Veterinary medical examinations, including both physical examination and diagnostic tests, are important to monitor the health of both managed-care and wild marine mammals. However, limited ...species-specific reagents and assays are available that may contribute to a broader medical examination. This project evaluated if commercially available human and porcine antibodies and reagents would cross-react with manatee (Trichechus manatus) cytokines as the first step to validate a new diagnostic tool for manatees. Overall, as a result of limited cross-reactivity, human and porcine commercial reagents did not allow for the quantification of manatee cytokines. At this point, caution must be exercised when using human or porcine immunoassay reagents to quantify manatee cytokines if the reagents have not been fully validated. Future efforts will continue to explore and test the cross-reactivity of reagents to measure manatee cytokines as new species-specific and commercial reagents become available.
•In vitro approach allowed for the detection of PbTx-3 induced immunotoxicity.•PbTx-3 modulated innate and adaptive immune functions in bottlenose dolphins.•PbTx-3 exposure may increase ...susceptibility to opportunistic infections.
Harmful algal blooms (HAB), commonly referred to as ‘red tides’, involving the dinoflagellate Karenia brevis produce a series of neurotoxins known as brevetoxins (PbTx). Brevetoxins have long been associated with extensive fish kills, adverse human health effects such as neurotoxic shellfish poisoning, and have been associated with mortality events in aquatic mammals such as bottlenose dolphins (Tursiops truncatus). The immunotoxicological effects of these brevetoxins have been studied in manatees, humans, and cell lines, however, the effects in bottlenose dolphins remain unclear. There are increasing concerns that dolphins may be exposed to repeated/chronic, sub-lethal concentrations, which may impact their overall health. The objectives of this study were to measure the changes in innate (phagocytosis, respiratory burst, NK cell activity) and adaptive (mitogen-induced B and T lymphocyte proliferation) immune functions upon in vitro exposure to increasing concentrations of brevetoxin (PbTx-3) (0, 0.01, 0.1, 1, 10, 100, 500, and 1000nM) using bottlenose dolphin peripheral blood immune cells. Brevetoxin significantly increased spontaneous lymphocyte proliferation at 0.1–1000nM compared to the unexposed control. Brevetoxin significantly increased T lymphocyte proliferation upon suboptimal (0.1μg/ml) and optimal (1.0μg/ml) Con-A stimulation at 0.01–100nM and 0.1nM of PbTx-3 respectively, as well as suboptimal (0.05μg/ml) and optimal (5.0μg/ml) LPS-induced B lymphocyte proliferation at 0.01–100nM and 0.01–500nM of PbTx-3, respectively. Both neutrophil and monocyte respiratory burst were significantly increased at 500 and 1000nM. There were no significant effects on neutrophil or monocyte phagocytosis or NK cell activity. Importantly, concentrations that modulated immune functions in vitro were within the range measured in the blood of dolphins during two unusual mortality events, suggesting that naturally exposed dolphins may be at risk for immunomodulation. Brevetoxin-induced immunomodulation may increase an animal's susceptibility to bacterial, viral, or fungal infections. Understanding the risk for immunomodulation upon HAB toxin exposure can contribute in the health assessment and management of marine mammals, as well as guide veterinarians and wildlife rehabilitators in caring for and treating afflicted animals.
To better elucidate the potential immune-related health effects of exposure to environmentally persistent organic pollutants (POP), such as polychlorinated biphenyls (PCBs) and perfluoroalkyl ...substances (PFASs), in ringed seals (Pusa hispida), a sentinel Arctic species, we assessed 1) associations between mitogen-induced lymphocyte proliferation and in vivo tissue contaminant burdens, and 2) the concentration-response effects of in vitro exposure to PFASs and PCB congeners on mitogen-induced lymphocyte proliferation. Upon in vitro contaminant exposure, the non-coplanar PCB congeners CB 138, 153, and 180, but not the coplanar CB 169, significantly reduced lymphocyte proliferation between 10 and 20µgg−1 ww. The respective in vitro EC50 values for these congeners were 13.3, 20.7, 20.8, and 54.6µgg−1 ww. No modulation of lymphocyte proliferation was observed upon in vitro exposure to two individual PFASs, perfluorooctane sulphonic acid (PFOS) and perfluorooctanoic acid (PFOA), at concentrations up to 1000ngg-1. In addition, no significant correlations were found between lymphocyte proliferation and any blood or blubber contaminant measured. Taken together, these data suggest this population of ringed seals is not currently at high risk of altered lymphocyte proliferation from exposure to the POPs or PFASs in this study.
