Non-viral precision T cell receptor replacement for personalized cell therapy.

T cell receptors (TCRs) enable T cells to specifically recognize mutations in cancer cells1-3. Here we developed a clinical-grade approach based on CRISPR-Cas9 non-viral precision genome-editing to simultaneously knockout the two endogenous TCR genes TRAC (which encodes TCRα) and TRBC (which encodes TCRß). We also inserted into the TRAC locus two chains of a neoantigen-specific TCR (neoTCR) isolated from circulating T cells of patients. The neoTCRs were isolated using a personalized library of soluble predicted neoantigen-HLA capture reagents. Sixteen patients with different refractory solid cancers received up to three distinct neoTCR transgenic cell products. Each product expressed a patient-specific neoTCR and was administered in a cell-dose-escalation, first-in-human phase I clinical trial ( NCT03970382 ). One patient had grade 1 cytokine release syndrome and one patient had grade 3 encephalitis. All participants had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease and the other eleven had disease progression as the best response on the therapy. neoTCR transgenic T cells were detected in tumour biopsy samples after infusion at frequencies higher than the native TCRs before infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs that recognize mutational neoantigens. Moreover, simultaneous knockout of the endogenous TCR and knock-in of neoTCRs using single-step, non-viral precision genome-editing are achieved. The manufacture of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene-edited neoTCR T cell products and the ability of the transgenic T cells to traffic to the tumours of patients are also demonstrated.

Relationship between red blood cell lifespan and endogenous carbon monoxide in the common bottlenose dolphin and beluga.

Certain deep-diving marine mammals [i.e., northern elephant seal (Mirounga angustirostris), Weddell seal (Leptonychotes weddellii)] have blood carbon monoxide (CO) levels that are comparable with those of chronic cigarette smokers. Most CO produced in humans is a byproduct of heme degradation, which is released when red blood cells (RBCs) are destroyed. Elevated CO can occur in humans when RBC lifespan decreases. The contribution of RBC turnover to CO concentrations in marine mammals is unknown. Here, we report the first RBC lifespans in two healthy marine mammal species with different diving capacities and heme stores, the shallow-diving bottlenose dolphin (Tursiops truncatus) and deep-diving beluga whale (Delphinapterus leucas), and we relate the lifespans to the levels of CO in blood and breath. The belugas, with high blood heme stores, had the longest mean RBC lifespan compared with humans and bottlenose dolphins. Both cetacean species were found to have three times higher blood CO content compared with humans. The estimated CO production rate from heme degradation indicates some marine mammals may have additional mechanisms for CO production, or delay CO removal from the body, potentially from long-duration breath-holds.NEW & NOTEWORTHY This is the first study to determine the red blood cell lifespan in a marine mammal species. High concentrations of carbon monoxide (CO) were found in the blood of bottlenose dolphins and in the blood and breath of belugas compared with healthy humans. Red blood cell turnover accounted for these high levels in bottlenose dolphins, but there may be alternative mechanisms of endogenous CO production that are contributing to the CO concentrations observed in belugas.

Evolved increases in hemoglobin-oxygen affinity and the Bohr effect coincided with the aquatic specialization of penguins.

Dive capacities of air-breathing vertebrates are dictated by onboard O2 stores, suggesting that physiologic specialization of diving birds such as penguins may have involved adaptive changes in convective O2 transport. It has been hypothesized that increased hemoglobin (Hb)-O2 affinity improves pulmonary O2 extraction and enhances the capacity for breath-hold diving. To investigate evolved changes in Hb function associated with the aquatic specialization of penguins, we integrated comparative measurements of whole-blood and purified native Hb with protein engineering experiments based on site-directed mutagenesis. We reconstructed and resurrected ancestral Hb representing the common ancestor of penguins and the more ancient ancestor shared by penguins and their closest nondiving relatives (order Procellariiformes, which includes albatrosses, shearwaters, petrels, and storm petrels). These two ancestors bracket the phylogenetic interval in which penguin-specific changes in Hb function would have evolved. The experiments revealed that penguins evolved a derived increase in Hb-O2 affinity and a greatly augmented Bohr effect (i.e., reduced Hb-O2 affinity at low pH). Although an increased Hb-O2 affinity reduces the gradient for O2 diffusion from systemic capillaries to metabolizing cells, this can be compensated by a concomitant enhancement of the Bohr effect, thereby promoting O2 unloading in acidified tissues. We suggest that the evolved increase in Hb-O2 affinity in combination with the augmented Bohr effect maximizes both O2 extraction from the lungs and O2 unloading from the blood, allowing penguins to fully utilize their onboard O2 stores and maximize underwater foraging time.

