International Journal of Systematic and Evolutionary Microbiology (2000), 50, 1973–1979 Printed in Great Britain Bartonella birtlesii sp. nov., isolated from small mammals (Apodemus spp.) Delphine Bermond,1 Re! my Heller,1 Francine Barrat,2 Gilles Delacour,3 Christoph Dehio,4 Annie Alliot,2 Henri Monteil,1 Bruno Chomel,5 Henri-Jean Boulouis2 and Yves Pie! mont1 Author for correspondence: Delphine Bermond. Tel: j33388211970.Fax:j33388251113. e-mail: delphine.bermond!medecine.u-strasbg.fr 1 Institut de Bacte! riologie de Three strains isolated from Apodemus spp. were similar to Bartonella species la Faculte! de Me! decine, on the basis of phenotypic characteristics. Futhermore, genotypic analysis Universite! Louis Pasteur, Ho# pitaux Universitaires de based on sequence analysis of the 16S rRNA and gltA genes and on DNA–DNA Strasbourg, 3 rue hybridization showed that the three isolates represented a distinct and new Koeberle! , 67000 species of Bartonella. The name Bartonella birtlesii is proposed for the new Strasbourg, France species. The type strain of B. birtlesii sp. nov. is IBS 325T (l CIP 106294T l CCUG 2 UMR 956 44360T). INRA/AFSSA/ENVA, Microbiologie/IIAC, Ecole Nationale Ve! te! rinaire, 94704 Maisons-Alfort, Keywords: Bartonella birtlesii, rodents, taxonomy, citrate synthase, 16S rDNA gene France 3 Office Nationale de la Chasse, Gerstheim, France 4 Max-Planck-Institut fu$ r Biologie, Tu$ bingen, Germany 5 Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616, USA INTRODUCTION pennsylvanicus is the host for Bartonella vinsonii (Baker, 1946), Apodemus spp. are the hosts for Barton- Prior to 1993, Bartonella bacilliformis was the only ella taylorii (Birtles et al., 1995), Clethrionomys glare- member of the Bartonella genus. Now, after only a few olus is the host for Bartonella grahamii (Birtles et al., years of research and taxonomic study, this genus 1995) and Microtus agrestis is the host for Bartonella includes 12 approved species available in the inter- doshiae (Birtles et al., 1995). Furthermore, B. taylorii, national bacterial collections (Brenner et al., 1993). B. grahamii and B. doshiae are able to infect numerous For two species [B. bacilliformis (Weinman, 1968) and species of small woodland mammals and there is no Bartonella quintana (Vinson & Fuller, 1961)] the narrow animal host spectrum of infection. The 12th human is the natural host, for three others [Bartonella approved species is Bartonella elizabethae (Daly et al., henselae (Regnery et al., 1992; Welch et al., 1992), 1993), for which only a single isolate, originating from Bartonella clarridgeiae (Lawson & Collins, 1996) and a human case of endocarditis, was described. The 16S Bartonella koehlerae (Droz et al., 1999)] it is the cat rRNA gene sequence of this species is close to that of (Felis domesticus) and for one other (Bartonella alsa- the rat Bartonella sp., B. tribocorum (Heller et al., tica; Heller et al., 1999) it is the rabbit (Oryctolagus 1998) and to that of other isolates not yet validly cuniculus). For five Bartonella species, small mammals described, but all were isolated from small wild are the hosts: the rat (Rattus rattus) is the host for mammals (Birtles & Raoult, 1996; Kosoy et al., 1997). Bartonella tribocorum (Heller et al., 1998), Microtus In addition to these 12 species, two others have been described in Peromyscus spp. [Bartonella peromysci ................................................................................................................................................. (Birtles et al., 1995; Ristic & Kreier, 1984)] and in The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene the mole [Bartonella talpae (Birtles et al., 1995; Ristic sequence and the partial 3h-end gene sequence of strain IBS 325T (l CIP & Kreier, 1984)], but type strains for these species are 106294T l CCUG 44360T) are AF204274 and AF204272, respectively. no longer extant. Moreover, B. vinsonii (Baker, 1946; 01459 # 2000 IUMS 1973 D. Bermond and others Breitschwerdt et al., 1995), now includes three sub- Electron microscopy. Bacteria were grown on solid medium species, one of which, B. vinsonii subsp. arupensis, was and submitted to electron microscopy as described pre- isolated from humans and mice (Peromyscus leucopus) viously (Fusseneger et al., 1996). Briefly, bacteria were and is therefore presumptively considered as patho- suspended in PBS (0n145 M NaCl, 0n15 M sodium phos- genic for humans (Welch et al., 1999; Hofmeister et al., phate), spread onto a water surface, absorbed onto Form- var-coated nickel grids, stained in 1% (w\v) uranyl acetate 1998). and then air-dried. Samples were examined with a JEOL Thus, several Bartonella species have been isolated 100CX2 electron microscope. from numerous