Repeatability and sources of variation of the bacteria-killing assay in the common snapping turtle

Research on reptile ecoimmunology lags behind that on other vertebrates, despite the importance of such studies for conservation and evolution. Because the innate immune system is highly conserved across vertebrate lineages, assessments of its performance may be particularly useful in reptiles. The bacteria-killing assay requires a single, small blood sample and quantifies an individual's ability to kill microorganisms. The assay's construct validity and interpretability make it an attractive measure of innate immunity, but it requires proper optimization and sample storage. We optimized this assay for the common snapping turtle (Chelydra serpentina) to assess the repeatability of the assay and the effects of freezing and thawing on bactericidal capacity. We determined whether age (adult female and hatchlings) or incubation temperature influenced bactericidal capacity. We found that the assay was repeatable and that freezing plasma samples for 6 weeks at -80°C did not decrease bactericidal capacity nor did a single 30-min thaw and subsequent refreezing. However, we detected subtle interassay variation and results from one assay were 5-6% greater than those from the other two. Adult females had significantly greater bactericidal ability than hatchlings and we found no relationship between incubation temperature and bactericidal capacity. This assay is a useful tool in snapping turtles and may have applicability in other reptiles. However, species-specific optimization is required to ensure that variation among individuals exceeds interassay variation. Consideration should be given to optimization conditions that facilitate comparisons between or within groups, particularly groups that differ considerably in bactericidal capacity.