Shawn P. Johnson

CHARACTERIZATION AND CLINICAL MANIFESTATIONS OF ARCANOBACTERIUM PHOCAE INFECTIONS IN MARINE MAMMALS STRANDED ALONG THE CENTRAL CALIFORNIA COAST

Between 1994 and 2000, 141 Arcanobacterium phocae isolates were recovered from marine mammals that stranded along the central California coast (USA). Arcanobacterium phocae was cultured from tissue sites with abnormal discharge or evidence of inflammation in 66 California sea lions (Zalophus californianus), 50 Pacific harbor seals (Phoca vitulina richardii), 19 northern elephant seals (Mirounga angustirostris), five southern sea otters (Enhydra lutris nereis), and one common dolphin (Delphinus delphis). The overall prevalence of A. phocae among cultured stranded marine mammals was 8%. This is the first report of A. phocae in animals from the Pacific Ocean. Sequence analysis of a portion of the 16S ribosomal RNA gene confirmed recent isolates as A. phocae. Prior to phylogenetic testing and the routine use of the esculin hydrolysis and motility tests, A. phocae isolates may have been misidentified as Listeria ivanovii. Arcanobacterium phocae was commonly isolated from superficial abscesses, was often present in mixed infections, and was susceptible to all antimicrobial agents tested.

The sun, moon, wind, and biological imperative-shaping contrasting wintertime migration and foraging strategies of adult male and female northern fur seals (Callorhinus ursinus).

Adult male and female northern fur seals (Callorhinus ursinus) are sexually segregated in different regions of the North Pacific Ocean and Bering Sea during their winter migration. Explanations for this involve interplay between physiology, predator-prey dynamics, and ecosystem characteristics, however possible mechanisms lack empirical support. To investigate factors influencing the winter ecology of both sexes, we deployed five satellite-linked conductivity, temperature, and depth data loggers on adult males, and six satellite-linked depth data loggers and four satellite transmitters on adult females from St. Paul Island (Bering Sea, Alaska, USA) in October 2009. Males and females migrated to different regions of the North Pacific Ocean: males wintered in the Bering Sea and northern North Pacific Ocean, while females migrated to the Gulf of Alaska and California Current. Horizontal and vertical movement behaviors of both sexes were influenced by wind speed, season, light (sun and moon), and the ecosystem they occupied, although the expression of the behaviors differed between sexes. Male dive depths were aligned with the depth of the mixed layer during daylight periods and we suspect this was the case for females upon their arrival to the California Current. We suggest that females, because of their smaller size and physiological limitations, must avoid severe winters typical of the northern North Pacific Ocean and Bering Sea and migrate long distances to areas of more benign environmental conditions and where prey is shallower and more accessible. In contrast, males can better tolerate often extreme winter ocean conditions and exploit prey at depth because of their greater size and physiological capabilities. We believe these contrasting winter behaviors 1) are a consequence of evolutionary selection for large size in males, important to the acquisition and defense of territories against rivals during the breeding season, and 2) ease environmental/physiological constraints imposed on smaller females.

Inflation and deflation pressure-volume loops in anesthetized pinnipeds confirms compliant chest and lungs

We examined structural properties of the marine mammal respiratory system, and tested Scholanders hypothesis that the chest is highly compliant by measuring the mechanical properties of the respiratory system in five species of pinniped under anesthesia (Pacific harbor seal, Phoca vitulina; northern elephant seal, Mirounga angustirostris; northern fur seal Callorhinus ursinus; California sea lion, Zalophus californianus; and Steller sea lion, Eumetopias jubatus). We found that the chest wall compliance (CCW) of all five species was greater than lung compliance (airways and alveoli, CL) as predicted by Scholander, which suggests that the chest provides little protection against alveolar collapse or lung squeeze. We also found that specific respiratory compliance was significantly greater in wild animals than in animals raised in an aquatic facility. While differences in ages between the two groups may affect this incidental finding, it is also possible that lung conditioning in free-living animals may increase pulmonary compliance and reduce the risk of lung squeeze during diving. Overall, our data indicate that compliance of excised pinniped lungs provide a good estimate of total respiratory compliance.

