Molecular detection of Rickettsia felis in common fleas in Greece and comparative evaluation of genotypic methods

Introduction: Rickettsia felis is the causative agent of flea-borne spotted fever (FBSF), an emerging zoonosis. Although there is evidence of FBSF in Greece, fleas, the classic vectors of R. felis, have not been adequately studied. Thus, the aim of this study was to detect and characterize bacteria of genus Rickettsia and especially R. felis from common fleas parasitizing domestic cats and dogs in Greece and evaluate the efficiency of established molecular techniques. Materials and methods: DNA of flea-pools (samples) by animal-host was investigated by quantitative real-time PCRs (qPCR), and 16S metagenomics (16S). Determination of Rickettsia spp., Rickettsia felis-like organisms (RFLOs), and R. felis was based on a combination of qPCRs targeting gltA and ompB genes, 16S automated metagenomics and manual comparison of 16S sequences for >99% similarity with the publicly available 16S R. felis GenBank sequences using the Basic Local Alignment Search Tool (BLAST >99). Information for the animal-hosts was available and statistically analyzed. Results: Among 100 flea-pools, R. felis was detected in 14 samples with a combination of six, five and three assays in 10, two and two samples, respectively. The sensitivity of the assays for Rickettsia genus (16S, and genus specific qPCRs) ranged from 62.5% to 93.8% and the specificity from 65.0% to 100%. R. felis-targeting qPCRs for gltA and ompB demonstrated sensitivity and specificity of 92.9% and 100%, and 100.0% and 87.5%, respectively. 16S metagenomics using the assay software was not able to identify R. felis positive specimens, although manual BLAST >99 did identify the species, but demonstrated sensitivity of 92.9% and specificity of 65.0%. No association of the detection rate of Rickettsia genus or R. felis, with the epidemiological data collected, was identified. Conclusions: These observations suggest the occurrence of R. felis in fleas from pets in Attica, Greece, but PCR and sequencing assays varied considerably in sensitivity and specificity and a consensus methodology for assigning the positivity status is required to be established.