Molecular Detection of Bartonella Species Infecting Rodents in Slovenia

Molecular detection of Bartonella species infecting rodents in Slovenia Natasˇa Knap1, Darja Duh1, Richard Birtles2, Tomi Trilar3, Miroslav Petrovec1 & Tatjana Avsˇicˇ-Zˇ upanc1

1Institute of Microbiology and Immunology, Medical Faculty, Ljubljana, Slovenia; 2Centre for Comparative Infectious Diseases, Faculty of Veterinary Science, University of Liverpool, Cheshire, UK; and 3Slovenian Museum of Natural History, Ljubljana, Slovenia

Correspondence: Tatjana Avsˇicˇ-Zˇ upanc, Abstract Institute of Microbiology and Immunology, Downloaded from https://academic.oup.com/femspd/article/50/1/45/505104 by guest on 01 October 2021 Medical Faculty, Zalosˇka 4, 1000 Ljubljana, Rodents, collected in three zoogeographical regions across Slovenia, were tested for Slovenia. Tel.: 1386 1 543 7450; the presence of bartonellae using direct PCR-based amplification of 16S/23S rRNA fax: 1386 1 543 7401; gene intergenic spacer region (ITS) fragments from splenic DNA extracts. e-mail: [email protected] Bartonella DNA was detected in four species of rodents, Apodemus flavicollis, Apodemus sylvaticus, Apodemus agrarius and Clethrionomys glareolus, in all three Received 21 November 2006; revised 27 zoogeographic regions at an overall prevalence of 40.4%. The prevalence of December 2006; accepted 15 January 2007. infection varied significantly between rodent species and zoogeographical regions. First published online 21 March 2007. Comparison of ITS sequences obtained from bartonellae revealed six sequence variants. Four of these matched the ITS sequences of the previously recognized DOI:10.1111/j.1574-695X.2007.00226.x species, Bartonella taylorii, Bartonella grahamii, Bartonella doshiae and Bartonella

Editor: Ewa Sadowy birtlesii, but one was new. The identity of the bartonellae from which the novel ITS sequences was obtained were further assessed by sequence analysis of cell division Keywords protein-encoding gene (ftsZ) fragments. This analysis demonstrated that the strain Bartonella ; rodents; direct molecular is most likely a representative of possible new species within the genus. characterization; Slovenia.

represent additional rodent-associated species but, to date, Introduction they have only been partially characterized, usually on the Until 1995, parasites from the family Bartonellaceae of non basis of sequence analysis of citrate synthase-encoding gene human mammalian hosts, who were thought to be of no (gltA) fragments (Bermond et al., 2000; Birtles et al., 2001; medical relevance, formed a species called Grahamella. The Holmberg et al., 2003; Engbæk & Lawson, 2004; Tea et al., other species in the family was Bartonella, and these were 2004). considered human pathogens. Because of their fastidious nature, bartonellae are difficult The application of polyphasic methods to characterize to differentiate on the basis of phenotypic properties; for and compare members of each taxon indicated that they example, all are more or less inert in the standard biochem- would be better accommodated in a single genus, and ical tests used in bacterial identification. However, a number therefore their unification was proposed (Birtles et al., of PCR-based methods for the identification and delineation 1995). The availability of improved molecular techniques of Bartonella species have now been described, targeting a for the characterization of bartonellae led not only changes range of genetic loci including fragments of 16S rRNA gene, in taxonomy but also promoted the discovery of several new the 16S/23S rRNA gene intergenic spacer region (ITS), the species. Today, an ever increasing number of bartonellae, citrate synthase-encoding gene (gltA), the 60-kDa heat- infecting a wide range of mammalian hosts, continue to be shock protein-encoding gene (groEL), a cell division encountered (Birtles et al., 1999; Breitschwerdt & Kordick, protein-encoding gene (ftsZ), the RNA polymerase b sub- 2000). In Europe, surveys of small woodland mammals in unit-encoding gene (rpoB) and others (Birtles et al., 2000; the UK, Sweden, Denmark and Greece (Birtles et al., 2001; Haupikian & Raoult, 2001a, 2001b). The ITS region, which Holmberg et al., 2003; Engbæk & Lawson, 2004; Tea et al., lies between the 16S and 23S rRNA genes within the rRNA- 2004) have shown that Bartonella infections are common, encoding operon, has been characterized in several Barto- and that at least four different species, Bartonella birtlesii, nella species and has been found to be somewhat larger than Bartonella doshiae, Bartonella grahamii and Bartonella tay- observed among other bacteria, ranging between 906 and lorii, circulate in rodent communities. Several other Barto- 1529 base pairs (bp). As most of the ITS is noncoding, it nella strains have also been encountered. They probably is prone to hypervariability and thus interspecies and

