Bartonellaspp. in Rats and Zoonoses, Los Angeles, California, USA

Bartonella spp.were detected in rats (Rattus norvegicus) trapped in downtown Los Angeles, California, USA.Of 200 rats tested, putative human pathogens, B. rochalimae and B. tribocorum were found in 37 (18.5%) and 115 (57.5%) rats, respectively.These bacteria among rodents in a densely populated urban area are a public health concern. B artonella spp. are vector-borne bacteria associated withan increasing array of emerging zoonotic infections in humans and animals (1).Some Bartonella spp.are widely distributed among small mammals in the United States and potentially cause human health concerns because these bacteria may be associated with human diseases (2).However, limited surveys have been conducted to identify infectious agents involved in zoonotic infections in rodents within urban areas of the United States (3).Norway rats (Rattus norvegicus) have been shown to harbor several Bartonella species, including B. tribocorum, B. elizabethae, B. rattimassiliensis, B. phoceensis, B. queenslandensis, and a strain closely related to B. rochalimae (3-6), of which B. elizabethae, B. rochalimae, and B. tribocorum were implicated in human diseases (7-11).Our study had 3 purposes.The fi rst purpose was to investigate prevalence of Bartonella spp.infections in rodent populations in downtown Los Angeles, California, USA.The second purpose was to evaluate genetic diversity of Bartonella spp. in blood of urban rats by analyzing variations of the citrate synthase (gltA) gene.The third purpose was to compare rates of detection of Bartonella spp.infections in rats between culture and molecular assays. The StudyRats were sampled during 2003-2007 at 16 sites in downtown Los Angeles in a rodent management/ disease surveillance program.The rats were captured by using Tomahawk live traps (Tomahawk Live Trap Co., Hazelhurst, WI, USA) and anesthetized with CO 2 .Blood samples were obtained by cardiac puncture, transferred to sterile cryovials, placed on dry ice, and preserved at -70°C until testing at the Bartonella Laboratory of the Centers for Disease Control and Prevention (Fort Collins, CO, USA).Genomic DNA was extracted from animal blood according to the blood protocol of the QIAamp DNA Mini Kit (QIAGEN, Valencia, CA, USA).Primers CS140f and CS443f (12) and CS781f and CS1137n (13), including newly designed primers CS120f-5′-TTTCACTTATGATCCTGGCTT-3′ and CS1210r-5′-GAT CYTCAATCATTTCTTTCCA-3′, which amplify DNA fragments ranging from 380 to 1,091 bp encompassing a 327-bp-specifi c zone (between bp positions 801 and 1127) of the gltA gene ( 14), were used in different combinations.Eight PCRs were performed for each sample.PCR products were purifi ed by using the QIAquick PCR Purifi cation Kit (QIAGEN, Germantown, MD, USA).The same primers were used for DNA sequencing.The obtained DNA sequences were trimmed to 327 bp between positions 801 and 1127 because most Bartonella spp.gltA sequences available in GenBank are limited to this size.Details of procedures used for isolation of Bartonella spp.from mammalian blood have been described (2).Rat blood was diluted 1:4 in brain-heart infusion medium containing 5% fungizone and pipetted onto heart infusion agar plates containing 10% rabbit blood.Plates were incubated aerobically at 35°C in an atmosphere of 5% CO 2 for <4 weeks and monitored for bacterial growth at least 1× per week after initial plating.Colonies were subpassaged onto fresh agar plates until a pure culture, free from contamination, was obtained.Analysis of DNA sequences and phylogenetic relationships was performed by using MEGA4 (www. megasoftware.net/)and the neighbor-joining method with the Kimura 2-parameter distance model.Stability of inferred phylogeny was assessed by using bootstrap analysis of 1,000 randomly generated trees.DNA sequences obtained in this study were deposited in GenBank under accession nos.JF429450-JF429625.A total of 200 R. norvegicus rats were trapped in 16 sites in downtown Los Angeles.Bartonella DNA was detected in 135 (67.5%) of 200 rat blood samples.PCR-positive blood samples were subsequently cultured for viable Bartonella organisms.Fifty-nine (43.7%) of 135 blood samples were confi rmed as bacteremic for bartonellae by culturing.A total of 176 sequences were obtained either directly from blood samples (117 sequences) or from pure cultures