•Assess relationships between tissue contaminants and changes in immune function.•Risk for contaminant-induced immunotoxicity in East Greenland ringed seal is low.•Weight of evidence suggest non-coplanar PCBs are immunotoxic at high concentrations.
Threatened loggerhead sea turtles (Caretta caretta) face numerous environmental challenges, including exposure to anthropogenic chemicals such as polychlorinated biphenyls (PCBs). Despite being ...banned by the USA in the 1970s, PCBs persist in the environment and produce immunotoxic effects in a wide range of marine vertebrate species. This is of particular concern, as the modulation of the immune system may enhance the susceptibility to a variety of pathogens. Blood samples were collected from 19 immature, captive-reared loggerhead sea turtles. Functional immune assays phagocytosis and natural killer (NK) cell activity were used to quantify the direct effects of PCB congeners 105, 138, and 169 on innate immune functions upon in vitro exposure of sea turtle cells to increasing concentrations (control (0), 0.5, 1, 2.5, 5, 10, 15, or 20 ppm) of each PCB. PCB 105 significantly elevated eosinophil phagocytosis at 10 and 15 ppm and PCB 138 at 15 ppm compared to unexposed (0 ppm). The effects of PCB 169 on phagocytosis were not evaluated. PCB 138 and 105 significantly decreased NK cell activity at 15 and 20 ppm, compared to unexposed (0 ppm) controls. PCB 169 did not markedly modulate NK activity. This constitutes the first study to investigate the in vitro effects of these three PCBs on sea turtle innate immune functions. These results add to our understanding of PCB-induced immunotoxicity in sea turtles and may provide a framework for establishing the relationships between chemical levels and turtle immunity.
Following the Deepwater Horizon (DWH) oil spill, the Natural Resource Damage Assessment conducted comprehensive health assessments on common bottlenose dolphins Tursiops truncatus in Barataria Bay ...(BB), Louisiana, in 2011, 2013 and 2014, as well as in Mississippi Sound (MS) in 2013, to assess potential health effects from exposure to oil compared to Sarasota Bay (SB), Florida dolphins not exposed to DWH oil. Immune functions demonstrated a consistent increase in T (BB 2011) and B (BB 2011 and 2013) lymphocyte proliferation compared to SB. Cytokine concentrations varied considerably in wild dolphin populations, and no significant differences were found; however, interesting trends were observed. The Th1 cytokines IL-2, IL-12, and IFNγ, and the Th2 cytokines IL-5, IL-10, and IL-13, were 2- to 50-fold lower, and IL-4 was 3-fold higher, in BB 2011 compared to SB. Overall, the changes observed were compatible with those documented in other species following exposure to oil or PAHs and were most pronounced in BB 2011, at the place and time most affected by oil. The changes in T cell functions, and the trend towards a cytokine balance tilted towards a Th2, rather than a Th1 response, are compatible with intra-cellular bacterial infections such as Brucella, which has been identified as one of the potential contributory factors to perinatal dolphin mortalities, and changes in B cell functions are compatible with an increase in extra-cellular bacterial infections and primary bacterial pneumonia.
Sea turtles face numerous environmental challenges, such as exposure to chemical pollution and biotoxins, which may contribute to immune system impairment, resulting in increased disease ...susceptibility. Therefore, a more thorough assessment of the host's immune response and its susceptibility is needed for these threatened and endangered animals. In this study, the innate and acquired immune functions of sixty-five clinically healthy, immature, captive loggerhead sea turtles (Caretta caretta) were assayed using non-lethal blood sample collection. Functional immune assays were developed and/or optimized for this species, including mitogen-induced lymphocyte proliferation, natural killer (NK) cell activity, phagocytosis, and respiratory burst. Peripheral blood mononuclear cells (PBMC) and phagocytes were isolated by density gradient centrifugation on Ficoll–Paque and discontinuous Percoll gradients, respectively. The T lymphocyte mitogens ConA significantly induced lymphocyte proliferation at 1 and 2μg/mL while PHA significantly induced lymphocyte proliferation at 5 and 10μg/mL. The B lymphocyte mitogen LPS significantly induced proliferation at 1μg/mL. Monocytes demonstrated higher phagocytic activity than eosinophils. In addition, monocytes exhibited respiratory burst. Natural killer cell activity was higher against YAC-1 than K-562 target cells. These optimized assays may help to evaluate the integrity of loggerhead sea turtle's immune system upon exposure to environmental contaminants, as well as part of a comprehensive health assessment and monitoring program.
