Absence of zoonotic Bartonella species in questing ticks: first detection of Bartonella clarridgeiae and Rickettsia felis in cat fleas in the Netherlands.

BACKGROUND: Awareness for flea- and tick-borne infections has grown in recent years and the range of microorganisms associated with these ectoparasites is rising. Bartonella henselae, the causative agent of Cat Scratch Disease, and other Bartonella species have been reported in fleas and ticks. The role of Ixodes ricinus ticks in the natural cycle of Bartonella spp. and the transmission of these bacteria to humans is unclear. Rickettsia spp. have also been reported from as well ticks as also from fleas. However, to date no flea-borne Rickettsia spp. were reported from the Netherlands. Here, the presence of Bartonellaceae and Rickettsiae in ectoparasites was investigated using molecular detection and identification on part of the gltA- and 16S rRNA-genes. RESULTS: The zoonotic Bartonella clarridgeiae and Rickettsia felis were detected for the first time in Dutch cat fleas. B. henselae was found in cat fleas and B. schoenbuchensis in ticks and keds feeding on deer. Two Bartonella species, previously identified in rodents, were found in wild mice and their fleas. However, none of these microorganisms were found in 1719 questing Ixodes ricinus ticks. Notably, the gltA gene amplified from DNA lysates of approximately 10% of the questing nymph and adult ticks was similar to that of an uncultured Bartonella-related species found in other hard tick species. The gltA gene of this Bartonella-related species was also detected in questing larvae for which a 16S rRNA gene PCR also tested positive for "Candidatus Midichloria mitochondrii". The gltA-gene of the Bartonella-related species found in I. ricinus may therefore be from this endosymbiont. CONCLUSIONS: We conclude that the risk of acquiring Cat Scratch Disease or a related bartonellosis from questing ticks in the Netherlands is negligible. On the other hand fleas and deer keds are probable vectors for associated Bartonella species between animals and might also transmit Bartonella spp. to humans.

Proposals to unify the genera Grahamella and Bartonella, with descriptions of Bartonella talpae comb. nov., Bartonella peromysci comb. nov., and three new species, Bartonella grahamii sp. nov., Bartonella taylorii sp. nov., and Bartonella doshiae sp. nov.

Polyphasic methods were used to examine the taxonomic positions of three newly identified Grahamella species. A comparison of the 16S rRNA gene sequences of these organisms with the sequences available for other bacteria revealed that these three species form a tight monophyletic cluster with members of the genus Bartonella. This cluster is only remotely related to other members of the order Rickettsiales. Determinations of the levels of DNA relatedness between Grahamella species and Bartonella species (by using a modified hydroxyapatite method) revealed that all of the species belonging to these two genera are distinct but closely related. On the basis of these data and the results of guanine-plus-cytosine content and phenotypic characterization studies, we propose that the genera Grahamella and Bartonella should be unified and that the latter name should be retained. Bartonella talpae and Bartonella peromysci, new combinations for former Grahamella species, are created, and the following three new Bartonella species are described: Bartonella grahamii, Bartonella taylorii, and Bartonella doshiae. A taxonomic analysis of Grahamella species complete the study of all members of the family Bartonellaceae, and the results of this study support the proposal that the family should be transferred out of the order Rickettsiales.

Proposals to unify the genera Bartonella and Rochalimaea, with descriptions of Bartonella quintana comb. nov., Bartonella vinsonii comb. nov., Bartonella henselae comb. nov., and Bartonella elizabethae comb. nov., and to remove the family Bartonellaceae from the order Rickettsiales.

DNA hybridization data (hydroxyapatite method, 50 to 70 degrees C) indicate that Rickettsia prowazekii, the type species of the type genus of the family Rickettsiaceae, is substantially less closely related to Rochalimaea species than was previously thought. The levels of relatedness of Rickettsia prowazekii to Rochalimaea species and to Bartonella bacilliformis under optimal conditions for DNA reassociation were 0 to 14%, with 25.5% or greater divergence in related sequences. When stringent reassociation criteria were used, the levels of relatedness were 0 to 2%. The genera Bartonella and Rochalimaea are currently classified in different families (the Bartonellaceae and the Rickettsiaceae) in the order Rickettsiales. On the basis of DNA relatedness data, previous 16S rRNA sequence data, guanine-plus-cytosine contents, and phenotypic characteristics, neither Bartonella bacilliformis nor Rochalimaea species are closely related to other organisms currently classified in the order Rickettsiales. In fact, the closest relative of these organisms is Brucella abortus. It is therefore proposed that the family Bartonellaceae should be removed from the order Rickettsiales. Previous 16S rRNA sequence data and DNA hybridization data revealed high levels of relatedness between Bartonella bacilliformis and the four Rochalimaea species, indicating that these species are members of a single genus. It is proposed that the genus Rochalimaea should be united with the genus Bartonella in the family Bartonellaceae. The name Bartonella is retained as the genus name since it has nomenclatural priority over the name Rochalimaea. This means that new combinations for the Rochalimaea species must be created. Proposals are therefore made for the creation of Bartonella quintana comb. nov., Bartonella vinsonii comb. nov., Bartonella henselae comb. nov., and Bartonella elizabethae comb. nov.