Cold stress induces an adrenocortical response in bottlenose dolphins (Tursiops truncatus)

Two adult bottlenose dolphins (Tursiops truncatus) were individually housed in aboveground pools over a 10-day period and exposed to decreasing water temperatures to determine whether cold stress activated the hypothalamic-pituitary-adrenal axis. To serve as controls, two additional adult dolphins were similarly housed for the same duration but at ambient water temperatures (16.8-19.6 degrees C). Across all subjects, water temperatures ranged from 4.2 to 19.6 degrees C. Voluntary blood draws were made from each dolphin every 2-3 days, and serum was analyzed via radioimmunoassay for cortisol and aldosterone. Dolphins exposed to cold water showed an increase in serum cortisol and aldosterone as temperature declined; at the coldest water exposure, cortisol was more than three times and aldosterone more than two times the levels measured at ambient temperature. Elevations occurred before the water temperature declined below the individual animal's lower critical temperature, the point at which the metabolic rate increases to compensate for the loss of body heat. Variations in corticosteroids were unrelated to the 10-day isolation period, suggesting that the response was related to the cold stress and not impacted by the isolation. Elevations in cortisol and aldosterone were lower than those observed in force captured and stranded dolphins. Although potentially related to the general adaptive stress response, elevations in cortisol and aldosterone may have other adaptive functions related to mitigating impacts resulting from cold environmental temperatures.     

Relationship between plasma iron concentration and gastric pH in captive adult bottlenose dolphins (Tursiops truncatus)

OBJECTIVE: To determine the correlation between plasma iron concentrations and gastric pH in a population of captive Atlantic bottlenose dolphins (Tursiops truncatus). ANIMALS: 6 adult female dolphins that ranged from 16 to 30 years of age. PROCEDURES: Blood and gastric samples were collected from each dolphin to allow measurement of plasma iron concentrations and gastric pH, respectively. Samples were collected each month for 12 months. RESULTS: Within each dolphin, plasma iron concentrations and gastric pH did not differ significantly over time. There was a strong negative correlation (r = -0.85) between plasma iron concentration and gastric pH, which suggested that dolphins with a lower gastric pH had a higher plasma iron concentration. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis of results reported here suggested that gastric pH may play an important role in iron absorption in dolphins.     

Evaluation of the polymorphonuclear cell functions of bottlenose dolphins

The functions of polymorphonuclear cells (PMN) are the important non-specific defense mechanisms in the immune system. Especially marine mammals are protected by these mechanisms from the aquatic environment with a large variety of microorganisms. Therefore, we examined the PMN functions of bottlenose dolphins in order to obtain the normal ranges and to standardize the techniques. PMNs were isolated by using lymphocyte isolate solution whose density was 1.077; superoxide production was assessed by nitroblue tetrazolium reduction test (NBT) and phagocytosis was tested by using polystyrene latex beads. We showed that the optimal incubation time was 30 min in NBT assay and 12 hr in phagocytosis assay for dolphin PMNs.     

Brucella-induced abortions and infection in bottlenose dolphins (Tursiops truncatus).

Two bottlenose dolphins (Tursiops truncatus) aborted fetuses that died as a result of Brucella infection. Brucella placentitis occurred in both cases. Infected placenta and vaginal/uterine fluids may transmit Brucella species to other cetaceans. In a third case, an identical organism was cultured from lung necropsy tissue of an adult female T. truncatus. Microbiology, specific polymerase chain reaction, and pulsed-field gel electrophoresis results supported the designation of an additional genomic group(s), Brucella delphini, for isolates adapted to T. truncatus. Current serologic diagnostic tests reliable for known Brucella species are unreliable in detecting dolphin brucellosis. Our findings, together with previous reports, suggest that dolphin brucellosis is a naturally occurring disease that can adversely impact reproduction in cetaceans. The zoonotic significance of cetacean brucellosis is unknown, although the disease has not been reported in people who have frequent contact with dolphins. Further studies on the zoonotic aspects, distribution, prevalence, virulence, and impact of this disease in cetaceans and other marine mammal species are needed.     

Hematologic, biochemical, and endocrine effects of dexamethasone on bottlenose dolphins (Tursiops truncatus).