EFFICACY OF HUMAN RECOMBINANT GRANULOCYTE COLONY-STIMULATING FACTOR (G-CSF, FILGRASTIM; NEUPOGEN®) IN NEUTROPENIC CETACEANS.

Neutrophils are one of the initial cell lines of protection against pathogens, and when their concentrations in the blood are low, animals are highly susceptible to infections. Neutropenia has been reported in cetaceans secondary to administration of systemic sulfa antibiotics or antifungal medications and severe, overwhelming infection. Filgrastim was administered to treat neutropenia over a 15-y period in 11 cetaceans comprising four species-beluga (Delphinapterus leucas, n = 1), bottlenose dolphin (Tursiops truncatus, n = 4), killer whale (Orcinus orca, n = 5), and short-finned pilot whale (Globicephala macrorhynchus, n = 1)] ranging in age from 1 wk to >24 y. Seven study animals received multiple doses (2-6). All animals responded to at least one dose (1-7 µg/kg) of parenteral filgrastim characterized by an increase in peripheral immature (band) neutrophils, segmented neutrophils, or both. In most cases (9/11), neutrophil counts increased within 48 h of a single dose. Duration of response varied but was at least 2 wk in eight of the 11 animals and 5-9 d in the remaining animals. No adverse reactions were observed in any cases.

Campylobacter pinnipediorum subsp. caledonicus and C. pinnipediorum subsp. pinnipediorum recovered from abscesses in pinnipeds.

Campylobacter pinnipediorum was described recently for isolates recovered from pinnipeds. The novel species was further split into 2 subspecies based on host and geography, with C. pinnipediorum subsp. pinnipediorum recovered from otariid seals in California (USA) and C. pinnipediorum subsp. caledonicus recovered from phocid seals in Scotland. We report details of the infections of 7 pinnipeds from which C. pinnipediorum was isolated: C. pinnipediorum subsp. caledonicus was isolated from 2 harbour seals Phoca vitulina and a single grey seal Halichoerus grypus, and C. pinnipediorum subsp. pinnipediorum was isolated from California sea lions Zalophus californianus. Six of the isolates were recovered from samples collected at post-mortem investigation. In 2 of the Scottish seals and in 3 of the California seals, C. pinnipediorum was the sole bacterial isolate recovered from abscesses present and suggests they may have resulted from conspecific or intraspecific bite wounds.

EVALUATION OF PLASMA 25-HYDROXYVITAMIN D, IONIZED CALCIUM, AND PARATHYROID HORMONE IN GREEN SEA TURTLES (CHELONIA MYDAS) EXPOSED TO DIFFERENT INTENSITIES OF ULTRAVIOLET B RADIATION.

For many reptile species, adequate ultraviolet B (UVB) radiation is essential for proper calcium metabolism. In this study, the effects of UVB radiation on calcium metabolism were evaluated in green sea turtles (Chelonia mydas). 25-Hydroxyvitamin D, parathyroid hormone (PTH), and ionized calcium (iCa) were measured in juvenile (n = 18, 9 indoor, 9 outdoor) and adult (n = 8, 4 indoor, 4 outdoor) turtles. All animals were fed an identical diet. Outdoor animals had access to unfiltered sunlight, whereas indoor animals were housed under artificial lighting without UVB. Mean values for 25-hydroxyvitamin D for the outdoor and indoor groups were 34.33 ± 7.98 nmol/L and 7.11 ± 1.69 nmol/L for juveniles and 73.25 ± 30.34 nmol/L and 14.0 ± 11.52 nmol/L for adults respectively. Mean values for iCa for the outdoor and indoor groups were 0.98 ± 0.07 mmol/L and 0.99 ± 0.06 mmol/L for juveniles and 1.18 ± 0.22 mmol/L and 0.97 ± 0.18 mmol/L for adults respectively. UVB exposure (P < 0.001) and age (P < 0.001) had a significant effect on 25-hydroxyvitamin D as well as a significant interaction between the two variables (P = 0.008), with highest values in adult outdoor turtles. There was a significant interaction between age group and UVB status for iCa (P = 0.036), with greater values in older outdoor turtles. 25-Hydroxyvitamin D and total calcium were positively correlated, rs = 0.39, P = 0.042. iCa and calcium-to-phosphorus ratios were also positively correlated, rs = 0.42, P = 0.027. These results suggest that UVB exposure is an important source of 25-hydroxyvitamin D for green sea turtles and has significant effects on calcium metabolism in this species. PTH values in this study were near the minimum limits of detection and suggest that current mammalian-based PTH assays are not valid for reptiles.

