Bartonella australis sp. nov. from Kangaroos, Australia

To the Editor: During April–May 1999, 3 Bartonella isolates (AUST/NH1, AUST/NH2, AUST/NH3) were cultivated and established from the blood of 5 Macropus giganteus gray kangaroos from central coastal Queensland, Australia. We used multigene sequencing to evaluate whether these Bartonella isolates fulfill the minimum requirements for classification as a new species. 
 
DNA from each Bartonella isolate was extracted by using the QIAamp tissue kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Partial PCR amplification and sequencing of the genes encoding the 16S rDNA (rrs), citrate synthase (gltA), β-subunit of the RNA polymerase (rpoB), and cell division protein (ftsZ), as well as for the 16S–23S rDNA intergenic spacer (ITS) were attempted by using previously described primers and conditions (1). Bartonella sp. isolates AUST/NH1 to AUST/NH3 exhibited identical sequences for all 4 genes and the spacer studied, and isolate AUST/NH1 was selected as type strain among kangaroo isolates. Similarity rates between strain Aust/NH1 and validated Bartonella species (Appendix Table) ranged from 84.7% to 91.6%, from 97.5% to 98.5%, from 79.6% to 87.2%, from 85.4% to 95.0%, and from 83.5% to 87.1% for the ITS and rrs, gltA, rpoB, and ftsZ genes, respectively. Therefore, for each of these 4 genes or the spacer, strain AUST/NH1 exhibited similarity rates with all other species lower than the cutoffs published to classify Bartonella isolates within a validated species (1). It may thus be regarded as a new species. 
 
To estimate the genomic G+C content of strain AUST/NH1, we amplified and sequenced its ftsY gene as described (2) by using the BartftsyF (5′-ATGACAAAAYCYTTTATMAA-3′) and BartftsyR (5′-TCATGAGTGTCTTCCTGC-3′) primers. The ftsY G+C content was 37.7%; the calculated genomic G+C content was 39.51%. The ftsY sequence was deposited in GenBank under accession no. {"type":"entrez-nucleotide","attrs":{"text":"DQ538398","term_id":"109649511","term_text":"DQ538398"}}DQ538398. 
 
The phylogenetic relationships among the studied bartonellae were inferred from sequence alignments of each gene and from concatenated gene sequences by using the maximum parsimony and neighbor-joining methods within the MEGA version 2.1 software package (3) and the maximum-likelihood method within the PHYLIP software package (4). Using rrs, gltA, and rpoB sequences, the phylogenetic position of strain AUST/NH1 was supported by bootstrap values <70%. In contrast, by using the ITS, ftsZ, and concatenated sequences, strain AUST/NH1 clustered with a group of B. tribocorum, B. grahamii, and B. elizabethae, with elevated bootstrap values according to the 3 analysis methods (Figure). 
 
 
 
Figure 
 
Unrooted dendrogram showing the phylogenetic position of Bartonella sp. strain AUST/NH1 among Bartonella species inferred from the comparison of concatenated sequences from the rrs, gltA, intergenic spacer, rpoB, and ftsZ genes by the neighbor-joining ... 
 
 
 
The Bartonella strains we describe are the first, to our knowledge, obtained from kangaroos and, more generally, from marsupials. Before this study, the only 2 Bartonella species found in Australia were B. henselae (5) and B. quintana (6). We demonstrated that strain AUST/NH1 was reliably associated with a well-established cluster, including the rodent-associated B. elizabethae, B. grahamii, and B. tribocorum (7). Therefore, we are confident that the phylogenic position of the new Bartonella, which was similar according to 3 analysis methods and supported by high bootstrap values, is reliable. Although B. grahamii (8) and B. elizabethae (9), members of the same phylogenetic cluster as strain AUST/NH1, cause human infections, the pathogenicity of B. tribocorum is as yet unknown. Its pathogenicity should therefore be investigated, especially for persons who come in contact with kangaroos. 
 
