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.     

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.     

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.     

Effects of in vitro hemolysis on serum biochemistry values of the bottlenose dolphin (Tursiops truncatus).

The effects of in vitro hemolysis on 23 biochemical analytes were assessed in sera from 14 clinically healthy Atlantic bottlenose dolphins (Tursiops truncatus). Each serum sample was divided into three portions for analysis: 1) nonhemolyzed control; 2) moderate hemolysis, simulated by adding hemolyzed serum to a final concentration of approximately 150 mg/dl Hb; and 3) severe hemolysis, simulated by adding hemolyzed serum to a final concentration of approximately 500 mg/dl Hb. Moderate hemolysis resulted in statistically significant increases in the mean values of iron, lactate dehydrogenase, potassium, and uric acid and a decrease in creatinine (P < 0.001). Severe hemolysis resulted in statistically significant changes in the mean values of the above analytes in addition to the following increases: alanine aminotransferase, calcium, and serum globulins (P < 0.001) and albumin and total protein (P < 0.01). Total bilirubin and gamma glutamyl transferase levels were lower in the severely hemolyzed sample (P < 0.001). Differences in mean values for alkaline phosphatase between nonhemolyzed and hemolyzed serum were not significant but did show a downward trend in the hemolyzed sera. The presence and severity of hemolysis must be considered in the interpretation of the serum chemistry values.     

Altrenogest and progesterone therapy during pregnancy in bottlenose dolphins (Tursiops truncatus) with progesterone insufficiency

Progesterone production is essential for growth and development of the conceptus during pregnancy. Abnormal development of the corpus luteum (CL) after conception can result in early embryonic loss or fetal abortion. Routine monitoring of bottlenose dolphin (Tursiops truncatus) pregnancy after artificial insemination or natural conception with ultrasonography and serum progesterone determination has allowed for the establishment of expected fetal growth rates and hormone concentrations. Using these monitoring techniques, we revealed four pregnant dolphins (12-24 yr old) with abnormally low progesterone production indicative of luteal insufficiency. Once diagnosed, animals were placed on altrenogest (0.044-0.088 mg/kg once daily) alone or with oral progesterone (50-200 mg twice daily). Doses of hormone were increased or decreased in each animal based on how fetal skull biparietal and thoracic growth rates compared with published normal values. Hormones were withdrawn starting from day 358 of gestation in animals 1 and 2, with labor occurring 6 and 7 days after withdrawal and at 376 and 373 days of gestation, respectively. Both deliveries were dystocic, with each calf requiring manual extraction and fetotomy for calf 1. The fetuses in animals 3 and 4 died at 348 and 390 days of gestation, respectively. Induction of labor was attempted in both animals, after fetal death, by using a combination of rapid progesterone withdrawal and steroid and prostaglandin F2alpha administration. The calf of animal 4 had to be removed with manual cervical dilation and fetotomy All adult females survived the procedures. These data provide the first in vivo evidence that the CL is the primary source of progesterone throughout pregnancy in the bottlenose dolphin. Until further characterization of hormones required during pregnancy and at parturition has been accomplished, the exogenous progestagen supplementation protocol described here cannot be recommended for treatment of progesterone insufficiency in bottlenose dolphins.     

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.     

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.     

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.     

Relationship between transportation stress and polymorphonuclear cell functions of bottlenose dolphins, Tursiops truncatus

Dolphins in a captive environment are exposed to various kinds of stresses. Handling and transportation are stressful events for terrestrial mammals, and such stress may affect immune system function and increase susceptibility to infectious diseases. The same phenomenon could occur in dolphins, however, few studies have reported this in dolphins. The objective of this study was to evaluate the relationship between stress and polymorphonuclear (PMN) cell function of dolphins during transportation. Four bottlenose dolphins (Tursiops truncatus) were transported for 6 hr by truck. Serum cortisol levels, leukograms, phagocytosis, and superoxide production of PMN cells were evaluated during handling and transportation compared to resting values. The mean serum cortisol level was significantly increased during handling and transportation (p<0.05) when compared with the resting values. White blood cell (WBC) counts, eosinophil counts, phagocytosis, and superoxide production of PMN cells during handling and transportation stages decreased significantly in comparison with the resting stage (p<0.05). The concentration of serum cortisol was significantly correlated with the results of the WBC counts, eosinophil counts, superoxide production, and phagocytosis (p<0.01, p<0.05, p<0.05, and p<0.001, respectively). The present results indicate that handling and transportation are stressful events for dolphins and could affect their PMN cell functions, thereby leading to the impairment of the immune system.     

