Refined methodology for quantifyingvirulence using.

Abstract

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 ofto studypathogenesis, however, is challenging because of the extreme sensitivity of larvae to this bacterium. For somestrains, as few as 1-5 colony-forming units are sufficient to killwhich poses challenges for determining LDvalues. For this reason, some groups have used time-to-death as a measure ofvirulence, 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 ofvirulence. We also describe a quality control metric for enhancing the reproducibility of LTvalues. This approach provides an accurate and reproducible methodology for usinglarvae to measure and compare the virulence ofstrains.IMPORTANCEis a significant cause of morbidity and mortality. The invertebrateis used as a model to determine the virulence ofstrains. We provide a protocol and analytical approach for using a time-to-death metric to accurately quantify the virulence ofstrains inlarvae. This methodology, which has several advantages over 50% lethal dose approaches, is a useful resource for the study ofpathogenicity.