B. australis is a facultative intracellular gram-negative bacterium. It grows on Columbia agar with 5% sheep blood at 32°C to 37°C in a moist atmosphere containing 5% CO2. A primary culture was obtained after 7 days, and subculture was obtained after 4 days under the same conditions. Colonies are homogeneous, smooth, round, and gray-white. The 3 strains tested were oxidase negative, catalase negative, and nonmotile. Pathogenicity for humans is, as yet, unknown. 
 
The type strain is strain AUST/NH1. The new species is distinguished from other Bartonella species by its 16S rRNA, gltA, rpoB, ftsZ gene sequences, as well as its 16S–23S rRNA ITS sequence. The estimated G+C content is 38%. The type strain exhibits a specific serotype (10) and was susceptible to amoxicillin, ceftriaxone, imipenem, erythromycin, clarithromycin, ofloxacin, ciprofloxacin, rifampin, and tetracycline (unpub. data). The type strain AUST/NH1 has been deposited in the Collection of the World Health Organization Collaborative Center for Rickettsioses, Borrelioses and Tick-borne Infections (CSUR), Marseille, France, under reference CSUR B1; in the Collection de l’Institut Pasteur (CIP) under reference CIP 108978T; and in the Culture Collection of the University of Goteborg (CCUG), Sweden, under reference CCUG 51999. The strains AUST/NH2 and AUST/NH3 have been deposited in CSUR under references CSUR B2 and CSUR B3, in the CIP under references CIP 108980 and CIP 108979, and in CCUG under references CCUG 52000 and CCUG 52001, respectively.

DNA from each Bartonella isolate was extracted by using the QIAamp tissue kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Partial PCR amplifi cation and sequencing of the genes encoding the 16S rDNA (rrs), citrate synthase (gltA), β-subunit of the RNA polymerase (rpoB), and cell division protein (ftsZ), as well as for the 16S-23S rDNA intergenic spacer (ITS) were attempted by using previously described primers and conditions (1). Bartonella sp. isolates AUST/NH1 to AUST/NH3 exhibited identical sequences for all 4 genes and the spacer studied, and isolate AUST/NH1 was selected as type strain among kangaroo isolates. Similarity rates between strain Aust/NH1 and validated Bartonella species (online Appendix Table, available from www.cdc.gov/EID/ content/13/12/1961-appT.htm) ranged from 84.7% to 91.6%, from 97.5% to 98.5%, from 79.6% to 87.2%, from 85.4% to 95.0%, and from 83.5% to 87.1% for the ITS and rrs, gltA, rpoB, and ftsZ genes, respectively. Therefore, for each of these 4 genes or the spacer, strain AUST/NH1 exhibited similarity rates with all other species lower than the cutoffs published to classify Bartonella isolates within a validated species (1). It may thus be regarded as a new species.
The phylogenetic relationships among the studied bartonellae were inferred from sequence alignments of each gene and from concatenated gene sequences by using the maximum parsimony and neighbor-joining methods within the MEGA version 2.1 software package (3) and the maximumlikelihood method within the PHYLIP software package (4). Using rrs, gltA, and rpoB sequences, the phylogenetic position of strain AUST/NH1 was supported by bootstrap values <70%. In contrast, by using the ITS, ftsZ, and concatenated sequences, strain LETTERS AUST/NH1 clustered with a group of B. tribocorum, B. grahamii, and B. elizabethae, with elevated bootstrap values according to the 3 analysis methods (Figure).
The Bartonella strains we describe are the fi rst, to our knowledge, obtained from kangaroos and, more generally, from marsupials. Before this study, the only 2 Bartonella species found in Australia were B. henselae (5) and B. quintana (6). We demonstrated that strain AUST/NH1 was reliably associated with a well-established cluster, including the rodent-associated B. elizabethae, B. grahamii, and B. tribocorum (7). Therefore, we are confi dent that the phylogenic position of the new Bartonella, which was similar according to 3 analysis methods and supported by high bootstrap values, is reliable. Although B. grahamii (8) and B. elizabethae (9), members of the same phylogenetic cluster as strain AUST/NH1, cause human infections, the pathogenicity of B. tribocorum is as yet unknown. Its pathogenicity should therefore be investigated, especially for persons who come in contact with kangaroos.
B. australis is a facultative intracellular gram-negative bacterium. It grows on Columbia agar with 5% sheep blood at 32°C to 37°C in a moist atmosphere containing 5% CO 2 . A primary culture was obtained after 7 days, and subculture was obtained after 4 days under the same conditions. Colonies are homogeneous, smooth, round, and gray-white. The 3 strains tested were oxidase negative, catalase negative, and nonmotile. Pathogenicity for humans is, as yet, unknown.
The type strain is strain AUST/ NH1. The new species is distinguished from other Bartonella species by its 16S rRNA, gltA, rpoB, ftsZ gene sequences, as well as its 16S-23S rRNA ITS sequence. The estimated G+C content is 38%. The type strain exhibits a specifi c serotype (10) and was susceptible to amoxicillin, ceftriaxone, imipenem, erythromycin, clarithromycin, ofl oxacin, ciprofl oxacin, rifampin, and tetracycline (unpub. data).