small mammals. For one species that is Biochemical analysis. The following biochemical assays were potentially pathogenic for humans (B. elizabethae) performed with diagnostic tablets (Rosco Diagnostica): the small woodland mammals are suspected as a reservoir; Voges–Proskauer reaction, tributyrine hydrolysis and pyra- B. vinsonii subsp. arupensis (Welch et al., 1999), how- zinamidase, proline aminopeptidase and trypsin-like ac- ever, was isolated from mice and humans. B. grahamii tivities. Pre-formed bacterial enzyme activity was tested DNA was detected from the anterior chamber fluid of using the MicroScan Rapid Anaerobe Panel (Dade In- ternational) according to the manufacturer’s instructions the eye in an HIV-negative woman who presented with and as described previously (Welch et al., 1993; Birtles et al., bilateral eye inflammatory disease. This detection was 1995). performed by using PCR amplification and sequence analysis of the 16S rRNA gene (Kerkhoff et al., 1999). Amplification of 16S rRNA and citrate synthase genes. DNA Thus, the members of the bacterial genus Bartonella, was extracted from bacterial suspensions via the Chelex particularly species isolated from small woodland extraction technique (De Lamballerie, 1992). An approxi- mately 1500 bp fragment of the 16S rRNA gene was mammals, appear to be a group of emerging human amplified from the extracted DNA by using the eubacterial pathogens. Better knowledge of the bacteria harboured universal primers specific for the 16S rRNA gene, i.e. primer by wild small mammals is therefore necessary. P8 (5h-AGAGATTTGATCCTGGCTCAG-3h) and primer Pc1544(5h-AAGGAGGTGATCCAGCCGCA-3h)(Helleret The aim of this work was to characterize a new al., 1998). PCR amplification of the citrate synthase gene Bartonella group, members of which were isolated in was performed, as described by Birtles & Raoult (1996), with France and the UK from the blood of small mammals two primers, i.e. primer CS.140f (5h-TTACTTATGATCCK- belonging to the genus Apodemus. GGYTTTA-3h, where K represents equimolar amounts of G and T and Y represents equimolar amounts of C and T) and primer BhCS.1137n (5h-AATGCAAAAAGAACAGTAA- METHODS ACA-3h). Standard procedures for preventing sample DNA cross-contamination were undertaken (Kwok & Higuchi, Animals, blood sampling and culture conditions. Small 1989). Each set of reactions also included negative controls mammals were caught in February 1997 by a professional to confirm the absence of cross-contamination between trapper in a wetland conservation area (at point kilometre samples and previously amplified DNA or field samples. The PK 275, from the Bodensee in Germany), near the River presence of the desired amplicons was controlled by electro- Rhine, in Gerstheim, France. Identification of the small phoresis on a 1n5% agarose gel, followed by ethidium mammals was based on morphology and dentition. Animals bromide staining and visualization on a UV transillumin- were anaesthetized by diethylether inhalation and then ator. 0n2 ml blood was collected from each animal by intracardiac puncture. Blood was transferred to a paediatric lysis–centri- Purification of the amplicons and DNA sequencing. The fugation tube (Wampole Laboratories). After centrifuga- amplified fragments were purified via phenol extraction and tion, the pellet was plated onto Columbia-base agar plates 2-propanol precipitation (Brow, 1990). Sequencing of the containing 5% fresh, defibrinated rabbit blood. The plates complete 16S rRNA gene was performed on the coding and were incubated at 35 mC in a moist atmosphere containing complementary strands by using the following four primer 5% CO# for 4 weeks. pairs 5h-labelled with FITC (Eurogentec): P8 and Pc535 (5h- GTATTACCGCGGCTGCTGGCAC-3h);P515(5h-GTGC- Bacterial strains. Two isolates obtained from Apodemus spp. T CAGCAGCCGCGGTAAKAC-3h) andPc804 (5h-GACTA- and named IBS 325 and IBS 358 were examined. (We were CCAGGGTATCTAATCC-3h);P784(5h-GGATTAGATA- unable to identify the Apodemus spp. precisely, because the CCCTGGTAGTC-3h) and Pc1198 (5h-ACTTGACGTTAT- animals captured were too young.) In addition, strain N40, CCCCACCTTCC-3h); and P1174 (5h-GAGGAAGGTGG- kindly provided by R. Birtles (Bristol, UK) and isolated in GGATGACGTC-3h) and Pc1544. the UK from Apodemus sylvaticus, was included in the study. Partial sequencing of the 3h-end of the citrate synthase gene Reference strains used for DNA–DNA hybridization. B. (gltA) was performed on coding and complementary strands elizabethae ATCC 49927T, B. henselae ATCC 49882T and B. by using primer pairs BhCS.1137n and BhCS.781p (5 - T h quintana ATCC VR-358 were purchased from the American GGGGACCAGCTCATGGTGG-3h). All primers were 5h- Type Culture Collection (ATCC; Manassas, VA, USA).