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.     

Congenital diffuse hyperplastic goiter associated with perinatal mortality in 11 captive-born bottlenose dolphins (Tursiops truncatus).

Diffuse hyperplastic goiter was diagnosed by histopathology in 11 perinatal bottlenose dolphins (Tursiops truncatus) that died at four separate zoos and aquaria. Thyroid morphology of these animals was compared with the histologically normal thyroids of two stranded wild bottlenose dolphin calves, a neonate and a 2-mo-old calf. Histologic changes included reduced follicular luminal diameter, markedly reduced or absent luminal colloid, hypertrophy of follicular epithelium, and follicular dysplasia. The etiology of the thyroid gland lesion was not identified. Cause of death was not determined for most of these animals, but they were presumed to have died from metabolic derangements associated with the thyroid lesion, drowning, or dystocia.     

Infection with Brucella ceti and high levels of polychlorinated biphenyls in bottlenose dolphins (Tursiops truncatus) stranded in south-west England

Eight bottlenose dolphins (Tursiops truncatus) that stranded in Cornwall, south-west England, between June 2004 and December 2007 were examined using standardised postmortem examination and bacteriological methods. Evidence of Brucella species infection was found in four of these dolphins on culture. In addition, of the eight dolphins, four were positive and two were weakly positive for antibodies to Brucella species on serological analyses of pericardial and other fluids using a competitive ELISA and two indirect ELISAs. High or very high levels of the sum of 25 individual chlorobiphenyl congeners (∑25CBs) were also determined in blubber samples from two of the dolphins (45.5 and 446.6 mg/kg lipid weight).     

Characterization of a parainfluenza virus isolated from a bottlenose dolphin (Tursiops truncatus)

A novel member of the parainfluenza virus family was identified in a bottlenose dolphin with respiratory disease. The case animal was a 19-year old male Atlantic bottlenose dolphin (Tursiops truncatus) that presented with signs of respiratory illness, including raspy, foul-odored breaths and cream-colored exudate from the blowhole. Focally extensive pyogranulomatous bronchointerstitial pneumonia with moderate numbers of intralesional yeast organisms was identified on histopathological examination. Other significant microscopic findings included multifocal erosive and ulcerative tracheitis and laryngitis consisting of active laryngeal lymphatic tissue and dilated glands with eosinophilic fluid. The cause of death was attributed to respiratory disease of unknown etiology. In addition to the postmortem isolation of Candida glabrata and mixed bacteria from lung tissue, a virus was isolated from two antemortem affected lung aspirates collected over a 2-month period and two postmortem samples (mediastinal lymph node and left lung tissue homogenate). The morphology of the virions on negative staining and transmission electron microscopy was consistent with that of paramyxoviruses. Two genomic fragments, comprising 532 and 419 nucleotides from the open reading frames that code for the viral polymerase and fusion protein, respectively, were amplified by polymerase chain reaction using degenerate primers. Phylogenetic analyses of the two viral RNA segments showed that the isolate comprised a novel virus strain, tentatively named T. truncatus parainfluenza virus type 1 (TtPIV-1). The virus is monophyletic with, but genetically distinct from, the various bovine parainfluenza virus type 3 strains.     

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.     

Feeding and growth in a captive-born bottlenose dolphin Tursiops truncatus

The feeding and growth of a captive-born bottlenose dolphin Tursiops trunÄtus calf were studied for 30 months post partum. Changes in the behaviours associated with suckling were monitored and suggested that the mammary glands need tactile stimulation before the calf can feed. The calf exhibited no teat preference and mean duration of each suckling bout (4 s) remained constant throughout the suckling period. Suckling frequency declined rapidly during the first two months and continued decreasing steadily for the remainder of the study. A reduced growth rate from seven months suggests that nutrition obtained only from milk is insufficient; however, growth rate increased from 11 months, after the calf started feeding on fish. Solid food intake increased rapidly until completion of the study, by which time the calf was eating 16 kg of fish daily. Short-term reductions in food intake after weaning resulted in corresponding reductions in weight, but appeared to have no effect on linear growth.     

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.