Coelomic Fluid Evaluation in Pisaster ochraceus Affected by Sea Star Wasting Syndrome: Evidence of Osmodysregulation, Calcium Homeostasis Derangement, and Coelomocyte Responses.

Sea Star Wasting Syndrome (SSWS) is one of the largest marine wildlife die-offs ever recorded, killing millions of sea stars from more than 20 Asteroid species from Alaska to Mexico from 2013 to 2015 from yet undetermined cause(s). Coelomic fluid surrounds the sea star's organs, playing critical roles in numerous systemic processes, including nutrient transportation and immune functions. Coelomocytes, which are cellular components of coelomic fluid and considered functionally equivalent to vertebrate leukocytes, are responsible for innate cell-mediated immunity. The objectives of this study were to (1) evaluate changes in coelomic fluid chemistry, coelomocyte counts, and cytology from ochre sea stars (Pisaster ochraceus) (n = 55) with clinical signs consistent with SSWS at varying intensity (SSWS score 1: n = 4, score 2: n = 2, score 3: n = 3, score 4: n = 18, score 5: n = 26) in comparison to coelomic fluid from clinically normal sea stars (n = 26) and to (2) correlate SSWS score with cellular and biochemical analytes. SSWS-affected sea stars had wider ranges of all electrolytes, except calcium; statistically significantly higher chloride, osmolality, and total protein; lower calcium; and higher coelomocyte counts when compared to clinically normal sea stars maintained under identical environmental conditions. Free and/or phagocytized bacteria were noted in 29% (16 of 55) coelomic fluid samples from SSWS-affected sea stars but were absent in clinically normal sea stars. SSWS score correlated significantly with increasing chloride concentration, osmolality, and coelomocyte counts. These chemistry and cytological findings in coelomic fluid of SSWS-affected sea stars provide insight into the pathophysiology of SSWS as these results suggest osmo- and calcium dysregulation, coelomocyte responses, and presumptive opportunistic bacterial infection in SSWS-affected sea stars. This information provides potential future research applications for the development of treatment strategies for sea stars in managed care and for understanding the complexity of various biochemical and cellular pathophysiological mechanisms involved in sea star wasting.

Coelomic Fluid Evaluation in Clinically Normal Ochre Sea Stars Pisaster ochraceus: Cell Counts, Cytology, and Biochemistry Reference Intervals.

Coelomic fluid sampling is a noninvasive technique that is used to access the body fluid of sea stars for diagnostics and research. Given recent mortality events including sea star wasting disease, which has killed millions of sea stars along the Pacific coast since 2013, there is a need for validated diagnostic tests to evaluate sea star health. The objectives of this study were to establish coelomic fluid reference intervals for clinically normal ochre sea stars Pisaster ochraceus in an open system aquarium, to describe the cytologic findings, and to compare the chemistries of coelomic fluid with open system tank water. Coelomic fluid from 26 clinically normal sea stars was sampled for coelomocyte counts, cytologic evaluation, and biochemical analysis including magnesium, sodium, potassium, chloride, calcium, and total protein. The number of coelomocytes and total protein did not fit normal distribution and were excluded from analyses. Reference intervals were established for other chemistry analytes. There was no statistical difference in biochemistries between sea star coelomic fluid and water from five open system tanks, which supports previous evidence that sea stars are osmoconformers. Very low numbers of coelomocytes were observed cytologically. These results provide a useful baseline and diagnostic tool for health assessments of sea stars.

Population pharmacokinetics of enrofloxacin in purple sea stars (Pisaster ochraceus) following an intracoelomic injection or extended immersion.

OBJECTIVE To determine population pharmacokinetics of enrofloxacin in purple sea stars (Pisaster ochraceus) administered an intracoelomic injection of enrofloxacin (5 mg/kg) or immersed in an enrofloxacin solution (5 mg/L) for 6 hours. ANIMALS 28 sea stars of undetermined age and sex. PROCEDURES The study had 2 phases. Twelve sea stars received an intracoelomic injection of enrofloxacin (5 mg/kg) or were immersed in an enrofloxacin solution (5 mg/L) for 6 hours during the injection and immersion phases, respectively. Two untreated sea stars were housed with the treated animals following enrofloxacin administration during both phases. Water vascular system fluid samples were collected from 4 sea stars and all controls at predetermined times during and after enrofloxacin administration. The enrofloxacin concentration in those samples was determined by high-performance liquid chromatography. For each phase, noncompartmental analysis of naïve averaged pooled samples was used to obtain initial parameter estimates; then, population pharmacokinetic analysis was performed that accounted for the sparse sampling technique used. RESULTS Injection phase data were best fit with a 2-compartment model; elimination half-life, peak concentration, area under the curve, and volume of distribution were 42.8 hours, 18.9 µg/mL, 353.8 µg•h/mL, and 0.25 L/kg, respectively. Immersion phase data were best fit with a 1-compartment model; elimination half-life, peak concentration, and area under the curve were 56 hours, 36.3 µg•h/mL, and 0.39 µg/mL, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that the described enrofloxacin administration resulted in water vascular system fluid drug concentrations expected to exceed the minimum inhibitory concentration for many bacterial pathogens.