Population Pharmacokinetics of Danofloxacin After Single Intramuscular Dose Administration in California Brown Pelicans (Pelecanus occidentalis californicus).

The pharmacokinetics of danofloxacin was investigated in rehabilitated California brown pelicans (Pelecanus occidentalis californicus) after a single intramuscular injection at a dose of 15 mg/kg body weight. The concentration of the drug in plasma was assayed by high-pressure liquid chromatography. A sparse-sampling design was used to reduce the number of samples (1-4 venipunctures) obtained from 24 brown pelicans. A population pharmacokinetic analysis with nonlinear mixed-effects modeling was used to accommodate the sparse-sampling strategy. The nonlinear mixed-effects modeling approach measured both fixed effects (typical values for the population) and random effects (between-subject variability) for this population. A 1-compartment model best represented the concentration-versus-time data after injection. After injection, the elimination half-life, peak concentration, area under the curve, and volume of distribution were 2.76 hours, 2.5 µg/mL, 13.75 µg/h/mL, and 4.35 L/kg, respectively. Rate of absorption was highly variable among the birds. The intramuscular injection of danofloxacin in pelicans at this dose produced plasma concentrations that meet therapeutic targets for bacteria with a minimum inhibitory concentration of ≤0.25 µg/mL. This dose can be used for future studies to evaluate the efficacy of danofloxacin for treating susceptible bacteria.

MAMMARY FIBROADENOMATOUS HYPERPLASIA IN A JUVENILE CALIFORNIA SEA LION ( ZALOPHUS CALIFORNIANUS).

A juvenile female California sea lion ( Zalophus californianus) presented with a firm subcutaneous mass in the right caudoventral pelvic region, measuring approximately 15 × 15 cm, and edema in the right hind flipper. The initial diagnostic workup included an ultrasound, blood analysis, and fine-needle aspirate of the mass under manual restraint. While the blood analysis was within normal limits, the ultrasound revealed a large discrete hyperechoic homogeneous mass with anastomosing hypoechoic fluid pockets that, when aspirated, was suggestive of a reactive lymph node. Short-term empiric antibiotic therapy prescribed to address possible lymphadenitis yielded no response, and edema of the right hind flipper and perianal region worsened. The sea lion was anesthetized to facilitate a more thorough exam, repeat blood analysis, obtain survey radiographs, evaluate with ultrasound, and perform biopsy of mass. The thoracic radiographs were normal; however, the large radiopaque mass was observed cranial to the pelvis on lateral abdominal view and appeared solitary on ultrasound. An exploratory surgery was performed to completely excise the mass. On histopathologic review, the mass demonstrated diffuse fibrous hyperplasia of lobular ducts of the mammary gland and mild mastitis, characterizing it as a mammary fibroadenoma. The surgical wound healed within 6 wk, and there was no recurrence of mammary hyperplasia.

SERUM COBALAMIN AND FOLATE CONCENTRATIONS AS INDICATORS OF GASTROINTESTINAL DISEASE IN KILLER WHALES ( ORCINUS ORCA).

