International Journal of Systematic Bacteriology Printed in Great Britain -~ - __ (1 998), 48, 1333-1 339

Bartonella tribocorum sp. nov., a new species isolated from the blood of wild rats

Remy Heller,’ Philippe Riegel,’ Yves Hansmann,’ Gilles Delacour,’ Delphine Bermond,’ Christoph Dehi~,~Franqois Lamarque,’ Henri Monteil,’ Bruno Chome14 and Yves Piemontl

Author for correspondence: Remy Heller. Tel: +33 3 88 21 19 70. Fax: +33 3 88 25 11 13. e-mail: yves.piemont(a1medecine.u-strasbg.fr

lnstitut de Bacteriologie de Two Bartonella strains from blood of two wild rats (Rattus norvegicus) living la Faculte de Medecine, in a rural environment were isolated. These strains were distinct from all Universite Louis-Pasteur, HBpitaux Universitaires de previously known Bartonella species based on phenotypic and genotypic Strasbourg, France characteristics. This new species is distinguished by its trypsin-like activity, the Office National de la absence of the ability to hydrolyse proline and tributyrin, its 165 rRNA and Chase, Gerstheim, France citrate synthase gene sequences and by whole-DNA hybridization data. This Max-Plank-lnstitut fur new species, for which the name Bartonella tribocorum sp. nov. is proposed, Biologie, Tubingen, seems to be genetically related to Bartonella elizabethae, an agent isolated in Germany a case of human endocarditis. The type strain of Bartonella tribocorum sp. nov. Department of Population is IBS 506T(CIP 105476T). Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CAI USA Keywords: Bartonella tribocorum sp. nov., rat, zoonoses

INTRODUCTION Species of this genus have been demonstrated to be vector-borne, but can also be transmitted by animal Since the beginning of this century, red blood cells of scratches or bites. It is of importance to evaluate the various animal species have been known to harbour presence of Bartonella species in animals living close to intra-erythrocytic which were referred to as humans. It has been shown that 14% of pet cats in the Graham~lltr(Weinman & Kreier, 1977). Most of these Paris area and more than 50 YOof urban stray cats are bacteria were not cultivated and were only detected by bacteraemic with or Bartonella observation of stained blood smears. Birtles et al. clarridgeiae (Gurfield et al., 1997; Heller et al., 1997a). (1995) demonstrated that species of the genus Pet dogs can also be infected by Bartonella; Bartonella Grahamelltr should be reclassified and included in the vinsonii subsp. berkhofii was isolated from a dog genus Birrtonella. Therefore, the Bartonella presently suffering from endocarditis (Breitschwerdt et al., includes species which were formerly classified in 1995). We have investigated the presence of Bartonella the genera Bartonella, Rocl?alimaea and Grahamella in wild rats (Rattus norvegicus) as these animals are (Birtles et al., 1995; Brenner et al., 1993). Nine found in urban and rural environments. They share Burtonella species are currently available from human habitats, especially those of people of very low international collections : , socio-economic status. Contacts can be direct or Bart on ellu cla r r idg e iae , Bart on ella dosh iae, Bart one lla indirect via haematophagous arthropods such as the elizabethae, , Bartonella henselue, rat (Xenopsylla cheopis). We report the isolation Bartonellu quintana, Bartonella taylorii and Bartonella and description of a new Bartonella species from the vinsonii. TWOadditional species have been described blood of wild rats. This new species seems to be by Birtles et al. (1 995), and Bartonella genetically related to Bartonella elizabethae, an agent peromj*sci,but no strains or 16s rRNA sequence data isolated in a case of human endocarditis (Daly et al., are available. 1993). Several oft hese Bartonella species have been identified as emerging pathogens for humans in the 1990s. METHODS Reference strains. Bartonella elizabethae ATCC 49927T, The EMBUGenBank accession numbers for the sequences of the 165 rRNA Bartonella henselae ATCC 49882’, gene and gltA of IBS 506T are AJ003070 and AJ005494, respectively. ATCC VR-358’ and Bartonella vinsonii ATCC VR- 1 52T