POPULATION PHARMACOKINETICS OF ENROFLOXACIN AND ITS METABOLITE CIPROFLOXACIN IN THE GREEN SEA URCHIN (STRONGYLOCENTROTUS DROEBACHIENSIS) FOLLOWING INTRACOELOMIC AND IMMERSION ADMINISTRATION.

Sea urchin mass mortality events have been attributed to both infectious and noninfectious etiologies. Bacteria, including Vibrio spp. and Pseudoalteromonas spp., have been isolated during specific mortality events. Aquarium collection sea urchins are also subject to bacterial infections and could benefit from antimicrobial treatment, but pharmacokinetic studies have been lacking for this invertebrate group until recently. This study evaluated the pharmacokinetics of enrofloxacin and its active metabolite ciprofloxacin in the green sea urchin (Strongylocentrotus droebachiensis) after intracoelomic injection and medicated bath immersion administration. The utility of a population pharmacokinetic method using nonlinear mixed effects modeling (NLME) was also evaluated. Thirty sea urchins were assigned to either the injection or immersion group. Twelve study animals and three untreated controls were utilized for each administration method: enrofloxacin 10 mg/kg intracoelomic injection or a 6-hr enrofloxacin 10 mg/L immersion. Each animal was sampled four times from 0 to 120 hr. Water samples were collected during immersion treatment and posttreatment time points in both groups. Hemolymph and water sample drug concentrations were analyzed using high-performance liquid chromatography, and pharmacokinetic parameters were determined using an NLME population pharmacokinetic method. Enrofloxacin concentrations were fit to a two-compartment model with first-order input for the intracoelomic injection group. The enrofloxacin elimination half-life (t½), peak hemolymph concentration (CMAX), and area under the curve (AUC) were 38.82 hr, 90.92 µg/ml, and 1,199 hr·µg/ml, respectively. Enrofloxacin was modeled to a one-compartment model with first-order input for the immersion treatment. The enrofloxacin t½, CMAX, and AUC were 33.46 hr, 0.48 µg/ml, and 32.88 hr·µg/ml, respectively. Ciprofloxacin was detected in trace concentrations in all hemolymph samples, indicating minimal production of this metabolite. The concentrations of enrofloxacin achieved far exceeded minimum inhibitory concentrations reported for teleost pathogens. No adverse effects were associated with enrofloxacin administration by either treatment method or from hemolymph sampling.

Densovirus associated with sea-star wasting disease and mass mortality.

Populations of at least 20 asteroid species on the Northeast Pacific Coast have recently experienced an extensive outbreak of sea-star (asteroid) wasting disease (SSWD). The disease leads to behavioral changes, lesions, loss of turgor, limb autotomy, and death characterized by rapid degradation ("melting"). Here, we present evidence from experimental challenge studies and field observations that link the mass mortalities to a densovirus (Parvoviridae). Virus-sized material (i.e., <0.2 µm) from symptomatic tissues that was inoculated into asymptomatic asteroids consistently resulted in SSWD signs whereas animals receiving heat-killed (i.e., control) virus-sized inoculum remained asymptomatic. Viral metagenomic investigations revealed the sea star-associated densovirus (SSaDV) as the most likely candidate virus associated with tissues from symptomatic asteroids. Quantification of SSaDV during transmission trials indicated that progression of SSWD paralleled increased SSaDV load. In field surveys, SSaDV loads were more abundant in symptomatic than in asymptomatic asteroids. SSaDV could be detected in plankton, sediments and in nonasteroid echinoderms, providing a possible mechanism for viral spread. SSaDV was detected in museum specimens of asteroids from 1942, suggesting that it has been present on the North American Pacific Coast for at least 72 y. SSaDV is therefore the most promising candidate disease agent responsible for asteroid mass mortality.