Two Atlantic bottlenose dolphins (Tursiops truncatus) were given 0.11 mg/kg dexamethasone p.o., and complete blood count and serum chemistry analyses, including insulin, thyroxine (T4) adrenocorticotrophic hormone (ACTH), and cortisol level determinations, were performed at 0 hr, 24 hr, 36 hr, 48 hr, 7 days, and 17 days. Significant changes included neutrophilia, eosinopenia, lymphopenia, elevated insulin, and depressed ACTH and cortisol levels within 24 hr of dexamethasone administration. These effects were rapid, and values returned to normal within 48 hr.     

Increased number of whistles of bottlenose dolphins, Tursiops truncatus, arising from interaction with people

The acoustic mode is the most reasonable means for social animals such as dolphins to maintain contact in the underwater habitat, and has been developed since they moved to the sea. This study investigates variations in dolphin vocalizations under the following conditions in a captive environment: 1) before feeding (Pre-feeding), 2) during feeding (Feeding), 3) during free time without the presence of people (Free), 4) during interaction with people located upon a float (Float), 5) during interaction with people in the water (Water). During the experiments, a total of 2642 whistles were extracted from sonogram data using a spectrogram. About 44% of the total whistles were observed during Pre-feeding (1171/2642), and the number recorded during Free, when people were absent, was the smallest. The acoustic contours of dolphin whistles differed in different situations: convex, wave, and trill whistles were made repeatedly during Pre-feeding, thereby being more common at this time than at other times. The situation of Feeding saw an increased number of Upsweeps, which might be related to the use of echolocation. The lower frequencies were recorded during Pre-feeding, reflecting the emotion related to the dolphin's hunger. The results of this study indicate that dolphins increase their vocalization during interaction with people, suggesting that interactions with dolphins provide an effective treatment for human health problems, which is discussed with a reference article in this study. Vocal data obtained during contact with humans might serve as an important index for the dolphin-assisted therapy.     

Paternity determination of captive bottlenose dolphins (Tursiops truncatus) using microsatellite DNA analysis

We applied previously published PCR primer pairs to amplify alleles at three polymorphic microsatellite loci to determine the genetic relationship of 6 bottlenose dolphins (Tursiops truncatus) that were living together in a Japanese aquarium. The three microsatellite loci were sufficient to determine the haplotype relationships of the six dolphins, which represented three different generations. It was confirmed that this genotyping method is simple and economical for assessing, establishing and maintaining genetic diversity in captive populations and will become a very effective technique for ex situ conservation in aquariums and zoos.     

The seasonal fluctuation of plasma amino acids in aquarium-maintained bottlenose dolphins (Tursiops truncatus)

Although there has been extensive research on plasma amino acid profiles of mammals, there is currently a lack of information on seasonal differences in the concentrations of plasma amino acids specifically in cetaceans. The present study examined the response of the plasma amino acids to seasonal changes in the culture environment after controlling for the effect of sex and age. Significant seasonal changes in plasma carnosine (P=0.012), cystine (P=0.0014), isoleucine (P=0.0042), methionine (P=0.002), ornithine (P=0.0096), and taurine (P=0.032) were observed. These amino acids were mainly related to capacity for exercise, ammonia detoxification, thermoregulation, and osmoregulation. We proposed that optimizing plasma amino acids levels by supplementation of amino acids should be of considerable benefit for aquarium-maintained bottlenose dolphins. This study constitutes a first step towards improving our understanding of the metabolism of aquarium-maintained bottlenose dolphins. We also revealed that the ratio of tryptophan to large neutral amino acids significantly declined (P=0.0076), suggesting reduction in serotonin synthesis in winter and autumn. Although further studies are needed, this finding implied that bottlenose dolphins could produce behavioral changes seasonally by the alteration of serotonin activity. To better understand the metabolic machinery for amino acids that facilitate the adaptation of marine mammals to their environments, it is essential to continue monitoring of and further investigations into relationships between plasma amino acids and specific environmental factors.     