Cobalamin and folate are water-soluble vitamins that are useful indicators of chronic gastrointestinal (GI) function in humans and some animal species. Serum cobalamin and folate concentrations in an ex situ population of killer whales ( Orcinus orca) were measured and factors that may affect their serum concentrations were identified. Serum samples ( n = 104) were analyzed from killer whales ( n = 10) both while clinically healthy and during periods of clinical GI disease as defined by clinical signs and fecal cytology. To characterize serum cobalamin and folate concentrations in clinically healthy animals, a mixed-model regression was used, with cobalamin and folate both significantly affected by weight (cobalamin: P < 0.0001, folate: P = 0.006) and season (cobalamin: P < 0.0001, folate: P < 0.0001). The marginal mean concentrations for cobalamin and folate across weight and season were 742 ± 53.6 ng/L and 30.2 ± 2.6 µg/L, respectively. The predicted 95% confidence intervals (CI) for these analytes were then compared with samples collected during periods of GI disease. Across individuals, 22% (2/9) of the folate and 80% (8/10) of the cobalamin samples from the animals with GI disease fell outside the 95% CI for the population. When comparing samples within an individual, a similar pattern presented, with 100% of cobalamin of the observed abnormal samples reduced compared to healthy animal concentration variability. The same was not true for folate. These results suggest that serum concentrations of cobalamin and folate may be useful minimally invasive markers to identify GI disease in killer whales, especially when values are compared within an individual.

ESTABLISHING MARGINAL LYMPH NODE ULTRASONOGRAPHIC CHARACTERISTICS IN HEALTHY BOTTLENOSE DOLPHINS ( TURSIOPS TRUNCATUS).

Pulmonary disease has been well documented in wild and managed dolphin populations. The marginal lymph nodes of the dolphin thorax provide lymphatic drainage to the lungs and can indicate pulmonary disease. This study standardized a technique for rapid, efficient, and thorough ultrasonographic evaluation of the marginal lymph nodes in bottlenose dolphins ( Tursiops truncatus). Thoracic ultrasonography was performed on 29 clinically healthy adult bottlenose dolphins. Reference intervals for lymph node dimensions and ultrasonographic characteristics of marginal lymph nodes were determined from four transducer orientations: longitudinal, transverse, oblique, and an orientation optimized to the ultrasonographer's eye. The relationship between lymph node dimensions and dolphin age, sex, length, weight, origin, and management setting (pool versus ocean enclosure) were also evaluated. The mean marginal lymph nodes measured 5.26 cm in length (SD = 1.10 cm, minimum = 3.04 cm, maximum = 7.61 cm, reference interval [10th to 90th percentiles per node dimension] 3.78-6.55 cm) and 3.72 cm in depth (SD = 0.59 cm, minimum = 2.64, maximum = 5.38 cm, reference interval 2.98-4.50 cm). Sex, dolphin length, weight, and management setting had no effect on lymph node dimensions. Dolphins >30 yr of age had longer node lengths than dolphins 5-10 yr old. Node dimensions did differ between dolphins from various origins. Most commonly, the lymph node was found to be hyperechoic relative to surrounding soft tissues (98%) and to have irregular caudal borders (84%), ill-defined deep borders (83%), flat superficial border (67%), triangular or rounded triangle shape (59%), irregular cranial border (55%), and moderate heterogeneity (34%). The data reported in this study serve as a baseline reference that may contribute to earlier detection of pleural and pulmonary disease of managed and wild cetacean populations.

Evaluation of anti-Erysipelothrix rhusiopathiae IgG response in bottlenose dolphins Tursiops truncatus to a commercial pig vaccine.

Erysipelothrix rhusiopathiae is the causative agent of erysipeloid in humans and of erysipelas in various animals, including bottlenose dolphins Tursiops truncatus, in which an infection has the potential to cause peracute septicemia and death. The purpose of this study was to evaluate the efficacy of using an off-label porcine (ER BAC PLUS®, Zoetis) E. rhusiopathiae bactrin in a bottlenose dolphin vaccination program by determining the anti-E. rhusiopathiae antibody levels in vaccinated dolphins over a 10 yr period. Serum samples (n = 88) were analyzed using a modified fluorescent microbead immunoassay from 54 dolphins, including 3 individuals with no history of vaccination and 51 dolphins with an average of 5 vaccinations, 3 of which had previously recovered from a natural E. rhusiopathiae infection. A mean 311-fold increase in the immunoglobulin G (IgG) antibody index was measured in a subsample of 10 dolphins 14 d after the first booster vaccination. Serum IgG antibody titers were influenced by number of vaccines received (r2 = 0.47, p < 0.05) but not by age, gender, history of natural infection, adverse vaccine reaction, vaccination interval or time since last vaccination. The commercial pig bacterin was deemed effective in generating humoral immunity against E. rhusiopathiae in dolphins. However, since the probability of an adverse reaction toward the vaccine was moderately correlated (p = 0.07, r2 = 0.1) with number of vaccines administered, more research is needed to determine the optimal vaccination interval.

