Mouse Skin Flea Feeding Assay

An adult mouse skin affixed to an artificial feeding device maintained at 37°C was used (23 (link)). In the infectious blood meal, fleas were allowed to feed for 1 h. To determine if Ail affected colonization insects fed blood containing the Δail mutant, Y. pestis KIM6⁺, or the Δail/ail⁺ complemented strain were collected, cold immobilized, and fleas that took a blood meal were identified under a dissecting microscope by the presence of fresh blood in the proventriculus or midgut. Three days after the infectious feed a 10 fleas/strain were collected and frozen at −80°C for later determination of bacterial numbers (designated 72 h). This experiment was repeated four times on separate days. Remaining insects were fed with sterile defibrinated Swiss Webster mouse whole blood (Bioreclamation) or defibrinated whole human blood (Bioreclamation) and selected as above. Fleas were kept for another 24 h in a maintenance chamber, collected, and frozen at −80°C (designated 96 h). To evaluate infection prevalence and infection rate, fleas were thawed, individually homogenized in 100 μL of a 10% glycerol solution in BHI, plated on BHI agar, and incubated 48 h at 28°C. Fleas were designated uninfected when no colonies grew from plating 15% of flea lysate. The latter experiment was repeated four times on separate days. All human blood used in these flea experiments was shown to have bactericidal activity against Y. pestis by using the serum resistance assay protocol described above.