Evaluation and comparison of the health status of Atlantic bottlenose dolphins from the Indian River Lagoon, Florida, and Charleston, South Carolina

OBJECTIVE: To conduct health assessments and compare outcomes in 2 populations of Atlantic bottlenose dolphins. Design-Repeated cross-sectional study. ANIMALS: 171 Atlantic bottlenose dolphins. PROCEDURES: During June and August of 2003 through 2005, 89 dolphins from the Indian River Lagoon (IRL), Florida, and 82 dolphins from estuarine waters near Charleston, SC, were evaluated. A panel of 5 marine mammal veterinarians classified dolphins as clinically normal, possibly diseased, or definitely diseased on the basis of results of physical and ultrasonographic examinations, hematologic and serum biochemical analyses, and cytologic and microbiologic evaluations of gastric contents and swab specimens. RESULTS: Prevalence of dolphins classified as definitely diseased did not differ significantly between the IRL (32%) and Charleston (20%) sites. Proportions of dolphins classified as possibly diseased also did not differ. Lobomycosis was diagnosed in 9 dolphins from the IRL but in none of the dolphins from Charleston. Proportions of dolphins with orogenital papillomas did not differ significantly between the IRL (12%) and Charleston (7%) sites. From 2003 through 2005, the proportion classified as definitely diseased tripled among dolphins from the Charleston site but did not increase significantly among dolphins from the IRL. Dolphins from the Charleston site were more likely to have leukocytosis, lymphocytosis, and low serum concentrations of total protein and total J-globulins than were dolphins from the IRL. CONCLUSIONS AND CLINICAL RELEVANCE: High prevalences of diseased dolphins were identified at both sites; however, the host or environmental factors that contributed to the various abnormalities detected are unknown.     

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.     

Seasonal changes of blood composition in captive bottlenose dolphins

To determine how blood values in bottlenose dolphins changed during the year, 504 blood samples were taken from 9 dolphins from 1991 to 1999 and clinical blood examinations were undertaken monthly including 3 hematological and 19 serum chemistry tests. In creatinine, significant seasonal changes were found among three groups of adult males, adult females and juveniles, and the average values in summer were 15-38% higher than those in winter. In two out of three groups the average total cholesterol value were highest in winter, and the lowest of all groups were in summer. In two other groups the peaks of average FFA value were recorded in summer, and the lows were in winter.     

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.     

海豚Prion基因的克隆与序列分析

动物海绵状脑病是一种动物中枢神经系统发生的一系列致命性神经退行性变化的疾病，包括人的新型变异型克雅氏病（v-CJD）、库鲁病（Kuru）等，动物的羊痒病（Scrapie）、牛海绵状脑病（BSE）、鹿慢性消耗性疾病（CWD）等多种疾病。 Prusiner教授提出，该类疾病的感染因子是一种不含有核酸的淀粉样蛋白颗粒，并命名为Prions（Proteinaceous infectious particles）。目前的研究表明，朊蛋白疾病的传播是由于哺乳动物食用了含有Prion蛋白感染因子的饲料而导致发病，如牛食用了含有痒病因子的肉骨粉发生疯牛病。然而，痒病致病因子只能传染给牛，不能传染给人类；但是经过中间宿主牛以后，该致病因子便可以传染给人类。对于这种现象的产生目前还不明确，有专家指出可能与Prion蛋白不同株系基因序列的变化有关。因此对动物海绵状脑病种间屏障的研究具有重要的意义。     

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.     

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.     

A DNA vaccine against dolphin morbillivirus is immunogenic in bottlenose dolphins

The immunization of exotic species presents considerable challenges. Nevertheless, for facilities like zoos, animal parks, government facilities and non-profit conservation groups, the protection of valuable and endangered species from infectious disease is a growing concern. The rationale for immunization in these species parallels that for human and companion animals; to decrease the incidence of disease. The U.S. Navy Marine Mammal Program, in collaboration with industry and academic partners, has developed and evaluated a DNA vaccine targeting a marine viral pathogen – dolphin morbillivirus (DMV). The DMV vaccine consists of the fusion (F) and hemagglutinin (H) genes of DMV. Vaccine constructs (pVR-DMV-F and pVR-DMV-H) were evaluated for expression in vitro and then for immunogenicity in mice. Injection protocols were designed for application in Atlantic bottlenose dolphins (Tursiops truncatus) to balance vaccine effectiveness with clinical utility. Six dolphins were inoculated, four animals received both pDMV-F and pDMV-H and two animals received a mock vaccine (vector alone). All animals received an inoculation week 0, followed by two booster injections weeks 8 and 14. Vaccine-specific immune responses were documented in all four vaccinated animals. To our knowledge, this is the first report of pathogen-specific immunogenicity to a DNA vaccine in an aquatic mammal species.     

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.     

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.     

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.     

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.     

Aspiration pneumonia as a cause of neonatal death in three captive bottlenose dolphins (Tursiops truncates)

Neonatal weakness of calves is one of the common reproductive-related problems in captive cetaceans; clinical signs can be observed in the first few hours after delivery. Three 3-day-old bottlenose dolphins died with history of weakness since birth. Pathological study demonstrated purulent bronchopneumonia associated with prominent bacterial colonies and foreign substances in alveoli, suggesting aspiration pneumonia as a cause of neonatal weakness and resultant death of the three calves.     

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.     

Effects of duration of capture and sample handling on critical care blood analytes in free-ranging bottlenose dolphins

OBJECTIVE: To determine effects of duration of capture and sample-handling procedures on blood analytes in free-ranging bottlenose dolphins. DESIGN: Cross-sectional study. ANIMALS: 154 free-ranging bottlenose dolphins of various ages and both sexes. PROCEDURES: Blood samples were drawn from each dolphin within 10 minutes of capture and before release and analyzed by use of a portable analyzer with a single-use 8-analyte disposable cartridge. Analyte values were compared according to duration between sample acquisition and analysis (time to run [TTR]) and duration between net encirclement and sample acquisition (time to bleed [TTB]). RESULTS: Neither TTB nor TTR significantly affected sodium or chloride concentration. Potassium concentration was not significantly affected by TTR, whereas the effect of TTB was significant. Glucose, total CO(2), HCO(3), Hct, and base excess of extracellular fluid values were significantly affected by TTR. Increased TTB resulted in significantly increased total CO(2), HCO(3), and base excess when TTR was kept within 10 minutes. CONCLUSIONS AND CLINICAL RELEVANCE: The effect of TTB on certain acid-base and electrolyte values was readily measured in free-ranging bottlenose dolphins, and such values may provide a reference range for those variables.     

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.     

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.