Refined Methodology for Quantifying Virulence Using

Larvae of (the greater wax moth) are being increasingly used as a model to study microbial pathogenesis. In this model, bacterial virulence is typically measured by determining the 50% lethal dose (LD) of a bacterial strain or mutant. The use of to study pathogenesis, however, is challenging because of the extreme sensitivity of larvae to this bacterium. For some strains, as few as 1-5 colony-forming units are sufficient to kill which poses challenges for determining LD values. For this reason, some groups have used time-to-death as a measure of virulence, but methodologies have not been standardized. We provide a detailed protocol for using the time at which 50% of larvae have died (LT) at a particular inoculum as a measure of virulence. We also describe a quality control metric for enhancing the reproducibility of LT values. This approach provides an accurate and reproducible methodology for using larvae to measure and compare the virulence of strains.IMPORTANCE is a significant cause of morbidity and mortality. The invertebrate is used as a model to determine the virulence of strains. We provide a protocol and analytical approach for using a time-to-death metric to accurately quantify the virulence of strains in larvae. This methodology, which has several advantages over 50% lethal dose approaches, is a useful resource for the study of pathogenicity.