1333 R. Heller and others were purchased from the American Type Culture Collection instructions. The sequence was obtained with an ALF DNA (Rockville, MD, USA). Bartonella grahamii NCTC 12860T, Sequencer (Pharmacia), according to the manufacturer’s Bartonella taylorii NCTC 12861T and instructions. Sequencing of the complete 16s rRNA gene NCTC 12862Twere kindly provided by R. Birtles (London). fragment was performed on coding and complementary strands with four pairs of primers (Eurogentec): P8 and Animals and blood sampling. Four wild rats (Rattus nor- Pc535 (5’ GTATTACCGCGGCTGCTGGCAC 3’), P5 15 vegicus) were trapped in February 1997 in a wetland GTGCCAGCAGCCGCGGTAAKAC 3’) and Pc804 conservation area near the Rhine river, in the eastern part of (5’ (5’ GACTACCAGGGTATCTAATCC 3’), P784 (5’ GGATT- France, by a professional trapper. The rats were caught with AGATACCCTGGTAGTC 3’) and Pcll98 ACTTGA- an automatic trap and had already drowned at collection (5’ CGTTATCCCCACCTTCC 3’), and PI 174 (5’ GAGG- time. Blood (0.5 ml) was collected from each dead animal by AAGGTGGGGATGACGTC 3’) and Pc 1544. Partial intracardiac puncture and placed into a Pediatric Isolator sequencing of gltA was performed with primer pair 1-5tube (Wampole Laboratories) for haemolysis. One drop BhCS. 1137n and BhCS.781~ GGGGACCAGCTCAT- of blood from one of the rats was smeared on a glass slide. (5’ GGTGG 3’). All primers were 5’-labelled with fluorescein Identification of the animal species was based on mor- is0 t hiocyanate. phology and dentition. Sequence analysis. The sequences obtained were compared Isolation of strains. Blood (200 pl) was streaked without with similar sequences from other bacteria, including all prior centrifugation onto two blood agar plates made with a spp., contained in the EMBL/GenBank data- Columbia agar base with 5% defibrinated rabbit blood. Bartonella Incubation of the plates was performed at 35 “C in a moist base. The sequences were aligned by using the CLUSTAL program (Higgins & Sharp, 1988) with DNASTAR software. atmosphere containing 5 O/O CO,. Three randomly selected control plates were incubated in parallel as a sterility control. DNA-DNA hybridization. Colonies were harvested from The count (c.f.u. ml-I) was determined for each blood agar blood agar plates and DNA was extracted as described plate. previously (Riegel et al., 1994). Hybridization between the Light microscopic examination. The blood smear was treated labelled DNA and the fragmented DNA preparation was with May-Griinwald-Giemsa stain and examined with an carried out at 58 “C for 16 h in 0-42 M NaCl by the nuclease- oil-immersion objective at a magnification of x 1000. The TCA method (Grimont et al., 1980; Riegel et al., 1994). cultured micro-organisms were also observed at a magnifi- Determination of DNA base composition. The G + C content cation x 1000 after enhanced Gram staining by using a of DNA was determined by a capillary electrophoresis counterstain of Kinyoun carbol fuchsin at a 1 : 20 dilution method as described previously (Riegel et al., 1995). (Larson 1994). et al., Nucleotide sequence accession numbers. The EMBL/ Electron microscopy. Bacteria grown on solid medium were GenBank accession numbers for the 16s rRNA gene prepared for electron microscopy as described by Fusseneger sequences are as follows : Bartonella bacilliformis, M65249 ; et al. (1996). Briefly, bacteria were resuspended in phos- Bartonella clarridgeiae, X8920; Bartonella doshiae, 23135 1 ; phate-buffered saline, spread on a water surface and ab- Bartonella elizabethae, LO 1260 ; Bartonella grahamii, sorbed to Formvar-coated nickel grids. The grids were 231349 ; Bartonella henselae Houston- 1, M73229 ; Bartonella stained in 1 Oh uranyl acetate and air-dried. The samples henselae BA-TF, 211684; Bartonella yuintana Fuller, were viewed under a Zeiss M109 electron microscope at M 1 1927 ; Bartonella taylorii, 231350 ; Bartoriella vinsonii, 80 kV. LO 1259; Bartonella vinsonii subsp. berkhqfii L35052 ; strain Biochemical analysis. The following biochemical tests were C5-rat, 270008 ; strain C7-rat, 270004; strain Cl-phy, performed with diagnostic tablets (Rosco Diagnostica) : 270006, strain C4-phy, 270007 ; strain R-phy 1, 270005 ; Voges-Proskauer reaction, tributyrine hydrolysis, pyrazi- strain R-phy2, 270001; strain N40, 270002. The EMBL/ namidase, proline aminopeptidase and trypsine-like activity. GenBank accession numbers for the gltA gene sequences, as The Microscan Rapid Anaerobe Panel (Baxter Diagnostics) given by Birtles & Raoult (1996), are as follows: Bartonellir was used to test the activity of preformed bacterial enzymes. bacilliformis LA6.3, 270021 ; Bartonella doshiae, 2700 17; Bartonella elizabethae, 270009 ; Bar toriella grahanzii, Amplification of 16s rRNA and citrate synthase (gltA) genes. 2700 16 ; Bartonella quintana Fuller, 2700 14 ; Barto~ellci Amplification of 16s rRNA and citrate synthase genes was taylorii, 2700 13 ; Bartonella vinsonii, 2700 15 ; strain Cl -phy, performed as described by Birtles & Raoult (1996) and 270022; strain C4-phy, 270019; strain C5-rat, 2700 18 ; Heller et al. (1997a). Briefly, DNA was extracted from strain C7-rat, 270020; strain R-phyl, 270010, strain R- several (3-10) colonies. In vitro amplification of a 1500 bp phy2, 27001 1 ; strain N40, 270012. The EMBL/GenBank DNA fragment was performed with two 16s rRNA gene accession numbers for the gltA gene sequences, as given by eubacterial universal primers, P8 and Pc1544 (Heller et al., Kosoy et a/. (1997), are as follows: Al, U84372; A2. 1997a). For gltA, the primers used for the amplification were U84373; A3, U84374; B1, U84375; B2, U84376; Cl, CS140f and BhCS.1137n (Birtles & Raoult, 1996). Standard U84377; C2, U84378; Dl, U84379; D2, U84380; D3, procedures to prevent sample DNA contamination were U84381; D4, U84382; D5, U84383; D6, U84384; D7, performed (Kwok & Higuchi, 1989). Negative controls were U84385. included in each experiment to check for the absence of cross-contamination between samples and between pre- viously amplified products and field samples. RESULTS Purification of the amplified product and DNA sequencing. Animals and culture The amplified DNA fragment was purified by phenol extraction and 2-propanol precipitation (Brow, 1990). Plates streaked with the blood of two of the four rats Sequencing was performed with a Thermo Sequenase had to be discarded because of heavy bacterial con- Fluorescent Labelled Primer Cycle Sequencing Kit with 7- tamination (various bacterial colonies grew within deaza-dGTP (Amersham), according to the manufacturer’s 24 h). From the other plates, small bacterial colonies

