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1 UMR 956 INRA/AFSSA/ENVA/UPVM, Microbiologie, Ecole Nationale Vétérinaire, 7 Avenue du Général de Gaulle, 94 704 Maisons-Alfort, France
2 Institut de Bactériologie de la Faculté de Médecine, Université Louis Pasteur, Hôpitaux Universitaires de Strasbourg, 3 rue Koeberlé, 67 000 Strasbourg, France
3 Unité de Pathologie du Bétail, Ecole Nationale Vétérinaire, 7 Avenue du Général de Gaulle, 94 704 Maisons-Alfort, France
Correspondence
Henri-Jean Boulouis
hjboulouis{at}vet-alfort.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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The GenBank accession numbers for the 16S rRNA and 3' end gltA sequences of Bartonella chomelii are AY254309 and AY254308, respectively.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Bartonella species are small Gram-negative bacteria, often piliated or flagellated. They are intracellular parasites of red blood cells and endothelial cells. In reservoir hosts, Bartonella infections usually result in asymptomatic bacteraemia. This bacteraemic phase is required for the long-term perpetuation of the bacterium, allowing its transmission by blood-sucking arthropods (Breitschwerdt & Kordick, 2000; Chomel et al., 1996; Alexander, 1995). Some species of Bartonella are able to infect not only reservoir hosts, but also accidental hosts for which the bacterium is not adapted or poorly adapted. In this case, infections are associated with human or animal diseases including cat scratch disease (CSD), bacillary angiomatosis, peliosis hepatitis and endocarditis, in either immunocompetent or immunocompromised patients (Breitschwerdt & Kordick, 2000).
To date, molecular characterization of Bartonella species has included PCR amplification of protein-coding genes (Houpikian & Raoult, 2001a), as well as the 16S rRNA gene (Birtles, 1995) and the 16S–23S rRNA intergenic spacer region (ITS) (Jensen et al., 2000). The degree of identity of these genes among Bartonella species has then been assessed both indirectly, by using RFLP analysis (Birtles, 1995; Joblet et al., 1995; Norman et al., 1995; Houpikian & Raoult, 2001b), and directly by base sequence comparisons. While 16S rDNA has been shown to be a powerful tool for appreciating genetic variability among Bartonella species, the protein-encoding genes appear to be good indicators of interspecies divergence and RFLP-PCR on the gltA gene is often used to identify Bartonella species in biological samples (La Scola et al., 2003).
Recently, three Bartonella species have been isolated from ruminants. Two of these (Bartonella schoenbuchensis and Bartonella capreoli) were recovered from wild roe deer (Capreolus capreolus) (Dehio et al., 2001; Bermond et al., 2002), and a third one (Bartonella bovis) from domestic cattle (Bermond et al., 2002). All of these three species appear to be adapted to ruminants. In view of the potential zoonotic pathogenicity of Bartonella and the high exposure of humans to ruminants through consumption of bovine-derived products or hunting practices, the risk of zoonotic transmission of ruminant-derived Bartonella to humans should not be underestimated.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Bacterial strains.
One hundred and fifty bacterial strains isolated from 75 cows were identified using PCR amplification followed by analysis of the RFLP-profile of the gltA gene (citrate synthase) using TaqI, MseI, HhaI and DdeI restriction enzymes as described previously (Birtles & Raoult, 1996; Norman et al., 1995), according to the manufacturer's instructions (New England Biolabs).
Electron microscopy.
Bacteria were grown on Columbia base agar (bioMérieux) plates containing 5 % fresh defibrinated rabbit blood for 5 days. Bacteria were then suspended in PBS, spread on a water surface and absorbed to Formvar-coated nickel grids, stained with 1 % (w/v) uranyl acetate and air-dried (Fusseneger et al., 1996). Samples were examined with a JEOL 100CX2 electron microscope.
Biochemical analysis.
