For this study, 546 exotic small mammals were purchased from trading companies. The animals represented 3 orders and included 6 families, 23 genera, and 28 species (Table 1). They had been imported into Japan as pets from June 2004 through October 2007 from 8 countries in 4 geographic regions: Asia (China, Thailand, and Indonesia), Europe (the Netherlands and Czech Republic), North America (United States), and the Middle and Near East (Egypt and Pakistan). Of the 546 animals, 367 had been captured in their natural environment and 179 had been bred in the exporting countries. Heparinized blood samples were aseptically collected from each animal (anesthetized with chloroform) and centrifuged at 3,000 rpm for 15 min. Plasma was removed and the blood sample pellets were kept at –80ºC until examination.

The blood sample pellets were thawed at room temperature, 100-μL supplemented Medium 199 (24) was added to each pellet, and each sample was mixed well. A 100-μL sample of each mixture was plated on 2 heart infusion agar (DIFCO, Sparks Glencoe, MI, USA) plates containing 5% defibrinated rabbit blood. The plates were incubated at 35ºC under 5% CO₂. After 2 weeks of incubation, 2 or 3 colonies with genus Bartonella morphologic characteristics (small, gray or cream-yellow, round colonies) were picked from each plate, confirmed to be gram-negative pleomorphic bacteria, and subcultured using the same conditions used for the original cultures.

The genomic DNA of each isolate was extracted by using InstaGene Matrix (Bio-Rad, Hercules, CA, USA). The extracted DNA was used for PCR analysis of a 312-bp part of the gltA gene to confirm that the bacteria were from the genus Bartonella. PCR was performed by using an iCycler (Bio-Rad) with a 20-μL mixture containing 20 ng extracted DNA, 200 μM of each deoxynucleoside triphosphate, 1.5 mmol/L MgCl₂, 0.5 U Taq DNA polymerase (Promega, Madison, WI, USA), and 1 pmol of each primer. The specific primer pair and PCR conditions for gltA were as previously reported (25).

The PCR products were purified by using a commercial kit (Spin Column PCR Products Purification Kit; Bio Basic, Markham, Ontario, Canada). Direct DNA sequencing of the purified PCR products was carried out by using dye terminator chemistry with specific primers (25) and a Model 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The 312-bp gltA sequences from the isolates and type strains of established Bartonella spp. in GenBank/EMBL/DDBJ were aligned with the Clustal X program (26), and a phylogenetic tree was drawn, based on the sequence data and using the neighbor-joining method (27) with the Kimura 2-parameter distance method (28) in MEGA 4 (29). Bootstrap analysis was carried out with 1,000 replications (30).

The results were analyzed in 2×2 tables. Chi-square tests were used to examine the statistical significance; p<0.05 was considered significant.

The prevalence of bartonellae in the exotic small mammals examined was 26.0% (142/546). A total of 407 isolates were obtained from the 142 bacteremic animals (Table 1). The prevalence by animal origin was 37.3% (137/367) in captive animals and 2.8% (5/179) in animals from breeder facilities. A significantly higher prevalence of bartonellae was observed in captive animals than in animals from breeder facilities (p<0.001). In the captive animals, the prevalence by region varied up to 47.2% in Asia, which is higher than the 39.7% prevalence in North America. The prevalence of bartonellae by corresponding taxonomic family of host animal ranged from 38.6% (49/127) in the family Muridae to 43.9% (69/157) in the family Sciuridae. No bartonellae were detected in animals in the families Octodontidae and Erinaceidae. Among animals from breeders, only 5 chipmunks (Tamias sibiricus) from China were found to be infected with bartonellae; no bartonellae were isolated from animals in the families Petauridae, Muridae, Octodontidae, or Dipodidae.

Bartonellae were isolated from 17 of the 28 animal species studied (Table 1). The prevalence by animal species varied from 9.7% (3/31) in the Cairo spiny mouse (Acomys cahirinus) to 100% (10/10) in the bushy-tailed jird (Sekeetamys calurus). Prevelances were considerably higher for the bushy-tailed jird, large Egyptian gerbil (Gerbillus pyramidum), greater Egyptian jerboa (Jaculus orientalis), and lesser Egyptian jerboa (J. jaculus) at 100% (10/10), 90.0% (9/10), 81.3% (13/16), and 75.0% (6/8), respectively.

