Adam M. Schaefer

Associations between perfluoroalkyl compounds and immune and clinical chemistry parameters in highly exposed bottlenose dolphins (Tursiops truncatus)

Perfluoroalkyl compounds (PFCs) are ubiquitous, persistent chemical contaminants found in the environment, wildlife, and humans. Despite the widespread occurrence of PFCs, little is known about the impact these contaminants have on the health of wildlife populations. The authors investigated the relationship between PFCs (including ∑perfluorocarboxylates, ∑perfluoroalkyl sulfonates, perfluorooctane sulfonate, perfluorooctanoic acid, and perfluorodecanoic acid) and the clinocopathologic and immune parameters in a highly exposed population (n = 79) of Atlantic bottlenose dolphins (mean ∑PFCs = 1970 ng/ml; range 574-8670 ng/ml) sampled from 2003 to 2005 near Charleston, South Carolina, USA. Age-adjusted linear regression models showed statistically significant positive associations between exposure to one or more of the PFC totals and/or individual analytes and the following immunological parameters: absolute numbers of CD2+ T cells, CD4+ helper T cells, CD19+ immature B cells, CD21+ mature B cells, CD2/CD21 ratio, MHCII+ cells, B cell proliferation, serum IgG1, granulocytic, and monocytic phagocytosis. Several PFC analyte groups were also positively associated with serum alanine aminotransferase, gamma-glutamyltransferase, creatinine, phosphorus, amylase, and anion gap and negatively associated with cholesterol levels, creatinine phosphokinase, eosinophils, and monocytes. Based on these relationships, the authors suggest that the PFC concentrations found in Charleston dolphins may have effects on immune, hematopoietic, kidney, and liver function. The results contribute to the emerging data on PFC health effects in this first study to describe associations between PFCs and health parameters in dolphins.

Stress response of wild bottlenose dolphins (Tursiops truncatus) during capture-release health assessment studies.

There is a growing concern about the impacts of stress in marine mammals as they face a greater array of threats. The stress response of free-ranging dolphins (Tursiops truncatus) was examined by measuring their physiologic response to capture and handling. Samples were collected from 168 dolphins during capture-release health assessments 2003-2007 at two study sites: Charleston, SC (CHS) and the Indian River Lagoon, FL (IRL). Adrenocorticotropic hormone (ACTH), cortisol, aldosterone (ALD) and catecholamines (epinephrine (EPI), norepinephrine (NOR), dopamine (DA)), were measured in blood and cortisol in urine. Mean time to collect pre-examination samples after netting the animals was 22min; post-examination samples were taken prior to release (mean 1h 37min). EPI and DA concentrations decreased significantly with increased time to blood sampling. ACTH and cortisol levels increased from the initial capture event to the post-examination sample. EPI concentrations increased significantly with increasing time to the pre-examination sample and decreased significantly with time between the pre- and post-examination sample. Cortisol concentrations increased between the pre- and post-examination in CHS dolphins. Age- and sex-adjusted mean pre-examination values of catecholamines were significantly higher in CHS dolphins; ALD was higher in IRL dolphins. Significant differences related to age or sex included higher NOR concentrations in males; higher ALD and urine cortisol levels in juveniles than adults. Wild dolphins exhibited a typical mammalian response to acute stress of capture and restraint. Further studies that relate hormone levels to biological and health endpoints are warranted.

Morbillivirus infection in free-ranging Atlantic bottlenose dolphins (Tursiops truncatus) from the Southeastern United States: seroepidemiologic and pathologic evidence of subclinical infection.

From 2003 to 2007, sera (n=234) from free-ranging Atlantic bottlenose dolphins (Tursiops truncatus) inhabiting two southeast Atlantic estuarine regions, the Indian River Lagoon (IRL), FL and Charleston, SC (CHS) were tested for antibodies to cetacean morbilliviruses as part of a multidisciplinary study of individual and population health. Positive morbillivirus titers were found on initial capture in 12 of 122 (9.8%) IRL dolphins in the absence of an epizootic. All CHS dolphins were seronegative. Positive fluctuating morbillivirus titers and seroconversion were found in IRL dolphins. Seropositivity was detected in dolphins 8-13 years of age as well as in dolphins that were alive during the 1987-1988 epizootic. During the study period, pathologic and immunohistochemical findings from stranded IRL dolphins (n=14) did not demonstrate typical morbillivirus-associated lesions or the presence of morbillivirus antigen. The findings suggest that morbillivirus infections are occurring in the absence of widespread mortality in IRL 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.

Lobomycosis: Risk of Zoonotic Transmission from Dolphins to Humans

Lobomycosis, a fungal disease of the skin and subcutaneous tissues caused by Lacazia loboi, is sometimes referred to as a zoonotic disease because it affects only specific delphinidae and humans; however, the evidence that it can be transferred directly to humans from dolphins is weak. Dolphins have also been postulated to be responsible for an apparent geographic expansion of the disease in humans. Morphological and molecular differences between the human and dolphin organisms, differences in geographic distribution of the diseases between dolphins and humans, the existence of only a single documented case of presumed zoonotic transmission, and anecdotal evidence of lack of transmission to humans following accidental inoculation of tissue from infected dolphins do not support the hypothesis that dolphins infected with L. loboi represent a zoonotic hazard for humans. In addition, the lack of human cases in communities adjacent to coastal estuaries with a high prevalence of lobomycosis in dolphins, such as the Indian River Lagoon in Florida (IRL), suggests that direct or indirect transmission of L. loboi from dolphins to humans occurs rarely, if at all. Nonetheless, attention to personal hygiene and general principals of infection control are always appropriate when handling tissues from an animal with a presumptive diagnosis of a mycotic or fungal disease.