International Journal of Systematic Bacteriology 48 1334 Bartonella tribocorum sp. nov. isolated from wild rats

-~ ~ -______

strains were catalase-negative, exhibited a negative Voges-Proskauer reaction, had negative pyrazina- midase and urease activities and did not produce acid from trehalose. They did not hydrolyse bis@-nitrophenyl) phosphate and p-nitropheny1-N- acetyl-p-u-glucosaminide. The strains showed amino acid arylamidase activity with leucine, methionine, lysine (alkaline as well as acid), glycine, arginine and tryptophan, but not with proline. They had glycyl- glycylarylamidase and trypsin-like activity but no pyrrolidonylarylamidase activity.

16s rRNA gene sequence analysis Sequencing of the complete 16s rRNA gene was performed for the two strains. The two sequences obtained were identical to each other but differed from those previously determined for other Bartonella species. The species with the closest sequence hom- ology was Burtonella elisabethae which only had six different bases (0.4 YO)over the whole 16s rRNA gene. The most distant sequences were those of Fig. 1. Negative staining of strain IBS 506T. Bar, 0.25 pm. Bartonella ~___~___~____ bacillifornzis and Bartonella clarridgeiae with 29 different bases (2.1 YO).The main differences between the sequences of the two rat strains and the 16s rRNA (100 and 150 c.f.u. rnl-l, respectively) appeared 10 d gene sequences of all other known Bartonella species after inoculation. The two strains were designated IBS are presented in Table 1 and the percentage similarities 500 and IBS 506'. are presented in Table 2. Sequencing of the 16s rRNA gene of the Bartonella elizabethae type strain was Phenotypic identification also performed. The sequence obtained was slightly different from the one in the GenBank/EMBL data- The colonies appeared after 5 d subculture as white- base. In the first 150 5' bases two gaps were filled by G cream, rough colonies with a diameter of less than residues at positions 82 and 195 ( 1 mm. The colonies were embedded in the agar surface, numbering). Furthermore, an ambiguity was resolved, appeared compact and were difficult to emulsify. Light as a 'B', corresponding to G, T or C, at position microscopic examination after enhanced Gram stain- 183 (E. coli numbering) was found to be a G. These ing revealed small Gram-negative rods. Electron three bases were identical for the two wild rat strains. microscopic examination showed small bacilli without Nevertheless, four of the six bases remained different flagella hut there was evidence of polar structures between the 16s rRNA gene sequences of the wild rat resembling fimbriae in both strains (Fig. 1). The two strains and the type strain of Burtonella elizahethae.

___-~ -~-_____.~ ~~ Table 1. Major differences between the 165 rRNA genes in Bartonella

0,Bases matching the consensus; -, missing bases.

Strain Consensus sequence and position (E. coli numbering)

A-TA1A A C - T T T G T C CA A G CA A G CCTACGCGT 86 89 90 94 98 107 137 139 191 192 I93 194 195 224 263 579 646 648 657 838 868 1007 1008 1019 1020 1021 1041 1136 1151 1263 1267 1335

______~______International Journal of Systematic Bacteriology 48 1335 R. Heller and others -~

Table 2. DNA-DNA hybridization and 16s rRNA gene similarity between Bartonella species and strain IBS 506T

Strain Hybridization Hybridization Similarity to G+C with IBS with ATCC 16s rRNA content 50(jT(Yo) 49927T(%) gene (YO) (mol Oh)

1BS 506T 100 IBS 500 100 Bartonella bacillifornzis 97.9 Bartonella clurridgeiae 97.9 Bartonella doshiue NCTC 12862'r 99.2 Bartonellu elizabethae ATCC 49927T 99-6 Bartonella grahamii NCTC 12860' 99.3 Bartonella henselae ATCC 49882T 98.9 Bartonella quiiztuna ATCC VR-35ST 98-3 Burtonella taylorii NCTC 1286 lT 99.1 Bartonella vinsonii ATCC VR- 152' 99.3 Bartonella vinsonii subsp. berklzqffri 98.6 C1-phy 99-I C4-phy 98-9 C5-rat 99.8 C7-rat 99-3 N40 99.1 R-phy 1 99.3 R-phy2 99.1

-, Not tested. * Daly et ul. (1993). -t Birtles et al. (1995).

Citrate synthase gene sequence analysis dness, respectively, with strain IBS 506T. No DNA relatedness over 70% was found between DNA of A partial sequence of 332 bases in the 3' end of gltA strain IBS 50fjT and the type strains of the other was determined for the two strains. The sequences Bartonella species tested, thus confirming that the two obtained were identical to each other but differed from rat isolates represented a single species distinct from all other 3' end sequences of Bartonella species avail- the other Bartonella species previously described. able in the GenBank/EMBL database. The closest gltA sequences were those of the cluster described by Blood smear examination Birtles & Raoult (1996), including Bartonella eliza- betlzae (94.9 YOsimilarity) Bartonella grahamii (96-1YO) No intraerythrocytic Bartonella-like bacterium and two strains isolated from rats in Peru, C5-rat (Weinman & Kreier, 1977) could be observed in the (98.3 YO)and C7-rat (94.9 YO).All other gltA sequences single blood smear from the wild rat, but we identified available in the GenBank/EMBL database had lower numerous trypanosomes, probably Trypanosomu similarity (< 92 YO). lertt~iwhich is known to infect wild rodents, especially rats (Laveran & Mesnil, 19 12). However, preliminary DN A-D NA hybridi za t io n experimental infections performed on laboratory rats by intravenous injection of a suspension of strain IBS DNA of strain IBS 506' and the type strain of 506T permitted the detection of intraerythrocytic Bartonella elizabethae was labelled and tested by bacteria in blood smears, showing the ability of these DNA-DNA hybridization against unlabelled DNA of bacteria to enter the red blood cells. strains IBS 506T and IBS 500 and of the type strains of Bartonella elizabethae, Bartonella doshiae, Bartonella DISCUSSION grahamii, Bartonella henselae, Bartonella quintana, Bartonella tuylorii and Bartonella vinsonii. The results The strains isolated from the blood of two wild rats are shown in Table 2 together with G+C content. had similar phenotypic characteristics to other Barto- DNA of strains IBS 506' and IBS 500 showed 46 and nella species. (1) The bacteria were observed within 44 % relatedness, respectively, with the labelled DNA erythrocytes. (2) They grew slowly and required blood- of the type strain of Bartonella elizabetliae. Conversely, enriched medium. (3) They appeared as small, fas- DNA of strain IBS 500 and the type strain of tidious, aerobic, oxidase-negative, Gram-negative Bartonella elizabethae presented 94 and 36 YO relate- rods. (4) They grew best on blood-enriched medium in

