Molecular typing of isolates of Rickettsia rickettsii by use of DNA sequencing of variable intergenic regions

Abstract

Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is found throughout the Americas, where it is associated with different animal reservoirs and tick vectors. No molecular typing system currently exists to allow for the robust differentiation of isolates of R. rickettsii. Analysis of eight completed genome sequences of rickettsial species revealed a high degree of sequence conservation within the coding regions of chromosomes in the genus. Intergenic regions between coding sequences should be under less selective pressure to maintain this conservation and thus should exhibit greater nucleotide polymorphisms. Utilizing these polymorphisms, we developed a molecular typing system that allows for the genetic differentiation of isolates of R. rickettsii. This typing system was applied to a collection of 38 different isolates collected from humans, animals, and tick vectors from different geographic locations. Serotypes 364D, from Dermacentor occidentalis ticks, and Hlp, from Haemaphysalis leporispalustris ticks, appear to be distinct genotypes that may not belong to the species R. rickettsii. We were also able to differentiate 36 historical isolates of R. rickettsii into three different phylogenetic clades containing seven different genotypes. This differentiation correlated well, but not perfectly, with the geographic origin and likely tick vectors associated with the isolates. The few apparent typing discrepancies found suggest that the molecular ecology of R. rickettsii needs more investigation.

FIG. 1.

Genotypes of sequenced IGRs. Each table shows individual genotypes identified for each IGR. The numbers in parentheses indicate the number of isolates in each genotype. The total number of nucleotides in the consensus sequence of each IGR is shown under the table, and the nucleotide positions indicated are relative to the aligned consensus sequence. Deleted nucleotides are indicated with dashes.

FIG. 2.

Phylogenetic relationships of R. rickettsii isolates. Maximum parsimony phylogenetic relationships of R. rickettsii isolates are based on the concatenated sequences of all six intergenic regions. Numbers at the nodes are bootstrap values based on 1,000 bootstrap replicates. Only bootstrap values of >50 are shown. R. conorii Malish 7 represents the outgroup. The scale bar corresponds to the number of steps.