Genomic sequencing of a virus representing a novel type within the species Dyopipapillomavirus 1 in an Indian River Lagoon bottlenose dolphin.

Fecal samples collected from free-ranging Atlantic bottlenose dolphins (BDs) in the Indian River Lagoon of Florida were processed for viral discovery using a next-generation sequencing (NGS) approach. A 693-bp contig identified in the NGS data was nearly identical to the partial L1 gene sequence of a papillomavirus (PV) previously found in a penile papilloma in a killer whale (Orcinus orca). Based on this partial bottlenose dolphin papillomavirus (BDPV) sequence, a nested inverse PCR and primer-walking strategy was employed to generate the complete genome sequence. The full BDPV genome consisted of 7299 bp and displayed a typical PV genome organization. The BDPV E6 protein contained a PDZ-binding motif, which has been shown to be involved in carcinogenic transformation involving high-risk genital human PVs. Screening of 12 individual fecal samples using a specific endpoint PCR assay revealed that the feces from a single female BD displaying a genital papilloma was positive for the BDPV. Genetic analysis indicated that this BDPV (Tursiops truncatus papillomavirus 8; TtPV8) is a new type of Dyopipapillomavirus 1, previously sequenced from an isolate obtained from a penile papilloma in a harbor porpoise (Phocoena phocoena). Although only a partial L1 sequence has been determined for a PV detected in a killer whale genital papilloma, our finding of a nearly identical sequence in an Atlantic BD may indicate that members of this viral species are capable of host jumping. Future work is needed to determine if this virus is a high-risk PV that is capable of inducing carcinogenic transformation and whether it poses a significant health risk to wild delphinid populations.

Clinicoimmunopathologic findings in Atlantic bottlenose dolphins Tursiops truncatus with positive Chlamydiaceae antibody titers.

Sera from free-ranging Atlantic bottlenose dolphins Tursiops truncatus inhabiting the Indian River Lagoon (IRL), Florida, and coastal waters of Charleston (CHS), South Carolina, USA, were tested for antibodies to Chlamydiaceae as part of a multidisciplinary study of individual and population health. A suite of clinicoimmunopathologic variables was evaluated in Chlamydiaceae-seropositive dolphins (n = 43) and seronegative healthy dolphins (n = 83). Fibrinogen, lactate dehydrogenase, amylase, and absolute numbers of neutrophils, lymphocytes, and basophils were significantly higher, and serum bicarbonate, total alpha globulin, and alpha-2 globulin were significantly lower in dolphins with positive Chlamydiaceae titers compared with seronegative healthy dolphins. Several differences in markers of innate and adaptive immunity were also found. Concanavalin A-induced T lymphocyte proliferation, lipopolysaccharide-induced B lymphocyte proliferation, and granulocytic phagocytosis were significantly lower, and absolute numbers of mature CD 21 B lymphocytes, natural killer cell activity and lysozyme concentration were significantly higher in dolphins with positive Chlamydiaceae antibody titers compared to seronegative healthy dolphins. Additionally, dolphins with positive Chlamydiaceae antibody titers had significant increases in ELISA antibody titers to Erysipelothrix rhusiopathiae. These data suggest that Chlamydiaceae infection may produce subclinical clinicoimmunopathologic perturbations that impact health. Any potential subclinical health impacts are important for the IRL and CHS dolphin populations, as past studies have indicated that both dolphin populations are affected by other complex infectious and neoplastic diseases, often associated with immunologic perturbations and anthropogenic contaminants.

Clinicopathologic findings from Atlantic bottlenose dolphins (Tursiops truncatus) with cytologic evidence of gastric inflammation.

