Differential detection of Bartonella species and strains in cat scratch disease diagnostics by polymerase chain reaction amplification of 16S ribosomal RNA gene

Cat scratch disease (CSD) has been difficult to diagnose in animals because of the protracted clinical course of infection and the quiescent phases wh en the microbial culprit lies dormant. The causative agent in CSD appears t o be multiple species and strains of Bartonella. Using polymerase chain rea ction (PCR) techniques for amplification of highly variable regions of the 16S ribosomal RNA (rRNA) gene sequence, a very sensitive species- and strai n-specific assay for CSD-causing Bartonella species was developed. PCR prim ers were designed to specifically amplify the 16S rRNA gene of Bartonella s pecies but not of other microbial pathogens. This initial PCR was multiplex ed with a universal primer set, based on conserved sequence regions in the 16S rRNA gene, that provides a 162-bp fragment in all species tested. Subse quently, 3 distinct nested PCR primer sets enabled the individual amplifica tion and specific detection of Bartonella henselae type I, B. henselae type II, and B. clarridgeae. Thus, this 2-step PCR procedure enabled the sensit ive detection and identification of these species and the B. henselae genot ype by exploiting minor sequences differences. Verification of these result s were demonstrated with both sequencing and ligase chain reaction techniqu es. The diagnostic usefulness of this CSD test has been demonstrated by the analysis of specimens from control and infected cats. The diagnosis was co nfirmed and the specific B. henselae strain was correctly identified in per ipheral blood specimens obtained from control and strain-specific CSD-infec ted cats. Such an accurate and sensitive diagnostic tool for the detection and identification of CSD causative agents should be a useful for the medic al, veterinary, and scientific communities.