FEMS Immunol Med Microbiol 50 (2007) 45–50 c 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 46 N. Knap et al. interstrain ITS sequence variation is markedly higher than DNA extraction and PCR amplification of that observed at other genetic loci (Roux & Raoult, 1995). Bartonella ITS and ftsZ fragments Birtles and colleagues described the use of PCR-based We amplified Bartonella DNA directly from spleen samples, amplification of ITS fragments to detect and identify without prior bacterial cultivation. DNA extracts were pre- bartonellae in the blood of rodents. Direct detection was of pared from a small piece (1 mm3) of spleen from individual particular use in the longitudinal survey of bartonella s animals using QIAmp DNA Mini Kit (Qiagen, Gmbh, bacteraemia that involved the field collection of very small Hilden, Germany). Each sample of undiluted extracted DNA amounts of blood from live wild-living rodents (Birtles was incorporated into a Bartonella-specific PCR targeting an et al., 2000). However, although comparison of ITS ITS fragment (Minnick & Barbian, 1997). The thermal sequences is useful for the allocation of detected organisms programme employed was 35 cycles of 94 1C for 1 min, into one of the recognized Bartonella species, detection of a 55 1C for 1 min and 72 1C for 1 min. The success of each novel ITS sequence can be problematic. The hypervariability Downloaded from https://academic.oup.com/femspd/article/50/1/45/505104 by guest on 01 October 2021 amplification reaction was assessed by electrophoresis of an of this region makes meaningful alignment of sequences aliquot of the reaction mix after the thermal programme in a from different Bartonella species difficult, compromising its 2% agarose gel containing ethidium bromide and examina- use as a basis for phylogenetic inference. Other more tion under UV illumination. Some DNA extracts were also conserved loci such as gltA or ftsZ can be used to comple- incorporated into a Bartonella-specific PCR targeting a ftsZ ment ITS analysis for strain identification and can also serve fragment, as previously described (Zeaiter et al., 2002). The as a basis for phylogenetic inference (Zeaiter et al., 2002). success of PCR was gauged as described above. In this study, an attempt was made to extend the survey of the prevalence and diversity of Bartonella species in European DNA sequencing and phylogenetic analysis rodents by determining the diversity of organisms infecting animals from three ecologically-distinct regions of Slovenia. Sequencing of PCR products was carried out using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (ABI PRISM, PE Applied Biosystems, CA), as per the Materials and methods manufacturer’s instructions. The products of each reaction were resolved and translated into crude sequence data on an Trapping of rodents automatic DNA sequencer (ABI 310 Genetic Analyser, PE Rodents were captured between 1996 and 2000 at six sites in Applied Biosystems). These data were combined and ana- three different zoogeographic regions of Slovenia: the Sub- lyzed using SEQMAN and EDITSEQ software within the LASERGENE pannonian, Prealpine and Mediterranean regions. Ecologi- (2001) package (Dnastar, Madison, WI). The new sequences cal conditions in Slovenia are heterogenous due to different obtained were compared with sequences deposited in Gen- abiotic (climatic, tectonic, edaphic, orographic and litholo- Bank using the nucleotide BLAST program (National Center gic) conditions across the country, and on the basis of these for Biotechnology Information). Alignment and phyloge- parameters, the country has been divided into five zoogeo- netic analyses of ITS and ftsZ amplicons were performed graphic regions that possess characteristic flora and fauna using PAUP software, employing parsimony and distance (Mrsˇicˇ, 1997). Differences between the fauna in each region, methods (Version 4.0b10, Sinauer Associates, Inc. Publish- particularly the diversity and population dynamics of ro- ers, Sunderland, MA). The stability of inferred phylogenies dents and their ectoparasites, may influence the ecology of was assessed by bootstrap analysis of 1000 randomly gener- the Bartonella infections they maintain. The three regions ated sample trees. included in the current study are predominantly covered with broad-leaved forest, with the addition of coniferous Statistical analysis forest of boreal type in the Prealpine, oak forest in the Data were analyzed using SPSS for Windows, version 13.0 Mediterranian region and elements of mountains in the (SPSS Inc., Chicago, IL). The association between preva- Mediterranean and Subpannonian regions (Mrsˇicˇ, 1997). lence of infection and small mammal species, and between Animals were caught in overnight strategically-placed Elliot prevalence of infection and zoogeographic region, was special and Sherman type traps and were euthanized with evaluated using the w2 test, Fisher’s exact test, as appropriate. ether. Each animal was weighed, sized, sexed and identified P-values were two-sided and considered significant at a level as to species. In the laboratory, each animal was dissected of o 0.05. and internal organs (heart, lung, liver, kidney, spleen, bladder) were collected and stored at 70 1C until ana- À Results lyzed. From the captured animals we selected approximately the same number of every rodent species from each zoogeo- A total of 146 rodents belonging to four species, A. agarius graphic region. (striped field mouse), A. flavicollis (yellow-necked field

