Comparative Innate and Adaptive Immune Responses in Atlantic Bottlenose Dolphins (Tursiops truncatus) With Viral, Bacterial, and Fungal Infections.

Free-ranging Atlantic bottlenose dolphins (n = 360) from two southeastern U.S. estuarine sites were given comprehensive health examinations between 2003 and 2015 as part of a multi-disciplinary research project focused on individual and population health. The study sites (and sample sizes) included the Indian River Lagoon (IRL), Florida, USA (n = 246) and Charleston harbor and associated rivers (CHS), South Carolina, USA (n = 114). Results of a suite of clinicoimmunopathologic tests revealed that both populations have a high prevalence of infectious and neoplastic disease and a variety of abnormalities of their innate and adaptive immune systems. Subclinical infections with cetacean morbillivirus and Chlamydiaceae were detected serologically. Clinical evidence of orogenital papillomatosis was supported by the detection of a new strain of dolphin papillomavirus and herpesvirus by molecular pathology. Dolphins with cutaneous lobomycosis/lacaziasis were subsequently shown to be infected with a novel, uncultivated strain of Paracoccidioides brasiliensis, now established as the etiologic agent of this enigmatic disease in dolphins. In this review, innate and adaptive immunologic responses are compared between healthy dolphins and those with clinical and/or immunopathologic evidence of infection with these specific viral, bacterial, and fungal pathogens. A wide range of immunologic host responses was associated with each pathogen, reflecting the dynamic and complex interplay between the innate, humoral, and cell-mediated immune systems in the dolphin. Collectively, these studies document the comparative innate and adaptive immune responses to various types of infectious diseases in free-ranging Atlantic bottlenose dolphins. Evaluation of the type, pattern, and degree of immunologic response to these pathogens provides novel insight on disease immunopathogenesis in this species and as a comparative model. Importantly, the data suggest that in some cases infection may be associated with subclinical immunopathologic perturbations that could impact overall individual and population health.

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.

Immune function in female B(6)C(3)F(1) mice is modulated by DE-71, a commercial polybrominated diphenyl ether mixture.

Polybrominated diphenyl ethers (PBDEs) are an important class of flame-retardants that are environmentally persistent and bioaccumulative. Toxicity of these compounds has become a concern because detectable levels of PBDEs are present in humans and wildlife and they are structurally similar to polychlorinated biphenyls (PCBs). This study examined the effects of the commercial penta-BDE mixture, DE-71, in adult female B(6)C(3)F(1) mice on hematology, serum clinical chemistry, thyroid hormones, tissue histology, and several immunotoxicity end-points (lymphocyte proliferation, NK cell activity, splenic immunophenotypes, and SRBC-specific-IgM production). Mice were exposed via oral gavage for 28 days to achieve total administered doses (TAD) of 0, 0.5, 5, 50, or 100 mg/kg. No changes in histology, clinical chemistry, body or organ weights were observed. Serum total T3 and T4 levels were not altered by any of the DE-71 treatments. Peripheral blood monocyte numbers were decreased by the 0.5, 5, and 50 mg/kg treatments, but not by the 100 mg/kg TAD concentration. Compared to controls, mitogen-stimulated T- and B-cell proliferation was increased by the 100 mg/kg TAD concentration (ED(50) = 60 mg/kg TAD [2.14 mg/kg/day] and 58 mg/kg TAD [2.57 mg/kg/day], respectively). NK cell activity was decreased compared to controls by the 100 mg/kg TAD concentration (ED(50) = 20 mg/kg TAD [0.7 mg/kg/day]). No alterations were noted in thymic T-cell populations or in SRBC-specific-IgM production. Numbers of CD19(+)CD21(-), CD19(+)CD21(+), CD4(+)CD8(-), CD4(-)CD8(+), CD4(-)CD8(-), and MHC-II(+) cells in the spleen were not affected. However, the numbers of splenic CD4(+)CD8(+) cells were decreased compared to the controls by 0.5, 5, and 100 mg/kg TAD. This study provides an assessment of the systemic toxicity and immunotoxicity of DE-71, and indicates that immune parameters are modulated at exposure concentrations lower than previously reported.

Effects of environmentally-relevant levels of perfluorooctane sulfonate on clinical parameters and immunological functions in B6C3F1 mice.

