Isolation, Cryopreservation, and Mitogenesis of Peripheral Blood Lymphocytes from Chickens (Gallus domesticus) and Wild Herring Gulls (Larus argentatus)

Monitoring the toxicity of environmental contaminants on the physiologic function of wild birds often includes measures of immune function. The purpose of this study was to apply methods of isolation, cryopreservation, and cell culture of chicken lymphocytes to blood samples from herring gulls, which are a sentinel species for biomonitoring studies in the Great Lakes and northern North America. Slow-spin centrifugation and density gradient isolation of lymphocytes were compared using chicken blood. Significant thrombocyte contamination of density gradient isolated samples (40% to 77% thrombocytes) led to the use of slow-spin centrifugation (2% thrombocytes) for blood from herring gulls. Cryopreserved blood samples were collected from adult and prefledgling herring gulls at sites of low environmental contamination around the Great Lakes and the Bay of Fundy between 1999 and 2002. Cryopreservation decreased the viability of lymphocytes from wild birds, but a high proportion of samples yielded enough live lymphocytes to assess function in culture. Cryopreserved lymphocytes from herring gulls proliferated in response to phytohemagglutinin (PHA), concanavalin-A, phorbol myristate acetate (PMA), PHA plus PMA, and lipopolysaccharide stimulation. Weber and Roswell Park Memorial Institute medium (RPMI) 1640 media were compared for culture of lymphocytes. Weber medium better supported chicken B-lymphocyte proliferation than RPMI 1640 and supported chicken T-lymphocyte proliferation of a similar magnitude as RPMI. Proliferation responses for lymphocytes from prefledgling herring gulls were stronger in Weber medium than RPMI medium, especially for PHA, for which there was no stimulation in RPMI. Proliferation responses of lymphocytes from adult herring gulls were up to twofold greater than that for prefledgling herring gulls. The magnitudes of proliferation responses were similar to that for chicken lymphocytes. These methods have subsequently proven useful in ecotoxicology studies that involve species in remote locations.