Sylvain De Guise

Health of Common Bottlenose Dolphins (Tursiops truncatus) in Barataria Bay, Louisiana, Following the Deepwater Horizon Oil Spill

The oil spill resulting from the explosion of the Deepwater Horizon drilling platform initiated immediate concern for marine wildlife, including common bottlenose dolphins in sensitive coastal habitats. To evaluate potential sublethal effects on dolphins, health assessments were conducted in Barataria Bay, Louisiana, an area that received heavy and prolonged oiling, and in a reference site, Sarasota Bay, Florida, where oil was not observed. Dolphins were temporarily captured, received a veterinary examination, and were then released. Dolphins sampled in Barataria Bay showed evidence of hypoadrenocorticism, consistent with adrenal toxicity as previously reported for laboratory mammals exposed to oil. Barataria Bay dolphins were 5 times more likely to have moderate-severe lung disease, generally characterized by significant alveolar interstitial syndrome, lung masses, and pulmonary consolidation. Of 29 dolphins evaluated from Barataria Bay, 48% were given a guarded or worse prognosis, and 17% were considered poor or grave, indicating that they were not expected to survive. Disease conditions in Barataria Bay dolphins were significantly greater in prevalence and severity than those in Sarasota Bay dolphins, as well as those previously reported in other wild dolphin populations. Many disease conditions observed in Barataria Bay dolphins are uncommon but consistent with petroleum hydrocarbon exposure and toxicity.

Anaemia, hypothyroidism and immune suppression associated with polychlorinated biphenyl exposure in bottlenose dolphins (Tursiops truncatus)

Polychlorinated biphenyls (PCBs), persistent chemicals widely used for industrial purposes, have been banned in most parts of the world for decades. Owing to their bioaccumulative nature, PCBs are still found in high concentrations in marine mammals, particularly those that occupy upper trophic positions. While PCB-related health effects have been well-documented in some mammals, studies among dolphins and whales are limited. We conducted health evaluations of bottlenose dolphins (Tursiops truncatus) near a site on the Georgia, United States coast heavily contaminated by Aroclor 1268, an uncommon PCB mixture primarily comprised of octa- through deca-chlorobiphenyl congeners. A high proportion (26%) of sampled dolphins suffered anaemia, a finding previously reported from primate laboratory studies using high doses of a more common PCB mixture, Aroclor 1254. In addition, the dolphins showed reduced thyroid hormone levels and total thyroxine, free thyroxine and triiodothyronine negatively correlated with PCB concentration measured in blubber (p = 0.039, < 0.001, 0.009, respectively). Similarly, T-lymphocyte proliferation and indices of innate immunity decreased with blubber PCB concentration, suggesting an increased susceptibility to infectious disease. Other persistent contaminants such as DDT which could potentially confound results were similar in the Georgia dolphins when compared with previously sampled reference sites, and therefore probably did not contribute to the observed correlations. Our results clearly demonstrate that dolphins are vulnerable to PCB-related toxic effects, at least partially mediated through the endocrine system. The severity of the effects suggests that the PCB mixture to which the Georgia dolphins were exposed has substantial toxic potential and further studies are warranted to elucidate mechanisms and potential impacts on other top-level predators, including humans, who regularly consume fish from the same marine waters.

A cDNA Microarray for Crassostrea virginica and C. gigas

The eastern oyster, Crassostrea virginica, and the Pacific oyster, C. gigas, are species of global economic significance as well as important components of estuarine ecosystems and models for genetic and environmental studies. To enhance the molecular tools available for oyster research, an international group of collaborators has constructed a 27,496-feature cDNA microarray containing 4460 sequences derived from C. virginica, 2320 from C. gigas, and 16 non-oyster DNAs serving as positive and negative controls. The performance of the array was assessed by gene expression profiling using gill and digestive gland RNA derived from both C. gigas and C. virginica, and digestive gland RNA from C. ariakensis. The utility of the microarray for detection of homologous genes by cross-hybridization between species was also assessed and the correlation between hybridization intensity and sequence homology for selected genes determined. The oyster cDNA microarray is publicly available to the research community on a cost-recovery basis.

Chemical and biological pollution contribute to the immunological profiles of free‐ranging harbor seals

