Tracy A. Romano

Neuropeptide-Y innervation of the rat spleen: Another potential immunomodulatory neuropeptide

Neuropeptide-Y (NPY) is a 36 amino acid peptide that acts as a chemical messenger in the central and peripheral nervous systems. NPY often is found colocalized with the classical neurotransmitter norepinephrine (NE) and can potentiate the effects of this neurotransmitter postsynaptically in many systems. Using immunocytochemistry for NPY and specific lymphoid cell markers, we mapped the distribution of NPY-positive nerve fibers in the rat spleen. NPY-positive nerve fibers were present along the vasculature, trabeculae, and capsule, and also were found associated with specific lymphoid parenchymal compartments of the spleen, in close contact with lymphocytes and macrophages. These contacts were investigated further at the electron microscopic level. NPY-positive nerve terminals were found in close apposition with lymphocytes in the periarteriolar lymphatic sheath, and with lymphocytes and macrophages in the marginal zone. Previous studies have reported that postganglionic noradrenergic nerve fibers innervate specific lymphoid compartments of the rat spleen, with nerve terminals forming direct appositions with cells of the immune system. The possible colocalization of NPY and NE in these nerve fibers was investigated by chemical sympathectomy with 6-hydroxydopamine, followed by immunocytochemical labeling of NPY and tyrosine hydroxylase (TH), the rate-limiting enzyme in norepinephrine synthesis. Colocalization also was investigated by labeling for NPY with a fluorescent label, eluting the NPY, and staining for TH with diaminobenzidine as the label. These studies demonstrate that norepinephrine and NPY are colocalized in the postganglionic sympathetic nerve fibers of the rat spleen.(ABSTRACT TRUNCATED AT 250 WORDS)

Immune dysfunction in Atlantic bottlenose dolphins (Tursiops truncatus) with lobomycosis

Lobomycosis (Lacaziosis) occurs only in humans and dolphins under natural conditions. We evaluated the immune status of eight dolphins with lobomycosis and 40 healthy dolphins from the Indian River Lagoon (IRL), Florida. Lobomycosis cases had multiple abnormalities in their immunologic parameters when compared to healthy dolphins. The absolute number of circulating lymphocytes and serum albumin concentration were reduced (P<0.05) while the segmented neutrophils, alpha 1, total beta, total gamma and total globulins were increased (P<0.05). Although innate immunity was relatively intact and phagocytosis and natural killer cell activity were not affected, the plasma lysozyme concentrations were elevated in dolphins with lobomycosis (P<0.05). Adaptive immunity was depressed with statistically significant decreases found in the absolute numbers of CD4(+) helper T cells and CD19(+) and CD21(+) B cells. The ratios of CD2(+) T cells to CD4(+) cells and CD2(+) to CD21(+) cells were increased (P=0.05 and P<0.05, respectively) and the numbers of lymphocytes expressing MHC class II molecules was decreased in dolphins with lobomycosis (P<0.05). Lymphocyte proliferation was reduced in response to stimulation with lipopolysaccharide and concanavalin A (P<0.05). Antibody titers to Erysipelas rhusiopathiae, a common marine micro-organism, were decreased (P<0.05). In summary, dolphins with lobomycosis exhibit significant impairment in adaptive immunity.

Molecular cloning and characterization of CD4 in an aquatic mammal, the white whale Delphinapterus leucas

Abstract Given the importance of the cell surface recognition protein, CD4, in immune function, the cloning and characterization of CD4 at the molecular level from an odontocete cetacean, the white whale (Delphinapterus leucas), was carried out. Whale CD4 cDNA contains 2662 base pairs and translates into a protein containing 455 amino acids. Whale CD4 shares 64% and 51% identity with the human and mouse CD4 protein, respectively, and is organized in a similar manner. Unlike human and mouse, however, the cytoplasmic domain, which is highly conserved, contains amino acid substitutions unique to whale. Moreover, only one of the seven potential N-linked glycosylation sites present in whale is shared with human and mouse. Evolutionarily, the whale CD4 sequence is most similar to pig and structurally similar to dog and cat, in that all lack the cysteine pair in the V2 domain. These differences suggest that CD4 may have a different secondary structure in these species, which may affect binding of class II and subsequent T-cell activation, as well as binding of viral pathogens. Interestingly, as a group, species with these CD4 characteristics all have high constituitive expression of class II molecules on T lymphocytes, suggesting potential uniqueness in the interaction of CD4, class II molecules, and the immune response. Molecular characterization of CD4 in an aquatic mammal provides information on the CD4 molecule itself and may provide insight into adaptive evolutionary changes of the immune system.

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.

Development, validation, and utilization of a competitive enzyme-linked immunosorbent assay for the detection of antibodies against Brucella species in marine mammals.