In the first part of a series of studies to account for perfluorooctane sulfonate (PFOS)-induced sheep red blood cell (SRBC)-specific immunoglobulin M (IgM) antibody suppression in mice, a survey of clinical and immunotoxicological endpoints was examined. Adult female B6C3F1 mice were exposed orally for 28 days to a total administered dose (TAD) of 0, 0.1, 0.5, 1, or 5 mg PFOS/kg. Uterus wet weight was significantly decreased compared with control at the 5 mg/kg dose. No indications of wasting syndrome, malnutrition, alteration of thyroid homeostasis, or signs of overt toxicity were observed. Numbers of splenic CD19+/CD21⁻, CD19+/CD21+, B220+/CD40+, CD4+/CD154⁻, CD4+/CD154+, and MHC-II+ cells were not altered. Additionally, ex vivo interleukin-4 (IL-4), IL-5, and IL-6 production by in vitro anti-CD3- or phorbol myristate acetate-stimulated CD4+ T-cells was not affected. Ex vivo IL-6 production by B-cells was significantly increased by in vitro stimulation with either anti-CD40 or lipopolysaccharide. Increased IL-6 production by B-cells was the most sensitive endpoint assessed resulting in alterations at the lowest dose tested (0.1 mg/kg TAD) following anti-CD40 stimulation. Further studies are required to characterize effects on inflammatory markers such as IL-6 at environmentally relevant concentrations of PFOS and to determine the key events associated with PFOS-induced IgM suppression to address potential human health risks.

Effects of perfluorooctane sulfonate (PFOS) exposure on markers of inflammation in female B6C3F1 mice.

Perfluorooctane sulfonate (PFOS; 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-heptadecafluoro-1-octanesulfonic acid) has been reported to alter humoral immune functions, but inflammatory processes following PFOS exposure have not been fully characterized. Therefore, the current study, assessed TNF-α and IL-6 concentrations in serum and peritoneal lavage fluid, numbers of splenoctyes expressing intracellular TNF-α, IL-6, IL-10 or IL-1, and ex vivo TNF-α and IL-6 production by peritoneal macrophages following either in vivo or in vitro LPS exposure. Adult female B6C3F1 mice were exposed orally for 28 days to 0, 1, 3, or 300 mg PFOS/kg total administered dose [TAD] (e.g., 0, 0.0331, 0.0993 or 9.93 mg/kg/day). Body and spleen masses were significantly reduced in the highest PFOS treatment group compared to the control group, whereas liver mass was significantly increased. Serum TNF-α levels were significantly decreased following exposure to 1 mg PFOS/kg TAD as compared to controls, while serum IL-6 levels were increased. IL-6 concentrations in peritoneal lavage fluid decreased with increasing dose. PFOS treatment did not alter numbers of splenocytes expressing intracellular levels of TNF-α, IL-10 or IL-1. Numbers of splenocytes expressing intracellular levels of IL-6 were significantly decreased in the 3 mg/kg treatment as compared to controls. Overall, these data suggest that PFOS exposure can alter some inflammatory processes, which could potentially lead to misdirected inflammatory responses.

Complex contaminant exposure in cetaceans: a comparative E-Screen analysis of bottlenose dolphin blubber and mixtures of four persistent organic pollutants.

