A case of stranded Indo-Pacific bottlenose dolphin (Tursiops aduncus) with lobomycosis-like skin lesions in Kinko-wan,Kagoshima, Japan

Lobomycosis is a chronic fungal disease caused by the etiologic agent, Lacazia loboi, in the skin and subcutaneous tissues in humans and dolphins in tropical and transitional tropical climates. An Indo-Pacific bottlenose dolphin (Tursiops aduncus) stranded in Kagoshima, Japan, had severe skin lesions characterized by granulomatous reactions and hyperkeratosis that were similar to those of the lobomycosis, but no fungal organism was observed in the skin lesion. In this paper, we report a stranded Indo-Pacific bottlenose dolphin with lobomycosis-like lesions based on pathological examinations in Japan.     

An insight into the epidemiology of dolphin morbillivirus worldwide

Serum samples from 288 cetaceans representing 25 species and originating from 11 different countries were collected between 1995 and 1999 and examined for the presence of dolphin morbillivirus (DMV)-specific antibodies by an indirect ELISA (iELISA) (N = 267) or a plaque reduction assay (N = 21). A total of 35 odontocetes were seropositive: three harbour porpoises (Phocoena phocoena) and a common dolphin (Delphinus delphis) from the Northeastern (NE) Atlantic, a bottlenose dolphin (Tursiops truncatus) from Kent (England), three striped dolphins (Stenella coeruleoalba), two Risso's dolphins (Grampus griseus) and a bottlenose dolphin from the Mediterranean Sea, one common dolphin from the Southwest (SW) Indian Ocean, three Fraser's dolphins (Lagenodelphis hosei) from the SW Atlantic, 18 long-finned pilot whales (Globicephala melas) and a bottlenose dolphin from the SW Pacific as well as a captive bottlenose dolphin (Tursiops aduncus) originally from Taiwan. The presence of morbillivirus antibodies in 17 of these animals was further examined in other iELISAs and virus neutralization tests. Our results indicate that DMV infects cetaceans worldwide. This is the first report of DMV-seropositive animals from the SW Indian, SW Atlantic and West Pacific Oceans. Prevalence of DMV-seropositives was 85.7% in 21 pilot whales from the SW Pacific and both sexually mature and immature individuals were infected. This indicates that DMV is endemic in these animals. The same situation may occur among Fraser's dolphins from the SW Atlantic. The prevalence of DMV-seropositives was 5.26% and 5.36% in 19 common dolphins and 56 harbour porpoise from the NE Atlantic, respectively, and 18.75% in 16 striped dolphins from the Mediterranean. Prevalence varied significantly with sexual maturity in harbour porpoises and striped dolphins; all DMV-seropositives being mature animals. The prevalence of seropositive harbour porpoise and striped dolphins appeared to have decreased since previous studies. These data suggest that DMV is not endemic within these populations, that they are losing their humoral immunity against the virus and that they may be vulnerable to new epidemics.     

Disseminated panniculitis in a bottlenose dolphin (Tursiops truncatus) due to Mycobacterium chelonae infection

A 14-year-old female bottlenose dolphin was diagnosed with mycobacterial panniculitis based on punch biopsy specimens. The necropsy revealed numerous pyogranulomas in the blubber, as well as marked acute multifocal necrosuppurative pneumonia and lymphadenitis. In addition, the animal had marked scoliosis, which had first been noted clinically in the dolphin at about 1 mo of age. The animal had been treated with low-dose dexamethasone for approximately the last 19 mo to reduce perceived discomfort from spondyloarthritis and with the progestational agent altrogenest for approximately 8 yr to prevent pregnancy. Acid-fast positive bacilli were detected in the dermis but not in lung or lymph nodes. Mycobacterium chelonae was isolated from pooled skin, lung, and peripheral lymph-node tissue. Mycobacterial infection may be considered as a differential diagnosis in bottlenose dolphins with generalized cutaneous inflammation, particularly if chronic steroid and progesterone treatments were administered, both of which may have an immunomodulatory effect.     

