoniae in wild-caught and stranded marine mammal populations on the US Pacific Coast. Samples were collected from 270 free-ranging California sea lions (CSLs; Zalophus californianus) captured at three discrete sampling sites and from 336 stranded marine mammals of various species. We recovered HMV K. pneumoniae only from CSLs, with a prevalence of 1.5% (4 of 275) in stranded animals, compared with 1.1% (3 of 270) in wild-caught animals. We assessed the phenotypic and genotypic variability of recovered HMV K. pneumoniae isolates recovered from CSLs ( n=11) and of archival HMV and non-HMV isolates from stranded marine mammals ( n=19). All but two HMV isolates were of the K2 serotype, whereas none of the non-HMV isolates belonged to this serotype. Of the HMV isolates, 96% (24 of 25) were PCR positive for the HMV-associated gene p- rmpA, whereas 92% (23 of 25) were PCR positive for p- rmpA2. Genetic fingerprinting by repetitive extragenic palindromic PCR showed four discrete clusters, demonstrating genotypic variability that loosely correlated with phenotype. Antimicrobial susceptibility testing revealed all isolates from stranded CSLs were susceptible to ceftiofur, indicating this antimicrobial agent is an appropriate choice for treatment of HMV K. pneumoniae infections in stranded CSLs. Our culture assay could reliably detect HMV K. pneumoniae from concentrations as low as 102 colony-forming units per milligram of feces. We identified the presence of HMV K. pneumoniae in both wild-caught and stranded CSLs from the US Pacific Coast and highlight the need for further studies to evaluate the potential impact of this pathogen on marine mammal health.

Distribution and Prevalence of Myxobolus cerebralis in Postfire Areas of Plumas National Forest: Utility of Environmental DNA Sampling.

Myxobolus cerebralis is a myxozoan parasite and the etiological agent of whirling disease in salmonids. The parasite's life cycle involves waterborne spores and requires both a salmonid fish and the benthic freshwater oligochaete worm Tubifex tubifex (Oligochaeta: Tubificidae). Wildfires can lead to the erosion of fine sediments into stream channels and have been implicated as promoting environmental conditions that are suitable for the survival and success of T. tubifex, whose presence in turn can affect the prevalence of M. cerebralis. Analysis of environmental DNA (eDNA) has the potential to be a powerful tool for evaluating the presence of microorganisms, for which direct observation is impossible. We investigated the presence of M. cerebraliseDNA in river water and river sediment samples collected from areas affected by recent fire activity in Plumas National Forest, California. We compared eDNA loads in the environment to M. cerebralis infection in T. tubifex and sentinel-exposed Rainbow Trout Oncorhynchus mykiss and the presence of T. tubifex lineages in the same environment. For the latter, we developed a multiplex quantitative PCR assay for detection of T. tubifex lineages I, III, and V. Lineage IIIT. tubifex and M. cerebralis (eDNA as well as DNA extracted from fish and worm tissues) were detected only in samples obtained from areas affected by the Moonlight wildfire. The association between M. cerebralis infection in sentinel-exposed fish and eDNA detection in environmental samples only approached significance at a P-value of 0.056. However, given the difference in relative effort between the two sampling methods (host versus nonhost environment), our data suggest that eDNA sampling of water and substrate is a promising approach for surveillance of myxozoan fish parasites.

Effects of temperature on Veronaea botryosa infections in white sturgeon Acipenser transmontanus and fungal induced cytotoxicity of fish cell lines.

Veronaea botryosa is a melanized mold and cause of systemic fungal infections in cultured sturgeon (Acipenser spp.). Mortality in adult female sturgeon caused by this emergent pathogen results in significant economic losses for the caviar industry. Little is known regarding environmental conditions conducive to V. botryosa infection. This study evaluated the effect of temperature on V. botryosa infectivity and dissemination following intramuscular injection challenge of white sturgeon maintained at 13 or 18 °C for 40 days. Daily mortality was recorded and persistence of the fungus in the livers of moribund and surviving fish was investigated using culture and histopathological analysis. Fish maintained at 18 °C developed systemic phaeohyphomycosis and had significantly greater mortality than controls and fish maintained at 13 °C (p < 0.05). Challenged fish, regardless of temperature, exhibited lesions in multiple organs. However, muscle lesions, angioinvasion, and systemic dissemination were more severe and widespread in fish challenged at the higher temperature. In vitro cytotoxicity of V. botryosa was evaluated in white sturgeon skin (WSSK-1) and epithelioma papulosum cyprini (EPC) cell lines inoculated at spore:cell ratios of 1:10, 1:1 and 10:1, then incubated 15, 20 and 25 °C. Cytotoxicity, as indicated by quantifying the release of lactate dehydrogenase into culture supernatants, increased with increasing spore dose and incubation temperature in both fish cell lines. Findings suggest that temperature significantly influences the development of systemic V. botryosa infection in white sturgeon and that WSSK-1 and EPC cells are suitable in vitro models for the study of host-pathogen interactions between V. botryosa and fish epithelial cells.