Cetaceans are federally protected species that are prone to accumulate complex mixtures of persistent organic pollutants (POPs), which individually may exert estrogenic or antiestrogenic effects. In the present study it was assessed whether contaminant mixtures harbored by cetaceans are estrogenic or antiestrogenic using a comparative approach. Interactions of antiestrogenic and estrogenic compounds were first investigated with the E-Screen assay using a mixture of four POPs (dichlorodiphenyldichloroethylene [4,4'-DDE], trans-nonachlor, and polychlorinated biphenyls [PCBs] 138 180) prevalent in cetacean blubber. Estrogenic/antiestrogenic activity was determined for the individual compounds and their binary, tertiary, and quaternary combinations. Significantly different responses were observed for the various POP mixtures, including enhanced estrogenic and antiestrogenic effects and antagonistic interactions. These results were then compared to the concentrations and estrogenic/antiestrogenic activity of contaminant mixtures isolated directly from the blubber of 15 bottlenose dolphins (Tursiops truncatus) collected from five U.S. Atlantic and Gulf of Mexico locations. The lowest observed effect concentrations (LOECs) determined for 4,4'-DDE (20 µmol/L), PCB 138 (20 µmol/L), PCB 180 (21 µmol/L), and trans-nonachlor (3 µmol/L) in the E-Screen were greater than estimated dolphin blood concentrations. Although estimated blood concentrations were below the LOECs, significant estrogenic activity was detected in diluted dolphin blubber from Cape May, NJ and Bermuda. Positive correlations between blubber estrogenicity and select POP concentrations (ΣDDTs, ΣPBDEs, ΣHCB, Σestrogenic PCBs, Σestrogenic POPs) were also observed. Collectively, these results suggest that select bottlenose dolphin populations may be exposed to contaminants that act in concert to exert estrogenic effects at biologically relevant concentrations. These observations do not necessarily provide direct evidence of endocrine disruption; however, they may indicate an environmental source of xenoestrogenic exposure warranting future research.

A dolphin peripheral blood leukocyte cDNA microarray for studies of immune function and stress reactions.

A microarray focused on stress response and immune function genes of the bottlenosed dolphin has been developed. Random expressed sequence tags (ESTs) were isolated and sequenced from two dolphin peripheral blood leukocyte (PBL) cDNA libraries biased towards T- and B-cell gene expression by stimulation with IL-2 and LPS, respectively. A total of 2784 clones were sequenced and contig analysis yielded 1343 unigenes (archived and annotated at ). In addition, 52 dolphin genes known to be important in innate and adaptive immune function and stress responses of terrestrial mammals were specifically targeted, cloned and added to the unigene collection. The set of dolphin sequences printed on a cDNA microarray comprised the 1343 unigenes, the 52 targeted genes and 2305 randomly selected (but unsequenced) EST clones. This set was printed in duplicate spots, side by side, and in two replicates per slide, such that the total number of features per microarray slide was 19,200, including controls. The dolphin arrays were validated and transcriptomic profiles were generated using PBL from a wild dolphin, a captive dolphin and dolphin skin cells. The results demonstrate that the array is a reproducible and informative tool for assessing differential gene expression in dolphin PBL and in other tissues.

Immunological function in mice exposed to JP-8 jet fuel in utero.

Immunological parameters, host resistance, and thyroid hormones were evaluated in F1 mice exposed in utero to jet propulsion fuel-8 (JP-8). C57BL/6 pregnant dams (mated with C3H/HeJ males) were gavaged daily on gestation days 6-15 with JP-8 in a vehicle of olive oil at 0, 1000, or 2000 mg/kg. At weaning (3 weeks of age), no significant differences were observed in body, liver, spleen, or thymus weight, splenic and thymic cellularity, splenic CD4/CD8 lymphocyte subpopulations, or T-cell proliferation. Yet, lymphocytic proliferative responses to B-cell mitogens were suppressed in the 2000 mg/kg treatment group. In addition, thymic CD4-/CD8+ cells were significantly increased. By adulthood (8 weeks of age), lymphocyte proliferative responses and the alteration in thymic CD4-/CD8+ cells had returned to normal. However, splenic weight and thymic cellularity were altered, and the IgM plaque forming cell response was suppressed by 46% and 81% in the 1000 and 2000 mg/kg treatment groups, respectively. Furthermore, a 38% decrease was detected in the total T4 serum hormone level at 2000 mg/kg. In F1 adults, no significant alterations were observed in natural killer cell activity, T-cell lymphocyte proliferation, bone marrow cellularity and proliferative responses, complete blood counts, peritoneal and splenic cellularity, liver, kidney, or thymus weight, macrophage phagocytosis or nitric oxide production, splenic CD4/CD8 lymphocyte subpopulations, or total T3 serum hormone levels. Host resistance models in treated F1 adults demonstrated that immunological responses were normal after challenge with Listeria monocytogenes, but heightened susceptibility to B16F10 tumor challenge was seen at both treatment levels. This study demonstrates that prenatal exposure to JP-8 can target the developing murine fetus and result in impaired immune function and altered T4 levels in adulthood.