ASSOCIATION BETWEEN POSITIVE CANINE HEARTWORM (DIROFILARIA IMMITIS) ANTIGEN RESULTS AND PRESENCE OF ACANTHOCHEILONEMA ODENDHALI MICROFILARIA IN CALIFORNIA SEA LIONS (ZALOPHUS CALIFORNIANUS)

Abstract This study establishes a relationship between positive canine heartworm (Dirofilaria immitis) test results frequently observed in California sea lions (Zalophus californianus) and infection with the filarid nematode Acanthocheilonema odendhali. Four commercially available canine heartworm antigen tests were evaluated for cross-reaction with A. odendhali in California sea lions. Sera were tested from fifteen California sea lions with A. odendhali-associated microfilaremia, confirmed by blood smear, and with no evidence of D. immitis infection at necropsy. Ninety-five percent of tests were falsely positive for D. immitis. This study also determined that the prevalence of A. odendhali infection in stranded California sea lions from central California is approximately 23% by comparing the number of findings of mircofilaremia to the total number of California sea lions sampled at The Marine Mammal Center between 2005 and 2011, inclusive. Acanthocheilonema odenhali microfilaremia in California sea lions is likely to cross-react with canine heartworm antigen tests, and clinicians should interpret results with caution.

PHARMACOKINETICS OF A SINGLE SUBCUTANEOUS DOSE OF SUSTAINED RELEASE BUPRENORPHINE IN NORTHERN ELEPHANT SEALS (MIROUNGA ANGUSTIROSTRIS)

Abstract:  Information regarding analgesics in pinnipeds is limited. This study aimed to establish the pharmacokinetic parameters of a single subcutaneous dose of sustained release buprenorphine (Buprenorphine SR®) in juvenile northern elephant seals (Mirounga angustirostris) with regard to its potential to provide long-lasting analgesia that requires infrequent dosing. Seals (n = 26) were administered a single dose of sustained release buprenorphine at 0.12 mg/kg s.c. Blood samples were collected from the extradural intervertebral vein at 0 hr and at three or four of the following time points: 0.5, 1, 2, 6, 12, 24, 36, 48, 60, 96, 120, and 144 hr. Seals were examined daily for systemic and local adverse reactions. Plasma was analyzed by liquid chromatography tandem–mass spectrometry for buprenorphine and norbuprenorphine concentrations. A noncompartmental analysis for pharmacokinetic parameters was calculated using standard methods and equations. An average maximum concentration of 1.21 ng/ml (0.3–2.9 ng/ml) was detected 12 hr postadministration. Concentrations were quantifiable up to 144 hr postadministration but were below those expected to provide analgesia in some other species. No systemic adverse effects were noted in healthy seals receiving sustained release buprenorphine. Cellulitis or abscesses at the injection site were observed in 6/26 (23%) seals between 24 and 168 hr postadministration. Adverse local effects suggest that this drug should be used with caution in northern elephant seals.

DIAGNOSIS AND SUCCESSFUL TREATMENT OF A LUNG ABSCESS ASSOCIATED WITH BRUCELLA SPECIES INFECTION IN A BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATUS)

Abstract:  This brief communication describes the clinical presentation, antemortem diagnosis, and successful treatment of a pulmonary abscess associated with a Brucella sp. in a 27-yr-old female bottlenose dolphin (Tursiops truncatus). Ultrasound revealed a 3-cm diameter hypoechoic mass deep to the pleural lining in the left lung field. Multiple ultrasound-guided fine-needle aspirates were performed and tested for bacterial and fungal etiology. All cultures were negative, but the infectious agent was identified by MicroSEQ analysis in two samples and confirmed with real-time polymerase chain reaction (PCR) amplification using known Brucella sp. primers. Amikacin was infused into the abscess and was followed by an oral doxycycline and rifampin protocol. Follow-up diagnostic imaging, including radiographs and computed tomography, revealed a resolved lesion with minimal mineralization within the affected lung fields. Brucellosis should be considered for pulmonary disease in dolphins, and personnel who interact with marine animals should use caution to prevent zoonotic brucellosis.

Best practice recommendations for the use of fully implanted telemetry devices in pinnipeds

Electronic telemetry devices have enabled many novel and important data collection and experimental opportunities for difficult to observe species. Externally attached devices have limited retention and may affect thermoregulation, energetics, social and reproductive behavior, visibility, predation risk and entanglement. Internally placed, surgically implanted devices can mitigate some of these effects and may open additional experimental opportunities. However, improper implementation can significantly affect animals and data. From a review of recent studies using fully implanted tags and studying their effects, we present 15 specific best practice recommendations for the use of such tags in pinnipeds. Recommendations address issues including device size, coating and sterilization, implantation surgery and effect assessment, within the framework of the Three R’s: Reduction, Refinement, Replacement. While developed for pinnipeds, these recommendations could apply to other aquatic mammals and vertebrates and to partially implanted or even external tags.