Isolation of culturable microorganisms from free-ranging bottlenose dolphins (Tursiops truncatus) from the southeastern United States

Reports of diseases in marine mammals are increasing worldwide, however our understanding of the microorganisms associated with marine mammals is still limited. In this study, we cultured bacteria and fungi isolated from the upper respiratory tract (blowhole), gastric fluid and anus of 180 wild bottlenose dolphins (Tursiops truncatus) from two estuarine locations along the southeastern Atlantic Coast of the United States. A total of 339 and 491 isolates from Charleston, SC (CHS) and Indian River Lagoon, FL (IRL) dolphins, respectively, were cultured from gastric (70 CHS/82 IRL), fecal (141 CHS/184 IRL), and blowhole (128 CHS/225 IRL) swabs on selective media used for routine clinical microorganisms of human concern. The most frequently cultured Gram-negative bacteria from all sample and study types were Plesiomonas shigelloides, Aeromonas hydrophila, Escherichia coli, and Pseudomonas fluorescens. Among the Gram-positive bacteria, Clostridium perfringens, Bacillus sp., and Staphylococcus Coag. Neg were the predominant organisms. For fungi, the most abundant species were Candida glabrata, budding yeasts, and Candida tropicalis. Of concern, the MRSA strain of Staphylococcus aureus was detected in the blowhole and gastric swabs from CHS dolphins. In general, a greater prevalence of bacteria and fungi (four-fold increase) were cultured from IRL than CHS animals. Together, these culture-dependent studies, coupled to on-going culture-independent approaches, should help establish a baseline of microorganisms associated with bottlenose dolphins and aid in the identification of organisms responsible for infectious diseases(s) in these animals.     

Enrofloxacin serum bioactivity in bottlenose dolphins, Tursiops truncatus, following oral administration of 5 mg/kg in whole fish

Eight adult bottlenose dolphins Tursiops truncatus (six male, two female) were employed in a single-dose study of orally administered enrofloxacin dosed at 5 mg/kg body weight. Blood samples were obtained from all animals at 0, 2, 4, 8, 12 and 24 h following administration of the dose in the animals morning ration of fish. Serum antimicrobial activity concentrations (SAAC) were determined using bioassay. The mean elimination half-life (t1/2) of enrofloxacin and its major metabolites was 6.4+/-2.0 h with a range of 3-9.4 h. The time of maximal serum concentration (tmax) occurred at approximately 4 h with a range of 2-8 h following a single oral dose of 5 mg/kg. This variation in tmax most likely resulted from individual differences in absorption because of variations in the storage and digestion of the fish ration containing the drug dose within the compartmentalized cetacean stomach.     

Echocardiographic evaluation of the bottlenose dolphin (Tursiops truncatus).

Safe and effective echocardiography would represent a valuable tool for marine mammal veterinarians and physiologists evaluating the dolphin heart. Unfortunately, conventional ultrasound technology (transthoracic echocardiography) has been limited by logistic, anatomic, and behavioral challenges. Five mature male Atlantic bottlenose dolphins (Tursiops truncatus) were trained for echocardiographic imaging (four for both transthoracic and transesophageal imaging, and one for only transthoracic imaging). It was noted that transesophageal image quality transiently improved when the dolphins spontaneously exhaled. Subsequently, dolphins were conditioned to hold their breath following forced exhalation, and imaging proceeded during such behavioral breath holds. Over 25 transthoracic and 100 transesophageal echocardiographic studies were performed, including both two-dimensional imaging and color flow mapping. Transthoracic imaging yielded poor-quality images of only small portions of the heart. In contrast, transesophageal imaging, which improved dramatically with behavioral breath holding following exhalation, yielded consistently high-quality images of the entire heart (mitral, tricuspid, aortic, and pulmonary valves, atrial and ventricular septa, left and right atria, left and right ventricles, and ascending aorta and main pulmonary artery). Color flow mapping demonstrated mild tricuspid regurgitation in all dolphins, and mild aortic regurgitation in one dolphin found to have a pedunculated mass arising from the sinutubular junction just above the aortic valve. There were no complications in nonsedated dolphins. The heart of the bottlenose dolphin can be safely, effectively, and reproducibly evaluated with the use of transesophageal echocardiography in conjunction with behavioral breath holding following forced exhalation. This approach, and the normative echocardiographic data generated from this work, lays the foundation for future echocardiographic studies of cetaceans.     

Development and validation of monoclonal and polyclonal antibodies for the detection of immunoglobulin G of bottlenose dolphins (Tursiops truncatus).