Bartonella species are biochemically inert in almost all conventional tests. Biochemical assays performed with diagnostic tablets (Rosco; Eurobio) were as follows: Voges–Proskauer reaction, tributyrin hydrolysis, pyrazinamidase, proline aminopeptidase and trypsin-like activity. Commercially available reagents (Rapid ID 32 A; bioMérieux) were used to test the activity of bacterial enzymes according to the manufacturer's instructions.
Reference strains used for DNA–DNA hybridization.
Bartonella grahamii (V2T, ATCC 49927T), Bartonella clarridgeiae (Houston-2T, ATCC 51734T), Bartonella elizabethae (F9251T, ATCC 49927T), Bartonella henselae (Houston-1 G5436T, ATCC 49882T), Bartonella koehlerae (C29T, ATCC 700693T), Bartonella quintana (FullerT, ATCC VR-358T), Bartonella doshiae (R18T, ATCC 700133T), Bartonella vinsonii subsp. vinsonii (BakerT, ATCC VR-152T), Bartonella vinsonii subsp. berkhoffii (ATCC 51672T) and Bartonella bacilliformis (ATCC 35685T) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Bartonella taylorii (M6T, CIP 107028T) was purchased from the Collection of the Pasteur Institute (CIP, Paris, France). Bartonella alsatica (IBS 382T, CIP 105477T), Bartonella birtlesii (IBS 325T, CIP 106294T), Bartonella bovis (91-4T, CIP 106692T), Bartonella capreoli (IBS 193T, CIP 106691T), Bartonella schoenbuchensis (R1T, NCTC 13165T) and Bartonella tribocorum (IBS 506T, CIP 105476T) were kindly given by one of the authors.
DNA–DNA hybridization.
DNA extraction and purification were performed as described by Riegel et al. (1994). DNA hybridization between labelled DNA and unlabelled fragmented DNAs was performed at 58 °C for 16 h in 0·42 M NaCl using the nuclease/trichloroacetic method (Grimont et al., 1980; Riegel et al., 1994). When the percentage hybridization was above 30 %, experiments were performed in triplicate. Therefore, the given value represents the mean of triplicate values±standard deviation.
Amplification and sequencing of the 16S rRNA and citrate synthase genes.
A fragment of approximately 1500 bp of the 16S rRNA gene was amplified from the extracted DNA by using eubacterial universal primers specific for 16S rRNA gene: P8 (5'-AGAGTTTGATCCTGGCTCAG-3') and Pc1544 (5'-AAGGAGGTGATCCAGCCGCA-3') (Heller et al., 1997). PCR amplification of a 350 bp fragment of the citrate synthase gene was performed as described by Norman et al. (1995) with primers BhCS.781p (5'-GGGGACCAGCTCATGGTGG-3') and BhCS.1137 (5'-AATGCAAAAAGAACAGTAAACA-3') (Norman et al., 1995). The presence and size of the amplicons were verified by electrophoresis on a 1·5 % agarose gel, followed by ethidium bromide staining and visualization on a UV transilluminator.
Amplified fragments were sent to Genome express (Meylan, France) for sequencing. Sequencing of the almost complete 16S rRNA gene was performed bidirectionally, using three primer pairs: P8 and PC535 (5'-GTATTACCGCGGCTGCTGGCA-3'), P350F (5'-CTCCTACGGGAGGCAGCAGT-3') and P785R (5'-GGGTATCTAATCC-3'), and P535F (5'-TGCCAGCAGCCGCGGTAATAG-3') and Pc1544.
Sequence alignment and tree construction.
