We obtained ≈200,000 high-quality sequences (average length 167 bp) from DNA sequencing of fecal material from a Daubenton’s bat (Technical Appendix Figure 1). Sequences (>50 bp) were assigned on the basis of best E-value BLASTN scores (http://blast.ncbi.nlm.nih.gov/blast.cgi) in GenBank. The most abundant non-metazoan sequence matches were with bacteria. The genera Leuconostoc, Enterobacter, Lactococcus, and Chlamydia dominated (Figure 1). Surprisingly, the fecal material also contained DNA of the ectoparasite-transmitted genera, such as the hemotrophic bartonellae (9). It was thought that this DNA originated either from bleeding into the intestine or from the insect prey of the bats that includes the abundant bloodfeeding bat ectoparasites. PCR verified the presence of Bartonella DNA in the bat fecal material. The transfer messenger RNA gene (ssrA) (10) could be amplified and was sequenced from the fecal material of 1 Daubenton’s bat, 1 northern bat (Eptesicus nilssonii), and 1 Brandt’s bat (Myotis brandtii) (no. 2771, no. 2788, and no. 2786, respectively; Technical Appendix Table 1). The obtained 218-bp ssrA sequences were 100% identical. The closest matches in GenBank, with a similarity score of 94.8% (183/193 bp), were B. tamiae Th339 (GenBank accession no. JN029780) and Th307 strains (GenBank accession no. JN029778) isolated from 2 humans in Thailand (11).

Bats belonging to the 4 most prevalent bat species in Finland were captured in August and September 2012 at 3 locations in southwestern Finland (Technical Appendix Table 1). Culturing of peripheral blood samples of 5 Daubenton’s bats and 1 northern bat yielded distinct colonies. The isolates were identified as Bartonella spp. by sequencing a PCR-amplified 485-bp fragment containing the hypervariable regions V6–V8 of the 16S rRNA gene. Overall health of the bats as analyzed by body condition indexing was not affected by the Bartonella infection (Technical Appendix Table 1).

The 16S rRNA gene sequences are highly conserved within the genus Bartonella and thus not robust in differentiating species (12). Therefore, we sequenced PCR-amplified fragments of the RNA polymerase β-subunit gene (rpoB), citrate synthase gene (gltA), filamenting temperature-sensitive mutant Z gene (ftsZ), VirB type IV secretion system VirB4 component gene (virB4), hypervariable region 2 of the 16S-23S rRNA intergenic spacer region (ISR), and ssrA (Technical Appendix Table 2). Sequencing of rpoB was first conducted on all 28 clonal isolates. Three distinct rpoB alleles were identified (Technical Appendix Table 1). The multilocus sequence analysis (MLSA) was completed on 1 rpoB-1 allele isolate (clone 3, bat no. 1157, referred to hereafter as 1157/3), 1 rpoB-2 allele isolate (clone 1, bat no. 2574, referred to hereafter as 2574/1), and 1 rpoB-3 allele isolate (clone 1, bat no. 1160, referred to hereafter as 1160/1). Thin-section transmission electron micrographs of these isolates are shown in the Technical Appendix Figure 2. No major pili or fimbriae-like structures were detected on the surface of the rod-shaped bacteria.

Results of BLASTN homology searches performed in January 2013 are shown in Technical Appendix Table 3. ISR is a robust species discriminatory marker within the genus Bartonella (13,14). ISR of the strain 2574/1 did not have any hits, whereas ISR of strains 1157/3 and 1160/1 had on1y 1 hit in GenBank Candidatus B. mayotimonensis (15), with high sequence similarity scores. Sequence analyses of the other MLSA markers (Technical Appendix Table 3) further indicate that isolates 1157/3 and 1160/1 belong to the Candidatus-status species B. mayotimonensis and that strain 2574/1 belongs to a new Bartonella species. Indeed, the lowest pairwise genetic distance values with the concatenated rpoB, gltA, 16S rRNA, and ftsZ sequence fragments of the bat strains 1157/3 and 1160/1 in the genus Bartonella were 0.040 and 0.038, respectively, with Candidatus B. mayotimonensis (Technical Appendix Table 4). Because the distance value 0.05 is the recommended cutoff value for species delineation (16), the bat isolates 1157/3 and 1160/1 classify as strains of the Candidatus-status species B. mayotimonensis. The bat strain 2574/1 belongs to a new Bartonella species because the lowest genetic distance value in the genus Bartonella was 0.070 with B. washoensis, above the 0.05 cutoff value (16).