As part of the Bottlenose Dolphin Health and Risk Assessment study, blood, gastric, fecal, and blowhole samples were collected from 114 Atlantic bottlenose dolphins (Tursiops truncatus) in the Indian River Lagoon, Florida (IRL), and from 73 dolphins from the estuarine waters around Charleston, South Carolina (CHS), to assess the presence and degree of cytologic evidence of gastric inflammation from 2003 to 2007. The prevalence of moderate and severe gastric inflammation was 9.6% in the IRL and 11.0% at CHS. A case-control study of 19 dolphins with cytologic evidence of gastric inflammation and 82 with normal cytology from the combined populations was conducted. Blood parameters evaluated included hematology, serum chemistry, serum protein electrophoresis, and stress hormones. Few differences of clinical or statistical significance were found between affected and unaffected dolphins. Serum norepinephrine and cortisol were significantly higher in cases compared to the controls, and aldosterone was marginally higher (P = 0.06) based on eight cases. None of the hematologic, serum chemistry, or serum electrophoresis results were significantly different. Gastric fluid pH was not significantly different between cases and controls. There were no clinically significant aerobic-anaerobic or fungal culture results from gastric contents; bacteria cultured from both groups were considered to represent normal flora. The prevalence of inflammation did not differ by gender. Historically, cytologic evidence of gastric inflammation has constituted a marker of systemic illness in dolphins; however, there was little evidence to indicate systemic illness among affected animals. The data obtained from this study provide a basis for further investigation and evaluation of gastric cytology in wild and managed bottlenose dolphins.

Clinicoimmunopathologic findings in Atlantic bottlenose dolphins Tursiops truncatus with positive cetacean morbillivirus antibody titers.

Sera from free-ranging Atlantic bottlenose dolphins Tursiops truncatus inhabiting the Indian River Lagoon (IRL), Florida were tested for antibodies to cetacean morbilliviruses from 2003 to 2007 as part of a multidisciplinary study of individual and population health. A suite of clinicoimmunopathologic variables were evaluated in morbillivirus-seropositive dolphins (n = 14) and seronegative healthy dolphins (n = 49). Several important differences were found. Serum alkaline phosphatase, creatine phosphokinase, chloride, albumin and albumin/globulin ratios were significantly lower in seropositive dolphins. Innate immunity appeared to be upregulated with significant increases in lysozyme concentration and marginally significant increases in monocytic phagocytosis. Adaptive immunity was also impacted in dolphins with positive morbillivirus antibody titers. Mitogen-induced T lymphocyte proliferation responses were significantly reduced in dolphins with positive morbillivirus antibody titers, and marginally significant decreases were found for absolute numbers of CD4+ lymphocytes. The findings suggest impairment of cell-mediated adaptive immunity, similar to the immunologic pattern reported with acute morbillivirus infection in other species. In contrast, dolphins with positive morbillivirus antibody titers appeared to have at least a partially upregulated humoral immune response with significantly higher levels of gamma globulins than healthy dolphins, which may represent an antibody response to morbillivirus infection or other pathogens. These data suggest that subclinical dolphin morbillivirus infection in IRL dolphins may produce clinicoimmunopathologic perturbations that impact overall health.

Associations between mercury and hepatic, renal, endocrine, and hematological parameters in Atlantic bottlenose dolphins (Tursiops truncatus) along the eastern coast of Florida and South Carolina.

We evaluated associations between total mercury (Hg) concentrations in blood and skin and endocrine, hepatic, renal, and hematological parameters in free-ranging bottlenose dolphins (Tursiops truncatus). Dolphins in Indian River Lagoon, FL had higher concentrations of Hg in blood (0.67 µg/l wet wt) and skin (7.24 µg/g dry wt) compared with those from Charleston Harbor, SC (0.15 µg/l wet wt, 1.68 µg/g dry wt). An inverse relationship was observed between blood and skin Hg concentrations and total thyroxine, triiodothyronine, absolute numbers of lymphocytes, eosinophils, and platelets. Adrenocorticotropic hormone (ACTH), blood urea nitrogen, and gamma-glutamyl transferase increased with increasing concentrations of Hg in blood and skin; lactate dehydrogenase and neutrophils increased with concentrations in skin only. Hemoglobin and mean corpuscular hemoglobin increased with increasing concentrations of Hg in blood. Selenium was negatively associated with free T4, progesterone, and absolute numbers of monocytes, and positively correlated with absolute numbers of eosinophils and lymphocytes, and mean corpuscular volume. The results suggest the potential for a deleterious effect of Hg in highly exposed dolphins.

