A combined approach for the enhanced detection and isolation of Bartonella species in dog blood samples:: Pre-enrichment liquid culture followed by PCR and subculture onto agar plates

Historically, direct plating, lysis centrifugation, or freeze-thaw approaches have proven to be highly insensitive methods for confirming Bartonella species infection in dogs. A prospective study was designed to compare diagnostic methods for the detection of Bartonella using samples submitted to the Vector-Borne Disease Diagnostic Laboratory at North Carolina State University. Methods included indirect immunofluorescence assay, PCR, direct inoculation of a blood agar plate (trypticase soy agar with 5% rabbit blood), and inoculation into a novel preenrichment liquid medium, Bartonello/alpha-Proteobacteria growth medium (BAPGM). Sequential research efforts resulted in the development of a combinational approach consisting of pre-enrichment culture of Bartonella species in BAPGM, sub-inoculation of the liquid culture onto agar plates, followed by DNA amplification using PCR. The multi-faceted approach resulted in substantial improvement in the microbiological detection and isolation of Bartonella when compared to direct inoculation of a blood agar plate, Importantly, this approach facilitated the detection and subsequent isolation of both single and co-infections with two Bartonella species in the blood of naturally infected dogs. The use of a combinational approach of pre-enrichment Culture and PCR may assist in the diagnostic confirmation of bartonellosis in dogs and other animals. (C) 2007 Elsevier B.V. All rights reserved.