Molecular identity of cat fleas (Ctenocephalides felis) from cats in Georgia, USA carrying Bartonella clarridgeiae, Bartonella henselae and Rickettsia sp. RF2125

• Molecular barcoding places the cat fleas from Georgia, US in a clade with African cat fleas
• Fleas are confirmed to carry Rickettsia and Bartonella, specifically B. clarridgeiae, B. henselae and Rickettsia sp. RF2125 corrected
• Individual, published Rickettsia and Bartonella assays are combined in a multiplex TaqMan real-time PCR assay

The cat flea (Ctenocephalides felis) is the most common ectoparasite of dogs and cats. Close association of humans with cats and dogs enables flea-borne disease transmission either directly, via flea bites, or indirectly, via pathogens excreted in flea faeces. The aim of this study was to evaluate molecular identity of C. felis from cats in Georgia, USA based on a molecular barcode marker gene (cox1) and the frequency at which the fleas were carriers of the vector-borne bacteria, Bartonella and Rickettsia. The multiplexed Bartonella and Rickettsia real-time PCR assay (targeting ssrA and gltA, respectively) adopted in this study, together with sequencing of the ssrA enabled rapid identification of Bartonella spp. in cat fleas. Eighteen out of 20 fleas examined were positive for Bartonella spp. and all fleas were positive for Rickettsia spp. DNA sequencing of the ssrA confirmed presence B. clarridgeiae and B. henselae. Amplification and DNA sequencing of gltA and ompA confirmed presence of Rickettsia sp. RF2125 (Rickettsia felis-like organism). All fleas from 21 cats in Georgia, USA were morphologically identical with C. felis. Sequencing of the flea cox1 revealed identity with C. felis from Seychelles, Africa recognised as a subspecies C. felis strongylus, the African cat flea. Analysis of the cox1 sequences of fleas improves understanding of global distribution of cat flea cox1 clades (C. felis) when compared with sequences from Ctenocephalides spp. from Asia, Africa, Europe, Asia and Australia. Coupling flea barcoding approach with the multiplexed real-time PCR assay followed by Bartonella sequencing represents a rational approach for screening and elucidation of fleas' capacity to vector infectious agents.