Rickettsial Isolation from Booklice

Ixodes scapularis-derived ISE6 cells, provided by T. Kurtti (University of Minnesota), were maintained in antibiotic-free L15B growth medium supplemented with 10% heat-inactivated fetal bovine serum (HyClone), 10% tryptose phosphate broth (Becton, Dickinson and Company) [40] (link), at pH 6.8–7.0 in a humidified 5% CO₂ incubator at 32°C. For rickettsial isolation, two pools of 25 L. bostrychophila were sequentially washed over filter paper as follows: three times in 70% ethanol, one time in 10% sodium hypochlorite, and rinsed in sterile distilled water. Booklice pools were either transferred by pipette tip to a sterile glass pestle tissue grinder and ground in 25 µl of L15B tick cell culture media or directly transferred to a 25-cm² tissue culture flask containing ISE6 cells (passage 134). ISE6 cells with booklice preparations were immediately placed at 32°C. For regular maintenance of Rickettsia-infected cells, they were passed at a ratio of 1∶5 every 21 to 28 days. Upon each passage of ISE6 cells exposed to booklice homogenates, a portion of the cells were prepared for staining using a Cytospin centrifuge (Wescor) and rickettsial infection was assessed by traditional PCR [15] (link) and/or Diff-Quik (Dade Behring) staining, according to the manufacturer's protocol.