Hypervirulent Klebsiella pneumoniae in California Sea Lions (Zalophus californianus): Pathologic Findings in Natural Infections:

Tissues of stranded California sea lions (Zalophus californianus) naturally infected with a hyperviruluent strain of Klebsiella pneumoniae were examined by histopathology and immunohistochemistry against the K. pneumoniae K2 capsular antigen. In 7 of 8 animals, there was severe purulent bronchopneumonia, sometimes complicated by fibrinonecrotizing pleuritis with pyothorax. In affected areas of lung, large numbers of degenerate neutrophils and macrophages were admixed with rare large extracellular and intracellular gram-negative bacilli surrounded by a prominent capsule. Through serotyping, polymerase chain reaction, sequencing, and immunohistochemistry, these bacteria were confirmed to be a K2 serotype of K. pneumoniae. The same bacteria were identified through double immunolabeling within macrophages in blood vessels, lymph nodes, spleen, and liver. Intact K. pneumoniae were identified on epithelial surfaces of the nasopharyngeal, tracheal, and small intestine mucosae and within distal renal tubules. Our findings indicate that hypervirulent K. pneumoniae causes severe respiratory disease and intrahistiocytic bacteremia in California sea lions.

PHARMACOKINETIC STUDY OF ORAL ε-AMINOCAPROIC ACID IN THE NORTHERN ELEPHANT SEAL (MIROUNGA ANGUSTIROSTRIS)

Abstract ϵ-Aminocaproic acid (EACA) is a lysine analogue antifibrinolytic drug used to treat bleeding disorders in humans and domestic animals. Its use in zoological medicine is rare, and dosage is anecdotal. One possible application of EACA is to treat bleeding associated with prepatent Otostrongylus arteritis in Northern elephant seals (Mirounga angustirostris) presenting to wildlife rehabilitation centers. This study used an in vitro model of hyperfibrinolysis and a thromboelastograph-based assay to estimate the therapeutic plasma concentration of EACA in elephant seals (85 μg/ml, 95% confidence interval = 73.8–96.8 μg/ml). A concurrent pharmacokinetic study of orally administered, single-dose EACA found that doses of 75 and 100 mg/kg achieved therapeutic plasma concentrations (>85 μg/ml), but the drug was rapidly eliminated and remained in the therapeutic range for only 0.4 and 1.5 hr, respectively. Models of repeated oral dosing at 100 mg/kg every 6 hr predict that therapeutic plasma concentration will be maintained for 31.7% (7.6 hr) of a 24-hr period. More frequent dosing would be required to maintain continuous therapeutic concentrations but would be impractical in a wildlife rehabilitation setting. Further pharmacodynamic studies to evaluate the duration of action of EACA in elephant seals and a prospective, placebo-controlled study are needed to determine if EACA is effective in decreasing bleeding associated with prepatent Otostrongylus arteritis and other bleeding disorders in this species.

DEVELOPMENT OF RETROBULBAR AND AURICULOPALPEBRAL NERVE BLOCKS IN CALIFORNIA SEA LIONS (ZALOPHUS CALIFORNIANUS)

Abstract Eye lesions are commonly observed in pinnipeds. Clinical assessment is challenging because animals are often blepharospastic and under inhalant anesthesia the globe rotates ventrally, making observation difficult. Retrobulbar and auriculopalpebral nerve block techniques have been developed in other species to alleviate these difficulties and allow for a more thorough ophthalmic exam. Ocular nerve block techniques were developed for California sea lions (CSLs) (Zalophus californianus) using lidocaine hydrochloride 2%. To develop the retrobulbar block, a variety of needle sizes, anatomic approaches, and volumes of methylene blue were injected into the orbits of 10 CSL cadavers. An optimal technique, based on desired distribution of methylene blue dye into periocular muscles and tissues, was determined to be a two-point (ventrolateral and ventromedial) transpalpebral injection with a 20-ga, 1 1/2-inch needle. This technique was then tested using lidocaine on 26 anesthetized animals prior to euthanasia, and on one case with clinical ocular disease. A dose of 4 mg/kg of lidocaine was considered ideal, with positive results and minimal complications. The retrobulbar block had a 76.9% rate of success (using 4 mg/kg of lidocaine), which was defined as the globe returning at least halfway to its central orientation with mydriasis. No systemic adverse effects were noted with this technique. The auriculopalpebral nerve block was also adapted for CSLs from techniques described in dogs, cattle, and horses. Lidocaine was injected (2–3 ml) by subcutaneous infiltration lateral to the orbital rim, where the auriculopalpebral nerve branch courses over the zygomatic arch. This block was used in five blepharospastic animals that were anesthetized for ophthalmic examinations. The auriculopalpebral nerve block was successful in 60% of the cases, which was defined as reduction or elimination of blepharospasm for up to 3 hr. Success appeared to be dependent more on the location of injection rather than on the dose administered.