title: Mitogen-induced lymphocyte proliferation in loggerhead sea turtles: comparison of methods and effects of gender, plasma testosterone concentration, and body condition on immunity.
abstract: A fully functioning immune system is vital to the survival of threatened and endangered sea turtles. Immunological protection against diseases in any organism can be reduced by a number of natural and anthropogenic factors, such as seasonal changes, malnutrition, disease states, and contaminant exposure. These factors are even more critical when they occur in endangered species or populations. To identify alterations in the immunological health of loggerhead sea turtles (Caretta caretta), the mitogen-induced lymphocyte proliferation (LP) assay was developed using peripheral blood leukocytes (PBLs). Collection and culture conditions were optimized for this assay using non-lethal blood samples collected from free-ranging turtles along the southeastern US coast. During the collection, two anticoagulants (sodium heparin and lithium heparin) were compared to determine effects of different ions on assay results. Optimal culture conditions were established for loggerhead PBLs while two different methods of measuring LP were compared: (1) the traditional radioactive (3)H-thymidine assay and (2) a non-radioactive, colorimetric method utilizing 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium (MTT). The results indicate that the (3)H-thymidine and the non-radioactive MTT methods did not correlate with each other and that the use of heparin type did not influence the results of the LP assay. Lastly, using these optimized methods, we investigated the effect of gender, plasma testosterone concentration, and body condition on LP in loggerhead turtles and found that none of the parameters largely influenced LP.
authors.affiliation: Coastal Systems Science and Policy, and Integrated Toxicology Program, Nicholas School of the Environment and Earth Sciences, Duke University, 135 Duke Marine Lab Road, Beaufort, NC 28516, USA. jennifer.keller@noaa.gov
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Keller
authors.forename: Patricia D
authors.initials: PD
authors.lastname: McClellan-Green
authors.forename: A Michelle
authors.initials: AM
authors.lastname: Lee
authors.forename: Mike D
authors.initials: MD
authors.lastname: Arendt
authors.forename: Philip P
authors.initials: PP
authors.lastname: Maier
authors.forename: Al L
authors.initials: AL
authors.lastname: Segars
authors.forename: J David
authors.initials: JD
authors.lastname: Whitaker
authors.forename: Deborah E
authors.initials: DE
authors.lastname: Keil
authors.forename: Margie M
authors.initials: MM
authors.lastname: Peden-Adams
mesh_terms: D000818:Animals; D000925:Anticoagulants; D001402:B-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D049109:Cell Proliferation; D003247:Conservation of Natural Resources; D005260:Female; D005562:Formazans / Q000378:metabolism; D008213:Lymphocyte Activation / Q000187:drug effects / Q000276:immunology; D008297:Male; D008934:Mitogens / Q000494:pharmacology*; D012737:Sex Factors; D015144:Southeastern United States; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D013739:Testosterone / Q000097:blood*; D013778:Tetrazolium Salts / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism; D014426:Turtles / Q000097:blood / Q000276:immunology*
chemical_list: D000925:Anticoagulants; D005562:Formazans; D008934:Mitogens; D013778:Tetrazolium Salts; C079782:MTT formazan; D013739:Testosterone; D013936:Thymidine