Isolation of Candidatus Rickettsia asemboensis from Ctenocephalides Fleas

Candidatus Rickettsia asemboensis was identified molecularly in fleas collected in 2009 from Asembo, Kenya. Multilocus sequence typing using the 17-kD antigen gene, rrs, gltA, ompA, ompB, and sca4 demonstrated that Candidatus R. asemboensis is closely related to Rickettsia felis but distinct enough to be considered for separate species classification. Following this molecular characterization of Candidatus R. asemboensis, the in vitro cultivation of this bacterium was then performed. We used Ctenocephalides canis and Ctenocephalides felis fleas removed from dogs in Kenya to initiate the in vitro isolation of Candidatus R. asemboensis. Successful cultures were obtained using Drosophila melanogaster S2 and Aedes albopictus C6/36 cell lines. Cytological staining and quantitative real-time PCR (qPCR) assays were used to visualize/confirm the culture of the bacteria in both cell lines. Sequencing of fragments of the 17-kD antigen gene, gltA, and ompB genes confirmed the identity of our Candidatus R. asemboensis isolates. To date, we have passaged Candidatus R. asemboensis 12 times through S2 and C6/36 cells, and active and frozen cultures are currently being maintained. This is the first time that a R. felis-like organism has been grown and maintained in culture and is therefore the first time that one of them, Candidatus R. asemboensis, has been characterized beyond molecular typing.