Characterizing Primary Immune Responses Against Three Antigens in a Reptile

Emerging diseases threaten wildlife worldwide and understanding immune function in the context of the ecology of an organism is critical in predicting disease outcomes. The immune response in reptiles is especially understudied, leaving major gaps in the ability to address disease threats. Freshwater turtles are especially imperiled due to a swath of anthropogenic impacts, including bacterial, viral, and fungal diseases. Because multiple novel pathogen types threaten turtles, understanding nuances in their immune responses may help predict how populations may respond to challenges and shape conservation decisions. We aimed to characterize immune responses following exposure to 3 commercially available antigens representing bacterial, viral, and fungal pathogens in adult red-eared slider turtles (Trachemys scripta elegans) housed in natural conditions. We collected blood samples from red-eared sliders at 0, 12, 24, 48, and 72 h following an antigen treatment (lipopolysaccharide; LPS, polyinosinic-polycytidylic acid; poly(I:C), zymosan, or saline). We created blood smears at 0 and 72 h and the collected serum and buffy layer (serum + BL) were subjected to 3 manipulations (fresh, frozen, frozen + heat) for use in microbial killing assays against Escherichia coli, Candida albicans, and Staphylococcus aureus. The combination of antigen treatments on the turtles and manipulations to the collected blood allowed us to examine different immune responses and components. We also quantified corticosterone using enzyme-linked immunosorbent assays to understand energy mobilization following antigen treatment. Microbial killing was generally stable against microbes, regardless of antigen exposure and serum manipulations reveal differences in relative leukocyte and protein-based contribution to killing microbe classes. Overall, the low killing of Gram-positive S. aureus relative to E. coli and C. albicans further suggests that red-eared sliders rely on a robust complement response. Though we did not observe substantial differences among treatments, our results suggest that LPS elicited an increased complement response. The other antigens did not consistently stimulate stronger responses, but more work is needed to understand dose-dependent responses to poly(I:C) and zymosan and in turtles and other reptiles. Overall, our research demonstrates that microbial killing assays can be enhanced through deliberate serum + BL manipulation and microbe usage as ecoimmunological tools to gain a more robust understanding of the immune response in wild organisms.