1336 International Journal of Systematic Bacteriology 48 Bartonella tribocorun-i sp. nov. isolated from wild rats an atmosphere containing 5% CO,. (5) When com- & Kreier, 1977). These two Grahamella species were pared to the literature, the enzymic activities tested described only on the basis of rod-shaped structures were similar to those of other species of the genus observed in erythrocytes in blood smears stained with Bartonellu (Birtles et a/., 1995; Daly et a/., 1993; a Giemsa solution. Therefore, no collection type strain Lawson & Collins, 1996), with the exception of proline was available to compare our isolates to these species. aminopeptidase activity which was negative, as seen Recently, Ellis et a/. (1997) described the isolation of also for Hartonella doshiae. Proline aminopeptidase Bartonella strains from blood samples of rats (Rattus activity was negative when tested with both diagnostic norvegicus and Rattus rattus) captured in the USA, tablets and the MicroScan Rapid Anaerobe Panel. Bolivia and Paraguay. These authors found an overall However, Bartonella doshiae and the two rat strains frequency of bacteraemia of 16 %, with rates ranging could be differentiated, as Bartonella doshiae hydro- from 0 to 51 YO,depending on the region where the lysed tributyrin (Birtles et a/., 1995) whereas the rat animals were trapped. Ellis et al. (1997) sequenced part strains did not. (6) The 16s rRNA gene sequence of the of the citrate synthase gene of new Bartonella isolates two rat strains was highly similar to the 16s rRNA obtained from rats and found high similarity with the gene sequence of all known Bartonella species. The sequence of Bartonella elizabethae, ranging from 93 to closest sequence was that of the type strain of Barto- 100%. However no sequence is available in the riella elixhethae (99.6 YO similarity) and the most databases. Another study was performed on wild divergent sequences within the genus Bartonella were rodents by Kosoy et a/. (1997). The sequences of gltA the sequences of Barfonella bacilliformis and Bartonella available in the GenBank/EMBL database corre- clarridgcim (97.9 YO).Furthermore, the high level of sponding to isolates from the Kosoy study were DNA-DNA hybridization shared by the two rat compared to the sequence of strain IBS 506T: the strains (93%) confirmed that both belonged to the similarity was less than 92 YO.This value is lower than same bacterial species. The hybridization rates be- that observed for the cluster described by Birtles & tween the two Bartonella strains isolated from the rats Raoult (1996), including Bartonella elizabethae, Bar- and Bartonella elizabethae were between 36 and 46 YO, tonella grahamii and the two strains isolated from rats indicating that these strains and Bartonella elizabethae in Peru in which the similarity was more than 94.9 %. belonged to two different species. DNA-DNA hybri- Among other reports of Bartonella isolation from wild dization was performed only with the type strains of rodents, two strains were obtained from rats in Peru the other Bartonella species having a 16s rRNA gene (Birtles & Raoult, 1996). The 16s rRNA gene se- sequence with a similarity over 98 YO. Therefore, quences of these strains were likewise highly related to and Bartorwlh bucilliforrnis Bartonella clarridgeiae the sequence of Bartonella elizabethae (994 and with only 97.9 YOsimilarity and also presenting a polar 99.2 Yo). One of these two strains had a citrate synthase tuft of flagella, were not tested. Based on criteria sequence identical to that of the type strain of defining ii species (Stackebrandt & Goebel, 1994), we suggesting that this strain could conclude that the two rat strains belong to a new Bartonella elizabethae, be an isolate of Bartonella elizabethae. The 16s rRNA species within the genus Bartonella. gene sequences of these different strains are highly For an additional characterization, 16s rRNA gene related and seem to constitute a cluster within the amplification and sequencing was used. This method Bartonella genus. Our isolates, IBS 500 and IBS 506T, was selected as it allows a universal characterization of also belong to this cluster. The differences between tested strains, especially for unknown bacteria. The these sequences and those of our isolates are reported 16s rRNA gene of the two Bartonella isolates obtained in Table 1. from the two wild rats had exactly the same sequence Birtles & Raoult (1 996) concluded from their isolation which diflered from sequences of all known Bartonella of Bartonella strains from Peruvian rodents that species. The species with the nearest 16s rRNA gene Bartonella elizabethae infections in humans, like Bar- sequence was Bartonella elizabethae. Bartonella eliza- infections, were zoonotic. We have no was described in 1993 with only a single strain tonella henselae bethae indication whether the Bartonella species carried by available. isolated from a patient with endocarditis the wild rats we tested can be transmitted to and cause (Daly et a/., 1993). The determination of a partial any pathology in humans. Moreover, experimental sequence of gltA and its comparison to other known infection of laboratory rats with a suspension of the gltA sequences enhanced the genotypic relationship type strain of Bartonella elizabethae or strain IBS 506T between the rat strains and Bartonella elizabethae led to bacteraemia lasting 15 d after injection, dem- formerly demonstrated by 16s rRNA sequencing. The onstrating the ability of these animals to be infected by closest gltA sequence corresponded to the strain C5- these Bartonella species. Rats are symbiotic animals rat (98.3%) isolated from blood of a rat in Peru which can live in the vicinity of humans and could (Birtles & Raoult, 1996). therefore transmit Bartonella species they harbour to The limited sample size of this study did not allow humans, either directly or indirectly via ectoparasites. epidemiological observations. Two different Graha- Such a hypothesis is supported by several studies mella species have previously been identified from rats, which tested the prevalence of antibodies against Graharziclla joyeuxi by Brumpt in 19 1 3 and Grahamella Bartonella elizabethae in humans. Out of 600 patients crocidurrre by Schwetz and Cabu in 1930 (see Weinman from 45 different states of the USA suspected of having