Antibodies directed against species-specific immunoglobulin G (IgG) have a broad range of applications in serologic and immunologic research and in the development of clinical assays. Validated anti-IgG antibodies for marine mammal species are in short supply. The objective of this study was to produce and validate antibodies with specificity for IgG of the common bottlenose dolphin (Tursiops truncatus). Bottlenose dolphin IgG was purified using protein G. Two mouse monoclonal antibodies and a rabbit polyclonal antibody were developed from mice and rabbits immunized with bottlenose dolphin IgG. The specificity of the monoclonal antibodies and the polyclonal antibody for bottlenose dolphin IgG was first verified by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). For further validation, both monoclonal antibodies and the polyclonal antibody were incorporated in an indirect ELISA for the detection of the immune response of bottlenose dolphins to a vaccine antigen. Three bottlenose dolphins were immunized with a commercial Erysipelothrix rhusiopathiae vaccine, and serial blood samples were collected from all dolphins for measurement of levels of circulating antibodies. Seroconversion was observed in all 3 dolphins by use of both monoclonal antibodies and the polyclonal antibody. Circulating antibodies were detectable as early as 6 days after immunization in 1 dolphin. Peak antibody levels were detected 14 days after the immunization. The ability to detect seroconversion in all 3 immunized bottlenose dolphins firmly establishes the specificity of the monoclonal antibodies and the polyclonal antibody for IgG of the common bottlenose dolphin.     

Simultaneous measurement of phagocytosis and respiratory burst of leukocytes in whole blood from bottlenose dolphins (Tursiops truncatus) utilizing flow cytometry

Phagocytic and respiratory burst activity was simultaneously measured by flow cytometry in polymorphonuclear leukocytes (PMN) and monocytes in whole blood from bottlenose dolphins (Tursiops truncatus). Blood was collected from 16 adult dolphins, 12 males (6-34 years of age) and 4 females (11-30 years) and subsequently incubated with a bacteria-to-leukocyte ratio of 25:1 and 10 μl of 500 μM 2',7'-dichlorofluorescein diacetate for 70 min at 37°C. PMN (44.5 ± 3.2%) and monocytes (33.5 ± 3.0%) were positive for propidium iodide-labeled Staphylococcus aureus, indicating phagocytosis. Respiratory burst activity after 70 min as measured by the mean fluorescence intensity (MFI) was 68.0 ± 14.4 in PMN and 47.0 ± 10.3 in monocytes. There were no significant differences in MFI or percentage of phagocytizing PMN (p > 0.094) or monocytes (p > 0.275) after storage at 4°C for 24h when compared to activity measured in fresh blood. Nor was there an effect of storage on respiratory burst activity (MFI or percentage) in PMN (p > 0.420) or monocytes (p > 0.301). This assay may be particularly useful to assess the ability of dolphins to effectively combat bacterial pathogen challenges with minimal amounts of blood.     

Molecular cloning and expression of bottlenose dolphin (Tursiops truncatus) interleukin-8

The bottlenose dolphin interleukin (IL)-8 cDNA was molecularly cloned. The dolphin IL-8 has an open reading frame of 303-bp encoding 101 amino acids. The homology of the amino acid sequence with that of other species was: sheep, 89.1%; cattle, 88.1%; pig, 85.1%; dog, 85.1%; horse, 79.2%; human, 74.5%; and macaque, 72.3%. The amino acid sequence suggested that dolphin IL-8 was a CXC chemokine. The recombinant dolphin IL-8 protein was recognized with anti-ovine IL-8 monoclonal antibody.     

