Antigen culture and IFAT was performed at the Australian Rickettsial Reference Laboratory, Geelong, Australia. The L929 cell line was selected to establish culture of the rickettsial organisms tested in this study. Once a confluent monolayer was achieved, live R. felis and R. typhi cultures were revived from -80 °C and used to infect separate flasks. Leibovitz-15 media (GIBCO, Rockville, MD, USA) supplemented with 10% foetal calf serum, 2 mM L-glutamine and 5% tryptose phosphate broth was used to maintain R. felis. RPMI media (GIBCO), supplemented with 10% foetal calf serum and 2 mM L-glutamine was used to maintain R. typhi. Infection levels were monitored using a semi-quantitative qPCR, with species confirmation verified using PCR and DNA sequencing (Australian Genomic Research Facility Ltd., Australia); both molecular techniques were based on the citrate synthase (gltA) gene [23 (link)].
Infected cell monolayers were harvested by physical detachment and heat inactivated at 56 °C for 30 min. Differential centrifugation at 3000× g for 10 min at room temperature was used to separate the host cell material from the rickettsiae; this pelleted Rickettsia was then resuspended in PBS.
Infected cell monolayers were harvested by physical detachment and heat inactivated at 56 °C for 30 min. Differential centrifugation at 3000× g for 10 min at room temperature was used to separate the host cell material from the rickettsiae; this pelleted Rickettsia was then resuspended in PBS.