Molecular Detection ofBartonellaspp. in China and St. Kitts

Abstract

Bartonellaare vector-borne hemotropic bacteria that infect a wide variety of hosts, including people. While there are PCR assays that can identify individual or groups ofBartonella, there is no reliable molecular method to simultaneously detect all species while maintaining genus specificity and sensitivity. By comparing highly conserved16S rRNAsequences of the better-recognizedBartonellaspp. on GenBank, we selected primers and probes for a genus-specific pan-BartonellaFRET-qPCR. Then, agltA-basedBartonellaPCR was established by selecting primers for a highly variable region ofgltA, of which the sequenced amplicons could identify individualBartonellaspp. The pan-BartonellaFRET-qPCR did not detect negative controls (Brucellaspp.,Anaplasmaspp.,Rickettsiaspp.,Coxiella burnetii, andWolbachia) but reliably detected as few as two copies of the positive control (Bartonella henselae) per reaction. There was complete agreement between the pan-BartonellaFRET-qPCR and thegltA-basedBartonellaPCR in detectingBartonellain convenience test samples from China and St. Kitts: cats (26%; 81/310),Ctenocephalides felis(20%; 12/60), cattle (24%; 23/98), and donkeys (4%; 1/20). Sequencing of thegltA-basedBartonellaPCR products revealedB. henselae(70%; 57/81) andB. clarridgeiae(30%; 24/81) in cats andC. felis(67%; 8/12, and 33%; 4/12, respectively) andB. bovisin cattle (23.5%; 23/98) and donkeys (4.0%; 1/24). The pan-BartonellaFRET-qPCR andgltA-basedBartonellaPCR we developed are highly sensitive and specific in detecting recognizedBartonellaspp. in a single reaction. The pan-BartonellaFRET-qPCR is convenient requiring no gel electrophoresis and providing copy numbers, while thegltA-basedBartonellaPCR reliably differentiates individualBartonellaspecies. The use of these PCRs should greatly facilitate large-scale surveillance studies and the diagnosis of infections in clinical samples.