Kai Inoue

Prevalence and Genetic Diversity of Bartonella Species Isolated from Wild Rodents in Japan

ABSTRACT Here, we describe for the first time the prevalence and genetic properties of Bartonella organisms in wild rodents in Japan. We captured 685 wild rodents throughout Japan (in 12 prefectures) and successfully isolated Bartonella organisms from 176 of the 685 rodents (isolation rate, 25.7%). Those Bartonella isolates were all obtained from the rodents captured in suburban areas (rate, 51.8%), but no organism was isolated from the animals captured in city areas. Sequence analysis of rpoB and gltA revealed that the Bartonella isolates obtained were classified into eight genetic groups, comprising isolates closely related to B. grahamii (A-I group), B. tribocorum and B. elizabethae (B-J group), B. tribocorum and B. rattimassiliensis (C-K group), B. rattimassiliensis (D-L group), B. phoceensis (F-N group), B. taylorii (G-O group), and probably two additional novel Bartonella species groups (E-M and H-P). B. grahamii, which is one of the potential causative agents of human neuroretinitis, was found to be predominant in Japanese rodents. In terms of the relationships between these Bartonella genetic groups and their rodent species, (i) the A-I, E-M, and H-P groups appear to be associated with Apodemus speciosus and Apodemus argenteus; (ii) the C-K, D-L, and F-N groups are likely implicated in Rattus rattus; (iii) the B-J group seems to be involved in Apodemus mice and R. rattus; and (iv) the G-O group is probably associated with A. speciosus and Clethrionomys voles. Furthermore, dual infections with two different genetic groups of bartonellae were found in A. speciosus and R. rattus. These findings suggest that the rodent in Japan might serve as a reservoir of zoonotic Bartonella infection.

Exotic Small Mammals as Potential Reservoirs of Zoonotic Bartonella spp.

To evaluate the risk for emerging human infections caused by zoonotic Bartonella spp. from exotic small mammals, we investigated the prevalence of Bartonella spp. in 546 small mammals (28 species) that had been imported into Japan as pets from Asia, North America, Europe, and the Middle and Near East. We obtained 407 Bartonella isolates and characterized them by molecular phylogenetic analysis of the citrate synthase gene, gltA. The animals examined carried 4 zoonotic Bartonella spp. that cause human endocarditis and neuroretinitis and 6 novel Bartonella spp. at a high prevalence (26.0%, 142/546). We conclude that exotic small mammals potentially serve as reservoirs of several zoonotic Bartonella spp.

Bartonella japonica sp. nov. and Bartonella silvatica sp. nov., isolated from Apodemus mice

Two bacterial strains, Fuji 18-1(T) and Fuji 23-1(T), were isolated from the blood of the small Japanese field mouse (Apodemus argenteus) and the large Japanese field mouse (Apodemus speciosus), respectively, specimens of which were captured in the forest of Mount Fuji, Japan. Phenotypic characterization (growth conditions, incubation periods, biochemical properties and cell morphologies), DNA G+C contents (40.1 mol% for strain Fuji 18-1(T) and 40.4 mol% for strain Fuji 23-1(T)) and sequence analyses of the 16S rRNA genes indicated that both strains were members of the genus Bartonella. Using rpoB and gltA sequencing analysis, the highest sequence similarities between strains Fuji 18-1(T), Fuji 23-1(T) and other recognized species of the genus Bartonella showed values considerably lower than 91.4 % and 89.9 % in the rpoB gene and 89.1 % and 90.4 % in the gltA gene, respectively. It is known that similarities of 95.4 % for the rpoB gene and 96.0 % for the gltA gene can be applied as cut-off values for the designation of novel species of the genus Bartonella. In a phylogenetic tree based on the merged set of concatenated sequences of seven loci [16S rRNA, ftsZ, gltA, groEL, ribC and rpoB genes and the intergenic spacer region (ITS)], strains Fuji 18-1(T) and Fuji 23-1(T) formed a distinct clade from other recognized species of the genus Bartonella. These data support the classification of strains Fuji 18-1(T) and Fuji 23-1(T) as novel species of the genus Bartonella. The names Bartonella japonica sp. nov. and Bartonella silvatica sp. nov. are proposed for these novel species. The type strains of Bartonella japonica sp. nov. and Bartonella silvatica sp. nov. are Fuji 18-1(T) (=JCM 15567(T)=CIP 109861(T)) and Fuji 23-1(T) (=JCM 15566(T)=CIP 109862(T)), respectively.