~~ - hternational Journal of Systematic Bacteriology 48 1337 R. Heller and others cat scratch disease, 21 % had Bartonella elizabethae National de la Chasse team, including G. Leonhart for their antibodies (Daly et al., 1993). Among a population of help in catching the wild rats). blood donors in Seattle, 15% were seropositive for Bartonella elizabethae (Comer et al., 1996) ;in addition, REFERENCES one-third of 630 individuals tested in a population of intravenous drug users in Baltimore were positive for Birtles, R. J. & Raoult, D. (1996). Comparison of partial citrate synthase gene (gltA) sequences for phylogenetic analysis of Bartonella elizabethae (Comer et al., 1996). The poor Bartonella species. Int J Syst Bacteriol46, 89 1-897. living conditions of these drug users could put them in contact with rats contaminated with a Bartonella Birtles, R. J., Harrison, T. G., Saunders, N. A. & Molyneux, D. H. to (1995). Proposals to unify the genera Grahamella and Bartonella, species antigenically and/or genetically related with descriptions of Bartonella talpae comb. nov., Bartonella Bartonella elizabethae. Moreover, several human cat perowzysci comb. nov. and three new species, Bartonellu scratch disease cases are not serologically positive for graharnii sp. nov., Bartonella taylorii sp. nov. and Bartoizellu Bartonella henselae antigens and some cases have doshiae sp. nov. Int J Syst Bacteriol45, 1-8. occurred without any history of cat contact. The rate Breitschwerdt, E. B., Kordick, D. L., Malarkey, D. E., Keene, B., of these cases has been estimated by some authors to be Hadfield, T. L. & Wilson, K. (1995). Endocarditis in a dog due to as high as 60% of clinical cat scratch disease cases infection with a novel Bartonella subspecies. J Clin Microhiol (Dupon et al., 1996; Yoshida et al., 1996). Many 33, 154-160. Bartonella species appear to be present in our en- Brenner, D. J., O’Connor, 5. P., Winkler, H. H. & Steigerwalt,A. G. vironment (Heller et d., 1997b) and should be carefully (1993). Proposals to unify the genera Bartonella and Rocha- investigated. These investigations should focus on limaea, with descriptions of Bartonella quintana comb. nov., updating diagnostic tools to determine the importance Bartonella vinsonii comb. nov., Bartonella henselae comb. nov. of these Bartonella species in human and animal and Bartonella elizabethae comb. nov. and to remove the family pathology. Bartonellaceae from the order . Int J Syst Bacteriol 43, 777-786. Brow, M. A. (1990). Sequencing with DNA polymerase. In Description of Bartonella tribocorum sp. nov. Taq PCR Protocols: A Guide to Methods and Application, pp. Bartonella tribocorum [tri.bo.co’rum. L. n. gen. pl. 189-196. Edited by M. A. Innis, D. H. Gelfand, J. J. Sninsky & Triboci named after the tribes mentioned by Caesar (51 T. J. White. San Diego, CA: Academic Press. BC) in his Cornmentarii de Bello Gallico; these tribes Caesar, C. J. (51 BC). Bellum Gallicum, Book 11, paragraph L1, where living in the region near the Rhine river in the 2nd sentence. Rome : Roman Republic. eastern part of present day France where the wild rats, Comer, 1. A., Flynn, C., Regnery, R. L., Vlahov, D. & Childs, 