Identification of Rickettsial Infections (Rickettsia sp. TH2014) in Ctenocephalides orientis Fleas (Siphonaptera: Pulicidae)

Rickettsia felis (Rickettsiales: Rickettsiaceae) is an emergent human pathogen that causes febrile illnesses in various parts of the world. This study describes the identification and growth characteristics of a R. felis-like organism (designated as Rickettsia sp. TH2014) cultured from Ctenocephalides orientis fleas in rural Malaysia. In this study, culturing of rickettsiae from filtered triturated flea lysates was performed in Aedes albopictus C6/36 cells. Cytopathic effects were observed from one of the samples 4 d post-inoculation. Electron microscopy revealed actively replicating intracytosolic coccobacillary organisms in the rickettsia-infected cells. Sequence analysis of amplified citrate synthase (gltA) gene fragment shows complete match of the rickettsia with Rickettsia sp. Rf31 in Southeast Asia, and 'Candidatus Rickettsia senegalensis' strain PU01-02 in Africa. The whole-genome sequence of Rickettsia sp. TH2014 was determined and assembled. The estimated genome size and guanine + cytosine content of the rickettsia are 1.37 Mb and 32.9%, respectively. The high values of average nucleotide identity and tetra-nucleotide signature correlation index obtained from pairwise genome comparison study suggest the identification of the rickettsia as R. felis. The whole-genome single-nucleotide polymorphism analysis demonstrates close genetic relatedness of the rickettsia with R. felis and Rickettsia asemboensis. However, based on sequence analyses of rickettsial genes (16S rDNA, gltA, ompB, and sca4), Rickettsia sp. TH2014 is found to be distinct from R. felis and R. asemboensis. The sequence analyses reveal that Rickettsia sp. TH2014 is highly similar to 'Ca. Rickettsia senegalensis' detected in fleas from Africa, Asia, and North America. Further investigation to provide insights on pathogenic potential and transmission dynamics of the rickettsia is warranted.