Figure 3

Quantification accuracy of canine blood ingestion by fleas. HMBS copies were quantified for individual fleas fed only bovine blood that were collected directly from the flea colony followed immediately by homogenization and nucleic acid extraction (flea colony; n = 10 fleas). Another group contained fleas that were immediately removed from the dog and DNA was immediately extracted (collection control; n = 16). For evaluation of HMBS DNA recovery, fleas collected after 4 hours on the dog were either stored for 7 days at room temperature in RNA/DNA Stabilization buffer followed by homogenization (RT - homogenize; n = 10) or were homogenized immediately after collection followed by 7-day RT storage (homogenize - RT; n = 10). HMBS amplification was not observed in fleas from the colony, while fleas in the collection control group did contain a small but significant amount of HMBS targets indicating a background level of contamination with canine DNA by contact of the fleas with the dog. Fleas of the homogenize-RT group carried significantly higher HMBS copy numbers than RT-homogenize fleas (P = 0.006) indicating incomplete preservation of DNA in non-homogenized fleas. Fleas of the homogenize-RT group (the final collection approach) contained highly significantly more HMBS copies than those of the collection control group (P = 0.001), indicating a significant amount of blood feeding. Thick bars indicate the mean HMBS, and error bars indicate minimum/maximum copies per 10 fleas.