1. E. from which the two strains were isolated, were trap- (1996). Antibodies to Bartonella species in inner-city intra- venous drug users in Baltimore, Md. Arch Intern Med 156, ped] * 2491-2495. Colonies grown on blood agar appear as small, white, Daly, J. S., Worthington, M. G., Brenner, D. J. & 7 other authors smooth, regular colonies (about 1 mm diam.) after (1993). Rochalimaea elizabethae sp. nov. isolated from a patient 10 d. Electron microscopic examination shows small with endocarditis. J Clin Microbiol31, 872-88 1. bacilli without flagella, approximately 1-2 pm long by Dupon, M., Delarclause, A. M. 5.. Brouqui, P., Drancourt, M., 0.5 pm wide. Gram-negative rods, aerobic and oxi- Raoult, D., Demascarel, A. & Lacut, J. Y. (1996). Evaluation of dase-negative. Best growth on blood-enriched medium serological response to Bartonella henselae, Bartonella quintana in a moist atmosphere containing 5 % CO,. Negative and Afipiafelis antigens in 64 patients with suspected cat scratch for catalase, Voges-Proskauer reaction, pyrazi- disease. Scand J Infect Dis 28, 361-366. namidase and urease activities and acidification of Ellis, B., Regnery, R., Maraton, E., Jones, D., Olson, J., Glass, G. E. trehalose. bis(p-Nitrophenyl) phosphate and p-nitro- & Childs, J. E. (1997). Recovery and characterization of Barto- phenyl-N-acetyl-P-D-glucosaminide not hydrolysed. nella from the genus Rattus. In Thirteenth Sesqui-Annual Amino acid arylamidase activity with leucine, meth- Meeting of the American Society for Rickettsiology, abstract 2 I. ionine, lysine (alkaline as well as acid), glycine, arginine Champion, PA : American Society for Rickettsiology . and tryptophan, but not proline. Glycylglycylary- Fusseneger, M., Kahrs, A. F., Facius, D. & Meyer, T. F. (1996). lamidase and trypsin-like activity but no pyrrolidony- Tetrapac (tpc), a novel genotype of Neisseria gonorrhoea larylamidase activity. Distinguishable from other affecting epithelial cell invasion, natural transformation com- Bartonella species by its ability to hydrolyse trypsin petence and cell separation. Mol Microbioll9, 1357-1372. but not proline and tributyrin, its 16s rRNA gene Grimont, P. A. D., Popoff, M. Y., Grimont, F., Coynault, C. & sequence and by whole-DNA hybridization. Type Lemelin, M. (1980). Reproductibility and correlation study of strain is IBS 506T isolated from the blood of a wild rat three deoxyribonucleic acid hybridization procedures. Curr (Rattus norvegicus). Deposited in the Collection des Microbiol4, 325-330. Bacteries de 1’Institut Pasteur as CIP 105476T. Gurfield, N., Boulouis, H. J., Chomel, B., Heller, R., Larsen, R. W., Yamamoto, K. & Piemont, Y. (1997). Co-infection by Bartonella clarridgeiae and Bartonella henselae and different Bartonellu ACKNOWLEDGEMENTS henselae strains in domestic cats. J Clin Microbiol 35, 2110- We thank C. Fritsch, S. Niedergang, B. Pichler-Brand and 21 13. C. Tosch for technical assistance and F. Decup for historical Heller, R., Artois, M., Xemar, V., De Briel, D., GBhin, H., Jaulhac, research on C. J. Caesar’s writings. We also thank the Office B., Monteil, H. & PiBmont, Y. (1997a). Prevalence of Bartonella

