Abstract
Co-infection refers to the simultaneous infection of a host by multiple pathogenic organisms. Experimental co-infection studies using a mutant and its isogenic wild type have proven to be profoundly sensitive to analysis of pathogen factor mutation-associated fitness effects in in vivo models of infectious disease. Here we discuss the use of such co-infection experiments in studying the interaction between Yersinia pestis and its flea vector to more sensitively determine the critical bacterial determinants for Y. pestis survival, adaptation, and transmission from fleas. This chapter comprises two main sections, the first detailing how to infect fleas with mutant and wild type Y. pestis strains, and secondly how to process infected fleas and specifically quantify distinct Y. pestis strain burdens per flea. The Y. pestis competitive fitness co-infection model in fleas is insightful in evaluating the consequence of a mutation which may not be obvious in single-strain flea infections where there is less selective pressure.