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.

CHARACTERIZATION OF ANTERIOR SEGMENT OPHTHALMOLOGIC LESIONS IDENTIFIED IN FREE-RANGING DOLPHINS AND THOSE UNDER HUMAN CARE.

Cetaceans in the wild and under human care develop a variety of ocular lesions. Although they have echolocation, cetacean species have good sight, making ocular health an important part of overall health care. The cornea is the primary site of abnormalities in both populations. Typical lesions of cetaceans under human care are characterized in this retrospective review of cases. One hundred eighty animals (n = 360 eyes) were chosen from the author's ophthalmologic examination reports from different geographic areas; they included Atlantic bottlenose dolphins (Tursiops truncatus), Pacific bottle nose dolphins (Tursiopstruncatus gilli), Indopacific bottlenose dolphins (Steno bredanensis), Indopacific humpback dolphins (Sousa chinensis), and roughtooth dolphins (Steno bredanensis). These animals were examined at least once, although most were examined numerous times over many years; lesions were categorized and are described. Seventy-seven eyes from 47 animals were normal. Medial keratopathy was the most common lesion and identified in 180 eyes from 97 animals, with 83 affected bilaterally. Horizontal keratopathy was identified in 69 eyes from 41 animals, with 28 affected bilaterally. Axial keratopathy and nonspecific axial opacities were identified in 67 eyes from 44 animals, with 21 affected bilaterally. Seventy-eight eyes from 50 animals, with 28 affected bilaterally, had more than one type of corneal lesion. Cataracts were identified in 32 eyes from 19 animals, with 13 affected bilaterally. Traumatic injuries were also common and involved eyelids and cornea. Sixteen eyes from 11 animals were blind; five dolphins were blind bilaterally due to phthisis bulbi secondary to corneal perforation or severe trauma. None of the diseases had a sex predisposition; however, medial keratopathy was significantly more common as a bilateral presentation than as a unilateral presentation. Cetaceans under human care with impaired sight can use echolocation; however, ocular health should definitely be a priority in their overall health plan.

Mucocutaneous lesions in free-ranging Atlantic bottlenose dolphins Tursiops truncatus from the southeastern USA.

Mucocutaneous lesions were biopsied from free-ranging Atlantic bottlenose dolphins Tursiops truncatus inhabiting the Indian River Lagoon (IRL), Florida, and estuarine waters of Charleston (CHS), South Carolina, USA, between 2003 and 2013. A total of 78 incisional biopsies from 58 dolphins (n=43 IRL, n=15 CHS) were examined. Thirteen dolphins had 2 lesions biopsied at the same examination, and 6 dolphins were re-examined and re-biopsied at time intervals varying from 1 to 8 yr. Biopsy sites included the skin (n=47), tongue (n=2), and genital mucosa (n=29). Pathologic diagnoses were: orogenital sessile papilloma (39.7%), cutaneous lobomycosis (16.7%), tattoo skin disease (TSD; 15.4%), nonspecific chronic to chronic-active dermatitis (15.4%), and epidermal hyperplasia (12.8%). Pathologic diagnoses from dolphins with 2 lesions were predominately orogenital sessile papillomas (n=9) with nonspecific chronic to chronic-active dermatitis (n=4), TSD (n=3), lobomycosis (n=1), and epidermal hyperplasia (n=1). Persistent pathologic diagnoses from the same dolphins re-examined and re-biopsied at different times included genital sessile papillomas (n=3), lobomycosis (n=2), and nonspecific dermatitis (n=2). This is the first study documenting the various types, combined prevalence, and progression of mucocutaneous lesions in dolphins from the southeastern USA. The data support other published findings describing the health patterns in dolphins from these geographic regions. Potential health impacts related to the observed suite of lesions are important for the IRL and CHS dolphin populations, since previous studies have indicated that both populations are affected by complex infectious diseases often associated with immunologic disturbances and anthropogenic contaminants.

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.

Patterns of population structure for inshore bottlenose dolphins along the eastern United States.

Globally distributed, the bottlenose dolphin (Tursiops truncatus) is found in a range of offshore and coastal habitats. Using 15 microsatellite loci and mtDNA control region sequences, we investigated patterns of genetic differentiation among putative populations along the eastern US shoreline (the Indian River Lagoon, Florida, and Charleston Harbor, South Carolina) (microsatellite analyses: n = 125, mtDNA analyses: n = 132). We further utilized the mtDNA to compare these populations with those from the Northwest Atlantic, Gulf of Mexico, and Caribbean. Results showed strong differentiation among inshore, alongshore, and offshore habitats (ФST = 0.744). In addition, Bayesian clustering analyses revealed the presence of 2 genetic clusters (populations) within the 250 km Indian River Lagoon. Habitat heterogeneity is likely an important force diversifying bottlenose dolphin populations through its influence on social behavior and foraging strategy. We propose that the spatial pattern of genetic variation within the lagoon reflects both its steep longitudinal transition of climate and also its historical discontinuity and recent connection as part of Intracoastal Waterway development. These findings have important management implications as they emphasize the role of habitat and the consequence of its modification in shaping bottlenose dolphin population structure and highlight the possibility of multiple management units existing in discrete inshore habitats along the entire eastern US shoreline.

Vocalization characteristics of North Atlantic right whale surface active groups in the calving habitat, southeastern United States.