The 16S rRNA and gltA sequences obtained in this study were aligned with sequences from other Bartonella species first with CLUSTAL X software (version 1.63b, December 1997; Thompson et al., 1997) and then by visual optimization. The alignments are available upon request. Nucleic acid sequences of gltA were then translated into amino acid sequences. The aligned sequences were converted to a distance matrix (percentage differences without weighting). A distance tree was generated using the neighbour-joining method (Saitou & Nei, 1987). Both the branch-and-bound and heuristic search options in the PAUP program version 4.0 (Swofford, 1993) were used for comparison of sequence alignments and generation of parsimonious trees. The statistical support of the internal branches from the resulting trees was statistically tested by bootstrap analysis (Felsenstein, 1985) from 500 bootstrap replications. The list of the Bartonella species used for the phylogenetic analysis is indicated in Fig. 2 and the 16S rDNA tree is available as supplementary data in IJSEM Online. The 16S rRNA and the gltA sequences of Rickettsia rickettsii were used as outgroups.
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and the 16S rDNA tree available as supplementary data in IJSEM Online. |
RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) and characterized by the presence of restriction sites for TaqI and MseI, but not HhaI and DdeI, and a second RFLP profile different from that of other known Bartonella species and characterized by the presence of restriction sites for MseI, but not TaqI, HhaI and Dde (Table 1).
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The high prevalence of Bartonella bovis bacteraemia in domestic cattle in the absence of any apparent pathological effect strongly suggests that domestic cows are a natural reservoir of Bartonella bovis (Chang et al., 2001). In contrast, the low prevalence of this new Bartonella species in cattle suggests that this animal species might not be its natural reservoir, but rather an accidental host. If this hypothesis proves to be true, the potential for pathogenicity of A828T in cows would warrant further investigation. Therefore, these two strains (A828T and A12.90) sharing the same new RFLP profile and originating from two different herds were selected for further species characterization.
Phenotypic characterization
The A828T and A12.90 isolates displayed similar morphological characteristics. They both grew slowly (10 days for primary isolation), required CO2 and produced smooth colonies, 2–3 mm in diameter with straight and slender Gram-negative bacilli. Both of them were able to grow on rabbit blood and sheep blood agar plates, but with a higher efficiency on rabbit blood agar plates. Like other Bartonella species, they were oxidase- and catalase-negative, had a negative Voges–Proskauer reaction and a positive reaction for tributyrin. Results of amino acid arylamidase activity, using the rapid ID 32A system, are given in the species description below.
Electron microscopic examination of a subculture of isolate A828T revealed bacilli with multiple unipolar flagella (Fig. 1).
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For gltA sequences, A828T and A12.90 had identical 3' end sequences, which differed from those of the 18 other type strains of Bartonella and the other unidentified Bartonella isolates contained in the EMBL/GenBank database.
As for the 16S rRNA gene, the 3'-end sequence of gltA of A828T was closest to species isolated from ruminants, i.e. Bartonella schoenbuchensis (96·2 %), Bartonella capreoli (94·8 %) and Bartonella bovis (92·9 %). The most distant gltA gene sequence was that of Bartonella bacilliformis (82·0 %). These data, together with the culture characteristics (colonial morphology, duration of bacterial growth, microscopic appearance of the bacteria) and the recovery of the bacteria from the blood of cows, all supported the classification of these two isolates within the genus Bartonella.
To investigate whether they defined a new Bartonella species, DNA–DNA hybridization was performed.
DNA–DNA hybridization
DNA of isolate A828T was labelled and hybridized against unlabelled DNA of A12.90 and 16 type/reference strains. The extent of hybridization between A828T DNA with heterologous DNA was 78 % with isolate A12.90, 56·5 % with Bartonella schoenbuchensis, 52 % with Bartonella capreoli, 31·5 % with Bartonella bovis and less than 20 % with other Bartonella species. Considering that when DNA–DNA hybridization exceeds 70 % two isolates belong to the same species, a novel species was identified (Grimont et al., 1980). A828T and A12.90 were shown to belong to the same species since their degree of hybridization exceeded 70 %. Isolate A828T, though phylogenetically close to other Bartonella ruminant-derived species, was clearly different from them. Among the ruminant-derived Bartonella species, the new species appeared to be more closely related to species recovered from wild ruminants (Bartonella capreoli and Bartonella schoenbuchensis) than to Bartonella bovis.
