Bartonella vinsonii subsp. arupensis in Humans, Thailand

We identified Bartonella vinsonii subsp. arupensis in pre-enriched blood of 4 patients from Thailand. Nucleotide sequences for transfer-messenger RNA gene, citrate synthase gene, and the 16S–23S rRNA internal transcribed spacer were identical or closely related to those for the strain that has been considered pathogenic since initially isolated from a human in Wyoming, USA.

M ore than 30 species of bartonellae that are highly prevalent in a wide variety of vertebrates have been described. Bartonella bacilliformis, B. henselae, and B. quintana are well-known human pathogens, and several other Bartonella species, including B. elizabethae, B. tamiae, B. vinsonii subsp. arupensis, have been associated with various clinical manifestations in humans (1)(2)(3). A link between some of these pathogenic strains and their animal hosts has been documented; for example, B. elizabethae is linked with Rattus rats. However, the reservoir host is unknown for other species, such as B. tamiae, which was isolated in patients from Thailand (2).
B. vinsonii subsp. arupensis was fi rst isolated from a bacteremic cattle rancher in Wyoming, USA, in 1999 (3). Later studies showed that strains identical to B. vinsonii subsp. arupensis were highly prevalent among deer mice (Peromyscus maniculatus), a strictly North American rodent species frequently found across a wide geographic area, including Wyoming. Similar strains of B. vinsonii subsp. arupensis have not been found in other animals in North America, suggesting that deer mice are natural hosts of this bacterium (4).
However, the proposed link between infected mice and B. vinsonii subsp. arupensis infection in humans was challenged when this bacterium was reported in an endocarditis patient in France (5) and 2 febrile patients in Russia (6). The link was further disputed after identifi cation of B. vinsonii subsp. arupensis infection in 2 humans in Thailand (7) and the subsequent inability to identify this strain or related species among the local rodent population, despite intensive investigation in different parts of Thailand (8). B. vinsonii subsp. arupensis was also identifi ed in stray dogs in Thailand (9). In addition, B. vinsonii subsp. arupensis-specifi c antibodies were reported in febrile patients from Nepal (10). Together, these reports suggest that the spectrum of animal hosts carrying B. vinsonii subsp. arupensis may be underestimated. We report the identifi cation of B. vinsonii subsp. arupensis in 4 more patients in Thailand.

The Study
The patients were enrolled in a febrile illness study in 4 rural hospitals in Chiang Rai and Khon Kaen Provinces, in northern and northeastern Thailand, respectively, during February 2002-March 2003. One of the patients (no. 45-00250) was enrolled as an afebrile patient. The 4 patients had some common clinical symptoms, such as headache, myalgias, dizziness, and fatigue. In addition, 3 of the patients had elevated levels of liver enzymes. All patients reported trapping or killing rats or seeing rats inside or around their houses during the 2 weeks before onset of symptoms, and all patients owned a dog and/or cat (Table).
To determine if the patients were infected with Bartonella species, we tested blood clots from each patient for the presence of the bacterium. For testing, we used a liquid growth medium (Bartonella α-Proteobacteria growth medium [BAPGM]) as a pre-enrichment step (11), improved molecular assays for Bartonella detection (12), and established molecular methods. The blood clots were inoculated into freshly prepared BAPGM. To avoid potential contamination, we did not supplement the medium with animal blood. Blank BAPGM controls were included for each inoculation. The inoculants and the blank controls were incubated aerobically at 35°C in 5% CO 2 for 7 days.
DNA was extracted from this pre-enriched medium by using the QIAamp DNA Mini Kit (QIAGEN, Valencia, CA, USA). DNA was analyzed following published protocols (12)(13)(14). In brief, real-time PCR, nested PCR, and conventional PCR were used to target Bartonella-specifi c regions in the transfer-messenger RNA (ssrA) gene, the citrate synthase (gltA) gene, and the 16S-23S rRNA internal transcribed spacer, respectively. Positive controls (B. doshiae) and negative controls were included within each PCR run to evaluate the presence of appropriately sized amplicons and contamination, respectively. Amplicons were recovered from PCRs by gel purifi cation (QIAGEN) and sequenced in both directions.

Conclusions
In Thailand, at least 2 Bartonella species, B. henselae and B. tamiae, have been reported in association with human diseases (2,15). In addition, Bartonella DNA was detected recently in acutely ill patients from Thailand (7). gltA sequences revealed a broad range of Bartonella species in these patients, including B. elizabethae, B. tribocorum, B. rattimassiliensis, and B. vinsonii subsp. arupensis. This fi nding suggests that numerous species of Bartonella may be associated with acute illness in Thailand.
In the current study, sequence data from 3 genetic targets provide additional evidence to confi rm infection with B. vinsonii subsp. arupensis in patients in Thailand.
Although the potential source(s) and mechanism(s) of transmission of this bacterium to humans remain unclear, the previous fi nding of B. vinsonii subsp. arupensis among stray dogs in Thailand (9) and the fact that 3 of the 4 patients in this report owned dogs could suggest dogs might be a source of the bacterium. Commonality of rat exposure may suggest that rats can also be potential reservoir hosts of B. vinsonii subsp. arupensis. Further investigation is needed in this regard.
It is known that diseases of humans and animals can translocate across the globe. For example, bubonic plague, caused by Yersinia pestis, was spread by infected rats on ships traveling from Asia to other continents. Bartonella elizabethae and other rat-associated Bartonella species that originated in the Old World have been similarly translocated to the New World and other regions (1,8). Reports of infections caused by B. vinsonii subsp. arupensis in humans in France, Russia, and Nepal, suggest that the role of this bacterium as a human pathogen may be more geographically widespread than previously believed.
Given the postulated New World origin of this pathogen, its ecology might serve as a model system to examine a possible translocation of Bartonella species across broad geographic regions and even between hosts. In addition, our fi nding of B. vinsonii subsp. arupensis in patients in Thailand suggests that this pathogen may be responsible for some undiagnosed cases of febrile illness and other types of illnesses in Thailand and possibly other countries in Asia.

Acknowledgments
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