Cutaneous malignant melanoma in a Haller's round ray Urobatis halleri.

Multiple black raised nodular masses were noted on the dorsal surface of an adult male Haller's round ray Urobatis halleri. Biopsy of 2 masses was performed, and histopathology revealed proliferative sheets of melanocytes exhibiting mild anisocytosis and anisokaryosis, supporting a diagnosis of malignant melanoma. Approximately 2 mo following the biopsy procedure, the round ray became acutely anorexic and was found dead in its enclosure. A full necropsy was performed, and tissues were submitted for histopathology. The black raised nodular masses again exhibited histologic features of a melanoma. In addition to the nodular masses present, multiple flat areas of increased pigmentation were also present throughout the course of the case and were not suggestive of neoplasia histologically. The transformation of benign to malignant neoplasia has been well described in other species and may have played a role in the development of multiple tumors in this case.

INTRAOCULAR PRESSURE IN SOUTHERN ROCKHOPPER (EUDYPTES CHRYSOCOME) AND MACARONI PENGUINS (EUDYPTES CHRYSOLOPHUS): EVALUATION OF INFLUENCING FACTORS.

Ophthalmic examinations were performed on 160 macaroni penguins (Eudyptes chrysolophus) and 90 southern rockhopper penguins (Eudyptes chrysocome) at eight North American zoos and aquaria. Intraocular pressure (IOP) was measured using rebound tonometry while penguins were held in two different body positions. Correlations between IOP and factors including age, body position, eye pathology, and housing parameters were evaluated. Normal macaroni penguins had a mean IOP of 42.0 ± 9.7 mm Hg. Normal rockhopper penguins had a mean IOP of 32.9 ± 6.2 mm Hg. Neither species had significantly different IOP between sexes or between left and right eyes of the same penguin. In both species, there was a negative linear correlation between age and IOP. In the macaroni population, IOP was significantly higher when IOP measurement was performed before ophthalmic exam; this was not true in rockhoppers. In both species, IOP measured in a horizontal body position was significantly higher than IOP measured in a vertical body position. In both species, eyes with corneal lesions had significantly lower IOP than normal eyes. In the macaroni penguin, eyes with rubeosis iridis had significantly lower IOP than normal eyes. In macaroni penguins, eyes with cataracts had significantly lower mean IOP than normal eyes; this was not true for rockhoppers.

EVALUATION OF POTENTIAL RISK FACTORS ASSOCIATED WITH CATARACT IN CAPTIVE MACARONI (EUDYPTES CHRYSOLOPHUS) AND ROCKHOPPER PENGUINS (EUDYPTES CHRYSOCOME).

Complete ophthalmic examinations were performed on 160 Macaroni penguins ( Eudyptes chrysolophus ) and 90 Rockhopper penguins ( Eudyptes chrysocome ) at eight North American zoological institutions. Cataract prevalence in the Macaroni population was 46.5% (n = 74) of penguins and 42.3% (135/319) of eyes. Cataract prevalence in the Rockhopper population was 45.5% (n = 40) of penguins and 40.6% (73/180) of eyes. The mean age of Macaroni penguins without ocular disease was 7.4 ± 5.8 yr, while that of Rockhoppers was 9.8 ± 6.4 yr. Risk factors for cataract were examined through husbandry surveys completed by each institution and by evaluation of light intensity and ultraviolet (UV) light measurements acquired in each penguin exhibit. Risk factors associated with cataract in Macaroni penguins included age, dietary smelt, hand-feeding, and fluorescent exhibit lighting. Risk factors associated with cataract in Rockhopper penguins included age, dietary capelin, increasing population density, and increasing length of minimum photoperiod. Factors associated with decreased odds of cataract in Macaroni penguins included saltwater pool, monitoring of water quality for salinity, pH, and alkalinity; use of water additives; presence of pool filtration and sterilization systems; use of metal halide lightbulbs; increasing light intensity; and UV spectrum lighting. Factors associated with decreased odds of cataract in Rockhoppers included dietary herring and krill, increasing exhibit land area, pool temperature monitoring, increasing maximum photoperiod, and increasing minimum UV light.