1338 International Journal of Systematic Bacteriology 48 Bartonella tribocovurn sp. nov. isolated from wild rats ____ .~- henselae and Bartonella clarridgeiue in stray cats. J Clin Laveran & F. Mesnil. Paris: Masson et C", Libraires de Microbiol35, 1327-1 33 1. 1'Academie de Midecine. Heller, R., Kubina, M., Delacour, G., Mahoudeau, I., Lamarque, F., Lawson, P. A. & Collins, M. D. (1996). Description of Bartonella Artois, M., Monteil, H., Jaulhac, B. & PiBmont, Y. (1997b). clarridgeiue sp. nov. isolated from the cat of a patient with Prevalence of Bartonella spp. in blood of wild small rodents. In Bartonella henselae septicemia. Med Microbiol Lett 5, 64-73. Program ond Abstracts of the 97th General Meeting of the Riegel, P., de Briel, D., Prhost, G., Jehl, F. & Monteil, H. (1994). American Society of Microbiology. Genomic diversity among Corynebacteriumjeikeium strains and Higgins, D. G. & Sharp, P. M. (1988). CLUSTAL: a package for comparison with biochemical characteristics and antimicrobial performing multiple sequence alignment on a microcomputer. susceptibilities. J Clin Microbiol32, 1860-1 865. Gene 73, 237-244. Riegel, P., Ruimy, R., de Briel, D., Prkvost, G., Jehl, F., Christen, R. Kosoy, M. Y., Regnery, R. L., Tzianabos, T., Marston, E. L., Jones, & Monteil, H. (1995). Corynebacterium seminale sp. nov. a D. C., Green, D., Maupin, G. O., Olson, J. G. & Childs, 1. E. (1997). species associated with genital infections in male patients. J Clin Distribution, diversity and host specificity of Bartonella in Microbiol32, 22442249. rodents from the Southeastern United States. AmJ Trop Med Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a Hyg 57, 578-588. place for DNA-DNA reassociation and 16s rRNA sequence Kwok, S. & Higuchi, R. (1989). Avoiding false positives with analysis in the present species definition in bacteriology. Int J PCR. Ntrture 339, 237-238. Syst Bacteriol44, 846849. Larson, A. M., Dougherty, M. J., Nowowiejski, D. J., Welch, D. F., Weinman, D. & Kreier, 1. P. (1977). Bartonellu and Grahamella. In Matar, G. M., Swaminathan, B. & Coyle, M. B. (1994). Detection Parasitic Protozoa, pp. 197-233. Edited by J. P. Kreier. of Bartonellu (Rochalimaea)quintana by routine acridine orange London : Academic Press. staining of broth blood cultures. J Clin Microbiol32,1492-1496. Yoshida, H., Kusaba, N., Omachi, K., Miyazaki, N., Yamawaki, M., Laveran, A. & Mesnil, F. (1912). Trypanosomes non pathogenes Tsuji, Y., Nakahara, K., Sumino, M., Noudomi, M., Shimokawa,Y. des petits Inammifires. Tr. lewisi, parasite special du rat. In & Tanikawa, K. (1996). Serological study of Bartonella henselae Trypanosoiizes et Trypanosomiases, pp. 249-303. Edited by A. in cat scratch disease in Japan. Microbiol Irnmuno140,671-673.

~ ~~ International Journal of Systematic Bacteriology 48 1339