Genomic characterization of a novel pegivirus species from free-ranging bottlenose dolphins (Tursiops truncatus) in the Indian River Lagoon, Florida.

We report the discovery of the first cetacean pegivirus (family Flaviviridae) using a next-generation sequencing approach. One of two infected bottlenose dolphins had elevated activities of liver enzymes, which may suggest hepatocellular injury. Further research is needed to determine the epidemiology and pathogenicity of dolphin pegivirus.

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.

Complete Genome Sequencing of a Novel Type of Omikronpapillomavirus 1 in Indian River Lagoon Bottlenose Dolphins (Tursiops truncatus).

The genome sequence of a papillomavirus was determined from fecal samples collected from bottlenose dolphins in the Indian River Lagoon, FL. The genome was 7,772 bp and displayed a typical papillomavirus genome organization. Phylogenetic analysis supported the bottlenose dolphin papillomavirus as being a novel type of Omikronpapillomavirus1.

Evaluation of cetacean and sirenian cytologic samples.

Cytology is a fundamental part of marine mammal veterinary medicine that is involved in preventive medicine programs in captive animals and in the health assessment of wild populations. Marine mammals often exhibit few clinical signs of disease; thus, the cost-effective and widely accessible nature of cytologic sampling renders it one of the most important diagnostic procedures with these species. Many of these mammals are endangered, protected, and located in developing nations in which resources may be scarce. This article can be used as a field guide to advise a veterinarian, biologist, or technician working with cetaceans or sirenians. A simplistic cost-effective staining technique is used, which is ideal for situations in which funds, facilities, or time may be a limiting factor in clinical practice.

Hematologic, biochemical, and cytologic findings from apparently healthy atlantic bottlenose dolphins (Tursiops truncatus) inhabiting the Indian River Lagoon, Florida, USA.

The objective of this study was to establish reference baseline data for hematologic, biochemical, and cytologic findings in apparently healthy Atlantic bottlenose dolphins (Tursiops truncatus) inhabiting the Indian River Lagoon, Florida, USA. Sixty-two dolphins were captured, examined, and released during June 2003 and June 2004. Mean, standard deviation, and range were calculated for each parameter, and values for which published data were available, were close to or within the ranges previously reported for free-ranging bottlenose dolphins. No pathologic abnormalities were found in fecal and blowhole cytologic specimens. However, 24% (7/29) of the dolphins examined in 2003 had evidence of gastritis, which was graded as severe in 14% (4/29) of the cases. In 2004, only 4% (1/24) of dolphins sampled had evidence of mild or moderate gastritis; no severe inflammation was present. Dolphins with evidence of gastritis were 8 yr of age or older and predominantly male. Several statistically significant differences were found between males and females, between pregnant and nonpregnant animals, and between juveniles (<6 yr) and adults (> or =6 yr). However, the values remained within the established ranges for this species, and the differences were not likely to be of clinical significance.

Lobomycosis in Atlantic bottlenose dolphins from the Indian River Lagoon, Florida.

OBJECTIVE: To determine the prevalence of lobomycosis, a mycotic infection of dolphins and humans caused by a yeastlike organism (Lacazia loboi), among dolphins in the Indian River Lagoon in Florida. DESIGN: Cross-sectional study. ANIMALS: 146 Atlantic bottlenose dolphins. PROCEDURE: Comprehensive health assessments of bottlenose dolphins in the Indian River Lagoon of Florida (n = 75) and in estuarine waters near Charleston, SC (71), were conducted during 2003 and 2004. Bottlenose dolphins were captured, examined, and released. Skin lesions were photographed and then biopsied. Tissue sections were stained with H&E and Gomori methenamine silver stains for identification of L. loboi. RESULTS: 9 of 30 (30%) dolphins captured in the southern portion of the Indian River Lagoon had lobomycosis, whereas none of the 45 dolphins captured in the northern portion of the lagoon or of the 71 dolphins captured near Charleston, SC, did. Affected dolphins had low serum alkaline phosphatase activities and high acute-phase protein concentrations. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that lobomycosis may be occurring in epidemic proportions among dolphins in the Indian River Lagoon. Localization of the disease to the southern portion of the lagoon, an area characterized by freshwater intrusion and lower salinity, suggests that exposure to environmental stressors may be contributing to the high prevalence of the disease, but specific factors are unknown. Because only dolphins and humans are naturally susceptible to infection, dolphins may represent a sentinel species for an emerging infectious disease.

