Culture Isolate of Rickettsia felis from a Tick

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Abstract

Although the cat flea, Ctenocephalides felis, has been identified as the primary vector of Rickettsia felis, additional flea, tick, mite, and louse species have also been associated with this bacterium by molecular means; however, the role of these arthropods in the transmission of R. felis has not been clarified. Here, we succeeded in culture isolation of R. felis from a host-seeking castor bean tick, Ixodes ricinus, the most common tick in Slovakia. The bacterial isolation was performed on XTC-2 cells at 28 °C using the shell-vial technique. An evaluation of the growth properties was performed for both the XTC-2 and Vero cell lines. We observed R. felis in the infected host cells microscopically by Gimenez staining and immunofluorescence assay. The R. felis isolate was purified by gradient ultracentrifugation and visualized by electron microscopy. Fragments of the genes gltA, ompA, ompB, htrA, rpoB, sca4, rffE, and rrs were amplified and compared with the corresponding sequences of the type strain URRWXCal2 and other R. felis culture -isolated strains. We did not detect any nucleotide polymorphisms; however, plasmid pRFδ, characteristic of the standard strain, was absent in our isolate. Herein, we describe the first successful isolation and characterization of a tick-derived R. felis strain “Danube”, obtained from an I. ricinus nymph.

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Phylogenetic analysis of members of the genus Rickettsia using the gene encoding the outer-membrane protein rOmpB (ompB).

Ana V. Diez Roux, D Raoult (2000)

To confirm the phylogenetic analysis previously inferred by comparison of the citrate synthase and rOmpA gene sequences (gitA and ompA, respectively), the rOmpB gene (ompB) of 24 strains of the genus Rickettsia was amplified and sequenced. rOmpB is an outer-membrane protein of high molecular mass, the presence of which can be demonstrated in most rickettsiae by immunological cross-reactivity in Western blots. No PCR amplification was obtained with Rickettsia bellii or Rickettsia canadensis. For the other rickettsiae, phylogenetic analysis was inferred from the comparison of both the gene and derived protein sequences by using parsimony, maximum-likelihood and neighbour-joining methods which gave the same organization. All nodes were well supported (>86% bootstrap values), except in the cluster including Rickettsia africae strain S and Rickettsia parkeri, and this analysis confirmed the previously established phylogeny obtained from combining results from gltA and ompA. Based on phylogenetic data, the current classification of the genus Rickettsia is inappropriate, specifically its division into two groups, typhus and spotted fever. Integration of phenotypic, genotypic and phylogenetic data will contribute to the definition of a polyphasic taxonomy as has been done for other bacterial genera.

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Citrate synthase gene comparison, a new tool for phylogenetic analysis, and its application for the rickettsiae.

Ana V. Diez Roux, E B Rydkina, M Eremeeva … (1997)

Using PCR and an automated laser fluorescent DNA sequencer, we amplified and sequenced a 1,234-bp fragment of the citrate synthase-encoding gene (gltA) of 28 bacteria belonging to the genus Rickettsia. Comparative sequence analysis showed that most of the spotted fever group (SFG) rickettsiae belonged to one of two subgroups. The first subgroup included Rickettsia massiliae, strain Bar 29, Rickettsia rhipicephali, "Rickettsia aeschlimanni," and Rickettsia montana, which have been isolated only from ticks. The second subgroup was larger and included the majority of the human pathogens and also rickettsiae isolated only from ticks; the members of this subgroup were strain S, Rickettsia africae, "Rickettsia monglotimonae," Rickettsia sibirica, Rickettsia parkeri, Rickettsia conorii, Rickettsia rickettsii, the Thai tick typhus rickettsia, the Israeli tick typhus rickettsia, the Astrakhan fever rickettsia, "Rickettsia slovaca," and Rickettsia japonica. The sequence analysis also showed that the tick-borne organisms Rickettsia helvetica and Rickettsia australis and the mite-borne organism Rickettsia akari were associated with the SFG cluster, that Rickettsia prowazekii and Rickettsia typhi, two representatives of the typhus group, clustered together, and that Rickettsia canada; Rickettsia bellii, and the AB bacterium probably represent three new groups. We compared the phylogenetic trees inferred from citrate synthase gene sequences and from 16S ribosomal DNA (rDNA) sequences. For rickettsial phylogeny, the citrate synthase approach was more suitable, as demonstrated by significant bootstrap values for all of the nodes except those in the larger subgroup defined above. We also compared phylogenetic analysis results obtained in a comparison of the sequences of both genes for all of the representatives of the domain Bacteria for which the gltA sequence was determined. We believe that comparison of gltA sequences could be a complementary approach to 16S rDNA sequencing for inferring bacterial evolution, especially when unstable phylogenetic models are obtained from ribosomal sequences because of high levels of sequence similarity between the bacteria studied.