Morphology of the lymph nodes in bottlenose dolphin (Tursiops truncatus) and striped dolphin (Stenella coeruleoalba) from the Adriatic Sea

Morphology of the lymph nodes was examined in six bottlenose dolphins (Tursiops truncatus) and three striped dolphins (Stenella coeruleoalba) from the Adriatic Sea. All animals had been found dead in nature. One group of the nodes was taken from the tracheal branching area and was marked as bifurcational lymph node, and the other group was taken from the mesenteric root and was marked as mesenteric lymph node. Microscopic analysis showed that the lymph nodes in both dolphin species were surrounded by a connective tissue capsule comprising smooth muscle cells. The parenchyma of the mesenteric and bifurcational lymph nodes in bottlenose dolphin was divided into the peripherally situated cortex with the lymphatic nodules and diffuse lymphatic tissue, and the centrally situated medulla structured of the medullary cords separated by the medullary sinuses. These lymph nodes structurally correspond to the lymph nodes in the majority of terrestrial mammals. The mesenteric lymph node of striped dolphin also had a peripherally situated cortex and a centrally positioned medulla as the majority of terrestrial mammals. In the bifurcational lymph nodes of striped dolphin, there was a central dense lymphatic tissue with the lymphatic nodules and a peripheral less dense lymphatic tissue structured of the cell cords and sinuses. The bifurcational lymph node in striped dolphin resembled porcine lymph nodes and belonged to the inverse lymph nodes.     

Sublingual squamous cell carcinoma in an Atlantic bottlenose dolphin (Tursiops truncatus).

A 22-yr-old captive-born Atlantic bottlenose dolphin (Tursiops truncatus) presented with a nonhealing sublingual mucosal ulcer that was diagnosed histologically as a squamous cell carcinoma, the first such report in a dolphin. The lesion was excised completely and has not recurred.     

Evaluation of two-dimensional and three-dimensional ultrasound in the assessment of thyroid volume of the Indo-Pacific bottlenose dolphin (Tursiops aduncus)

The assessment of thyroid volume plays an indispensable role in the diagnosis and management of different thyroid diseases. The present study evaluates the accuracy of dolphin thyroid volume measurement as determined by four two-dimensional (2D) ultrasound methods (A-D), with a standard of reference using three-dimensional (3D) ultrasound. The measurement accuracy for different recognized thyroid configuration is also evaluated. Inter- and intraoperator variability of the measurement methods was determined. Thyroid ultrasound examinations were conducted in 16 apparently healthy Indo-Pacific bottlenose dolphins (Tursiops aduncus) with 2D and 3D ultrasound under identical scanning conditions. All 2D ultrasound measurement methods yielded high accuracies (79.9-81.3%) when compared with the 3D ultrasound measurement, and had high measurement reproducibility (77.6-86.2%) and repeatability (78.1-99.7%). For 2D ultrasound measurements, Methods A and B were more accurate and reliable than Methods C and D, regardless of thyroid configuration. Ultrasound is useful in the measurement of thyroid volume in bottlenose dolphins. For the first time, a reliable ultrasound scanning protocol for measuring dolphin thyroid volume was developed, which provides a means to establish a normative reference for the diagnosis of thyroid pathologies and to monitor the thyroid volume during the course of treatment in living dolphins. Key words: 3D ultrasound, Indo-Pacific bottlenose dolphin, thyroid volume measurement, Tursiops aduncus.     

Pulmonary vascular proliferation and lungworm (Stenurus ovatus) in a bottlenose dolphin (Tursiops turncatus)

A female adult bottlenose dolphin suddenly died at 17 days after the capture. Macroscopically, severe pulmonary congestive edema was found. Histopathology revealed many lungworms in the bronchioli and the worms were identified as Stenurus ovatus. Variously sized vessels proliferated around the lesioned bronchioli. Based on these findings, chronic bronchopneumonia due to the lungworm was diagnosed and vascular proliferation was similar to angiomatosis recently reported in Atlantic bottlenose dolphin.     

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.     

Escherichia coli septicemia associated with lack of maternally acquired immunity in a bottlenose dolphin calf

Stillbirth and neonatal mortality are substantial problems in captive bottlenose dolphins (Tursiops truncatus). The cause of these problems often is unknown. We report a case of Escherichia coli septicemia in a male 3-day-old bottlenose dolphin calf. Lesions included omphalitis, synovitis, and hepatic necrosis associated with the presence of Gram-negative bacilli. E. coli was isolated in pure culture from multiple organs. A serum gammaglobulin level of 1.5 g/L indicated a lack of maternally acquired immunity. The observed failure to nurse may have resulted from brain injury due to perinatal asphyxia. Evidence for perinatal asphyxia was the diffuse presence of a moderate amount of meconium in the lungs.     

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.     