Acute phase protein quantitation in serum samples from healthy Atlantic bottlenose dolphins (Tursiops truncatus).

Acute phase proteins (APPs) have been studied in many companion and large animals and have been reported to have a differential sensitivity to traditional markers of inflammation. Studies have been performed indicating the conservation of these proteins as well as the application and cross-reactivity of automated assays among different species, but few reports have detailed APPs in marine mammal species. In the present study, automated assays were utilized to generate reference intervals for C-reactive protein, haptoglobin, and serum amyloid A using 44 serum samples from healthy Atlantic bottlenose dolphins (Tursiops truncatus). A total of 25 samples were obtained from dolphins under human care and 19 samples were obtained from free-ranging dolphins. Mild yet statistically significant differences were observed in levels of haptoglobin and serum amyloid A between these groups. The reference intervals from the combined groups were as follows: C-reactive protein 3.1-19.7 mg/l, haptoglobin 0-0.37 mg/ml, and serum amyloid A 17.5-42.9 mg/l. These baseline data should provide an important foundation for future studies of the application of APP quantitation in monitoring the health and stressors of dolphins under human care and with live capture of free-ranging dolphins.

Antibiotic-resistant organisms cultured from Atlantic bottlenose dolphins (Tursiops truncatus) inhabiting estuarine waters of Charleston, SC and Indian River Lagoon, FL.

Bottlenose dolphins (Tursiops truncatus) from estuarine waters of Indian River Lagoon, FL (IRL) and Charleston, SC (CHS) were cultured to screen for microorganism colonization and to assess antibiotic sensitivity. Swabs (n = 909) were collected from the blowhole, gastric fluid, and feces of 171 individual dolphins The most frequently cultured organisms were Plesiomonas shigelloides (n = 161), Aeromonas hydrophila (n = 144), Escherichia coli (n = 85), and Pseudomonas fluorescens (n = 82). In descending frequency, organisms demonstrated resistance to erythromycin, ampicillin, and cephalothin. Human and animal pathogens resistant to antibiotics used in human and veterinary medicine were cultured. Escherichia coli (E. coli) more often was resistant in IRL dolphins. Three cases of methicillin-resistant Staphylococcus aureus (MRSA) were found at CHS. Emergence of antibiotic resistance is not confined to humans. Bottlenose dolphins may serve as sentinels for transfer of resistance from humans and animals or indicate that antibiotics are reaching the marine environment and causing resistance to emerge through selective pressure and genetic adaptation.

Beaked whale auditory evoked potential hearing measurements.

Several mass strandings of beaked whales have recently been correlated with military exercises involving mid-frequency sonar highlighting unknowns regarding hearing sensitivity in these species. We report the hearing abilities of a stranded juvenile beaked whale (Mesoplodon europaeus) measured with auditory evoked potentials. The beaked whale's modulation rate transfer function (MRTF) measured with a 40-kHz carrier showed responses up to an 1,800 Hz amplitude modulation (AM) rate. The MRTF was strongest at the 1,000 and 1,200 Hz AM rates. The envelope following response (EFR) input-output functions were non-linear. The beaked whale was most sensitive to high frequency signals between 40 and 80 kHz, but produced smaller evoked potentials to 5 kHz, the lowest frequency tested. The beaked whale hearing range and sensitivity are similar to other odontocetes that have been measured.