Sedation and physiologic response to manual restraint after intranasal administration of midazolam in Hispaniolan Amazon parrots (Amazona ventralis).

Administration of intranasal midazolam (2 mg/kg) was evaluated for sedation and effects on cloacal temperature, respiratory rate, and heart rate in manually restrained Hispaniolan Amazon parrots (Amazona ventralis). Adult parrots (n=9) were administered either midazolam (2 mg/kg) or an equal volume of saline solution intranasally before a 15-minute manual restraint in a complete crossover study. Respiratory rate and sedation scores were recorded before and during capture and during and after 15 minutes of manual restraint. Heart rate and cloacal temperature were recorded during manual restraint. After restraint, the parrots received intranasal flumazenil (0.05 mg/kg) or an equal volume of saline solution, and the recovery time was recorded. In those birds that received midazolam, sedation was observed within 3 minutes of administration, and vocalization, flight, and defense responses were significantly reduced during capture. During manual restraint, the mean rate of cloacal temperature increase was significantly slower and remained significantly lower in birds that received midazolam compared with controls. Mean respiratory rates were significantly lower for up to 12 minutes in parrots that received midazolam compared with those receiving saline solution. Flumazenil antagonized the effects of midazolam within 10 minutes. No overt clinical adverse effects to intranasal midazolam and flumazenil administration were observed. Further studies on the safety of intranasal midazolam and flumazenil in this species are warranted.

Antinociceptive efficacy of buprenorphine and hydromorphone in red-eared slider turtles (Trachemys scripta elegans).

Despite the frequent clinical use of buprenorphine in reptiles, its antinociceptive efficacy is not known. In a randomized, complete cross-over study, the antinociceptive efficacy of buprenorphine (0.2 mg/kg s.c.) was compared with hydromorphone (0.5 mg/kg s.c.), and saline (0.9% s.c. equivalent volume) in 11 healthy red-eared slider turtles (Trachemys scripta elegans). Additionally, buprenorphine at 0.1 and 1 mg/kg was compared with saline in six turtles. Hindlimb withdrawal latencies were measured after exposure to a focal, thermal noxious stimulus before and between 3 hr and up to 96 hr after drug administration. Buprenorphine did not significantly increase hindlimb withdrawal latencies at any time point compared with saline. In contrast, hydromorphone administration at 0.5 mg/kg significantly increased hindlimb withdrawal latencies for up to 24 hr. These results show that hydromorphone, but not buprenorphine, provides thermal antinociception in red-eared slider turtles.

Copper pellets simulating oral exposure to copper ammunition: absence of toxicity in American kestrels (Falco sparverius).

To evaluate the potential toxicity of copper (Cu) in raptors that may consume Cu bullets, shotgun pellets containing Cu, or Cu fragments as they feed on wildlife carcasses, we studied the effects of metallic Cu exposure in a surrogate, the American kestrel (Falco sparverius). Sixteen kestrels were orally administered 5 mg Cu/g body mass in the form of Cu pellets (1.18-2.00 mm in diameter) nine times during 38 days and 10 controls were sham gavaged on the same schedule. With one exception, all birds retained the pellets for at least 1 h, but most (69%) regurgitated pellets during a 12-h monitoring period. Hepatic Cu concentrations were greater in kestrels administered Cu than in controls, but there was no difference in Cu concentrations in the blood between treated and control birds. Concentration of the metal-binding protein metallothionein was greater in male birds that received Cu than in controls, whereas concentrations in female birds that received Cu were similar to control female birds. Hepatic Cu and metallothionein concentrations in kestrels were significantly correlated. Histopathologic alterations were noted in the pancreas of four treated kestrels and two controls, but these changes were not associated with hepatic or renal Cu concentrations, and no lesions were seen in other tissues. No clinical signs were observed, and there was no treatment effect on body mass; concentrations of Cu, hemoglobin, or methemoglobin in the blood; or Cu concentrations in kidney, plasma biochemistries, or hematocrit. Based on the parameters we measured, ingested Cu pellets pose little threat to American kestrels (and presumably phylogenetically related species), although the retention time of pellets in the stomach was of relatively short duration. Birds expected to regurgitate Cu fragments with a frequency similar to kestrels are not likely to be adversely affected by Cu ingestion, but the results of our study do not completely rule out the potential for toxicity in species that might retain Cu fragments for a longer time.