Molecular Typing of Isolates ofRickettsia rickettsiiby 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.

Phylogeography of Rickettsia rickettsii Genotypes Associated with Fatal Rocky Mountain Spotted Fever

“…26 Samples were tested for segments of the R. rickettsii genome by using multiple PCR assays (Table 1), including a broad-range, genusspecific real-time assay for the citrate synthase (gltA) gene, 27 a real-time assay targeting a R. rickettsii-specific hypothetical protein, 28 and nested PCR assays targeting the RR0155-rpmB, cspA-ksgA, and RR1240-tlc5 intergenic regions of R. rickettsii. 19,29 The locus spo0J-abcT1 was additionally assessed for a case-patient infected with an Hlp genotype by using a nested PCR assay. 19,29 Gel-purified PCR products were sequenced using a PRISM BigDye Terminator Cycle 3.1 sequencing kit (Applied Biosystems, Foster City, CA) and an ABI 3130xl genetic analyzer (Applied Biosystems), or a Dye Terminator Cycle Sequencing Quick Start Kit (Beckman Coulter, Indianapolis, IN) and a GenomeLab GeXP Genetic Analysis System (Beckman).…”

“…19,29 The locus spo0J-abcT1 was additionally assessed for a case-patient infected with an Hlp genotype by using a nested PCR assay. 19,29 Gel-purified PCR products were sequenced using a PRISM BigDye Terminator Cycle 3.1 sequencing kit (Applied Biosystems, Foster City, CA) and an ABI 3130xl genetic analyzer (Applied Biosystems), or a Dye Terminator Cycle Sequencing Quick Start Kit (Beckman Coulter, Indianapolis, IN) and a GenomeLab GeXP Genetic Analysis System (Beckman). DNA sequence analyses.…”

“…364D) as a distinct species. 19 Rocky Mountain spotted fever is a relatively rare and sporadic disease and R. rickettsii is infrequently isolated from clinical samples. In this context, existing genetic assessments of strains infecting humans have been limited predominantly to historical isolates passaged repeatedly in cell culture, including some strains of uncertain provenance or unknown clinical circumstances.…”

“…At least three molecular typing strategies have been developed that provide strain separation using nucleotide polymorphisms. [17][18][19][20][21] One of these systems, based on an analysis of six intergenic regions, divides the extant strains of R. rickettsii into four genetically discrete clades, and separates a previously identified strain (Rickettsia sp. 364D) as a distinct species.…”

Experimental infection of dogs with a Brazilian strain of Rickettsia rickettsii: clinical and laboratory findings

“…However, nothing is known about the pathogenicity of South American strains of R. rickettsii for dogs. Since ecological differences and genetic polymorphisms between strains of R. rickettsii from the United States and South America have been reported (Eremeeva et al 2003, Karpathy et al 2007, it is not known how pathogenic South American strains of R. rickettsii are for dogs. For this purpose, the present study evaluated experimental infection of dogs with a Brazilian strain of R. rickettsii.…”