Trauma-Induced Uveitis and Free Air in the Anterior Chamber of Three Eastern Screech Owls (Megascops asio).

Unusual ocular abnormalities were documented in 3 wild eastern screech owls (Megascops asio) presented to a wildlife rehabilitation hospital after vehicular strike-induced trauma to the head. All 3 had anterior uveitis and free air bubbles in the anterior chamber, but none of the cases had any discernable corneal damage, either grossly or with fluorescein stain technique. Perforation of the globe at the level of the scleral ossicle was considered a possible cause. All 3 cases recovered with standard treatment for anterior uveitis, and the free air was absorbed within 10-14 days and did not appear to cause any lingering complications. The owls were later released back into the wild after demonstrating the ability to navigate obstacles in a flight cage and capture live prey.

Diversity of Veronaea botryosa from different hosts and evaluation of laboratory challenge models for phaeohyphomycosis in Acipenser transmontanus.

Veronaea botryosa has been identified as a pathogen of cultured white sturgeon Acipenser transmontanus. In 2015, samples from 19 white sturgeon were received for diagnosis, of which 14 cultured positive for V. botryosa. Intraspecific variability among V. botryosa isolates from different clinically affected hosts and geographic regions was investigated using repetitive extragenic palindromic PCR fingerprinting (rep-PCR). The rep-PCR profiles of 16 V. botryosa isolates from a human, sea turtles, and cultured fish were distinct from those of other phaeoid fungi belonging to the genera Cladophialophora and Exophiala. To gain a better understanding of the pathogenesis of V. botryosa mycosis, 5 laboratory challenge methods were evaluated in white sturgeon fingerlings. Intramuscular (IM) and intracoelomic (IC) injection challenges produced cumulative mortalities of 13.3% (8/60) and 3.3% (2/60), respectively, and V. botryosa was recovered from 100% (10/10) of dead fingerlings. Affected fish exhibited abnormal orientation and/or failure to maintain neutral buoyancy, emaciation, coelomic distension, exophthalmos, cutaneous erythema, and ulcerated skin. After 6 wk, surviving fish were euthanized, and samples of liver were taken for mycological evaluation. Viable fungus was detected in 90% and 100% of fish surviving IM and IC challenge, respectively. No V. botryosa-associated mortality was detected in other groups challenged by immersion, immersion with abrasion, or orally. Both IM and IC challenge routes appear suitable for the induction of V. botryosa infection in white sturgeon and can serve as models for the study of disease pathogenesis associated with this emergent pathogen.

Co-infection of Acipenserid herpesvirus 2 (AciHV-2) and Streptococcus iniae in cultured white sturgeon Acipenser transmontanus.

A mortality event in cultured white sturgeon Acipenser transmontanus (Richardson, 1836) sub-adults was investigated. After transfer between farms, high mortality was observed in fish, associated with back arching, abnormal swimming, and ulcerative skin lesions. Necropsy of moribund individuals revealed hemorrhagic ascites and petechial hemorrhages in the coelomic peritoneum and serosa of internal organs. Acipenserid herpesvirus 2 (AciHV-2) was isolated from external tissue samples, then identified and genotyped by sequencing of the terminase and polymerase genes. In addition, Streptococcus iniae was recovered from internal organs of affected fish. Histologic changes were limited to interstitial hematopoietic areas of the kidney and consisted of small foci of necrosis accompanied by fibrin deposition, minimal inflammatory response, and small numbers of bacterial cocci compatible with streptococci. Identity was confirmed by partial sequencing of the 16S rRNA, rpoB, and gyrB genes. Genetic fingerprinting demonstrated a genetic profile distinct from S. iniae isolates recovered from previous outbreaks in wild and cultured fish in North America, South America, and the Caribbean. Although the isolates were resistant to white sturgeon complement in serum killing assays, in vivo challenges failed to fulfill Koch's postulates. However, the clinical presentation, coupled with consistent recovery of S. iniae and AciHV-2 from moribund fish, suggests viral and bacterial co-infection were the proximate cause of death. To our knowledge, this represents the first report of AciHV-2 and S. iniae co-infection in cultured white sturgeon.