Quantifying Innate Immune Responses in Reptiles

Natural antibody-mediated haemagglutination and complement-mediated haemolysis (NAbs, Lysis) ability are reported to be the first line of defense against pathogens in vertebrates (reviewed in [94 (link)]), and these measures of innate immune function have been studied in many reptile species (e.g., [4 (link), 5 (link), 32 , 80 , 82 (link), 95 (link)]). We completed the assays for NAbs and lysis ability according to the haemolysis–haemagglutination assay adapted from [96 (link)] for use in painted turtles [5 (link), 97 (link)]) using rabbit red blood cells. We used two bottles of rabbit red blood cells (HemoStat HemoStat Laboratories, Dixon, CA, USA) to complete all assays. We ran all plates with positive and negative controls and samples in duplicate. Higher titres for haemagglutination indicate greater abundance of natural antibodies in the plasma sample, as high titres are an indication that natural antibodies are at high concentrations even in increasingly diluted plasma [96 (link)]. Similarly, higher titres for haemolysis indicate the plasma is able to lyse RRBCs even at more dilute concentrations [96 (link)]. Thus, increased natural antibody levels and lysis ability are associated with increased immune function. We assessed bactericidal competence (BC) of plasma by quantifying its ability to inhibit growth of Escherichia coli using our published protocol for painted turtles [5 (link), 97 (link)], adapted from [98 ]. Five lyophilized pellets of E. coli (Microbiologics, ATCC#8739) were used in the present experiment, with each new pellet used to generate a new control solution as we progressed through samples. Increased bactericidal competence corresponds to increased immune function. All immune assays were conducted in spring of 2019 on samples collected in spring of 2018.