Proliferative dermatitis associated with a novel alphaherpesvirus in an Atlantic bottlenose dolphin (Tursiops truncatus).

Herpesviruses and herpes-like viruses have been reported in only a small number of species of cetaceans, and, to date, clinical manifestations have been either as a life-threatening, disseminated infection or as a non-life-threatening dermatitis. A stranded juvenile Atlantic bottlenose dolphin, Tursiops truncatus, was admitted to the Dolphin and Whale Hospital for rehabilitation. On initial physical examination, the rostral skin had multifocal regions of hyperplasia, and the skin of the dorsum contained a large number of small papules. Histologically, epithelial hyperplasia was evident, and clusters of epithelial cells contained 5-15-microm intranuclear inclusion bodies. Transmission electron microscopic investigation revealed numerous 170-190-nm enveloped virions in both the intracellular spaces and the cytoplasm of epithelial cells, with numerous nucleocapsids noted in epithelial cell nuclei. Consensus primer polymerase chain reaction identified the presence of a novel herpesvirus associated with the lesions. Phylogenetic analysis of the deduced amino acid sequences of the herpesvirus DNA polymerase gene fragment showed it to align with alphaherpesvirus sequences from humans and domestic animals. Although clearly distinct, it was most closely related to two previously described alphaherpesviruses of dolphins. This case represents the first documentation of herpesvirus dermatitis in the Atlantic bottlenose dolphin.     

Bottlenose dolphin (Tursiops truncatus) papillomaviruses: vaccine antigen candidates and screening test development

Papillomaviruses (PVs) have been shown as being the etiologic agents of various benign and malignant tumours in many vertebrate species. In dolphins and porpoises, a high prevalence of orogenital tumours has recently been documented with at least four distinct novel species-specific PV types detected in such lesions. Therefore, we generated the immunological reagents to establish a serological screening test to determine the prevalence of PV infection in Atlantic bottlenose dolphins [(Tursiops truncatus (Tt)]. Using the baculovirus expression system, virus-like particles (VLPs) derived from the L1 proteins of two TtPV types, TtPV1 and TtPV2, were generated. Polyclonal antibodies against TtPV VLPs were produced in rabbits and their specificity for the VLPs was confirmed. Electron microscopy and enzyme-linked immunosorbent assay (ELISA) studies revealed that the generated VLPs self-assembled into particles presenting conformational immunodominant epitopes. As such, these particles are potential antigen candidates for a TtPV vaccine. Subsequently, the VLPs served as antigens in initial ELISA tests using sera from six bottlenose dolphins to investigate PV antibody presence. Three of these sera were derived from dolphins with genital tumour history and showed positive PV ELISA reactivity, while the remaining sera from lesion-free dolphins were PV antibody-negative. The results suggest that the developed screening test may serve as a potential tool for determining PV prevalence and thus for observing transmission rates in dolphin populations as the significance of PV infection in cetaceans starts to unfold.     

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.     

Bartonella species detection in captive, stranded and free-ranging cetaceans

We present prevalence of Bartonella spp. for multiple cohorts of wild and captive cetaceans. One hundred and six cetaceans including 86 bottlenose dolphins (71 free-ranging, 14 captive in a facility with a dolphin experiencing debility of unknown origin, 1 stranded), 11 striped dolphins, 4 harbor porpoises, 3 Risso's dolphins, 1 dwarf sperm whale and 1 pygmy sperm whale (all stranded) were sampled. Whole blood (n = 95 live animals) and tissues (n = 15 freshly dead animals) were screened by PCR (n = 106 animals), PCR of enrichment cultures (n = 50 animals), and subcultures (n = 50 animals). Bartonella spp. were detected from 17 cetaceans, including 12 by direct extraction PCR of blood or tissues, 6 by PCR of enrichment cultures, and 4 by subculture isolation. Bartonella spp. were more commonly detected from the captive (6/14, 43%) than from free-ranging (2/71, 2.8%) bottlenose dolphins, and were commonly detected from the stranded animals (9/21, 43%; 3/11 striped dolphins, 3/4 harbor porpoises, 2/3 Risso's dolphins, 1/1 pygmy sperm whale, 0/1 dwarf sperm whale, 0/1 bottlenose dolphin). Sequencing identified a Bartonella spp. most similar to B. henselae San Antonio 2 in eight cases (4 bottlenose dolphins, 2 striped dolphins, 2 harbor porpoises), B. henselae Houston 1 in three cases (2 Risso's dolphins, 1 harbor porpoise), and untyped in six cases (4 bottlenose dolphins, 1 striped dolphin, 1 pygmy sperm whale). Although disease causation has not been established, Bartonella species were detected more commonly from cetaceans that were overtly debilitated or were cohabiting in captivity with a debilitated animal than from free-ranging animals. The detection of Bartonella spp. from cetaceans may be of pathophysiological concern.     

