An Exploratory Look at Functional Responses to a Second Antigen Injection in a Freshwater Turtle

Existing and emerging diseases threaten wildlife populations worldwide and population resilience in the face of disease depends on immune responses. To apply conservation strategies to populations threatened by disease, it is critical to know not only how individuals will respond to the initial exposure of the pathogen but also to determine risks when the pathogen becomes endemic or is reintroduced. Immune responses following a subsequent exposure to a pathogen may vary from initial responses due to several immunological memory mechanisms such as adaptive immune function and innate immune priming/training and tolerance. Alternatively, immune responses may vary as a consequence of resource limitation. Regardless of outcome, these altered responses could impact how individuals respond to successive pathogen exposures in their environment. Disease threatens reptiles worldwide but research on reptilian immunology has lagged behind other taxonomic groups, resulting in large gaps in our understanding of both mechanistic and functional immune responses. Reptiles possess traditionally considered "innate" and "adaptive" immune components, but current literature seems to agree that reptiles depend largely on innate immune components as adaptive responses are slow. We present an exploratory study in which we measured functional immune responses in male red-eared slider turtles (Trachemys scripta elegans) to 2 antigen injections representing bacterial (lipopolysaccharide), viral (polyinosinic-polycytidylic acid; poly(I:C), fungal infections (zymosan), and control (saline), administered 2 weeks apart. We separated serum and buffy layer (serum + BL) from blood samples and manipulated the serum + BL (fresh, frozen, frozen + heat) to systematically inactivate immune components. We conducted microbial killing assays using the manipulated serum + BL with Gram-negative Escherichia coli, Gram-positive Staphylococcus aureus, and the diploid yeast Candida albicans, which allowed us to examine immune responses across various contexts. Although sample sizes were small, we observed varied responses across treatments and serum + BL/microbe assay combinations, suggesting that several mechanisms of immune memory may have occurred after the first treatment injection. Given the time frame of our exploratory study and previous research on acquired antibody production timing in reptiles, we suggest that our observations may be products of immune training/priming, tolerance, and resource reallocation. However, more work is necessary to examine these processes in reptiles and we make suggestions for future research directions. Our work further demonstrates the role that diverse immunological tools have in understanding immune strategies across taxa to enhance our knowledge of reptilian immunology and inform conservation decisions.