PHAEOHYPHOMYCOSIS ASSOCIATED WITH OSSIFICATION OF THE SKULL AND CERVICAL VERTEBRAE IN A SWELL SHARK (CEPHALOSCYLLIUM VENTRIOSUM).

A female, captive bred, juvenile swell shark ( Cephaloscyllium ventriosum ) was observed swimming in tight circles and rolling. Radiographs and computed tomography of this individual revealed extensive cartilage mineralization of the skull and cranial cervical vertebrae compared with diagnostic images of clinically healthy conspecifics. Gross necropsy and histopathologic examination revealed ossification and fibrosis of the cartilaginous matrix of the skull and cervical vertebrae with deep invasion by a pigmented hyphal fungus. There was no growth on fungal culture, but fungal polymerase chain reaction identified a DNA sequence compatible with Exophiala sp. (99%). Radiographs and computed tomography were helpful to determine a prognosis and course of action for this individual. This case emphasizes the need to include fungal infections as a differential diagnosis when evaluating elasmobranchs with abnormal swimming behaviors and mineralization of the skeletal structures.

MALE REPRODUCTIVE PHYSIOLOGY AND THE DEVELOPMENT OF ARTIFICIAL INSEMINATION IN THE MAGELLANIC PENGUIN (SPHENISCUS MAGELLANICUS) USING CHILLED-STORED SEMEN.

Research was performed to increase our understanding of male Magellanic penguin (Spheniscus magellanicus) reproductive biology and to develop artificial insemination (AI) technology to assist with maintaining the species' genetic diversity. Seminal traits were characterized from seven males with noncontaminated ejaculates (n = 123) displaying high in vitro motion parameters, membrane integrity, and morphology. Seven females were maintained in nest sites that permitted visual, auditory, and tactile contact with their paired male but not copulation for 18.3 ± 2.4 days before egg lay. After cloacal AI (2.6 ± 0.4 inseminations/female) with semen chilled for up to 20.5 hr at 5°C, all females produced one to two fertile eggs, with the first oviposition occurring within 7 days of plasma progesterone concentrations exceeding 0.8 ng/ml. Overall fertility was 91.7%, hatchability was 63.6%, and genetic analyses confirmed that all embryos and hatchlings were sired by AI males. The heterospermic AI design demonstrated that eggs were fertilized by spermatozoa chilled for 1.5-19.8 hr before AI and were laid 4.5-11.5 days post AI. These results contribute new data on Magellanic penguin sperm biology and demonstrate that high fertility rates after AI of chilled semen can be achieved with females remaining in proximity to their paired mate.

CHARACTERIZATION OF A FOLLICULAR CELL CARCINOMA OF THE THYROID IN A YELLOWBAR ANGELFISH (POMACANTHUS MACULOSUS).

Histopathologic diagnosis of thyroid neoplasia in teleosts is complicated, because thyroid tissue is unencapsulated, and normal tissue can have wide ectopic extensions. Assessment of thyroid hormone concentrations in conjunction with histologic evaluation of thyroid carcinoma has not been reported in teleosts, even though routinely performed in other species. A yellowbar angelfish (Pomacanthus maculosus) presented with a mass that extended beyond the gill arches. Partial surgical resection was performed, and a histologic diagnosis of thyroid carcinoma was made. Plasma from the affected fish and two control Pomacanthus were used to assess thyroid hormone concentrations using a domestic mammalian assay. Thyroid-stimulating hormone and throxine were undetectable in two of three fish. Triiodothyronine was lower in the affected fish than in controls. The tumor did not appear to be actively secreting hormone. This is the first characterization of thyroid tumors and corresponding thyroid hormones in teleosts, which may assist in diagnosis of this disease.