c 2007 Federation of European Microbiological Societies FEMS Immunol Med Microbiol 50 (2007) 45–50 Published by Blackwell Publishing Ltd. All rights reserved Molecular detection of bartonellae in rodents in Slovenia 47 mouse), A. sylvaticus (long-tailed field mouse) and (Table 2). Four of these sequence variants were closely C. glareolus (bank vole) were surveyed for bartonellae. All related to bartonella ITS sequences held on GenBank (Table rodents were captured in each of the three regions surveyed, 2). However, two (AF-s2, AA-m), although almost identical with the exception of A. agarius, which is not encountered at to one another (99.7% identity), were distinct from the ITS the Prealpine site. When possible, 15 representatives of each sequences of either the recognized Bartonella species or species in each region and approximately equal numbers of other partially characterized strains. The novel ITS region each gender were selected. Bartonella DNA was detected in sequences could be aligned with that of Bartonella clarrid- 59 rodents. The number of infected animals varied accord- geiae, although they were somewhat longer, sharing only ing to the species of the rodent (the differences are statisti- about 68% similarity. cally significant, w2 with exact P = 0.004) (Table 1). Partial (781 bp) ftsZ sequences were obtained from five Apodemus flavicollis had the highest overall prevalence of samples that yielded different ITS sequences. These se- infection (62.7%) and the highest prevalence in all three quences were aligned with one another and with other Downloaded from https://academic.oup.com/femspd/article/50/1/45/505104 by guest on 01 October 2021 regions. The lowest overall and regional prevalence was bartonella ftsZ sequences available in GenBank. One Barto- found in A. agrarius (26.6%). Regionally, the highest pre- nella variant was identical to B. grahamii and the other two valence was found in the Subpanonnian region (51.7%) were 99.4% and 96.4% similar to B. doshiae and B. taylorii, (P = 0.021, Fisher’s exact test) (Table 1). respectively. However, two novel genotype sequences were Fourteen ITS fragments were selected for sequencing on identical to each other and 95.6% similar to B. clarridgeiae. the basis of size differences observed on 2% agarose electro- A phylogenetic tree, constructed using parsimony and dis- phoresis and the provenance of the DNA extract from which tance methods, derived from ftsZ sequence alignment the product was obtained. Comparison of the sequence data showed a topology consistent with a phylogenetic tree obtained demonstrated six sequence variants among them inferred from ITS sequence alignment (Figs 1 and 2).