Polychlorinated biphenyls and other persistent organic pollutants have been associated with immunotoxicity and outbreaks of (infectious) disease in marine mammals by rendering them vulnerable to infection by pathogens such as viruses and bacteria. In an immunotoxicological study of free-ranging harbor seals (Phoca vitulina), we obtained samples of blood and blubber from seal pups that were live-captured from two remote and two near-urban sites in British Columbia, Canada, and Washington state, USA. Using these samples, we quantified hematology, innate immune function, adaptive immune function, and polychlorinated biphenyl accumulation. While controlling for confounding factors (age, sex, and condition), univariate correlations between phagocytosis (r2 = 0.30, p = 0.002), respiratory burst (r2 =0.45, p= 0.000), T-lymphocyte function (r2 = 0.16, p = 0.028), lymphocyte signaling (r2 = 0.17, p = 0.025), and lymphocyte counts (r2 = 0.29, p = 0.002), and polychlorinated biphenyl concentrations suggested chemical-associated immunotoxicity. Principal component analysis of immunological endpoints provided additional evidence of immunotoxic effects in seals. However, principal component analysis also identified a noncontaminant-related factor by distinguishing between seals inhabiting urban versus remote sites, with results being consistent with increased pathogen exposure. Elevated fecal coliform concentrations in water, and observations of terrestrial spill-over pathogens in local seals, further support the notion of biological pollution at these sites. Although our study highlights the role that environmental contaminants might play in rendering marine mammal populations vulnerable to disease through immunotoxicity, it also suggests that biological pollution represents an emerging conservation concern.

Immunotoxic effects of environmental pollutants in marine mammals.

Due to their marine ecology and life-history, marine mammals accumulate some of the highest levels of environmental contaminants of all wildlife. Given the increasing prevalence and severity of diseases in marine wildlife, it is imperative to understand how pollutants affect the immune system and consequently disease susceptibility. Advancements and adaptations of analytical techniques have facilitated marine mammal immunotoxicology research. Field studies, captive-feeding experiments and in vitro laboratory studies with marine mammals have associated exposure to environmental pollutants, most notable polychlorinated biphenyls (PCBs), organochlorine pesticides and heavy metals, to alterations of both the innate and adaptive arms of immune systems, which include aspects of cellular and humoral immunity. For marine mammals, reported immunotoxicology endpoints fell into several major categories: immune tissue histopathology, haematology/circulating immune cell populations, functional immune assays (lymphocyte proliferation, phagocytosis, respiratory burst, and natural killer cell activity), immunoglobulin production, and cytokine gene expression. Lymphocyte proliferation is by far the most commonly used immune assay, with studies using different organic pollutants and metals predominantly reporting immunosuppressive effects despite the many differences in study design and animal life history. Using combined field and laboratory data, we determined effect threshold levels for suppression of lymphocyte proliferation to be between b0.001-10 ppm for PCBs, 0.002-1.3 ppm for Hg, 0.009-0.06 for MeHg, and 0.1-2.4 for cadmium in polar bears and several pinniped and cetacean species. Similarly, thresholds for suppression of phagocytosis were 0.6-1.4 and 0.08-1.9 ppm for PCBs and mercury, respectively. Although data are lacking for many important immune endpoints and mechanisms of specific immune alterations are not well understood, this review revealed a systemic suppression of immune function in marine mammals exposed to environmental contaminants. Exposure to immunotoxic contaminants may have significant population level consequences as a contributing factor to increasing anthropogenic stress in wildlife and infectious disease outbreaks.

Cetacean Morbillivirus: Current Knowledge and Future Directions

We review the molecular and epidemiological characteristics of cetacean morbillivirus (CeMV) and the diagnosis and pathogenesis of associated disease, with six different strains detected in cetaceans worldwide. CeMV has caused epidemics with high mortality in odontocetes in Europe, the USA and Australia. It represents a distinct species within the Morbillivirus genus. Although most CeMV strains are phylogenetically closely related, recent data indicate that morbilliviruses recovered from Indo-Pacific bottlenose dolphins (Tursiops aduncus), from Western Australia, and a Guiana dolphin (Sotalia guianensis), from Brazil, are divergent. The signaling lymphocyte activation molecule (SLAM) cell receptor for CeMV has been characterized in cetaceans. It shares higher amino acid identity with the ruminant SLAM than with the receptors of carnivores or humans, reflecting the evolutionary history of these mammalian taxa. In Delphinidae, three amino acid substitutions may result in a higher affinity for the virus. Infection is diagnosed by histology, immunohistochemistry, virus isolation, RT-PCR, and serology. Classical CeMV-associated lesions include bronchointerstitial pneumonia, encephalitis, syncytia, and lymphoid depletion associated with immunosuppression. Cetaceans that survive the acute disease may develop fatal secondary infections and chronic encephalitis. Endemically infected, gregarious odontocetes probably serve as reservoirs and vectors. Transmission likely occurs through the inhalation of aerosolized virus but mother to fetus transmission was also reported.

Immunomodulatory effects of in vitro exposure to organochlorines on T-cell proliferation in marine mammals and mice.