Evolutional and Geographical Relationships of Bartonella grahamii Isolates from Wild Rodents by Multi-locus Sequencing Analysis

To clarify the relationship between Bartonella grahamii strains and both the rodent host species and the geographic location of the rodent habitat, we have investigated 31 B. grahamii strains from ten rodent host species from Asia (Japan and China), North America (Canada and the USA), and Europe (Russia and the UK). On the basis of multi-locus sequencing analysis of 16S rRNA, ftsZ, gltA, groEL, ribC, and rpoB, the strains were classified into two large groups, an Asian group and an American/European group. In addition, the strains examined were clearly clustered according to the geographic locations where the rodents had been captured. In the phylogenetic analysis based on gltA, the Japanese strains were divided into two subgroups: one close to strains from China, and the other related to strains from Far Eastern Russia. Thus, these observations suggest that the B. grahamii strains distributed in Japanese rodents originated from two different geographic regions. In the American/European group, B. grahamii from the North American continent showed an ancestral lineage and strict host specificity; by contrast, European strains showed low host specificity. The phylogenetic analysis and host specificity of B. grahamii raise the possibility that B. grahamii strains originating in the North American continent were distributed to European countries by adapting to various rodent hosts.

Prevalence of Bartonella infection in cats and dogs in a metropolitan area, Thailand

The prevalence of Bartonella infection was studied in 312 cats and 350 dogs in the Bangkok metropolitan areas, Thailand, between June 2001 and February 2003. Bartonella was isolated from 47 (16.3%) of 288 stray cats, but from none of the 24 pet cats studied. Of the 47 Bartonella-positive cats, 45 animals were infected with only B. henselae, one was infected with only B. clarridgeiae, and one with both B. henselae and B. clarridgeiae. 16S rRNA typing showed that 40 cats were infected with B. henselae type I, four with B. henselae type II, and one with both B. henselae types I and II. These results indicated that B. henselae, especially type I, was prevalent in stray cats that constituted a large Bartonella reservoir in Bangkok. B. clarridgeiae was isolated for the first time in Asia from one of 350 dogs.

Detection of multiple Bartonella species in digestive and reproductive tissues of fleas collected from sympatric mammals

At least 12 species in the genus Bartonella are zoonotic pathogens that may be transmitted among mammalian hosts by fleas or other arthropods. Apparent host specificity by some Bartonella species to mammalian hosts has been observed, and the detection of multiple Bartonella species in mammalian fleas suggests that fleas take bloodmeals from a variety of host species. However, many flea species are observed to parasitize a narrow host range. Therefore, we suspect that fleas may acquire Bartonella by a mechanism other than ingesting infectious blood. We found that detection of multiple Bartonella genotypes and species is apparently common in fleas and that the majority of fleas tested (5/9) carried Bartonella species atypical of their hosts. We also detected Bartonella DNA in flea reproductive tissues, suggesting that vertical transmission of this organism in vectors is possible, potentially leading to the accumulation of Bartonella diversity over time within fleas.

Multi-locus sequence analysis reveals host specific association between Bartonella washoensis and squirrels.

To clarify phylogenetic relationships and genetic diversity among Bartonella washoensis strains obtained from squirrels, multi-locus sequence analysis (MLSA) with the 16S rRNA, ftsZ, gltA, groEL, ribC, and rpoB genes was applied for 20 strains of B. washoensis isolated from five genera of squirrels (Tamias, Tamiasciurus, Glaucomys, Sciurus, and Spermophilus) within the family Sciuridae. Sequence similarities in the concatenated sequences of B. washoensis strains from squirrels of different genera ranged from 94.7% (Sciurus vs. Spermophilus) to 98.4% (Tamiasciurus vs. Glaucomys). Phylogenetic trees based on the concatenated sequences revealed that B. washoensis strains formed five distinct clades and each clade correlated with the genus of squirrel from which the strains were originally obtained. The discrimination was supported by 100% bootstrap values and posterior probabilities, respectively. These results suggest that B. washoensis strains may have co-speciated with their squirrel hosts and provide new insights into the application of the MLSA to identify sources of B. washoensis infection with accuracy.