Figure 2 shows the phylogenetic position of the bat Bartonella isolates based on comparisons of concatenated sequences of rpoB, gltA, 16S rRNA and ftsZ, available for Candidatus B. mayotimonensis (15) and all type strains of the Bartonella species (Technical Appendix Table 5). The neighbor-joining and maximum-likelihood trees demonstrate that bat isolates 1157/3 and 1160/1 cluster with Candidatus B. mayotimonensis with high bootstrap values in a distinct phylogenetic position. The new Bartonella species (strain 2754/1) clearly diverges from the other bat isolates.

We sequenced a PCR-amplified fragment of the mitochondrial cytochrome c oxidase subunit I (17) and also used visual inspection to identify the ectoparasites of 18 bats (o Technical Appendix Table 1). Ectoparasite DNA preparations of 2 fleas and 10 flies were analyzed with a PCR protocol targeting the Bartonella rpoB. The blood isolate rpoB alleles 1 and 2 were detected in samples from 1 flea and 2 flies, respectively (Technical Appendix Table 1). In addition, 2 novel rpoB alleles were detected. The rpoB-5 allele detected in a fly sample is distantly related to the currently known Bartonella rpoB sequences. The highest BLASTN sequence identity score with the rpoB-4 allele detected in a flea sample, and from 1 blood DNA preparation of a culture-negative whiskered bat (no. 1156, Technical Appendix Table 1), was 97.8% (397/406 bp) with the corresponding fragment (FJ376736) of Candidatus B. mayotimonensis. This is a higher value than with the rpoB-1 and rpoB-2 alleles. Moreover, a partial 338-bp gltA fragment could be amplified from the rpoB-4–positive flea sample. The highest BLASTN sequence identity score with Candidatus B. mayotimonensis was 93.2% (315/338 bp), which is higher than with the isolates 1157/3 (92.0%, 311/338 bp) and 1160/1 (92.3%, 312/338 bp). The data further support the conclusion that bats are reservoir hosts of B. mayotimonensis and indicate that the bat flies and fleas transmit Bartonella spp. to new hosts.

A maximum composite likelihood–based neighbor-joining tree (Figure 3) was constructed on the basis of 253-bp gltA sequences obtained from Bartonella that infect bats in the United Kingdom (18), Kenya (19), Guatemala (20), Taiwan (21), and Peru (22). The 5 Bartonella-like bacteria detected in minced heart tissues in the United Kingdom (18), the B. mayotimonensis isolates from Finland, and the strain detected in 1 bat flea in Finland clustered in a distinct phylogenetic position away from the bat isolates and strains of the Southern Hemisphere. The new Bartonella species (strain 2574/1) does not belong to the Northern Hemisphere B. mayotimonensis cluster. Remarkably, the 253-bp gltA fragment of 1 of the Bartonella-like bacteria detected in minced heart tissue of a common noctule (Nyctalus noctule) (Cornwall-M451, AJ871615) yielded a 95.3% (241/253 bp) sequence identity score, compared with the corresponding fragment (FJ376732) of Candidatus B. mayotimonensis. This value is significantly higher than those obtained with corresponding gltA fragments of Finland bat isolates 1157/3 (92.9%, 235/253) or 1160/1 (94.1%, 238/253). The UK gltA data further support the conclusion that bats are reservoir hosts of B. mayotimonensis. Most importantly, bats appear to be reservoir hosts of B. mayotimonensis only in the Northern Hemisphere.

Bartonella naantaliensis (naan.tali´en.sis. N.L. fem. adj. n. naantaliensis of or belonging to Naantali) is the name proposed to highlight the municipality where the bat was trapped from which the type strain was isolated. The type strain is 2574/1. Its partial 16S rRNA gene nucleotide sequence is deposited in GenBank (accession no. KF003116).