Humoral immune responses differ according to antigen in Children’s pythons (Antaresia childreni)

Humoral immunity provides a robust defense against pathogens through the production of antibodies with diverse functions, yet it remains poorly understood in ectotherms. In this study, we compared the extent and time course of the humoral immune response to two commonly used non-pathogenic antigens in male and female Children’s pythons ( Antaresia childreni ). Two separate experimental cohorts of adult snakes from the same colony were used, each consisting of 10 males and 10 females. One cohort received either lipopolysaccharide (LPS) or a control injection, while the other cohort received keyhole limpet hemocyanin (KLH) or a control injection. Blood samples and blood smears were collected prior to treatment and at set intervals following injection. Humoral immune responses were assessed by indirect ELISAs for antigen-specific IgY. White blood cell differentials were performed on stained blood smears to evaluate the early innate immune response. KLH injections elicited a robust anti-KLH IgY response, increasing from day 14 through day 100 and remaining elevated through day 183. Additionally, males had higher anti-KLH IgY titers than females at days 14 and 21. In contrast, LPS injections failed to cause IgY production throughout the time frame of the study. Leukocyte responses were only observed following KLH injections, with increased monocyte counts at day 7. These results demonstrate that KLH may be a more effective antigen for studies investigating humoral responses in reptiles due to its novelty to these animals. In contrast, snakes likely had variable prior exposure to LPS-like antigens, since they are in the walls of gram-negative bacteria, which are ubiquitous. Overall, our results provide important context for understanding the variability of the humoral immune time course in reptiles.