APHAKIC PHACOEMULSIFICATION AND AUTOMATED ANTERIOR VITRECTOMY, AND POSTRETURN MONITORING OF A REHABILITATED HARBOR SEAL (PHOCA VITULINA RICHARDSI) PUP.

A female harbor seal pup rescued along the coast of San Diego on 13 June 2012 was diagnosed with bilateral mature cataracts, apparently congenital, in association with vitreal herniation in the anterior chamber of each eye. The cataracts were surgically removed on 1 August 2012 with single-port aphakic phacoemulsification and automated anterior vitrectomy. Postoperative monitoring during the next several weeks indicated that vision had been functionally repaired and that she could visually orient to and capture live fish in three different environments and in the presence of other animals. Consequently, we equipped the seal with a satellite-linked radio transmitter and returned her to the Pacific Ocean on 21 November 2012, and then monitored her movements until radio contact ended on 2 March 2013. She remained along the San Diego coast from 21 November until 5 December 2012 when she relocated to the Coronado Islands and remained there until 26 December. She then traveled directly to San Clemente Island and remained foraging in the near-shore kelp beds there through 2 March 2013, when radio contact ended. To our knowledge, this is the first published report of cataract treatment in a marine mammal using high-frequency ultrasound to emulsify the lenses followed by suction removal of the emulsified microfragments (i.e., phacoemulsification). Moreover, the rapid postoperative recovery of the seal and its quick acclimation, orientation, navigation, and foraging in marine habitats after return to the Pacific Ocean indicates that these surgical procedures can be safe and effective treatments for cataracts in seals, with substantially reduced postsurgical complications relative to other types of lens fragmentation and removal procedures.

Coccidioidomycosis in a Pacific walrus (Odobenus rosmarus divergens).

An 11 yr-old female Pacific walrus (Odobenus rosmarus divergens) demonstrated decreased appetite and weight loss approximately 4 wk after truck transport from a northern California facility to a southern California facility. An initial blood analysis revealed a leukocytosis of 22,800 white blood cells (WBC)/microl, with a left shift, low iron (58 microg/dl), and mild hyperglobulinemia (4.3 g/dl). Empiric antibiotic therapy was started with amoxicillin and clavulanic acid (14 mg/kg p.o. b.i.d.). Clinical improvement was observed initially; however, follow-up blood analysis demonstrated a persistent leukocytosis (24,000 WBC/microl), with left shift and progressive hyperglobulinemia (6.7 mg/dl). As a result of the relapse of clinical signs on antibiotic therapy, aggressive antifungal therapy was initiated with voriconazole (1.8 mg/kg p.o. s.i.d.). Concurrent fungal immunodiffusion antibody assays and complement fixation were repetitively positive for coccidioidomycosis. The walrus improved clinically over the next 3 mo and is currently stable on antifungal therapy at its originating facility in northern California.

Pharmacokinetics of single dose oral meloxicam in bottlenose dolphins (Tursiops truncatus).

The objective of this study was to investigate the pharmacokinetics of meloxicam in bottlenose dolphins (Tursiops truncatus). Ten adult bottlenose dolphins were used for the study. Each animal received a single oral dose of meloxicam at 0.1 mg/kg. Two to seven serial blood samples were collected per animal, at one of fourteen time points between T = 0 and T = 240 hr. Complete blood count and serum chemistry analysis were performed prior to drug administration, as well as at the final time point for each individual. Plasma drug concentrations were determined by high-pressure liquid chromatography. No adverse hematological, biochemical or clinical changes were noted during the study period. After oral administration, a peak plasma concentration of 1.03 microg/mL was achieved at approximately 11 hr. This suggests that a single oral dose of 0.1 mg/kg provides a peak plasma level similar to what is considered therapeutic in other species. However, the elimination of meloxicam in cetaceans was slower than in other species, with an elimination half-life of almost 70 hr, and detectable drug concentrations up to 7 days. A single oral dose of 0.1 mg/kg appears safe for use in this species, but caution in repeated dosing must be used, due to the prolonged elimination, until multi-dose pharmacokinetic studies are determined.