Development of a novel genus-specific real-time PCR assay for detection and differentiation of Bartonella species and genotypes

Abstract

The genus Bartonella includes numerous species with varied host associations, including several that infect humans. Development of a molecular diagnostic method capable of detecting the diverse repertoire of Bartonella species while maintaining genus specificity has been a challenge. We developed a novel real-time PCR assay targeting a 301-bp region of the ssrA gene of Bartonella and demonstrated specific amplification in over 30 Bartonella species, subspecies, and strains. Subsequent analysis of ssrA sequences was sufficient to discriminate Bartonella species and provided phylogenetic data consistent with that of gltA, a commonly used gene for differentiating Bartonella genotypes. Using this assay, we identified Bartonella DNA in 29% and 47% of blood specimens from elk in Wyoming and cattle in the Republic of Georgia, respectively. Sequence analysis of a subset of genotypes from elk specimens revealed a cluster most closely related to Bartonella capreoli, and genotypes from cattle were identified as Bartonella bovis, both Bartonella species commonly found in wild and domestic ruminants. Considering the widespread geographic distribution and infectivity potential to a variety of hosts, this assay may be an effective diagnostic method for identification of Bartonella infections in humans and have utility in Bartonella surveillance studies.

Fig 1

Phylogenetic relationships between ssrA sequences of all Bartonella species, subspecies, and isolates tested. GenBank accession numbers are shown for each genotype. Both ssrA genotypes obtained from ruminant blood were closely related to Bartonella species found in wild and domestic ruminants. The genotype identified from elk blood (JN982717) clustered closely with B. capreoli (96.2% similarity), and the single genotype identified in cattle blood was identical to B. bovis (99.7%). Only bootstrap replicates of >70% are noted.