Table 1. Prevalence of infection with Bartonella in small mammals captured in three zoogeoraphic regions in Slovenia Zoogeographic region

Species Mediterranian Prealpine Subpannonian Total Apodemus ﬂavicollis 10/15 (66.7%) 7/13 (53.8%) 10/15 (66.7%) 27/43 (62.7%) Apodemus sylvaticus 3/13 (23.0%) 1/4 (25.0%) 7/15 (46.7%) 11/32 (34.4%) Apodemus agrarius 2/15 (13.3%) NPRa 6/15 (40.0%) 8/30 (26.6%) Clethrionomys glareolus 2/11 (18.2%) 3/15 (20.0%) 8/15 (53.3%) 13/41 (31.7%) Total 17/54 (31.4%) 11/32 (34.3%) 31/60 (51.7%) 59/146 (40.4%)

The number of infected individuals and the number tested by species and locality are provided; prevalence of infection and percent are provided in parentheses. Overall prevelence in a zoogeographic region and in rodent species are presented in bold. aNot present in this region.

Table 2. Description of selected samples for sequencing, according to small mammal species, zoogeographic region and length of amplicons Isolate Host species Region of trapping Species of Bartonella GenBank accession no. ITS/ftsZ AF-m A. flavicollis Mediterranean B. taylorii DQ155374 AF-p A. flavicollis Prealpine B. taylorii DQ155391/DQ155390 AF-s1 A. flavicollis Subpannonian B. taylorii DQ155387 AF-s2 A. flavicollis Subpannonian unknown Bartonella DQ155386/DQ155378 AS-m A. sylvaticus Mediterranian B. taylorii DQ155385 AS-p A. sylvaticus Prealpine B. taylorii DQ155384 AS-s A. sylvaticus Subpannonian B. taylorii DQ155383 AA-m A. agrarius Mediterranian unknown Bartonella DQ155376/DQ155388 AA-s A. agrarius Subpannonian B.grahamii DQ155375/DQ155389 CG-m C. glareolus Mediterranian B. taylorii DQ155382 CG-p C. glareolus Prealpine B. birtlesii DQ155381 CG-s1 C. glareolus Subpannonian B. taylorii DQ155379 CG-s2 C. glareolus Subpannonian B.doshiae DQ155380/DQ155377

Accession numbers are assigned by GenBank. AA, A. agrarius;AF,A. ﬂavicollis;AS,A. sylvaticus; CG, C. glareolus. – m, mediterranean; -p, Prealpine; -s, subpannonian. 1, ﬁrst isolate; 2, second isolate. For example, CG-s2 is the second isolate from the Subpannonian region in the C. glareolus species of small mammals.

B. elizabethae European rodents has been determined only in the UK, 100 AA-s Sweden, Denmark and Greece. No such study has yet been 100 64 B. grahamii carried out in Central Europe (Birtles et al., 2001; Holmberg AF-p 100 et al., 2003; Engbæk & Lawson, 2004; Tea et al., 2004). B. taylorii 100 Although Slovenia represents a small part of central Europe, B. henselae it is topographically extremely diversiﬁed and zoologically 88 CG-s2 rich. 100 B. doshiae The prevalence of Bartonella infections in European AA-m rodents has been found to range from about 17% in Sweden 100 AF-s2 100 to over 60% in the UK (Birtles et al., 2001; Holmberg et al., B. Clarridgeiae 2003; Engbæk & Lawson, 2004; Tea et al., 2004). The overall Fig. 1. Phylogenetic tree including Bartonella sequences from this study prevalence of Bartonella infection in Slovenian rodents was Downloaded from https://academic.oup.com/femspd/article/50/1/45/505104 by guest on 01 October 2021 and sequences found in GenBank generated from the analysis of 40.4%, although it varied between the three zoogeographic N-terminal region sequences of the ftsZ gene. The GenBank accession regions examined and the rodent species surveyed, from as numbers for ftsZ sequences of the representatives of the established high as 66.7% in A. ﬂavicollis in two regions to as low as Bartonella taxa used: B. taylorii–AF467756, B. elizabethae–AF467760, 13.3% in A. agrarius in the Mediterranian region. The B. grahamii–AF467753, B. doshiae–AF467754, B. clarridgeiae–AF141018, difference in the prevalence of infection might be due to B. henselae–AF061746. Statistical support, as shown by the percentage of 1000 bootstrap samples, is given for each clade. The names of different habitat preference and abundance of the rodent samples are explained in the footnote to Table 2. species, which is related to population size and density. Apodemus agrarius does not share the same habitat as