The 407 isolates in this study were classified into 53 genotypes on the basis of DNA sequence analysis of a 312-bp fragment of their gltA genes. The sequence of a genotype from a Cairo spiny mouse was identical to that of the B. elizabethae type strain (GenBank accession no. Z70009) isolated from a human patient with endocarditis (10). The other 52 genotypes were found to be novel genotypes after comparison with known Bartonella spp. The phylogenetic tree of the gltA sequences shows that the 52 novel genotypes are clearly clustered in 10 genogroups, designated A to J (Figure).

Of the 52 novel genotypes, genogroup A, which consisted of 21 genotypes (AB444954 to AB444974) isolated from 7 squirrel species, was related to B. washoensis strain Sb944nv (AF470616), which was isolated from a California ground squirrel (Spermophilus beecheyi) and was genetically identical to an isolate from a human patient with myocarditis (9). The sequence similarities of these genotypes and B. washoensis strain Sb944nv ranged from 94.2% to 97.4%. Genogroup A contained B. washoensis–like genotypes; the genotypes from each squirrel species formed a separate clade, except for the genotypes from Richardson’s ground squirrels (Sp. richardsonii) and Columbian ground squirrels (Sp. columbianus), which formed a mixed clade (Figure).

In this study, 18 genotypes formed the 6 unique genogroups B to G. The DNA sequences of the genotypes in each genogroup showed relatively low similarity (82.4%–94.6%) to the type strains of known Bartonella spp., and sequence similarities between genogroups B to G were also low (87.5%–93.6%). The novel Bartonella genogroups B, C, D, and E were isolated from greater Egyptian jerboas, tricolored squirrels (Callosciurus notatus), fat-tailed gerbils (Pachyuromys duprasi), and golden spiny mice (A. russatus), respectively. The genotypes in group F were isolated from 6 animal species: large Egyptian gerbils, fat-tailed gerbils, fat sand rats (Psammomys obesus), lesser Egyptian jerboas, greater Egyptian jerboas, and bushy-tailed jirds; those in genogroup G were isolated from a bushy-tailed jird and a large Egyptian gerbil (Figure). In genogroup F, 3 of the 9 isolates from fat-tailed gerbils and the 14 isolates from fat sand rats had identical gltA DNA sequences. Furthermore, 5 of the 7 isolates from greater Egyptian jerboas and 2 of the 3 isolates from a lesser Egyptian jerboa also had identical sequences.

The 2 novel genotypes (AB444993 and AB444994) in genogroup H were also isolated from Siberian chipmunks, a Hokkaido squirrel (Sciurus vulgaris subsp. orientis), and Eurasian small flying squirrels (Pteromys volans). Their sequences showed high similarity (98.4%–98.7%) to B. grahami type strain (V2) (Figure).

The 10 novel genotypes in genogroup I were isolated from 9 animal species, and the sequence similarities between the genotypes (AB444995 to AB445005) and B. elizabethae type strain (F9251) ranged from 95.5% to 98.7%. The DNA sequences of gltA of the 3 isolates from a Cairo spiny mouse (AB445000) were identical to that of B. elizabethae (F9251). The sequences of the 13 isolates from lesser Egyptian jerboas were identical to those of the 16 isolates from greater Egyptian jerboas.

In genogroup J, a unique genotype (AB445006) was isolated from an American red squirrel (Tamiasciurus hudosonicus); it had 96.2% sequence similarity to B. clarridgeiae type strain (Houston-2), whose natural reservoir is cats (Figure).

Of the 142 Bartonella-positive animals, 25 (17.6%) were found to be infected with different Bartonella genogroups or genotypes (Table 2). A lesser Egyptian jerboa carried 3 different genotypes in 2 genogroups; the other 24 animals carried 2 different genogroups or genotypes. Of these 24 animals, an American red squirrel carried a B. washoensis–like strain in genogroup A and B. clarridgeiae–like strains in genogroup J; 11 animals were infected with B. elizabethae–like strains in genogroup I and strains in genogroups B, C, D, E, F, or G, and the remaining 12 carried different genotypes in the same genogroup (Table 2).