Q Fever in Migrant Workers, Scotland
To the Editor: Q fever is a zoonosis caused by infection with Coxiella burnetii and is most commonly associated with occupational exposure to animal-slaughtering facilities. C. burnetii is an obligate intracellular bacterium and causes highly variable disease, ranging from asymptomatic infection to fatal chronic infective endocarditis. In June 2006, the United Kingdom experienced its largest outbreak of Q fever with 138 cases associated with a slaughterhouse near Stirling in Scotland. The slaughterhouse had been processing post-parturition ewes in the lairage (place for keeping livestock temporarily) at the end of May. These animals were thought to be among the most likely to shed the organism (1). Further investigation showed that a ewe had aborted in the lairage toward the end of May. Although the sheep lairage was the most likely source of the infection, no microbiologic evidence confi rmed this, as C. burnetii was not isolated from environmental samples.
The outbreak was neither remarkable for its putative mode of transmission nor for the industry involved, but both the number and nationalities of migrant workers infected was noteworthy. Since 2004, 12 member states have joined the European Union and this has led to an infl ux of immigrants to the United Kingdom. The increase in migrant numbers has partly been a result of the government's managed migration policy, expanding migration to fi ll vacancies in skilled and lowwage occupations. Employers have diffi culty recruiting UK workers because of the jobs' physical demands, long hours that limit social activities, and low pay. They therefore recruit international workers with a good work ethic and reliability; central and Eastern European workers are compared favorably with UK nationals (2). Migrants from Eastern and central Europe are now more likely to be found in low-wage occupations in agriculture, construction, hospitality, and au pair employment. Of the 138 cases of Q fever, 48 were immigrants from the following countries: Slovakia (41), Poland (3), Czech Republic (2), and Lithuania (2). Unsurprisingly, epidemiologic case interviews were beset with linguistic and logistic problems.
The diagnosis of Q fever relies predominantly on its serologic legacy since asymptomatic seroconversion occurs in up to 60% of patients (3). Analysis of our cohort found that non-UK patients were signifi cantly less likely than their UK counterparts to have symptoms (fever, muscle pain, joint pain, headache, and cough) and to subsequently have Q fever confi rmed (Table, p<0.001). Twenty-two patients (15 UK, 7 non-UK) did not complete epidemiologic questionnaires and were therefore not included in this analysis.
Furthermore, analysis of cases registered with general practitioners (GPs) identifi ed a signifi cant difference (Table, p<0.001) between UK and non-UK patients with the latter group less likely to be registered with a GP. Although most UK residents were registered with a general practice, only 11 of 43 non-UK cases were registered. Information on GP registration was not known for 17 patients, and these were not included in the analysis.
Although the investigating health board took stringent steps to ensure follow-up of all patients, we believe that some asymptomatic non-UK patients may have permanently returned to their native countries with undiagnosed illness, and subsequently, cannot be traced. This unfortunate scenario has potentially catastrophic implications for these patients because proper follow-up clinical management of Q fever is necessary to prevent possible endocarditis (4), unnecessary surgery, and premature death.
Persons with known occupational hazards have benefi ted from an effective Q fever vaccine; abattoir workers and farmers are routinely vaccinated