Bartonella spp. 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.


The Study
Rats 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).
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.

Conclusions
In this study, 3 novel Bartonella genogroups identifi ed in 4 R. norvegicus rats were not genetically related to any known Bartonella spp. and might represent novel Bartonella spp. Genotypes 18 (JF429586) and 19 (JF429587) formed a Bartonella genogroup with 95% similarity to B. tribocorum as the closest species. Likewise, genotypes 20 (JF429584) and 21 (JF429585) have 91.4% and 92.6% sequence identities, respectively, with B. clarridgeiae, which is their closest related species (online Appendix Figure).
Our study reports detection and identifi cation of Bartonella spp. in urban rats in downtown Los Angeles, California. We demonstrated that R. norvegicus in downtown Los Angeles can serve as reservoirs of several Bartonella spp., such as B. rochalimae, B. tribocorum, B. queenslandensis, and possibly 3 additional novel species. Some genotypes identifi ed in rats showed a high level of similarity (<98.8%) with a B. tribocorum isolate obtained from a febrile patient in Thailand (GenBank accession no. GQ225706) (11). Another species (B. rochalimae) was isolated from a patient with splenomegaly who had traveled to South America (14). This bacterium has also been isolated from dogs, foxes, rats, shrews, gerbils, and raccoons, suggesting that multiple reservoirs may be involved in maintenance of this species. One Bartonella genotype found in 4 rats in this study was 99.0% similar to B. queenslandensis (GenBank accession no. EU111800), which was originally isolated from R. fuscipes rats in Australia (15).
Because most identifi ed Bartonella species have been reported as human infectious agents elsewhere, the fi nding that they were circulating among rodents in a densely populated urban area is of serious public health concern. In this context, further studies should be conducted on a larger collection of rodents and clinical human samples to determine evolutionary, genetic, and pathogenic relationships.