Cellular immune responses in cetaceans immunized with a porcine erysipelas vaccine.

Clinical erysipelas represents a significant health problem in managed cetacean species. Vaccination was suspended in many oceanariums in the past due to losses associated with vaccine-induced hypersensitivities which were deemed to be a greater threat than clinical erysipelas. A perceived shift in clinical presentation of erysipelas from a chronic dermatologic form to an acute systemic form in dolphins sparked interest in re-initiating vaccination with improved subunit vaccines of Erysipelothrix rhusiopathiae. This manuscript describes the development and application of in vitro correlates of immunity (T(H)1, T(H)2 and T(REG)) in Tursiops truncatus induced by immunization with a commercial porcine 65 kDa subunit E. rhusiopathiae vaccine. Variable degrees of pre-existing T cell memory were identified prior to vaccination. Vaccine-induced IFN gamma responses were consistent with a T(H)1 response and associated with elimination of erysipelas in all vaccinated animals. Comparative analysis between six-month and 12-month vaccination booster regimes demonstrated maintenance of superior memory in the six-month group; however, anamnestic responses induced by booster were only identified in the 12-month group. To our knowledge, this is the first study to develop and apply advanced immunologic analyses for assessing vaccine efficacy in captive or free-ranging wildlife.

Hypocitraturia in common bottlenose dolphins (Tursiops truncatus): assessing a potential risk factor for urate nephrolithiasis.

Numerous cases of urate nephrolithiasis in managed collections of common bottlenose dolphins (Tursiops truncatus) have been reported, but nephrolithiasis is believed to be uncommon in wild dolphins. Risk factors for urate nephrolithiasis in humans include low urinary pH and hypocitraturia. Urine samples from 94 dolphins were collected during April 2006 through June 2009 from 4 wild populations (n = 62) and 4 managed collections (n = 32). In addition, urine uric acid and pH were tested in a subset of these animals. Our null hypothesis was that wild and managed collection dolphins would have no significant differences in urinary creatinine, citrate, and uric acid concentrations and pH. Among urine samples from all 94 dolphins, the urinary levels (mean +/- SEM) for creatinine, citrate, uric acid, and pH were 139 +/- 7.6 mg/dL, 100 +/- 20 mg citrate/g creatinine, 305 +/- 32 mg uric acid/g creatinine, and 6.2 +/- 0.05, respectively. Of the 4 urinary variables, only citrate concentration varied significantly between the 2 primary study groups; compared with wild dolphins, managed collection dolphins were more likely to have undetectable levels of citrate in the urine (21.0% and 81.3%, respectively). Mean urinary citrate concentrations for managed collection and wild dolphin populations were 2 and 150 mg citrate/g creatinine, respectively. We conclude that some managed collections of dolphins, like humans, may be predisposed to urate nephrolithiasis due to the presence of hypocitraturia. Subsequent investigations can include associations between metabolic syndrome, hypocitraturia, and urate nephrolithiasis in humans and dolphins; and the impact of varying levels of seawater ingestion on citrate excretion.

Pure tone audiograms and possible aminoglycoside-induced hearing loss in belugas (Delphinapterus leucas).

A behavioral response paradigm was used to measure pure-tone hearing sensitivities in two belugas (Delphinapterus leucas). Tests were conducted over a 20-month period at the Point Defiance Zoo and Aquarium, in Tacoma, WA. Subjects were two males, aged 8-10 and 9-11 during the course of the study. Subjects were born in an oceanarium and had been housed together for all of their lives. Hearing thresholds were measured using a modified up/down staircase procedure and acoustic response paradigm where subjects were trained to produce audible responses to test tones and to remain quiet otherwise. Test frequencies ranged from approximately 2 to 130 kHz. Best sensitivities ranged from approximately 40 to 50 dB re 1 microPa at 50-80 kHz and 30-35 kHz for the two subjects. Although both subjects possessed traditional "U-shaped" mammalian audiograms, one subject exhibited significant high-frequency hearing loss above 37 kHz compared to previously published data for belugas. Hearing loss in this subject was estimated to approach 90 dB for frequencies above 50 kHz. Similar ages, ancestry, and environmental conditions between subjects, but a history of ototoxic drug administration in only one subject, suggest that the observed hearing loss was a result of the aminoglycoside antibiotic amikacin.

Expression and functional characterization of killer whale (Orcinus orca) interleukin-6 (IL-6) and development of a competitive immunoassay.

Interleukin-6 (IL-6) is a cytokine that can reach detectable systemic levels and is a major inducer of the acute phase response. As such, clinical assays to identify this cytokine in mammalian sera are of diagnostic value. A 558 base-pair (bp) fragment of killer whale IL-6 was cloned and expressed as a 21 kDa protein in Escherichia coli. Biological activity of the recombinant killer whale IL-6 (rkwIL-6) was demonstrated using the IL-6-dependent B9 mouse hybridoma cell line; acute phase sera from a killer whale and supernatants from lipopolysaccharide (LPS)-stimulated killer whale peripheral blood mononuclear cells (PBMCs) also supported the proliferation of the B9 hybridoma. Rat anti-mouse IL-6 receptor antibody effectively blocked biological activity of all three sources of IL-6. Polyclonal antisera, specific for the recombinant protein, were obtained by successive immunization of a rabbit with rkwIL-6. The polyclonal antibody was capable of neutralizing the biological activity of both recombinant and native kwIL-6. A competitive enzyme-linked immunosorbent assay (ELISA) was developed using the polyclonal rabbit anti-rkwIL-6 and the recombinant protein; sensitivity of the assay was in the range of 1 ng/ml. The ELISA was subsequently used to identify the presence of native IL-6 in acute phase sera of two species of delphinidae, a killer whale and a bottlenose dolphin. The application of quantitative cytokine assays as diagnostic tools for monitoring cetacean health are becoming feasible as many animals are now being trained for fluke presentation, making blood collection a routine procedure.