Marine mammals bioaccumulate various environmental contaminants such as organochlorines (OCs), which biomagnify via the food web. While the immunomodulatory effects of individual OCs have been studied, the effects of mixtures are not well understood. The immunomodulatory effects of polychlorinated biphenyl (PCB) 138, 153, 169, and 180 as well as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and all possible mixtures were examined in marine mammals and mice. Lymphocyte proliferation was significantly modulated by OCs in all species tested, mostly by non-coplanar PCBs, as shown using regression analyses. Correlation analyses showed significant correlations (interpreted as additive effects) between OCs in mice, killer whales, and Steller sea lions. Nonadditive synergistic and antagonistic interactions between OCs were detected in most of the species tested. Toxic equivalency (TEQ) values used for OC toxicity assessment failed to predict the immunomodulatory effects measured in mice and marine mammals. The commonly used mouse model failed to predict immunomodulatory effects in other species. Clustering data suggested that phylogeny does not predict toxicity of OCs. Overall, our data suggest the presence of species-specific sensitivities to different mixtures, in which OCs interactions may be complex and that may exert their effects through dioxinlike or dioxin-independent pathways. Lastly, lymphocyte proliferation, an important part of adaptive immunity, was significantly modulated in mice and marine mammals, suggesting the possibility of increased susceptibility to diseases. These findings will be useful to better characterize the risk associated with OC exposure and possibly lead to new conservation and management strategies.

Immune functions in beluga whales (Delphinapterus leucas) : Evaluation of phagocytosis and respiratory burst with peripheral blood leukocytes using flow cytometry

Flow cytometric assays using peripheral blood were developed to study phagocytosis and respiratory burst, the two major functions of neutrophils and among the most important non-specific defense mechanisms, in beluga whales. The use of flow cytometry avoids the problems associated with the isolation and purification of different cell types, and allows the measurement of a large number of cells (10,000) in a very short period of time. The methods described will be used to compare these functions in blood samples from highly contaminated beluga whales from the St. Lawrence and from relatively clean arctic beluga whales.

Eosinophilia and biotoxin exposure in bottlenose dolphins (Tursiops truncatus) from a coastal area impacted by repeated mortality events

Bottlenose dolphins (Tursiops truncatus) inhabiting coastal waters in the northern Gulf of Mexico have been impacted by recurrent unusual mortality events over the past few decades. Several of these mortality events along the Florida panhandle have been tentatively attributed to poisoning from brevetoxin produced by the dinoflagellate Karenia brevis. While dolphins in other regions of the Florida coast are often exposed to K. brevis blooms, large-scale dolphin mortality events are relatively rare and the frequency and magnitude of die-offs along the Panhandle raise concern for the apparent vulnerability of dolphins in this region. We report results from dolphin health assessments conducted near St. Joseph Bay, Florida, an area impacted by 3 unusual die-offs within a 7-year time span. An eosinophilia syndrome, manifested as an elevated blood eosinophil count without obvious cause, was observed in 23% of sampled dolphins. Elevated eosinophil counts were associated with decreased T-lymphocyte proliferation and increased neutrophil phagocytosis. In addition, indication of chronic low-level exposure to another algal toxin, domoic acid produced by the diatom Pseudo-nitzschia spp., was determined. Previous studies of other marine mammal populations exposed recurrently to Pseudo-nitzschia blooms have suggested a possible link between the eosinophilia and domoic acid exposure. While the chronic eosinophilia syndrome could over the long-term produce organ damage and alter immunological status and thereby increase vulnerability to other challenges, the significance of the high prevalence of the syndrome to the observed mortality events in the St. Joseph Bay area is unclear. Nonetheless, the unusual immunological findings and concurrent evidence of domoic acid exposure in this sentinel marine species suggest a need for further investigation to elucidate potential links between chronic, low-level exposure to algal toxins and immune health.

Non-coplanar PCB-mediated modulation of human leukocyte phagocytosis: a new mechanism for immunotoxicity.

Organochlorine (OC) contaminants, notably polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are ubiquitous in all ecosystems and found in the tissues of humans and wildlife. Although the immunotoxicity of coplanar, dioxinlike PCBs is well documented, the adverse effects exerted by non-coplanar, non-dioxinlike PCBs have received little attention. Direct causal relationship between PCB and dioxin exposure and the observed detrimental effects on the immune system has yet to be fully established in humans. The immunomodulatory potential of toxic coplanar PCB 169 and TCDD and abundant non-coplanar PCBs 138, 153, and 180 on human leukocyte phagocytosis, an important innate immune function that initiates the clearance of pathogens, was tested upon in vitro exposure. Mixture and concentration-response experiments demonstrated a suppression of phagocytosis by non-coplanar PCBs suggesting a previously unrecognized aryl hydrocarbon receptor (AhR)-independent pathway. Regression analysis revealed that reduction of phagocytosis was mostly explained by the non-coplanar congeners. The effects on phagocytosis could not be accurately predicted by either the currently used toxic equivalence (TEQ) approach or the mouse model, thus undermining the use of the traditional models in the risk assessment for OC mixtures containing non-coplanar congeners. Our results are cause for concern as they suggest an AhR-independent pathway through which non-coplanar PCBs modulate phagocytosis, the immune systems first line of defense, possibly increasing the risk to developing infectious disease.