AF-m A. flavicollis and C. glareolus. Whereas the latter can be 63 AS-s found mainly in the forest, A. agrarius prefers humid AF-p territories and forest borders and it can only be found in AF-s1 three isolated locations nationally (Krysˇtufek, 1991). Apode- AS-m 70 mus flavicollis is the most common rodent in Slovenia and 88 AS-p was found to have the highest prevalence of infection, an CG-m 68 observation in concordance with the hypothesis of Kosoy CG-s1 100 et al. that the dominant species in a community has the B. birtlesii highest prevalence of infection (Kosoy et al., 2000). Long- CG-p 54 B. taylorii itudinal studies have suggested that seasonal dynamics could AA-s influence the prevalence in bartonellae infection in rodents 100 100 B. grahamii (Bajer et al., 2001, Kosoy et al., 2004a). However, the animals CG-s2 tested in this study were all collected during late summer or 95 CG-s3 early fall. It is most likely that several factors including the 100 B. doshiae host–parasite interactions contribute to the observed differ- AF-s2 100 ences in the prevalence of infection among species. 100 AA-m The prevalence of Bartonella infection in rodents in this B. clarridgeiae study, as in others conducted in Europe, is remarkably high Fig. 2. Phylogenetic tree including Bartonella sequences from this study (Birtles et al., 1995, 1999, 2001; Breitschwerdt & Kordick, and sequences found in GenBank generated from the analysis of partial 2000; Tea et al., 2004). One hypothesis suggests that the ITS sequences. The GenBank accession numbers for ITS sequences of cause of such a high prevalence might be long-term bacter- the representatives of the established Bartonella taxa used: B. taylorii– aemia, as seen in rodent models and in cats infected with AJ269788, B. birtlesii–AJ269791, B. grahamii–AJ269785, B. doshiae– AJ269786, B. clarridgeiae–AF167989. Statistical support, as shown by Bartonella henselae (Bouloius et al., 2001; Schulein et al., the percentage of 1000 bootstrap samples, is given for each clade. The 2001; Kosoy et al., 1997). To some extent, the high pre- names of samples are explained in the footnote to Table 2. valence of infection observed in our study could also be explained with the detection method used. As PCR does not only detect viable bacteria, it is possible that the prevalence Discussion of infection is overestimated, due to possible detection of remaining DNA from destroyed bacteria. However, genus- Rodents are important reservoirs of different bartonellae, specific PCR performed directly on DNA extracted from including some human pathogens (Birtles et al., 2001; samples and further sequencing of the amplified products Holmberg et al., 2003; Engbæk & Lawson, 2004; Tea et al., has been very successful for the purpose of this study. We 2004). Nevertheless, the presence of bartonellae infection in were able to bypass a labour-intensive and time-consuming

