Multispacer typing of Rickettsia isolates from humans and ticks in Tunisia revealing new genotypes

Abstract

Background: Rickettsioses are important remerging vector born infections. In Tunisia, many species have been described in humans and vectors. Genotyping is important for tracking pathogen movement between hosts and vectors. In this study, we characterized Rickettsia species detected in patients and vectors using multispacer typing (MST), proposed by Founier et al. and based on three intergenic spacers (dksA-xerC, rmpE- tRNA(fMet), mppA-pruC) sequencing.

Methods: Our study included 25 patients hospitalized during 2009. Ticks and fleas were collected in the vicinity of confirmed cases. Serology was performed on serum samples by microimmunofluorescence using Rickettsia conorii and Rickettsia typhi antigens. To detect and identify Rickettsia species, PCR targeting ompA, ompB and gltA genes followed by sequencing was performed on 18 obtained skin biopsies and on all collected vectors. Rickettsia positive samples were further characterized using primers targeting three intergenic spacers (dksA-xerC, rmpE- tRNA(fMet) and mppA-purC).

Results: A rickettsial infection was confirmed in 15 cases (60%). Serology was positive in 13 cases (52%). PCR detected Rickettsia DNA in four biopsies (16%) allowing the identification of R. conorii subsp israelensis in three cases and R. conorii subsp conorii in one case. Among 380 collected ticks, nine presented positive PCR (2.4%) allowing the identification of six R. conorii subsp israelensis, two R. massiliae and one R. conorii subsp conorii. Among 322 collected fleas, only one was positive for R. felis. R. conorii subsp israelensis strains detected in humans and vectors clustered together and showed a new MST genotype. Similarly, R. conorii subsp conorii strains detected in a skin biopsy and a tick were genetically related and presented a new MST genotype.

Conclusions: New Rickettsia spotted fever strain genotypes were found in Tunisia. Isolates detected in humans and vectors were genetically homogenous despite location differences in their original isolation suggesting epidemiologic circulation of these strains.

Figure 1

Phylogenetic tree of MST genotypes of Rickettsia conorii isolates from humans and ticks, in Tunisia. The tree was constructed using the Neighbor joining method based on 3 concatenated intergenic spacer sequences in 34 MST genotypes. Bootstrap values (100 replicates), shown in MEGA5.2 software, were used for phylogenetic analysis. Filled triangles indicate R. conorii strains detected in the skin biopsies of Tunisian patients in this study. Filled circles indicate R. conorii strains detected in ticks.