The immune system is an arrangement of biological structures and processes within an organism that protects against infection and disease by identifying and riding the body of pathogens. It detects a ...wide variety of agents,including bacteria, viruses, parasites, fungal and neoplastic cells (1).The function of the immune system is divided into two major mechanisms:innate, and acquired immunity. Innate immunity is the body’s first line of defense. It is classified as non-specific and includes physical and chemical barriers such as the skin, respiratory tract and gastrointestinal tract, as well as immune cells within the body (1,2). These cells include phagocytic cells such as macrophages and neutrophils, which are able to engulf and kill pathogens, and natural killer cells which, can lyse tumor and virus-infected cells (2). Most of the microorganisms encountered daily in life of a normal healthy individual are detected and destroyed within hours by defense mechanisms that are not antigen specific and do not require a prolonged period of induction (2)If the innate immune system is unable to remove a pathogen, acquired immunity is activated. Specific immune responses are induced which generates antigen specific cells that prevent subsequent infection with the same microorganism. The key features that distinguish innate from acquired immunity are specificity, memory and the ability to distinguish self from non-self (3).However, the innate and adaptive immune systems do not operate independently. The activation of the innate immune response produces signals that stimulate and direct subsequent acquired responses. In addition, acquired immunity generates signals that stimulate and increase the effectiveness of innate immune responses (3).Acquired responses involve the proliferation of antigen specific B and Tlymphocytes (1). A fundamental characteristic of acquired immunity is that memory develops and secondary contact with the same antigen provokes a faster response. Two arms of acquired immunity include humoral and cell mediated immunity (2). In humoral immunity, B cells are stimulated following recognition of antigen by cell surface receptors. Mature B cells start the production of antigen specific immunoglobulins to act as antibodies. T cells mediate cellular immunity. These cells also have antigen receptors and function as helper cells for various immune responses and can act as cytotoxic cells killing target cells (2)
The complement and neutrophil defence systems, as major components of innate immunity, are activated during inflammation and infection. For neutrophil migration to the inflamed region, we ...hypothesized that the complement activation product C5a induces significant changes in cellular morphology before chemotaxis. Exposure of human neutrophils to C5a dose‐ and time‐dependently resulted in a rapid C5a receptor‐1 (C5aR1)‐dependent shape change, indicated by enhanced flow cytometric forward‐scatter area values. Similar changes were observed after incubation with zymosan‐activated serum and in blood neutrophils during murine sepsis, but not in mice lacking the C5aR1. In human neutrophils, Amnis high‐resolution digital imaging revealed a C5a‐induced decrease in circularity and increase in the cellular length/width ratio. Biomechanically, microfluidic optical stretching experiments indicated significantly increased neutrophil deformability early after C5a stimulation. The C5a‐induced shape changes were inhibited by pharmacological blockade of either the Cl−/HCO3−‐exchanger or the Cl−‐channel. Furthermore, actin polymerization assays revealed that C5a exposure resulted in a significant polarization of the neutrophils. The functional polarization process triggered by ATP–P2X/Y‐purinoceptor interaction was also involved in the C5a‐induced shape changes, because pretreatment with suramin blocked not only the shape changes but also the subsequent C5a‐dependent chemotactic activity. In conclusion, the data suggest that the anaphylatoxin C5a regulates basic neutrophil cell processes by increasing the membrane elasticity and cell size as a consequence of actin‐cytoskeleton polymerization and reorganization, transforming the neutrophil into a migratory cell able to invade the inflammatory site and subsequently clear pathogens and molecular debris.
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