title: 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.
abstract: 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.
authors.affiliation: Intracellular Pathogens Research Laboratory, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606, USA.
authors.forename: Ashlee W
authors.initials: AW
authors.lastname: Duncan
authors.forename: Ricardo G
authors.initials: RG
authors.lastname: Maggi
authors.forename: Edward B
authors.initials: EB
authors.lastname: Breitschwerdt
mesh_terms: D000362:Agar; D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D001474:Bartonella Infections / Q000097:blood / Q000382:microbiology / Q000662:veterinary*; D018416:Bartonella henselae / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D018929:Cell Culture Techniques / Q000379:methods / Q000662:veterinary; D003470:Culture Media; D004269:DNA, Bacterial / Q000097:blood / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000737:chemistry / Q000235:genetics; D004283:Dog Diseases / Q000097:blood* / Q000175:diagnosis / Q000382:microbiology*; D004285:Dogs; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect / Q000662:veterinary; D007074:Immunoglobulin G / Q000097:blood; D008297:Male; D016133:Polymerase Chain Reaction / Q000662:veterinary; D011446:Prospective Studies; D017422:Sequence Analysis, DNA
chemical_list: D000907:Antibodies, Bacterial; D003470:Culture Media; D004269:DNA, Bacterial; D021903:DNA, Ribosomal Spacer; D007074:Immunoglobulin G; D000362:Agar