Determination of the immunotoxic potential of heavy metals on the functional activity of bottlenose dolphin leukocytes in vitro

Heavy metals may affect the immune system of cetaceans. But no information exists on their effects on the bottlenose dolphin (Tursiops truncatus) immune system, although this species is a coastal top predator which can bioaccumulate high concentrations of them. This work studies the effects of Hg (1, 5 and 10mg/L), Al (2,5, 25 and 50mg/L), Cd (1, 10, 20 and 40mg/L), Pb (1, 10, 20 and 50mg/L) and Cr (1 and 10mg/L), on the function of phagocytes and lymphocytes isolated from the peripheral blood of bottlenose dolphins under in vitro conditions. Cell viability, apoptosis, lymphocyte proliferation and phagocytosis were evaluated. Viability and lymphoproliferation were measured with Alamar Blue assay, and apoptosis and phagocytosis were evaluated with flow cytometry. Apoptosis was detected as mechanism of cell death after cadmium and mercury exposure. A significant reduction in the lymphoproliferative response was registered by exposure to 1mg/L of mercury, 10mg/L of cadmium and 50mg/L of lead. Decreased phagocytosis was also observed at 5mg/L of mercury, 50mg/L of aluminium and 10mg/L of cadmium. Chromium did not present any effects on any immune assay at the concentrations tested. The concentrations of heavy metals that were found to affect the functional activity of bottlenose dolphin leukocytes are within the environmental ranges reported in the tissues of bottlenose dolphins. These results support the hypothesis that exposure to these contaminants, particularly mercury and cadmium could lead to a reduction in host resistance to disease in these animals.     

Fatal necrotizing fasciitis and myositis in a captive common bottlenose dolphin (Tursiops truncatus) associated with Streptococcus agalactiae.

A common bottlenose dolphin (Tursiops truncatus) was presented for necropsy after acute onset of gastrointestinal signs and cutaneous lesions that rapidly progressed to death. Gross and microscopic findings were characterized by locally extensive severe necrohemorrhagic fasciitis and cellulitis, and severe necrotizing myositis in the head and dorsocranial thorax, with numerous disseminated gram-positive cocci. Streptococcus agalactiae was isolated from the lesions and from visceral organs (liver and lung), and it was identified by standard microbiology techniques. This communication is the first report of necrotizing fasciitis in a marine mammal associated with S. agalactiae.     

Histological Structure of the Adrenal Gland of the Bottlenose Dolphin (Tursiops truncatus) and the Striped Dolphin (Stenella coeruleoalba) from the Adriatic Sea

The structure of the adrenal gland was studied in 11 bottlenose dolphins ( Tursiops truncatus ), and five striped dolphins ( Stenella coeruleoalba ). These species are legally protected in Croatia. All examined animals died of natural causes and were found stranded along eastern Adriatic coast. In both species the adrenal gland consists of a cortex and a medulla; the cortex is divided into three zones. Whereas in the bottlenose dolphin, there is a zona arcuata which contains columnar cells arranged in the form of arches; in the striped dolphin this zone is replaced by zona glomerulosa containing rounded clusters of polygonal cells. In both species, the zona fasciculata consists of radially oriented cords of polygonal cells, whereas in zona reticularis cells are arranged in branching and anastomosing cords. The adrenal medulla in both species contains dark, epinephrine-secreting cells and light norepinephrine-secreting cells. Epinephrine-secreting cells are localized in the outer part of the medulla, whereas norepinephrine-secreting cells are found in the inner part, arranged in clusters and surrounded by septa of thin connective tissue. The gland is surrounded by a thick connective-tissue capsule, from where thick trabeculae extend towards the interior. In the bottlenose dolphin, group of cells resembling both medullar and cortical cells can be seen within the capsule; whereas only groups of cells resembling cortical cells are found within the capsule of the striped dolphin. In the bottlenose dolphin invagination of the adrenal cortex into the medulla is obvious as well as medullary protrusions extending through cortex to the connective tissue capsule.     

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 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.