c 2007 Federation of European Microbiological Societies FEMS Immunol Med Microbiol 50 (2007) 45–50 Published by Blackwell Publishing Ltd. All rights reserved Molecular detection of bartonellae in rodents in Slovenia 49 cultivation step. The sample needed was very small. This is causes Carrion’s disease, and B. henselae, which causes cat particularly important because it indicates that a small scratch disease (Karem et al., 2000; Chomel et al., 2004). sample (blood) could be taken without the need to sacrifice However, in the last 15 years several other Bartonella species or maim the animal (Birtles et al., 2000). However, a have also been implicated as human pathogens, albeit to a drawback in PCR performed directly on DNA extracted far lesser degree than the three species mentioned above from samples is that not all primers are able to detect (Birtles et al., 1999). Among these is the European rodent- coinfections or even mixed infections, as the PCR products associated species B. grahamii, which has been associated from different species can be of the same length. It was with human neurotenitis and retinal artery occlusion (Ker- shown that the course of bartonellae infections could result khoff et al., 1999). We confirmed the presence of B. grahamii from a single, persistent and potentially multi-genogroup/ in Slovenia and extended its host range to include variant infection, during which variants differentially dom- A. agrarius. inate the detectable bacteraemia (Kosoy et al., 2004b). Comparison of phylogenetic information resulting from Downloaded from https://academic.oup.com/femspd/article/50/1/45/505104 by guest on 01 October 2021 We have been able to confirm the presence of four ITS – and ftsZ-based genotypic studies resulted in phyloge- recognized Old World rodent-associated bartonellae in netic trees comparable to phylogenetic results yielded in Slovenia: B. taylorii, B. grahamii, B. doshiae and B. birtlesii. other studies (Haupikian & Raoult, 2001a, b). Some of the Furthermore, on the basis of comparative analysis of ITS bootstrap values in the ITS phylogenetic tree are lower, due and ftsZ fragments, one additional genotype that may to a higher sequential diversity. However, FtsZ protein plays represent a new Bartonella species was encountered. The an important role in bacterial cell division, therefore its genotype was most similar to B. clarridgeiae. However, the nucleotide sequence is much more conserved and better degree of dissimilarity between the new genotype and the suited for phylogenetic analysis. It is important to note the published sequences of recognized species was well above close phylogenetic relationship of both sequences of the that recently proposed as a cut-off value for Bartonella novel genotype with the recognized human pathogen species definition (La Scola et al., 2003). B. clarridgeiae. To date, only one genotype, Af82Up, isolated A large number of genotypes from only 15 sequenced from rodents in Sweden clusters with B. clarridgeiae based samples confirms the great natural diversity of the genus. on gltA gene sequence analysis (Kordick et al., 1997; Holm- New species of Bartonella are demonstrated regularly from berg et al., 2003). Cultivation of the novel strain described animals such as rabbits and cattle to a well studied reservoir here would permit a more comprehensive phenotypic and like the cat (Droz et al., 1999; Heller et al., 1999; Maillard genotypic characterization and would also permit the devel- et al., 2004). This confirms previous suspicions about the opment of assays that could be used in investigating human lack of knowledge concerning the diversity of bartonellae exposure to the strain. (Birtles et al., 2000). Many authors have implied that the host specificity between bartonellae and their rodent hosts is imperfect References (Kosoy et al., 2000; Holmberg et al., 2003; Engbæk & Lawson, 2004). As we did not sequence all the amplified Bajer A, Pawelczyk A, Behnke JM, Gilbert FS & Sinski E (2001) products it is difficult to make predictions of our own. 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The other three Birtles RJ, Harrison TG, Saunders NA & Molyneux DH (1995) Bartonella species produced fragments of approximately the Proposals to unify the genera Grahamella and Bartonella, with same length (about 650 bp) differing by 1–30 bps, therefore descriptions of Bartonella talpae comb. nov., Bartonella it is impossible to predict their distribution in rodents. peromysci comb. nov, and three new species, Bartonella However, as such a high percentage of the sequenced grahamii sp. nov., Bartonella taylorii sp. nov., and Bartonella amplicons turned out to be B. taylorii, it is likely that at the doshiae sp. nov. Int J Syst Bacteriol 45: 1–8. time of the survey this was the most common Bartonella Birtles RJ, Canales J, Ventosilla P, Alvarez E, Guerra H, Llanos- species in rodents in Slovenia. 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