Polychlorinated biphenyls (PCBs) modulate both phagocytosis and NK cell activity in vitro in juvenile loggerhead sea turtles (Caretta caretta).

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.

Establishment, characterization, and toxicological application of loggerhead sea turtle (Caretta caretta) primary skin fibroblast cell cultures.

Pollution is a well-known threat to sea turtles but its impact is poorly understood. In vitro toxicity testing presents a promising avenue to assess and monitor the effects of environmental pollutants in these animals within the legal constraints of their endangered status. Reptilian cell cultures are rare and, in sea turtles, largely derived from animals affected by tumors. Here we describe the full characterization of primary skin fibroblast cell cultures derived from biopsies of multiple healthy loggerhead sea turtles (Caretta caretta), and the subsequent optimization of traditional in vitro toxicity assays to reptilian cells. Characterization included validating fibroblast cells by morphology and immunocytochemistry, and optimizing culture conditions by use of growth curve assays with a fractional factorial experimental design. Two cell viability assays, MTT and lactate dehydrogenase (LDH), and an assay measuring cytochrome P4501A (CYP1A) expression by quantitative PCR were optimized in the characterized cells. MTT and LDH assays confirmed cytotoxicity of perfluorooctanoic acid at 500 µM following 72 and 96 h exposures while CYP1A5 induction was detected after 72 h exposure to 0.1-10 µM benzo[a]pyrene. This research demonstrates the validity of in vitro toxicity testing in sea turtles and highlights the need to optimize mammalian assays to reptilian cells.

Evaluation of immune functions in captive immature loggerhead sea turtles (Caretta caretta).

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 2 µg/mL while PHA significantly induced lymphocyte proliferation at 5 and 10 µg/mL. The B lymphocyte mitogen LPS significantly induced proliferation at 1 µg/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.

Hematology and plasma biochemistry analytes in five age groups of immature, captive-reared loggerhead sea turtles (Caretta caretta).

Abstract: Blood samples of 85 immature, apparently healthy, captive-reared loggerhead sea turtles (Caretta caretta) were analyzed for 13 hematologic variables and total solids of 5 age groups (8, 20, 32, 44, and 56 mo old) and for 20 plasma biochemical analytes of 4 age groups (20 to 56 mo old). Each individual turtle was sampled under similar conditions during a blood collection period of 3 days. Hematologic analytes included packed cell volume, white blood cell (WBC) counts, WBC estimates, and leukocyte differentials. Biochemical analysis included albumin, alanine aminotransferase, alkaline phosphatase, amylase, aspartate aminotransferase, blood urea nitrogen, calcium, chloride, cholesterol, creatine kinase, creatinine, gamma glutamyltransferase, globulins, glucose, phosphorous, potassium, sodium, total bilirubin, total protein, total solids, and uric acid. In due consideration of small sample size in all five age groups, the results of hematologic and biochemical analysis were used to determine ranges for these analytes and to compare values among consecutive age groups. Several significant differences in some hematologic and biochemical variables were identified and need to be considered in the interpretation of blood work of immature, growing sea turtles in human care.