A competitive enzyme-linked immunosorbent assay (cELISA) was developed by using a whole-cell antigen from a marine Brucella sp. isolated from a harbor seal (Phoca vitulina). The assay was designed to screen sera from multiple marine mammal species for the presence of antibodies against marine-origin Brucella. Based on comparisons with culture-confirmed cases, specificity and sensitivity for cetacean samples tested were 73% and 100%, respectively. For pinniped samples, specificity and sensitivity values were 77% and 67%, respectively. Hawaiian monk seal (Monachus schauinslandi; n = 28) and bottlenose dolphin (Tursiops truncatus; n = 48) serum samples were tested, and the results were compared with several other assays designed to detect Brucella abortus antibodies. The comparison testing revealed the marine-origin cELISA to be more sensitive than the B. abortus tests by the detection of additional positive serum samples. The newly developed cELISA is an effective serologic method for detection of the presence of antibodies against marine-origin Brucella sp. in marine mammals.

SEASONAL HEMATOLOGY AND SERUM CHEMISTRY OF WILD BELUGA WHALES (DELPHINAPTERUS LEUCAS) IN BRISTOL BAY, ALASKA, USA

We collected blood from 18 beluga whales (Delphinapterus leucas), live-captured in Bristol Bay, Alaska, USA, in May and September 2008, to establish baseline hematologic and serum chemistry values and to determine whether there were significant differences in hematologic values by sex, season, size/age, or time during the capture period. Whole blood was collected within an average of 19 min (range=11–30 min) after the net was set for capture, and for eight animals, blood collection was repeated in a later season after between 80–100 min; all blood was processed within 12 hr. Mean hematocrit, chloride, creatinine, total protein, albumin, and alkaline phosphatase were significantly lower in May than they were in September, whereas mean corpuscular hemoglobin concentration, monocytes, phosphorous, magnesium, blood urea nitrogen, alanine aminotransferase, aspartate aminotransferase, γ-glutamyltranspeptidase, and creatinine kinase were significantly higher. Mean total protein, white blood cell count, neutrophils, and lymphocytes were significantly higher early in the capture period than they were later. No significant differences in blood analyte values were noted between males and females. Using overall body length as a proxy for age, larger (older) belugas had lower white blood cell, lymphocyte, and eosinophil counts as well as lower sodium, potassium, and calcium levels but higher creatinine levels than smaller belugas. These data provide values for hematology and serum chemistry for comparisons with other wild belugas.

Blow collection as a non-invasive method for measuring cortisol in the beluga (Delphinapterus leucas).

Non-invasive sampling techniques are increasingly being used to monitor glucocorticoids, such as cortisol, as indicators of stressor load and fitness in zoo and wildlife conservation, research and medicine. For cetaceans, exhaled breath condensate (blow) provides a unique sampling matrix for such purposes. The purpose of this work was to develop an appropriate collection methodology and validate the use of a commercially available EIA for measuring cortisol in blow samples collected from belugas (Delphinapterus leucas). Nitex membrane stretched over a petri dish provided the optimal method for collecting blow. A commercially available cortisol EIA for measuring human cortisol (detection limit 35 pg ml−1) was adapted and validated for beluga cortisol using tests of parallelism, accuracy and recovery. Blow samples were collected from aquarium belugas during monthly health checks and during out of water examination, as well as from wild belugas. Two aquarium belugas showed increased blow cortisol between baseline samples and 30 minutes out of water (Baseline, 0.21 and 0.04 µg dl−1; 30 minutes, 0.95 and 0.14 µg dl−1). Six wild belugas also showed increases in blow cortisol between pre and post 1.5 hour examination (Pre 0.03, 0.23, 0.13, 0.19, 0.13, 0.04 µg dl−1, Post 0.60, 0.31, 0.36, 0.24, 0.14, 0.16 µg dl−1). Though this methodology needs further investigation, this study suggests that blow sampling is a good candidate for non-invasive monitoring of cortisol in belugas. It can be collected from both wild and aquarium animals efficiently for the purposes of health monitoring and research, and may ultimately be useful in obtaining data on wild populations, including endangered species, which are difficult to handle directly.

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.

Neuroimmunological response of beluga whales (Delphinapterus leucas) to translocation and a novel social environment.

This study assessed changes in phagocyte function and activation of the sympatho-adrenal medullary and hypothalamo-pituitary adrenal axes of beluga whales (Delphinapterus leucas) in response to translocation and introduction to a novel social environment. Transported belugas exhibited increases in epinephrine (E), norepinephrine (NE), and cortisol levels in response to the translocation process. In response to the introduction of the transported belugas, resident belugas exhibited an increase in E and NE but not cortisol. Moreover, the increase in E and NE shown by the transported belugas was significantly greater than the increase exhibited by the resident belugas. Resident belugas exhibited a concomitant decrease in neutrophil and monocyte phagocytosis associated with the introduction of the transported belugas. In contrast, transported belugas exhibited an attendant increase in phagocytosis and respiratory burst activity immediately following transport. Differences in phagocyte response may derive from differences in hormonal milieu, stressor modality and/or intensity, or phagocyte priming. Investigating the complex interactions between types of stressors, neuroendocrine response, and immunocompetence will lead to a better understanding of the impacts of environmental challenges, including anthropogenic perturbations, on the health of cetacean populations.