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. 2009 Aug 7;191(20):6231–6241. doi: 10.1128/JB.00450-09

FIG. 5.

FIG. 5.

Spirochetes lacking GuaAB are attenuated for growth in fed ticks. I. scapularis larvae were artificially infected by immersion in liquid cultures of B. burgdorferi clone A3-M9 (wt), A3-M9 guaA::flgBp-kan/pBSV2G (GuaAB−), or A3-M9 guaA::flgBp-kan/pBSV2G guaAB (GuaAB+). (A) Total DNA was isolated from unfed larvae, and the spirochete density was assessed by qPCR. The symbols indicate the average numbers of spirochetes per larval tick calculated from the number of genome equivalents in the pooled DNA isolated from separate groups of 40 unfed larval ticks. (B and C) Seven to 21 days after the ticks were allowed to feed to repletion, the number of spirochetes per larval tick was determined by qPCR (B) and by plating dilutions of triturated whole fed larvae on solid BSK medium and determining the number of CFU (C). The symbols indicate the number of spirochetes per fed larval tick calculated from the number of genome equivalents in the pooled DNA isolated from separate groups of 20 larval ticks (B) or the number of colonies per individual fed larval tick (C). (D) Following molting, the spirochete densities in unfed nymphs were determined by qPCR. The symbols indicate the number of spirochetes per unfed nymph, calculated from the number of genome equivalents in the pooled DNA isolated from separate groups of four to seven unfed nymphs. (E and F) Seven to 21 days after the ticks were allowed to feed to repletion, the number of spirochetes per nymphal tick was determined by qPCR (E) and by plating dilutions of individual triturated whole fed nymphs on solid BSK medium and determining the number of CFU (F). The symbols indicate the number of spirochetes per fed nymphal tick calculated from the number of genome equivalents in the pooled DNA isolated from separate groups of one to three nymphal ticks (E) or the number of colonies per individual fed nymphal tick (F). The bars indicate the median values of the data sets. The data were analyzed using the Kruskal-Wallis nonparametric rank test followed by Dunn's multiple-comparison test.