Oral administration of sucrose solutions and measurement of serum sucrose concentrations to evaluate gastric permeability in adult bottlenose dolphins (Tursiops truncatus)

OBJECTIVE: To measure concentrations of sucrose in the serum of captive dolphins after oral administration of a sucrose solution and determine the suitability of this method for use as a test to detect gastric ulcers. ANIMALS: 8 adult captive bottlenose dolphins (Tursiops truncatus). PROCEDURES: Blood samples were collected from the ventral fluke vein of dolphins before and 45 minutes after oral administration of 500 mL of solution containing 25 or 50 g of sucrose; oral administration was achieved by use of gastric intubation. Serum was separated, diluted in a solution of 90% acetonitrile-to 10% water that contained 10 ng of an internal standard (trichlormethiazide)/microL, mixed, and centrifuged. Supernatant was analyzed by use of liquid chromatography-mass spectrometry-mass spectrometry (LC-MS-MS). RESULTS: Serum sucrose concentrations of dolphins were at or less than the limits of detection before oral administration. Values after administration of sucrose solution varied among dolphins and were higher and more variable after administration of 50 g, compared with concentrations after administration of 25 g. CONCLUSIONS AND CLINICAL RELEVANCE: Serum sucrose concentrations in samples collected during routine health evaluations of captive dolphins can be reliably measured by use of LC-MS-MS. Correlating serum sucrose concentrations with endoscopic observations of the gastric mucosa of dolphins will validate this approach for use in screening for the prevalence and severity of gastric ulcers and determining the efficacy of treatment regimens.     

Saksenaea vasiformis and Apophysomyces elegans zygomycotic infections in bottlenose dolphins (Tursiops truncatus), a killer whale (Orcinus orca), and pacific white-sided dolphins (Lagenorhynchus obliquidens).

During a 10-yr period, a killer whale (Orcinus orca), two Pacific white-sided dolphins (Lagenorhynchus obliquidens), and two bottlenose dolphins (Tursiops truncatus), all housed at SeaWorld of Texas from 1991 to 2001, were infected with fungi from the class Zygomycetes. In four out of five cases, the fungi were identified as either Saksenaea vasiformis or Apophysomyces elegans. All fungi in the class Zygomycetes aggressively invade the vascular system. Death occurred within 23 days after the initial clinical signs. The primary site of infection involved the s.c. tissue and skeletal musculature. In one case, infection originated in the placenta and uterus of a periparturient animal. All cases exhibited systemic spread of the organisms, including two to the central nervous system. The fifth and most recent case, a bottlenose dolphin, was treated with liposomal nystatin, an antifungal formulation with reduced nephrotoxicity. This animal initially responded to therapy; however, 14 days after cessation of therapy, fungal growth reoccurred. Thus, the animal was euthanatized 39 days after the initial clinical signs. This drug represents a promising treatment option if combined with early disease detection and aggressive tissue resection.     

Dermal-epidermal relationships in the skin of the bottlenose dolphin (Tursiops truncatus)

Using a variety of methods the outer surface of the dolphin dermis presents as a rather regularly arranged series of mainly longitudinal and approximately parallel ridges which are surmounted by laterally flattened, regularly distributed dermal papillae. At variable intervals groups of dermal ridges are supported by shallow elevations referred to here as basal dermal elevations. These tend to contain vessels and/or nerves for distribution to two or more dermal ridges. The opposing epidermal surface reveals an irregularly edged series of nearly parallel epidermal ridges alternating with the grooves which in the living state contain the dermal ridges. In the depths of these grooves are the crypts which in the intact skin contain the dermal papillae. Nothing is observable which could correspond to the epidermal pegs referred to in the literature. Small irregular epidermal projections (knobs) appear along the free edge of the epidermal ridge. Topographical variations in density and height of dermal ridges, height of basal dermal elevation plus dermal ridge and papillae as well as total epidermal thickness are illustrated by means of skin maps. Inconsistencies in the literature on Cetacean skin are discussed.     

Influence of season on the hematological and serum biochemical values of bottlenose dolphins (Tursiops truncatus) housed in a controlled environment in northern Italy

Season appears to influence the physiology of bottlenose dolphins (Tursiops truncatus). From 2000 to 2005, blood parameters of three captive bottlenose dolphins housed in northern Italy were examined to determine if seasonal variation was present. Seasonal variation was observed in the male dolphins, with both males exhibiting aminotransferase levels that were higher in autumn and lower in winter. Mean serum creatinine levels were higher during summer and lower during autumn in the adult male, and mean lactate dehydrogenase higher during summer and lower during spring in the juvenile male. Both males exhibited red blood cell counts and hemoglobin levels that were higher during autumn and lower during summer. This study contributes to the knowledge of baseline hematologic and biochemical values based on seasonality in bottlenose dolphins.