Sequence alignment and tree construction
The comparison of aligned 16S rRNA sequences was carried out on 1318 positions, including gaps, for a total of 20 taxa. Among those positions, 1090 were constant characters and only 38 were parsimony-informative characters. The phylogenetic analysis inferred from the 16S rRNA sequence comparison clearly demonstrated that A828T clustered with other ruminant-derived Bartonella species, Bartonella capreoli, Bartonella schoenbuchensis and Bartonella bovis (Bermond et al., 2002) (available as supplementary data in IJSEM Online). Phylogenetic analyses, neighbour-joining, and heuristic and branch-and-bound searches produced topologically similar trees, although statistical support for the branches varied (data not shown). All analyses distinguished four monophyletic groups; the first comprised A828T along with the three already described Bartonella species from ruminants, the second contained Bartonella taylorii, Bartonella birtlesii, Bartonella doshiae and Bartonella vinsonii subsp. vinsonii, the third was composed of Bartonella koehlerae and Bartonella henselae and the fourth contained Bartonella tribocorum and Bartonella elizabethae. These groups were clearly separated with branches supported by 79, 59, 79 and 82 % bootstrap values, respectively. The comparison of aligned citrate synthase sequences was carried out on 289 characters. Among these, 142 were constant and 81 were parsimony-informative characters. The phylogenetic analyses inferred from gltA amino acid sequences were in accordance with those inferred from 16S rRNA sequences (Fig. 2). The new species clustered with Bartonella capreoli, Bartonella bovis and Bartonella schoenbuchensis in a group supported by a 96 % bootstrap value. Additional groupings could be distinguished and the species Bartonella bacilliformis was in a paraphyletic position.
Description of Bartonella chomelii sp. nov.
Bartonella chomelii (cho.mel'i.i. N.L. gen. n. chomelii in honour of Bruno B. Chomel, who was the first to experimentally demonstrate the transmission of Bartonella henselae by fleas in cats, and to isolate Bartonella bovis from domestic cattle. His studies on Bartonella infection in animals contributed to an improved understanding of epidemiology and vectors of Bartonella-associated disease in animals.)
Colonies, grown on rabbit blood agar, appeared homogeneous, smooth, round and grey–white. Electron microscopic examination revealed small bacilli with unipolar flagellae. A primary culture strain of Bartonella chomelii was obtained after 10 days incubation at 35 °C in a moist atmosphere containing 5 % CO2. A subculture on blood agar was obtained after 6 days culture under the same conditions. The two strains tested were oxidase- and catalase-negative, had negative Voges–Proskauer reactions and had a positive reaction to tributyrin. Amino acid arylamidase activity was obtained with the following amino acids: arginine (ArgA), proline (ProA), phenylalanine (PheA), leucine (LeuA), tyrosine (TyrA), alanine (AlaA) and glycine (GlyA). The new species was distinguished from other Bartonella species by its 16S rRNA gene and gltA gene 3' end sequences as well as by whole-DNA hybridization. The type strain, A828T (isolated from the blood of Bos taurus), has been deposited in the CIP, Paris, France (CIP 107869T) and in the Culture Collection of the University of Göteborg, Sweden (CCUG 47497).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Bermond, D., Boulouis, H. J., Heller, R., Van Laere, G., Monteil, H., Chomel, B. B., Sander, A., Dehio, C. & Piemont, Y. (2002). Bartonella bovis Bermond et al. sp. nov. and Bartonella capreoli sp. nov., isolated from European ruminants. Int J Syst Evol Microbiol 52, 383–390.[Abstract]
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Chang, C. C., Hayashidani, H., Pusterla, N., Kasten, R. W., Madigan, J. E. & Chomel, B. B. (2002). Investigation of Bartonella infection in ixodid ticks from California. Comp Immunol Microbiol Infect Dis 25, 229–236.[CrossRef][Medline]
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