Molecular Detection of<i>Bartonella</i>spp. in China and St. Kitts

Abstract

<i>Bartonella</i> are vector-borne hemotropic bacteria that infect a wide variety of hosts, including people. While there are PCR assays that can identify individual or groups of <i>Bartonella</i>, there is no reliable molecular method to simultaneously detect all species while maintaining genus specificity and sensitivity. By comparing highly conserved <i>16S rRNA</i> sequences of the better-recognized <i>Bartonella</i> spp. on GenBank, we selected primers and probes for a genus-specific pan-<i>Bartonella</i> FRET-qPCR. Then, a <i>gltA</i>-based <i>Bartonella</i> PCR was established by selecting primers for a highly variable region of <i>gltA</i>, of which the sequenced amplicons could identify individual <i>Bartonella</i> spp. The pan-<i>Bartonella</i> FRET-qPCR did not detect negative controls (<i>Brucella</i> spp., <i>Anaplasma</i> spp., <i>Rickettsia</i> spp., <i>Coxiella burnetii</i>, and <i>Wolbachia</i>) but reliably detected as few as two copies of the positive control (<i>Bartonella henselae</i>) per reaction. There was complete agreement between the pan-<i>Bartonella</i> FRET-qPCR and the <i>gltA</i>-based <i>Bartonella</i> PCR in detecting <i>Bartonella</i> in convenience test samples from China and St. Kitts: cats (26%; 81/310), <i>Ctenocephalides felis</i> (20%; 12/60), cattle (24%; 23/98), and donkeys (4%; 1/20). Sequencing of the <i>gltA</i>-based <i>Bartonella</i> PCR products revealed <i>B. henselae</i> (70%; 57/81) and <i>B. clarridgeiae</i> (30%; 24/81) in cats and <i>C. felis</i> (67%; 8/12, and 33%; 4/12, respectively) and <i>B. bovis</i> in cattle (23.5%; 23/98) and donkeys (4.0%; 1/24). The pan-<i>Bartonella</i> FRET-qPCR and <i>gltA</i>-based <i>Bartonella</i> PCR we developed are highly sensitive and specific in detecting recognized <i>Bartonella</i> spp. in a single reaction. The pan-<i>Bartonella</i> FRET-qPCR is convenient requiring no gel electrophoresis and providing copy numbers, while the <i>gltA</i>-based <i>Bartonella</i> PCR reliably differentiates individual <i>Bartonella</i> species. The use of these PCRs should greatly facilitate large-scale surveillance studies and the diagnosis of infections in clinical samples.