Bartonella jaculi sp. nov., Bartonella callosciuri sp. nov., Bartonella pachyuromydis sp. nov. and Bartonella acomydis sp. nov., isolated from wild Rodentia

Four novel strains of members of the genus Bartonella, OY2-1(T), BR11-1(T), FN15-2(T) and KS2-1(T), were isolated from the blood of wild-captured greater Egyptian jerboa (Jaculus orientalis), plantain squirrel (Callosciurus notatus), fat-tailed gerbil (Pachyuromys duprasi) and golden spiny mouse (Acomys russatus). All the animals were imported to Japan as pets from Egypt, Thailand and the Netherlands. The phenotypic characterization (growth conditions, incubation periods, biochemical properties and cell morphologies), DNA G+C contents (37.4 mol% for strain OY2-1(T), 35.5 mol% for strain BR11-1(T), 35.7 mol% for strain FN15-2(T) and 37.2 mol% for strain KS2-1(T)), and sequence analyses of the 16S rRNA genes indicated that those strains belong to the genus Bartonella. Sequence comparisons of gltA and rpoB genes suggested that all of the strains should be classified as novel species of the genus Bartonella. In phylogenetic trees based on the concatenated sequences of five loci, including the 16S rRNA, ftsZ, gltA and rpoB genes and the ITS region, and on the concatenated deduced amino acid sequences of three housekeeping genes (ftsZ, gltA and rpoB), all strains formed distinct clades and had unique mammalian hosts that could be discriminated from other known species of the genus Bartonella. These data strongly support the hypothesis that strains OY2-1(T), BR11-1(T), FN15-2(T) and KS2-1(T) should be classified as representing novel species of the genus Bartonella. The names Bartonella jaculi sp. nov., Bartonella callosciuri sp. nov., Bartonella pachyuromydis sp. nov. and Bartonella acomydis sp. nov. are proposed for these novel species. Type strains of Bartonella jaculi sp. nov., Bartonella callosciuri sp. nov., Bartonella pachyuromydis sp. nov. and Bartonella acomydis sp. nov. are OY2-1(T) ( = JCM 17712(T) = KCTC 23655(T)), BR11-1(T) ( = JCM 17709(T) = KCTC 23909(T)), FN15-2(T) ( = JCM 17714(T) = KCTC 23657(T)) and KS2-1(T) ( = JCM 17706(T) = KCTC 23907(T)), respectively.

Serological Survey of Five Zoonoses, Scrub Typhus, Japanese Spotted Fever, Tularemia, Lyme Disease, and Q Fever, in Feral Raccoons (Procyon lotor) in Japan

We investigated the seroprevalence of five tick- or mite-borne zoonoses, scrub typhus (Orientia tsutsugamushi), Japanese spotted fever (Rickettsia japonica), tularemia (Francisella tularensis), Lyme disease (Borrelia afzelii and Borrelia garinii), and Q fever (Coxiella burnetii), in feral raccoons (Procyon lotor) captured in Hokkaido and Kanagawa Prefectures in Japan. Of the 559 raccoons captured in Hokkaido, 8 (1.4%), 3 (0.5%), 1 (0.2%), and 1 (0.2%) carried antibodies against O. tsutsugamushi (Gilliam type), F. tularensis, B. afzelii, and B. garinii, respectively. Of the 193 animals investigated in Kanagawa, 31 (16.1%) and 14 (7.3%) carried antibodies against O. tsutsugamushi and R. japonica, respectively, and the major serotype (27/31) of O. tsutsugamushi was Kuroki. No antibodies against C. burnetii were detected in either area examined. Therefore, feral raccoons could be an indicator of the prevalence of these four tick- or mite-borne zoonoses in the peridomestic environment in Japan.

PREVALENCE AND DIVERSITY OF BARTONELLA SPECIES IN WILD SMALL MAMMALS IN ASIA

Abstract We collected 641 small mammals belonging to 17 species of Rodentia and four species of Soricomorpha in Japan, Korea, Russia, Taiwan, and Thailand and investigated the prevalence and genetic diversity of Bartonella species. Apodemus (field mice) and Rattus (rats) were the most-common genera captured, making up 56.0% and 23.1% of the total specimens, respectively. Bartonellae were isolated from 54.6% of the collected animals, and the prevalence varied depending on the host species and the country of origin. The isolates were identified to the species level based on gltA and rpoB sequences. Although most Bartonella species were shared by more than two host species, the distribution patterns of Bartonella species clearly differed among the four most-common host genera: Apodemus, Rattus, Myodes (voles), and Suncus (shrews). The predominant Bartonella species were Bartonella grahamii in Apodemus, Bartonella tribocorum in Rattus, B. grahamii and Bartonella taylorii in Myodes, and an unclassified Bartonella sp. in Suncus.