Prevalence and Impacts of Motorized Vessels on Bottlenose Dolphins ( Tursiops truncatus ) in the Indian River Lagoon, Florida

Vessel-based anthropogenic impacts on bottlenose dolphins (Tursiops truncatus) in the Indian River Lagoon (IRL), Florida, were investigated by quantifying visible physical injuries to dorsal fins from photo-identification data collected from 1996 to 2006. Forty-three dolphins, 6.0% of the distinctly marked population, had injuries related to vessel impact. Impact was determined from previously published vessel-related wound definitions and the elimination of other possible wound sources. Spatial distribution was determined by dividing the IRL into six segments based on hydrodynamics and geographic features. Dolphins were assigned to a segment(s) and corresponding county according to ranging patterns. Segment 4, consisting of St. Lucie and Martin Counties, had the highest prevalence (9.9/100 distinct dolphins) of boatinjured dolphins and had the highest number of registered boaters per km of habitat. These preliminary data suggest that vessel impacts on dolphins occur disproportionally in the IRL and should be considered a high-priority management issue for local governments. Behavioral data collected during photo-identification surveys support the possibility of a low tolerance and sensitization to vessel interactions. Recommendations to reduce direct and indirect impacts from vessels on dolphins are discussed.

Serological Evidence of Exposure to Selected Viral, Bacterial, and Protozoal Pathogens in Free-Ranging Atlantic Bottlenose Dolphins (Tursiops truncatus) from the Indian River Lagoon, Florida, and Charleston, South Carolina

Sera from free-ranging Atlantic bottlenose dolphins (Tursiops truncatus) in the Indian River Lagoon, Florida (IRL) (n = 122), and the estuarine waters near Charleston, South Carolina (CHS) (n = 82) were collected from 2003 to 2007 and analyzed for antibodies to several bacterial and viral pathogens. Serological evidence of exposure to Chlamydophila psittaci; Eastern, Western, and Venezuelan equine encephalitis viruses; and West Nile virus represents the first reports of these pathogens in cetacean populations. Antibodies to Eastern and Venezualan encephalitis viruses and to West Nile virus were detected only in IRL dolphins. Positive titers to Toxoplasma gondii and Brucella abortus (rivanol and card tests) were identified in dolphins from both locations. The prevalence of antibodies to Brucella spp. on the card test was significantly higher in bottlenose dolphins sampled in the IRL compared to the CHS location. This study establishes baseline seroprevalence for several zoonotic pathogens in these two populations.

Atlantic Bottlenose Dolphins (Tursiops truncatus) as A Sentinel for Exposure to Mercury in Humans: Closing the Loop

Mercury (Hg) is a ubiquitous global contaminant with important public health implications. Mercury is released from a variety of anthropogenic, industrial processes, enters the earths atmosphere and is re-deposited onto the earth’s surface in rainfall. Much of this Hg enters the oceans which cover the majority of the earth’s surface. In the marine environment, inorganic Hg is converted to the most toxic form of the element, methylmercury, and biomagnified through the trophic levels of the food web. The bottlenose dolphin (Tursiops truncatus) is the apex predator in many estuarine and coastal ecosystems. Due to their long life span and trophic position, bottlenose dolphins bioaccumulate high concentrations of contaminants including Hg, thus making them an important sentinel species for ecosystem and public health. Bottlenose dolphins in Florida bioaccumulate high concentrations of Hg in their blood, skin and internal organs. The concentrations of Hg in blood and skin of bottlenose dolphins of the Indian River Lagoon, FL (IRL) are among the highest reported world-wide. In previous studies, we demonstrated associations between concentrations of total Hg in the blood and skin of IRL dolphins and markers of endocrine, renal, hepatic, hematologic and immune system dysfunction. The predominant manifestation of exposure to mercury in humans is neurotoxicity. During the 1950s and 1960s, residents of Minamata bay, Japan were exposed to high concentrations of methyl mercury as the result of ingestion of fish and shellfish that had become contaminated in this infamous environmental disaster. Affected adults had severe motor and sensory abnormalities often leading to death. Methyl mercury crosses the placenta during pregnancy. Children exposed in utero were born with multiple congenital anomalies and also suffered from neurologic disorders. Significantly, local cats that consumed Hg contaminated fish developed severe signs of neurotoxicity which led to their subsequent description as the “dancing cats of Minamata bay”. Unfortunately, the cause of these strange manifestations in cats was not recognized in time to prevent hundreds of additional cases from occurring. More recent studies have shown that exposure to mercury as a result of seafood consumption during pregnancy may result in multiple cognitive and neurodevelopmental effects in children. The levels of mercury found in bottlenose dolphins and the health effects we identified alerted us to the possibility of an important public health hazard. The IRL occupies 40 percent of the east coast of Florida and is bordered by counties with approximately 2.5 million human inhabitants. Therefore, we hypothesized that local inhabitants in communities bordering the IRL could be at risk of exposure to Hg from the consumption of fish and shellfish. We measured hair Hg in 135 local residents and found a mean concentration of 1.53 µg/g which was higher than that from previous studies of sport fishermen and coastal residents in other states. Over 50% of participants had a hair Hg concentration which exceeded the U.S. EPA exposure guideline. Hair Hg concentration was directly related to the frequency of seafood consumption and to the proportion of fish and shellfish obtained from local recreational sources. This study clearly exemplifies the importance of an animal sentinel in identifying a public health hazard and is virtually unique in “closing the loop” between animal and human health.

Cutaneous Granulomas in Dolphins Caused by Novel Uncultivated Paracoccidioides brasiliensis

Our findings could stimulate study of public health implications of diseases caused by this fungus.

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.