Identification of Bartonella henselae and B. quintana 16S rDNA sequences by branch-, genus- and species-specific amplification

Abstract

Given the controversy surrounding the aetiology of cat scratch disease and the association of both Bartonella henselae and B. quintana with bacillary angiomatosis, a method for the direct detection in clinical samples of 16S rRNA from the Proteobacteria alpha subgroup was developed. The primary structure of amplified 16S rDNA was determined by cloning and sequencing. Three sequences were identified: one corresponded exactly to GenBank accession number M73229 (B. henselae); the second was related to, but distinct from, GenBank accession number Z11684 (referred to as 'B. henselae variant'); and a third sequence was identical with GenBank accession number M73228 (B. quintana). No sequence corresponding to Afipia spp. was found. To speed identification and reduce the cost of analysis, a nested amplification method for B. henselae and B. quintana was devised. These techniques were applied to DNA extracted from 30 unfixed lymph node biopsies, two liver biopsies and 36 node pus samples from patients with suspected cat scratch disease, and from 17 skin biopsies from AIDS patients with suspected bacillary angiomatosis. B. henselae or B. henselae variant sequences were found in 42 (62%) of 68 samples from suspected cat scratch disease. B. quintana was not associated with cat scratch disease, but a B. quintana sequence was found in seven (41%) of 17 samples from suspected bacillary angiomatosis patients. B. henselae 16S rDNA sequences were not found in bacillary angiomatosis specimens.