Passive acoustic surveys were conducted to assess the vocal behavior of North Atlantic right whales (Eubalaena glacialis) in the designated critical calving habitat along the shallow coastal waters of southeastern United States. Underwater vocalizations were recorded using autonomous buoys deployed in close proximity to surface active groups (SAGs). Nine main vocalization types were identified with manual inspection of spectrograms, and standard acoustic descriptors were extracted. Classification trees were used to examine the distinguishing characteristics of calls and quantify their variability within the SAG vocal repertoire. The results show that descriptors of frequency, bandwidth, and spectral disorder are the most important parameters for partitioning the SAG repertoire, contrary to duration-related measures. The reported source levels and vocalization statistics provide sound production data vital to inform regional passive acoustic monitoring and conservation for this endangered species.

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.

Lacaziosis in bottlenose dolphins Tursiops truncatus along the coastal Atlantic Ocean, Florida, USA.

This study represents the first systematic study of lacaziosis (lobomycosis) in bottlenose dolphins Tursiops truncatus in the Atlantic Ocean along the east-central coast of Florida, USA. Lacaziosis is a chronic infection of the skin caused by the fungus Lacazia loboi, which affects only dolphins and humans. Previous studies have shown a high prevalence (6.8 to 12.0%) of lacaziosis in resident dolphins from the adjacent Indian River Lagoon Estuary (IRL), where the disease is endemic. We examined the prevalence of lacaziosis in this coastal area using photo-identification data collected between 2002 and 2008 to determine the prevalence of lacaziosis in coastal dolphins using photographic methodology shown to have high sensitivity and specificity in prior research. The prevalence of skin lesions compatible with lacaziosis estimated from photographic data was 2.1% (6/284), approximately 3 times lower than that described for the estuarine population using similar methods. To exclude potential bias introduced by differences in study duration and survey effort among areas, an 18 mo period when effort was most equal (January 2006 to June 2007) was chosen for statistical comparison. The prevalence of lacaziosis estimated from photographic data was significantly lower (3.8%: n = 6/160) in the Atlantic Ocean compared to the IRL (12.0%: n = 20/167) (risk ratio = 3.19, 95% CI 1.32 to 7.75, p < 0.01 by chi-square analysis). The lower prevalence of lacaziosis in dolphins found in the Atlantic Ocean and the overall lack of movement of dolphins between these habitats suggests that environmental conditions within the estuary may favor viability of L. loboi, and/or that immune compromise in resident estuarine dolphins is a precursor to the disease.

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.

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.

Lobomycosis in bottlenose dolphins (Tursiops truncatus) from the Indian River Lagoon, Florida: estimation of prevalence, temporal trends, and spatial distribution.

Lobomycosis (lacaziosis) is a chronic fungal disease of the skin that affects only dolphins and humans. Previous studies have shown a high prevalence of lobomycosis in bottlenose dolphins (Tursiops truncatus) from the Indian River Lagoon, Florida (IRL). We studied the occurrence and distribution of lobomycosis in the IRL using photo-identification survey data collected between 1996 and 2006. Our objectives were to (1) determine the sensitivity and specificity of photo-identification for diagnosis of lobomycosis in free-ranging dolphins; (2) determine the spatial distribution of lobomycosis in the IRL; and (3) assess temporal patterns of occurrence. Photographs from 704 distinctly marked dolphins were reviewed for skin lesions compatible with lobomycosis. The presumptive diagnosis was validated by comparing the results of photographic analysis with physical examination and histologic examination of lesion biopsies in 102 dolphins captured and released during a health assessment and 3 stranded dolphins. Twelve of 16 confirmed cases were identified previously by photography, a sensitivity of 75%. Among 89 dolphins without disease, all 89 were considered negative, a specificity of 100%. The prevalence of lobomycosis estimated from photographic data was 6.8% (48/704). Spatial distribution was determined by dividing the IRL into six segments based on hydrodynamics and geographic features. The prevalence ranged from <1% in the Mosquito Lagoon to 16.9% in the south Indian River. The incidence of the disease did not increase during the study period, indicating that the disease is endemic, rather than emerging. In summary, photo-identification is a useful tool to monitor the course of individual and population health for this enigmatic disease.

Home ranges of bottlenose dolphins (Tursiops truncatus) in the Indian River Lagoon, Florida: environmental correlates and implications for management strategies.

Photo-identification surveys conducted between 2002 and 2005 were used to determine dolphin home ranges and site fidelity within the Indian River Lagoon (IRL), Florida. The IRL was divided into six segments based on hydrodynamics and geographic features for purposes of characterization. Among the 615 dolphins with identifiable dorsal fins, 339 had > or =6 sightings and were used in segment and linear range analyses. The majority (98%) of dolphins were seen in < or =3 consecutive segments (331/339); of these, 44% (144/331) occurred in two segments, and 33% (109/331) in one segment. No dolphins were observed in all six segments. The largest number of dolphins was sighted in segment 1C (North Indian River). However, the highest density of dolphins was found in segment 2 (North-Central Indian River). Re-sighting rates for dolphins with > or =6 sightings ranged from 2.8 to 8.7 times observed. The mean linear home range varied from 22 to 54 km. Distributional analyses indicated that at least three different dolphin communities exist within the IRL: Mosquito Lagoon, and the North and South Indian River. No statistically significant correlations were found between the total number or density per km(2 )of dolphins and surface water area, salinity, or contaminant loads within segments of the lagoon. These results suggest that dolphins do not selectively avoid areas with relatively unfavorable water quality. IRL dolphins should be studied on smaller spatial scales than currently practiced, and potential anthropogenic impacts should be evaluated based on geographic partitioning.

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.