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 pre-enrichment liquid medium, Bartonella/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.